key: cord- -v xeqdz authors: izquierdo-suzán, mónica; zárate, selene; torres-flores, jesús; correa-morales, fabián; gonzález-acosta, cassandra; sevilla-reyes, edgar e.; lira, rosalia; alcaraz-estrada, sofía l.; yocupicio-monroy, martha title: natural vertical transmission of zika virus in larval aedes aegypti populations, morelos, mexico date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: v xeqdz we characterized natural vertical transmission of zika virus in pools of aedes aegypti larvae hatched from eggs collected in jojutla, morelos, mexico. of the pools analyzed, tested positive for zika virus rna; infectious zika virus was successfully isolated from of the larvae pools ( n) in c / cells. real-time quantitative pcr and indirect immunofluorescence assays confirmed the identity of the isolate, named zika virus isolate n; plaque assays in vero cells demonstrated the isolate’s infectivity in a mammalian cell line. we obtained the complete genome of zika virus isolate n by next-generation sequencing and identified single-nucleotide variants specific to zika virus isolate n using the meta-cats tool. these results demonstrate the occurrence of natural vertical transmission of zika virus in wild ae. aegypti mosquitoes and suggest that this transmission mode could aid in the spread and maintenance of zika virus in nature. we characterized natural vertical transmission of zika virus in pools of aedes aegypti larvae hatched from eggs collected in jojutla, morelos, mexico. of the pools analyzed, tested positive for zika virus rna; infectious zika virus was successfully isolated from of the larvae pools ( n) in c / cells. real-time quantitative pcr and indirect immunofluorescence assays confirmed the identity of the isolate, named zika virus isolate n; plaque assays in vero cells demonstrated the isolate's infectivity in a mammalian cell line. we obtained the complete genome of zika virus isolate n by next-generation sequencing and identified single-nucleotide variants specific to zika virus isolate n using the meta-cats tool. these results demonstrate the occurrence of natural vertical transmission of zika virus in wild ae. aegypti mosquitoes and suggest that this transmission mode could aid in the spread and maintenance of zika virus in nature. z ika virus is an enveloped, positive-sense, singlestranded srna, arthropod-borne virus (arbovirus) that is classified in the genus flavivirus, family flaviviridae. zika virus is closely related to other viruses of medical importance, such as dengue, west nile, and yellow fever viruses ( ) . zika virus was discovered in uganda in , but it has attracted the attention of specialists in the past few years because of its rapid spread through the pacific and into the americas in , as well as the severe neurologic manifestations associated with zika virus infections, such as neonatal microcephaly and guillain-barré syndrome ( ) . several studies carried out under laboratory conditions have demonstrated that zika virus can infect many different aedes mosquito species ( ) ; still, the key species for the transmission of zika virus to humans are ae. aegypti and ae. albopictus ( ) ( ) ( ) . in this study, we focused on the species ae. aegypti, which has an urban behavior and is usually in close contact with humans ( ) . to date, different mechanisms by which ae. aegypti mosquitoes can become infected with flaviviruses have been described: horizontal transmission and vertical transmission ( ) . horizontal transmission is considered the most common mode of transmission of arboviruses between mosquitoes and their vertebrate hosts and is responsible for the maintenance of arboviruses in nature, particularly during disease outbreaks. in contrast, the environmental maintenance of dengue virus (denv) during interepidemic periods is thought to be caused by vertical transmission of the virus from the infected adult mosquitoes to their offspring for > successive generations ( ) ( ) ( ) . both mechanisms together are thought to be essential for the survival of viral pathogens in their habitats, preventing their extinction during harsh environmental conditions or in populations in which the presence of susceptible mammal hosts is low. vertical transmission of zika virus in ae. aegypti mosquitoes has been evaluated under laboratory conditions by searching for the presence of zika virus rna in several organs from the offspring of infected mosquitoes, demonstrating the presence of viral rna in the guts and salivary glands of the offspring ( ) . additional studies have also demonstrated the presence of infectious zika virus in the offspring of artificially infected ae. aegypti and ae. albopictus mosquitoes, suggesting that vertical transmission can occur in laboratory-bred mosquitoes ( ) . however, evidence is insufficient to confirm that vertical transmission is a principal maintenance mechanism for zika virus in wild aedes mosquitoes. in this study, we sought to demonstrate natural vertical transmission in ae. aegypti mosquitoes by detecting viral rna and isolating infectious zika virus from larvae hatched from field-collected eggs. we also assessed the infectivity of the isolate in a mammalian cell line and obtained the complete genome of the virus by next-generation sequencing (ngs). we collected mosquito egg samples in the municipality of jojutla, located in the southern region of the state of morelos, mexico ( ° ′n, ° ′w). arboviral infections are common in this municipality, which is usually hot and dry during october-april and warm and humid during may-september. during , the incidence of zika virus infections increased in morelos; cases were reported by the end of the year, of which cases were from jojutla. the reported cases in jojutla were recorded as follows: in august, in september, in october, in november, and in december ( ) . we collected ae. aegypti eggs in collaboration with the national center of preventive programs and disease control (cenaprece), following regulations for entomological surveillance with ovitraps (nom- -ssa - ). in , ovitraps were placed and maintained throughout the year in different locations in jojutla ( figure ). we determined the locations of the ovitraps on the basis of the incidence of human arbovirus infections. we collected the eggs using filter paper, which we then air dried for days and stored in paper bags for further use. we placed the egg papers in water containers and incubated them at °c for wk under -h light/dark cycles. after the incubation period, we collected larvae in stages - and separated them into pools of - larvae. we macerated each of the pools in viral transport media ( % bsa, u/ml penicillin, µg/ml streptomycin, . µg/ml fungizone, and nahco in hank's solution) using a pestle mixer (thomas scientific, https://www.thomassci. com) and stored pools at - °c for further analysis. we incubated the macerated larvae pools with trizol (invitrogen, https://www.thermofisher.com) for min, extracted total rna from the lysates using zymo-spin rna extraction columns (zymo research, https://www.zymoresearch.com), and quantified the rna with a nano drop (thermo scientific, https://www.thermofisher.com). we detected the presence of zika virus rna by quantitative reverse transcription pcr (qrt-pcr) using the taq-man system (https://www.thermofisher.com) with primers zikv fw ′-ccgctgcccaacacaag- ′ and zikv c rv ′-ccactaacgttcttttgcagacat- ′; and probe zikv -fam probe ′-agcctacctt-gacaagcagtcagacactcaa- ′, as previously described by lanciotti et al. ( ) . we calculated the minimum infection rate (mir) of the larvae pools by dividing the number of positive larvae pools by the total number of larvae tested and multiplying by , . we selected the larvae pools that tested positive for zika virus rna and displayed the lowest quantitation cycle (cq) values (range . - . ) for viral isolation. in brief, we diluted macerated larvae pools in maintenance medium (emem medium with % fbs, u/ml penicillin, and µg/ml streptomycin) and filtered them through . µm membranes (millex, http://www.emdmillipore.com). twenty-four hours before infection, we seeded c / cells in -well plates at % of confluence in growth medium (emem medium supplemented with % fbs, u/ml penicillin, and µg/ml streptomycin) and incubated them at °c in a % co atmosphere. cells were adsorbed with the clarified larvae macerates for h at °c. after the incubation period, we added fresh maintenance medium to the cells and left the infection to proceed for d at °c and a % co atmosphere until a cytopathic effect was observable. we tested the supernatants of the infected cells for the presence of zika virus rna by qrt-pcr, as described earlier in this section. we performed passages of each isolate in c / cells to increase viral titers for ngs. for the indirect immunofluorescence assays, we grew vero cells over glass coverslips and then infected them with the zika virus isolate. at hours postinfection, we fixed cells with % paraformaldehyde and then incubated them with ice-cold methanol for min. we blocked the cells using % fbs in phosphate-buffered saline (pbs) for h and then incubated them overnight at °c with a : dilution of the monoclonal antibody g . after the incubation period, we washed the cells twice with pbs and then incubated them with an alexa fluor -conjugated goat antimouse igg antibody (jackson immunoresearch, https://www.jacksonimmuno.com) for h. we washed the cells twice with pbs and then mounted them with vectashield medium with dapi (vector laboratories, https://vectorlabs.com) for confocal microscopy (nikon, https://www.nikon.com). for the plaque assays, we seeded vero cells in -well plates until they reached a confluence of %. we used -fold serial dilutions of the zika virus isolate to infect the cell monolayers and left them to adsorb for h. after the adsorption period, we removed the virus and overlaid cell monolayers with ml of dmem (invitrogen) supplemented with % carboxymethyl-cellulose (sigma-aldrich, https://www.sigmaaldrich.com), % fbs, u/ml penicillin, µg/ml streptomycin, and mm l-glutamine (gibco, https://www.thermofisher.com) and incubated at °c for d. we fixed the cells with % paraformaldehyde in pbs and counterstained them with crystal violet-formaldehyde (sigma-aldrich). for the complete genome sequencing of the zika virus isolate, we depleted ng of total rna extracted from the supernatants of infected vero cell monolayers of rrna using the nebnext rrna depletion kit (human/mouse/rat) following the manufacturer's instructions (new england biolabs, https://www.neb.com). we constructed rnaseq illumina shotgun libraries at the unidad universitaria de secuenciación masiva y bioinformática-instituto de biotecnología and sequenced them using paired-end sequencing with a myseq system (illumina, https://www.illumina. com). we assessed the quality of reads using fastqc (http://www.bioinformatics.babraham.ac.uk/projects/ fastqc); low-quality positions and reads were eliminated using in-house scripts. we assembled the valid reads without reference using the program trinity . ( ) and evaluated the assembly using qualimap ( ) . finally, we verified the identity of the contig using blastn (http://blast.ncbi. nlm.nih.gov/blast.cgi). to determine the phylogenetic relatedness of the zika virus isolate, we retrieved relevant zika virus open reading frame (orf) rna sequences from the virus pathogen resource (vipr) database ( ) (http://www.viprbrc.org). we removed identical sequences and those with undetermined bases from alignment. we applied near-identity clustering ( . ) to the remaining sequences in cd-hit-test ( ) and used sequences for phylogenetic inference by maximum likelihood in raxml ( ) under a general time-reversible plus gamma substitution model. we reconstructed the tree with bootstrap replicas. we normalized branch lengths and condensed nodes with bootstrap values < % to emphasize tree topology. to determine the presence of variants in the larva-derived sequence, we used the alignment to identify positions that had mutations unique to our sequence or that were primarily shared with other mosquito-derived sequences and that were absent or rare in the human-derived genomes. to this end, we examined the alignment with the tool meta-cats ( ) , which performs a χ test to find positions with different polymorphism distribution between groups. during the study period, we analyzed larvae pools by qrt-pcr in search of zika virus rna. only ( . %) of the pools tested positive: ( . %) from the larvae raised from the eggs collected in june and ( . %) from larvae raised from the eggs collected in november. to determine the proportion of infected larvae in the population, we calculated mirs for the collection periods (june and november ) and found an increase in the mir observed from the larvae raised from the eggs collected in june ( . ) to the mir from the eggs collected in november ( . ) ( table ) . to confirm the presence of infectious zika virus in the larvae pools, we used of the pools that tested positive for zika virus rna that displayed the lowest cq values to attempt viral isolation. we were able to isolate infectious zika virus from only of the larvae pools ( n), as determined by the presence of cytopathic effect in c / cells characterized by monolayer detachment (figure , panel a) and the detection of zika virus rna in culture supernatants by qrt-pcr with a cq value of . . the ability of the n isolate to infect a mammalian cell line was confirmed by the appearance of the characteristic cytopathic effect of zika virus in infected vero cell cultures, characterized by cell rounding and detachment (figure , panel a) . moreover, immunofluorescent detection of the viral envelope (e) protein using the monoclonal antibody g revealed perinuclear staining (figure , panel b). the plaque assays carried out in vero cells revealed that the n isolate has the ability to produce lytic plaques; thus, this isolate can be considered cytopathic in mammalian cell culture (figure , panel c). we isolated viral rna from the supernatant of vero cells infected with the fourth passage of zika virus isolate n and processed it by rna sequencing by the ngs miseq illumina protocol. around % of the total reads ( , , ) were assembled in a single contig of , nt in length, with a mean depth of , reads. blast results of the assembled contig revealed that the strain zikv/aedes.sp/mex/mex_i- / , which belongs to a zika virus isolated from aedes mosquitoes obtained in the state of chiapas, mexico, had the highest identity ( . %) with our isolate. we identified single-nucleotide variants (snvs) in the zika virus isolate n genome by aligning the sequence with other mosquito-and human-derived sequences obtained from the vipr database and running the meta-cats analytic tool, as described in the materials and methods section. to carry out this analysis, we grouped the sequences by the host of origin. we identified snvs in positions , , and that were unique to the larva genome. on the other hand, the snvs in positions and were found in human-derived sequences but were not present in other mosquito genomes. the snv in position was shared only with a sequence from french polynesia, likely an example of parallel evolution, whereas the polymorphism in position was common with sequences from mexico and may correspond to a local variant. finally, we found snvs that were also present in other human and mosquito sequences but were more common in mosquitoes. only snv was found in the e gene, whereas the rest were found in nonstructural (ns) genes: in ns a, in ns , in ns , and in ns (table ) . to characterize the evolutionary relationship between the n isolate and other zika virus genomes that have been previously reported, we performed a phylogenetic analysis. the phylogenetic reconstruction of the complete genomes of zika virus sequences, including the n isolate, revealed that the n isolate belongs to the asian-american lineage of zika virus and clusters together with other sequences of human and mosquito origin from mexico ( figure ). natural vertical transmission of mosquito-carried viruses has been proposed as one of the main ecologic processes involved in the maintenance of these viruses in susceptible mosquito populations, particularly during interepidemic periods and harsh climate conditions when horizontal transmission becomes difficult ( , ) . in mexico, most of the studies regarding natural vertical transmission of arboviruses in mosquitoes have been carried out using denv as a study model ( ) ( ) ( ) , so the diversity of viruses that can be transmitted from infected adult mosquitoes to their offspring still needs to be characterized. only a few studies have addressed the natural vertical transmission of zika virus in wild mosquito populations; most of these have been carried out in brazil, where zika virus rna has been detected in male ae. aegypti mosquitoes and in adult ae. albopictus mosquitoes raised from field-collected eggs ( , ) . in this study, we demonstrated the occurrence of natural vertical transmission of zika virus in wild mosquito populations from the municipality of jojutla in the state of morelos, mexico. the rna of zika virus was detected in larvae pools; the rates of vertical transmission of zika virus in the wild ae. aegypti populations were estimated by calculating the mir. in morelos, the rainy season begins in late may and extends through the end of september, after which both the precipitation and the mosquito populations start to decrease. thus, the higher mir ( . ) calculated from the larvae hatched from the eggs collected in november , in contrast to the mir from the larvae hatched from the eggs collected in june ( . ), might be correlated with the increased number of human zika virus cases that were reported in jojutla during november, whereas during june no human cases were reported. the absence of reported zika virus human cases in june could be the result of asymptomatic cases, cases clinically misdiagnosed as dengue virus infections, or both. the higher number of persons infected with zika virus in jojutla during november reflects an increase in the number of infected mosquitoes resulting from horizontal transmission of the virus between mosquitoes and infected humans. it is possible that vertical transmission is contributing to the number of infected mosquitoes, which are, in turn, capable of transmitting this virus to a higher number of humans. however, the relative importance of vertical transmission for the maintenance and spread of the virus cannot be elucidated with the data available so far. the mir is usually affected by the number of mosquitoes that make up the pool of mosquitoes (or their immature stages) tested, which usually causes an underestimation of the real number of infected mosquitoes in a population ( ) . thus, the rate of vertical transmission in the mosquito populations from morelos might be even higher than estimated. previous studies performed with other flaviviruses, such as denv, have reported mirs as low as . ( ) and . ( ) or as high as ( ) in wild larvae, usually associated with the collection date and the place of sampling. under laboratory conditions, the filial infectious rate of zika virus in ae. aegypti mosquitoes ranged from . to . , depending on the strain of the virus tested ( ) , which corroborates that our results are comparable to the mirs reported for other zika viruses and other flaviviruses, including denv ( , ) . in this work, we were also able to demonstrate the natural vertical transmission of zika virus in ae. aegypti mosquitoes by the successful isolation of infectious zika virus ( n) from larvae raised from field-collected eggs. this evidence strongly suggests that infectious zika virus can be transmitted from adult female mosquitoes to their offspring and increases the evidence of the role of natural vertical transmission in the maintenance of zika virus in wild mosquito populations. the isolation of infective zika virus vertically transmitted from adult mosquitoes to their offspring suggests that this virus could be potentially transmissible between mosquitoes and their vertebrate hosts; nevertheless, this transmission still needs to be demonstrated. moreover, we were able to detect several snvs in the larvae-derived zika virus isolate n, of which were specific to this larvaederived genome, as well as others that were shared between the larvae and other mosquito and human sequences. although it is plausible that these snvs were acquired during virus culture, we think this is unlikely because of the sequences included in the alignment corresponded to cultured viruses and none of them presented these mutations; however, the sequences from more larvae-derived viruses need to be determined to establish the significance of these snvs. the larvae-specific snvs were located in the coding regions for the ns a and ns proteins, which are involved in the replication of the viral rna ( ) . nevertheless, whether the larvae-specific snvs are associated with the maintenance and vertical transmission of infectious zika virus from adult mosquitoes to their offspring still needs to be determined. in summary, we demonstrated natural vertical transmission of zika virus in wild ae. aegypti mosquitoes. our results suggest that this transmission mode could aid in the spread and maintenance of zika virus in nature, expanding the ongoing zoonotic threat from this virus to human health. structural biology of zika virus and other flaviviruses zika infection and the development of neurological defects vector competence of aedes aegypti, culex tarsalis, and culex quinquefasciatus from california for zika virus zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently dengue, chikungunya … and the missing entity-zika fever: a new emerging threat the global compendium of aedes aegypti and ae. albopictus occurrence. sci data transovarial transmission of denv in aedes aegypti 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mosquitoes and eggs, brazil patient-based dengue virus surveillance in aedes aegypti from recife, brazil transovarial transmission of dengue virus in aedes aegypti and aedes albopictus in relation to dengue outbreak in an urban area in malaysia. new delhi: who regional office for south-east asia effects of zika virus strain and aedes mosquito species on vector competence natural vertical transmission of dengue viruses in aedes aegypti in selected sites in cebu city first evidence of vertical infection of dengue virus in aedes aegypti mosquitoes from sinaloa, mexico. vector borne zoonotic dis biochemistry and molecular biology of flaviviruses we thank jorge luis peralta rodríguez and carlos marx ramírez huicochea for their help in the mosquito collection and identification and vadim pérez koldenkova for his support with the confocal microscopy at the laboratorio nacional de microscopia avanzada/centro medico nacional siglo xxi, imss. this work was supported by grants conacyt , conacyt , and conacyt . ms. izquierdo-suzán is a graduate student at universidad autónoma de la ciudad de méxico, mexico city, mexico. her primary research interests are molecular and ecological aspects of emerging infectious diseases in wildlife. key: cord- -xlk xpv authors: mögling, ramona; zeller, hervé; revez, joana; koopmans, marion; reusken, chantal title: status, quality and specific needs of zika virus (zikv) diagnostic capacity and capability in national reference laboratories for arboviruses in eu/eea countries, may date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: xlk xpv with international travel, zika virus (zikv) is introduced to europe regularly. a country's ability to robustly detect zikv introduction and local transmission is important to minimise the risk for a zikv outbreak. therefore, sufficient expertise and diagnostic capacity and capability are required in european laboratories. to assess the capacity, quality, operational specifics (guidelines and algorithms), technical and interpretation issues and other possible difficulties that were related to zikv diagnostics in european countries, a questionnaire was conducted among national reference laboratories in countries in the european union/european economic area (eu/eea) in may . while the coverage and capacity of zikv diagnostics in the eu/eea national reference laboratories were found to be adequate, the assessment of the quality and needs indicated several crucial points of improvement that will need support at national and eu/eea level to improve zikv preparedness, response and eu/eea zikv surveillance activities. zika virus (zikv) infections were historically not considered a significant public health concern [ ] . however, in the year following its first autochthonous transmission in the americas in , zikv has been linked to severe congenital anomalies in newborns and to other neurological disorders such as guillain-barré syndrome (gbs) [ ] . the world health organization (who) declared the cluster of microcephaly cases and other neurological disorders possibly associated with zikv a public health emergency of international concern (pheic) on february [ ] . by the end of , the unprecedented zikv outbreak affected countries and territories in the americas with more than half a million human cases [ ] . the pheic was declared over on november with the argument that zikvassociated congenital syndrome was considered to be a long-term public health challenge requiring a longterm commitment to control and prevent [ , ] . zikv is a mosquito-borne virus which belongs to the genus flavivirus, family flaviviridae [ ] . its genome can be detected in biological samples by reverse transcription pcr (rt-pcr) and the virus can be isolated in cell culture. however, viraemia is typically short-lived ( - days after onset of symptoms) [ ] [ ] [ ] , although an increased window of detection has been observed using urine (up to days after onset of symptoms) [ ] or whole blood (up to days after onset of symptoms) [ ] . consequently, the full spectrum of diagnostics includes serology, which is complex owing to extensive cross-reactivity with antibodies triggered by other flaviviral infections and/or vaccination [ ] . the majority of zikv infections are asymptomatic which complicates retracing the course of infection and increases the dependency on serology to confirm an infection [ ] . this is in particular an issue for pregnant women for whom correct diagnosis of a zikv infection, even if asymptomatic, is imperative [ , , ] . international travel and outbreaks of zikv in european overseas countries and territories are responsible for the regular introduction of zikv to europe [ ] [ ] [ ] [ ] . a risk assessment by the who regional office for europe (who/europe) indicated that the risk for an outbreak with zikv in europe should not be underestimated, in particular in countries with established presence of the vectors aedes aegypti and ae. albopictus [ , ] . a country's ability to robustly detect zikv introduction and local transmission is important to minimise the risk for a zikv outbreak. therefore sufficient expertise and diagnostic capacity and capability in european laboratories are required [ ] . to map zikv expertise and identify diagnostic capacity and capability gaps in europe during the initial phase of the pheic in february , the european commission (ec) asked the european centre for disease prevention and control (ecdc) for a rapid assessment of the capacity of laboratories in europe to detect zikv infections and the specific needs for support. the assessment was done by an in-depth questionnaire in may . here, the outcomes of the assessment are presented and discussed to identify knowledge and technical gaps to strengthen laboratory capacity and quality for zikv diagnostics in the european union/european economic area (eu/eea) and to support zikv preparedness and response as well as zikv surveillance activities in the eu/eea. a questionnaire was designed to address the capacity, quality, operational specifics (guidelines and algorithms), technical and interpretation issues and other possible difficulties related to zikv diagnostics. national reference laboratories for zikv diagnostics were asked to complete the online questionnaire through the eu/eea ecdc national focal points for microbiology (nmfps) [ ] . the questionnaire was sent to the nmfps on may and closed on may . as most questions in the questionnaire where not obligatory to answer, the sums in the presented data may vary as non-replies are not listed. in total, laboratories from eu/eea countries completed the questionnaire, of which laboratories in countries indicated that they were conducting zikv diagnostics by may (figure ). zikv infection was made notifiable in countries by may , and mandatory disease notification was planned for the near future in nine countries. for one country, the notification procedure was not specified. in agreement with the eu classification [ , ] , laboratories indicated that their operational biosafety level (bsl) for zikv diagnostics was bsl . eight laboratories indicated bsl as their operational biosafety level, while two laboratories performed zikv diagnostics at bsl . reasons given by laboratories to deviate from the official level bsl were (i) downgrading because diagnosis did not involve high titre virus culture (one laboratory), and (ii) upgrading based on inhouse assessment of biosafety and biosecurity issues or for logistical reasons such as aligning zikv diagnostics with other flavivirus diagnostic serology and virus isolation (three laboratories). the remaining six laboratories did not indicate reasons for the deviation. because of, at the time putative [ ] , teratogenic effects of zikv infection in pregnant women, laboratories conducting zikv diagnostics were asked whether special regulations for pregnant employees were in place. in six of the laboratories that answered this question, pregnant employees were not allowed to perform zikv diagnostic tests. in three additional laboratories, a restriction for pregnant employees was limited to handling zikv virus culture. the remaining laboratories did not have specific guidelines for pregnant employees. all laboratories conducting zikv diagnostics (n = , in countries) performed molecular testing, while laboratories in countries also conducted serology and one laboratory performed antigen detection. the laboratories accepted different types of patient samples, including plasma/serum, urine, semen, amniotic fluid, placenta, saliva and nasopharyngeal swabs. all the number of laboratories that participated in the questionnaire on invitation by their country nmfp in the period - may are indicated per country. one country indicated in january that they had implemented zikv diagnostics since may . two countries without implemented zikv diagnostics indicated to have access to diagnostics through an agreement with a laboratory in another country. laboratories received serum samples for primary diagnostics, and in addition, clinicians often provided urine as a second sample for testing ( / ). semen ( / ), saliva ( / ) and nasopharyngeal swabs ( / ) were received to a lesser extent. for an adequate choice of diagnostic tests to run and for a correct interpretation of results, a minimum of information on the patient history is required [ ] [ ] [ ] . laboratories were asked to indicate on a likert scale with - range ( = never, = always) how often certain essential background information about their patient samples was provided with the diagnostic requests ( figure ). overall, the provision of the date of sampling was scored the highest ( / laboratories scored this in category 'often' or 'always'), while the flavivirus vaccination status scored the lowest ( / scored it as 'often' or 'always'). other data essential for choice of testing algorithm and test interpretation such as 'first day of illness', 'country and dates of travel', 'clinical symptoms' and (in case of women) 'pregnancy status' were provided with requests in to of the laboratories. most laboratories ( / ) indicated that they could directly contact physicians to ask for more information about the requested samples or look up record sheets and laboratory reports. however, in practice the feasibility of this strongly depended on the daily sample load. twenty-one laboratories that conducted zikv molecular detection only used commercially available tests, laboratories only in-house tests, while nine laboratories indicated using both. the realstar zika virus [ ] . primary rt-pcrs were mostly carried out with a single zikv genome target ( / ). however, two laboratories performed a multiplex pcr targeting multiple viruses which were not specified further. positive results were confirmed independently in of laboratories. this was done either by a second rt-pcr targeting a different genome segment ( / laboratories) or through sequencing ( / ). forty-three of laboratories used a positive control, which was obtained through the european virus archive (https:// www.european-virus-archive.com) in six, through the robert koch institute (via former enivd expert laboratory network) in , from own patient materials in , or by using synthetic rna in four of laboratories. twenty-two laboratories indicated that they used another positive control, but not the specific source. eighteen of laboratories used zikv strains of the current outbreak in the americas as a control, while used strains belonging to the original (pre- ) asian lineage. african lineage strains were used in laboratories, had access to more than one zikv lineage as control. on both zikv igm and igg ( / ). ten laboratories in countries were able to assess the presence of zikv neutralising antibodies by plaque reduction neutralisation (prnt) and/or virus neutralisation (vnt) tests. thirty-one of laboratories carried out commercial serology assays. laboratories either used the euroimmun ag anti-zika virus elisa igm/igg ( / ), euroimmun arboviral fever mosaic if kit ( / ), both assays ( / ) or did not specify which assay they used ( / ). thirteen of laboratories used in-house assays, including elisa ( / ) and/or immunofluorescence assays (ifa) ( / ), as well as vnt ( / ) and prnt ( / ). the majority of these laboratories ( / ) indicated that they obtained their positive control material from own patient samples. four of laboratories were provided with zikv igm/igg positive control samples by collaborators. to gain insight in the level of quality control at the laboratories that performed zikv diagnostics, the laboratories were asked to specify their level(s) of laboratory accreditation. analysis at the laboratory level showed that international organization for standardization (iso) ('medical laboratories -requirements for quality and competence' [ ] another quality aspect concerns the extent of validation of the implemented diagnostics, in particular the serology in view of extensive cross-reactivity. the median size of in-house validation panels of confirmed zikv and wnv patients was small (table ) and serum samples from pregnant women and population panels from zikv-endemic regions were lacking in most laboratories (table ) . thirty-seven of laboratories indicated that they were willing to share validation data with other laboratories. however, of the zikv diagnostic laboratories indicated that their accreditation scheme did not accept validation done elsewhere, while this would be a possible option for of laboratories. laboratories were asked to indicate how many diagnostic samples they could process per week for the different types of test that they run ( figure ). diagnostic capacities of individual laboratories differed depending on the type of diagnostic test. in addition, the laboratories were asked how many samples they had processed and determined positive for molecular, igm, igg and neutralising antibody testing since january (table ). for of laboratories offering molecular testing, the cumulative number of total requests in the -week period covered in the questionnaire (including requests in support of other laboratories) remained below their indicated capacity per week. for of laboratories, the cumulative number of requests was approximately two or three times the indicated weekly capacity, while for three laboratories, it exceeded their capacity (five to -fold). for serology, a vast majority of the laboratories had a cumulative number of requests for the -week period that was two to three times their weekly capacity. fourteen of laboratories indicated that they supported other laboratories by performing molecular tests for them. fifteen laboratories supported others with serological testing and laboratories with provision of control materials. the majority of the laboratories ( / ) that had not offered laboratory support laboratories mainly implemented the zikv testing algorithms either as advised by ecdc ( / ) [ ] or by their national public health institutes ( / ). the remaining eight laboratories followed, among other algorithms, the zikv diagnostic algorithms advised by the pan american health organization (paho) [ ] . in case necessary background information such as first day of illness was not known, ten of the diagnostic laboratories performed both molecular and serological tests on the available samples, three always carried out serology, two always performed rt-pcr, while three only conducted the tests that were asked for by the clinician. ten laboratories either asked for more information or an additional sample from the patient for an interpretation based on kinetics. the remaining did not provide that answer. laboratories were also asked if there was a different testing algorithm for asymptomatic pregnant women with putative zikv exposure. ten laboratories indicated that they asked for paired serum samples of asymptomatic pregnant women for serology, while laboratories did both molecular and serological testing on the available samples. eight laboratories referred to their national zikv diagnostic algorithm and one laboratory indicated that asymptomatic patients were never tested regardless of pregnancy status. laboratories were asked to describe the main challenges they were faced with during the implementation of zikv molecular and/or serological diagnostics. the main indicated obstacles were the availability of a positive control and validation materials for molecular and serological tests, the availability of commercial serological tests and personnel capacity (figure ). in may , there was an eu/eea-wide coverage for zikv molecular diagnostics. only three countries were without in-country zikv molecular diagnostics but all had access to diagnostics through an agreement with a laboratory in another country and two had plans to implement zikv diagnostics in the near future. of these, one country had implemented zikv molecular diagnostics by january (figure ). in comparison to a february snapshot for the ec (data not shown), four more eu/eea countries had implemented zikv molecular diagnostics in may. the coverage for zikv serology increased from eu/eea countries to . access to zikv serology is particularly important because the confirmation or ruling out of a zikv infection during pregnancy is essential for medical followup regarding the teratogenicity of zikv [ , , ] . as an estimated % of zikv infections are asymptomatic and the genome detection window in serum is short [ ] , zikv diagnosis in pregnant women will often rely on zikv antibody detection in paired serum samples. an important aspect of conducting zikv serology is expertise about flaviviruses because the interpretation is complex [ , , ] . all laboratories conducting zikv serology indicated that they had experience with serodiagnostics for at least one other flavivirus. in addition, insight in the specificity and sensitivity of the serology tests used is required for proper test interpretation, but this appeared limited at the time of the questionnaire because adequate validation panels were lacking. validation data provided by commercial entities typically need to be confirmed in order to be acceptable for an accreditation scheme [ ] . similarly, validation of such assays performed in another laboratory may be insufficient to meet with accreditation requirements. indeed, the availability of validation panels for serology was indicated as the biggest challenge for implementation of zikv diagnostics by of zikv diagnostic laboratories. five of the zikv diagnostic laboratories did not have any kind of iso accreditation, while of laboratories did not have the most relevant iso accreditation [ ] . another concern is the broad reliance for zikv serology on one or two commercial tests, although it should be noted that in may , the commercial market for zikv serology offered very limited options. in december , no commercial serology test had been accepted for procurement through the who emergency use assessment and listing procedure, which requires extensive validation [ ] . this illustrates the importance of reference laboratory capacity. virus neutralisation is still considered the most specific flavivirus serology test, although cross-reactivity can be observed in patients with other flavivirus infections and research is ongoing to develop more specific assays [ , , , ] . broad implementation of this confirmatory test in eu/eea national reference laboratories is, however, expected to increase the reliability of serology results in returning travellers as the flavivirus background (in particular dengue) in european travellers is likely to be low. molecular testing, having capacity/capability to diagnose zikv, relied in of laboratories only on commercial assays, in laboratories only on in-house testing and in nine laboratories on both commercial and in-house testing. only one (lanciotti et al. [ ] ) of the two in-house tests that were most frequently used, was recommended based on in silico analysis [ ] and in an independent comparative study of zikv molecular tests [ ] , raising possible concerns about the performance of diagnostics in some laboratories. in november , the ecdc-funded emerging viral disease laboratory expert network (evd-labnet [ ] ) organised an external quality assessment (eqa) for member laboratories which included the majority of the national reference laboratories with zikv diagnostics that participated in this questionnaire. the results from such an eqa will provide points of improvement to the individual laboratories [ ] . for an adequate choice of which type of test to use and for correct interpretation of test results, a defined set of information on the patient history is essential. as shown for other diagnostics, the lack of information provided by clinicians requesting the diagnostics proved to be a major gap [ ] . although the date of sampling was given most often ( / scored 'often' or 'always'), this information is hardly meaningful without an indication of the first day of illness (only scored in the highest categories by laboratories). reliable interpretation of zikv serology is impeded without information on previous flavivirus infections or vaccinations that was often/always available in only of laboratories. reimbursements rules for the diagnostic tests differ between countries (data not shown). if the state covers the costs for the diagnostic tests, provision of necessary background information can be mandatory. the overall zikv diagnostic capacity in the eu/eea countries appeared to be sufficient, given the total number of reported requests vs the indicated capacities. at country level, the extent of under/overcapacities varied and were complicated by the fact that laboratories with certain iso accreditations are bound by strict regulations when sending a surplus of samples to a backup laboratory. the availability of validation materials, positive controls and personnel were indicated as the main challenges for implementation of zikv diagnostics in the reference laboratories of the eu/eea countries. that of laboratories indicated the cost of commercial tests as an obstacle for test implementation and the large dependency of the laboratories on commercial assays, while five of laboratories did not receive funding for development and/or implementation of in-house tests, illustrate an achilles' heel in the preparedness for emerging infections and needs careful consideration when defining strategies to strengthen laboratory preparedness and response for future outbreak situations. while the coverage and capacity of zikv diagnostics in eu/eea national reference laboratories were observed to suffice in may , the assessment of the quality and needs indicated several crucial points of improvement that will need support at national and eu/eea level. all reference laboratories should seek to have a relevant iso accreditation. awareness and facilitation of (temporary) acceptance of laboratory accreditation for novel diagnostics implemented in emerging situations is required, although it is currently hampered by lack of availability of well-defined validation panels. improved access is required to controls and validation panels for both molecular and serological tests. pending the development of more specific serology tests, a broader implementation of the current most specific neutralisation tests (prnt, vnt) is desirable, ideally in a comparative setting with other relevant flaviviruses. increased awareness is needed among clinicians to provide all necessary background information, and systems should be implemented that assure provision of necessary interpretation data. national and/ or eu contingency funding should be established to ensure adequate and robust laboratory preparedness and response. zika virus outside africa zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: systematic review who statement on the first meeting of the international health regulations : the year zika evolved from an emergency into a long-term public health challenge. washington: paho fifth meeting of the emergency commitee under the international health regulations ( ) regarding microcephaly, other neurological disorders and zika virus background review for diagnostic test development for zika virus infection detection of zika virus in saliva detection of zika virus in urine zika virus infection and prolonged viremia in whole-blood specimens zika virus: a new threat to human reproduction cdc guidelines for pregnant women during the zika virus outbreak travel-associated zika virus disease acquired in the americas through february : a geosentinel analysis assessing seasonal risks for the introduction and mosquito-borne spread of zika virus in rapid risk assessment. zika virus disease epidemic, tenth update rapid risk assessment. zika virus disease epidemic. ninth update world health organization regional office for europe (who europe). zika virus european centre for disease prevention and control (ecdc) coordinating competent bodies: structures, interactions and terms of reference on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of article l / the approved list of biological agents rapid risk assessment: zika virus disease epidemic: potential association with microcephaly and guillain-barré syndrome. fifth update mers coronavirus: data gaps for laboratory preparedness come fly with me: review of clinically important arboviruses for global travelers using routine diagnostic data as a method of surveillance of arboviral infection in travellers: a comparative analysis with a focus on dengue genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes #iso:std:iso-iec: :ed- :v :en . international organization for standardization (iso) ecdc technical document. zika virus disease epidemic: interim guidance for healthcare providers and zika virus laboratory diagnosis zika virus (zikv) surveillance in the americas: laboratory detection and diagnosis. algorithm for detecting zika virus (zikv) laboratory testing for zika virus infection emergency use assessment and listing (eual) update on submission of applications to the who eual for zika virus ivds european union invests € million into research to combat the zika disease assay optimization for molecular detection of zika virus emerging viral diseases-expert laboratory network (evd labnet). ecdc. stockholm: ecdc variable sensitivity in molecular detection of zika virus in european expert laboratories; external quality assessment this study was conducted with ecdc-funding srs- - -rs. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indicate if changes were made. key: cord- -v uevz l authors: zukor, katherine; wang, hong; siddharthan, venkatraman; julander, justin g.; morrey, john d. title: zika virus-induced acute myelitis and motor deficits in adult interferon αβ/γ receptor knockout mice date: - - journal: j neurovirol doi: . /s - - -z sha: doc_id: cord_uid: v uevz l zika virus (zikv) has received widespread attention because of its effect on the developing fetus. it is becoming apparent, however, that severe neurological sequelae, such as guillian-barrë syndrome (gbs), myelitis, encephalitis, and seizures can occur after infection of adults. this study demonstrates that a contemporary strain of zikv can widely infect astrocytes and neurons in the brain and spinal cord of adult, interferon α/β receptor knockout mice (ag strain) and cause progressive hindlimb paralysis, as well as severe seizure-like activity during the acute phase of disease. the severity of hindlimb motor deficits correlated with increased numbers of zikv-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. electrophysiological compound muscle action potential (cmap) amplitudes in response to stimulation of the lumbosacral spinal cord were reduced when obvious motor deficits were present. zikv immunoreactivity was high, intense, and obvious in tissue sections of the brain and spinal cord. infection in the brain and spinal cord was also associated with astrogliosis as well as t cell and neutrophil infiltration. cmap and histological analysis indicated that peripheral nerve and muscle functions were intact. consequently, motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or a gbs-like syndrome. thus, acute zikv infection of adult ag mice may be a useful model for zikv-induced myelitis, encephalitis, and seizure activity. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. abstract zika virus (zikv) has received widespread attention because of its effect on the developing fetus. it is becoming apparent, however, that severe neurological sequelae, such as guillian-barrë syndrome (gbs), myelitis, encephalitis, and seizures can occur after infection of adults. this study demonstrates that a contemporary strain of zikv can widely infect astrocytes and neurons in the brain and spinal cord of adult, interferon α/β receptor knockout mice (ag strain) and cause progressive hindlimb paralysis, as well as severe seizurelike activity during the acute phase of disease. the severity of hindlimb motor deficits correlated with increased numbers of zikv-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. electrophysiological compound muscle action potential (cmap) amplitudes in response to stimulation of the lumbosacral spinal cord were reduced when obvious motor deficits were present. zikv immunoreactivity was high, intense, and obvious in tissue sections of the brain and spinal cord. infection in the brain and spinal cord was also associated with astrogliosis as well as t cell and neutrophil infiltration. cmap and histological analysis indicated that peripheral nerve and muscle func-tions were intact. consequently, motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or a gbs-like syndrome. thus, acute zikv infection of adult ag mice may be a useful model for zikv-induced myelitis, encephalitis, and seizure activity. zika virus (zikv) is an emerging flavivirus that has received widespread attention because of its effect on the developing fetus. in utero infections cause congenital defects, most notably microcephaly along with other deformities (schuler-faccini et al. ) . while zikv infection of adults generally produces only a mild disease, it is becoming apparent that, as with other flavivirus infections, severe neurological sequelae can occur. recent outbreaks have been associated with higher incidences of peripheral neuropathies such as guillian-barrë syndrome (gbs) cao-lormeau et al. ; cardoso et al. ; samarasekera and triunfol ) and other neurological diseases such as myelitis (anaya et al. ; dirlikov et al. ; mecharles et al. ) , encephalitis (carteaux et al. ; nicastri et al. ; soares et al. ) , seizures (asadi-pooya ), and various ophthalmological conditions smith et al. ). this is not surprising considering that related flaviviruses such as west nile virus (wnv) and japanese encephalitis virus (solomon et al. ) can cause myelitis and motor deficits (sejvar et al. ) . given the emerging nature of zikv, however, it is likely that we do not fully understand the acute and long-term consequences of zikv infection on the nervous system. therefore, investigating the pathobiology of zikv in animal models will help to understand the potential for zikv to cause neurological disease in human subjects. we focus herein on adult models because zikv-related motor deficits have been primarily associated with adult infection, as opposed to in utero infection (anaya et al. ; dirlikov et al. ; mecharles et al. ). prior to the zikv outbreak, a few animal models of zikv infection existed, but infection of rodents was largely non-productive unless a lab strain of the virus that had undergone several serial passages in mice was used (dick ) . after the recent outbreaks, efforts to develop animal models with more clinically relevant isolates of the virus have been renewed. a rapid series of publications found that adult mice that lack type (αβ; a and ifnar −/− strains) or types and (αβ/γ; ag strain) interferon receptors are susceptible to lethal infection (aliota et al. ; lazear et al. ; manangeeswaran et al. ; rossi et al. ; zmurko et al. ) . these models may have some relevance to human zikv infections in that, like many viruses, zikv gains advantages in human hosts by inhibiting interferon responses (best ; bowen et al. ; schulz and mossman ) . because viruses may not be able to inhibit mouse-specific interferon pathways (aguirre et al. ) , blocking them by other means, as in ag mice, may more closely mimic what happens in humans. although ag mice are deficient in innate immune responses, they do elicit acquired immune responses, such as vaccine-elicited protective immunity (sumathy et al. ; weger-lucarelli et al. ) . while complete knockout of the interferon responses produces a more severe disease in mice, it can provide insights into what happens when zikv gains access to the adult central nervous system (cns). besides a lethal infection, these mice manifest neurological symptoms such as btoe walking,^tremors, loss of balance, paralysis, and hunched posture (aliota et al. ; lazear et al. ; manangeeswaran et al. ; rossi et al. ) . to better understand the consequences of zikv infection of the adult nervous system and the pathobiology of the resulting neurological disease, we infected ag mice with a contemporary, low-passage zikv isolate and evaluated the neurological motor deficits occurring during the acute phase of the disease. we characterized the onset, course, and severity of behavioral hindlimb deficits and used electrophysiology and immunohistochemistry (ihc) to determine if such deficits are due primarily to myelitis, peripheral neuropathy, myositis, or encephalitis. this included histological analysis of motor cortex, spinal cord, peripheral nerve, and muscle. male and female ag mice (van den broek et al. ) were bred in-house in sterilized cages and maintained in a / light cycle. mice were randomly assigned to treatment groups based on weight, gender, and baseline measurements. a puerto rican isolate of zikv (prvabc , human/ / puerto rico, genbank ku ) was obtained from bei resources (cat no. nr- , lot no. ) . the certificate of analysis confirmed that the sequence of this stock (genbank kx ) is % identical to prvabc (genbank ku ). the bei stock was passaged two times in vero cells to make a stock with a titer of × pfu/ml for use in all experiments. infected cells were frozen once, thawed, centrifuged to remove cell debris, and aliquoted in frozen stocks. dilutions were made in minimal essential medium supplemented with μg/ml gentamicin to deliver pfu subcutaneously in the inguinal area on the right side in a volume of . ml. uninfected cells were prepared and diluted similarly for sham infections. the purpose of this experiment was to assess the time course and severity of motor deficits and collect tissues for histological analysis. seventy-two to -day-old ( . weeks) male and female ag mice were infected with zikv (n = females and males) or sham (n = females and males) inoculum and monitored for weight loss and survival (fig. ) . behavioral motor assessments were performed before infection (baseline); once after infection, but before symptom onset ( days post-infection (dpi)); and twice a day after symptom onset ( - dpi) (fig. ) . seizure-like activity observed during behavioral assessments was also noted (fig. ) . videos of motor deficits and seizure-like activity were obtained to provide examples of symptoms seen. observations were made by researchers who were blind to the infection status of each group. moribund mice were perfused for histological analysis of the brain, lumbosacral spinal cord, sciatic nerve, and gastrocnemius muscle. the purpose of this experiment was to collect electrophysiological data on mice with motor deficits. male and female ag mice aged - days old ( weeks) or days old ( weeks) were divided into groups. the zikv-infected group contained three males ( weeks old), four males ( weeks old), and four females ( weeks old). the shaminfected group contained two males ( weeks old), two males ( weeks old), and two females ( weeks old). mice were monitored for weight loss and survival. behavioral assessments were performed before infection (baseline) and after symptom onset so that mice with moderate to severe deficits (scores - , see below) could undergo electrophysiological assessment. compound muscle action potential (cmap) of the gastrocnemius muscle was recorded before infection (baseline) and after moderate to severe deficits had developed. after infection, one sham-infected mouse was recorded for every one to two zikv-infected mouse. measurements were obtained by a researcher who was blind to the infection status and deficit score of each mouse. cmap responses were recorded in response to stimulation at the sciatic notch and then the lumbosacral spinal cord. because stimulation of the lumbar spinal cord was an invasive procedure, we did not obtain baseline measurements for cmap in response to spinal cord stimulation. mice were analyzed for signs of tail and hindlimb paresis/ paralysis using a sensitive, open-field assay modified from the basso mouse scale used to assess paralysis in spinal cord-injured mice (basso et al. ) and a test used to track paralysis in amyotrophic lateral sclerosis mouse models (hatzipetros et al. ) . each mouse was placed on a tabletop and allowed to roam freely for min. hindlimb function was scored on a seven-point scale detailed in table by researchers who were blind to the infection status of each group. scoring was based on four main categories: tail position during walking, miss-step severity, weight bearing, and joint movement. miss-step severity was scored only on assessable walking passes, which was defined as a pass in which the animal moved three body lengths at a consistent speed and without turning (basso et al. ) . separate scores were given for the left and right hindlimbs to assess if symptoms were bilateral or unilateral. seizure-like activity noted during viral paresis scale (vps) assessments was recorded as mild if the animal had mild shakes or tremors or severe if the animal was violently seizing in the cage. mice were perfused transcardially with phosphate-buffered saline (pbs) followed by % paraformaldehyde. the brain, kidney, liver, pancreas, hindlimbs, and lumbosacral spinal column were removed and post-fixed in the same fixative overnight, rocking at °c. tissues were rinsed twice in pbs, and the lumbosacral spinal cord, sciatic nerves, and gastrocnemius muscles were isolated. all tissues except the gastrocnemius muscle on the right were cryoprotected in % sucrose in pbs for - days at °c, embedded in oct compound (ted pella), and frozen with a dry ice/ethanol bath. five sets of adjacent sections were cut on a cryostat at μm, mounted on slides, and stored at − °c until ready for further processing. for immunohistofluorescent staining, sections were encircled with a hydrophobic barrier pen (immedge, vector labs), rinsed with pbs, and blocked with % normal serum and % triton x- in pbs for h. primary antibodies were diluted in blocking solution as shown in table and applied to sections for incubation overnight at room temperature. secondary antibodies conjugated to alexa- , alexa- , or alexa- (from invitrogen or jackson immunoresearch) were diluted to μg/ml in blocking solution. sections were rinsed three times in pbs, incubated with secondary antibody solution for h at room temperature, rinsed three times in pbs, incubated with hoechst (invitrogen, / in pbs with . % triton x- ), and rinsed twice in pbs. coverslips were mounted with fluoromount g (southern biotech). for cd labeling, the amount of triton x- was decreased to . % in the blocking solution and . % in the antibody diluent. gastrocnemius muscles from the right side were sectioned longitudinally at μm on a vibratome ( plus, vibratome co.), collected in pbs, and stored at °c. ten to sections were obtained for each muscle, and the third or fourth and seventh or eighth sections were chosen for neuromuscular junction (nmj) and neurofilament (nf-h) staining as described in itoh et al. ( ) . briefly, sections were incubated free floating in . m glycine in pbs, ph . for min; blocked with % donkey serum (jackson immunoresearch laboratories), . % triton x- , and % bsa in pbs containing tetramethylrhodamine (tmr)-conjugated alphabungarotoxin (α-btx) for h; permeablized with % methanol at − °c for min; rinsed with pbs with . % triton x- (pbst); and incubated with primary antibody diluted in % bsa and . % triton x- in pbs at °c for h. after rinsing in pbst three times, sections were incubated at room temperature for h with secondary antibody (anti- correlation between highest vps score and seizure severity for zikvinfected animals. each dot represents one animal. the line is the best fit line if , , and are used for seizure categories of none, mild, and severe, respectively. slope is not significantly different from zero chicken conjugated to alexa fluor® , / , jackson immunoresearch laboratories) diluted in pbs. after rinsing in pbst three times, sections were dehydrated through a methanol series ( min each in , , , %), cleared with benzyl alcohol/benzyl benzoate ( : ) (zukor et al. ) , and stored at °c until ready to image. fluorescent images were obtained at × or × with a laser scanning confocal microscope (zeiss, lsm ) equipped with , , , and laser lines. for images taken for pixel-based quantification, identical settings were used for all images in a set. for images chosen for publication, distracting artifacts were removed in imagej (schneider et al. ) and levels were adjusted in photoshop to maximize the signal-to-noise ratio so that relevant features could be seen more clearly. for images chosen to highlight pixel-based quantification, sham and zikv group images were adjusted identically to enable equitable comparison. imagej or fiji was used for image quantification (schneider et al. ). the cell counter plugin was used for manual cell counts. for pixel-based quantification of a given antibody pab polyclonal antibody, mab monoclonal antibody a summary of primary antibodies and dilutions used signal, the area to be measured was defined by a region of interest (roi); then, thresholds of single-channel images were adjusted to select pixels with signal above noise (positive pixels). thresholds of all images in a set were adjusted identically for equitable comparison. then, positive pixels within the whole area roi were measured to obtain the area occupied by positive pixels. the area of positive pixels was divided by the whole area of the main roi to determine what proportion of the roi was positive for the antibody. for co-localization analysis, pixels that were positive for two antibody signals were measured. sagittal sections of the brain containing motor cortex were identified based on the morphology of the lateral ventricle about mm lateral of bregma (paxinos and franklin ) . a × μm region containing the pyramidal cell layer of the motor cortex, in a region of the motor cortex just dorsal to the lateral ventricle, was measured for pixel-based quantifications (see box in fig. (d) for location of the region measured). two sections per animal were measured for each parameter. for analysis of zikv in the hippocampus, one section per animal was measured, and the area measured corresponded to pyramidal cell layer (see fig. (b) for location of region measured). spinal cord levels were identified using the mouse spinal cord atlas (watson et al. ) and the location and morphology of clusters of choline acetyltransferase (chat) positive neurons. because motor neurons for the gastrocnemius muscle are located at the l -l level (mchanwell and biscoe ) , sections from this level were chosen for analysis when possible. for ventral motor neuron, astrocyte, and zikv infection level analyses, two sections per animal were analyzed. zikv−, chat+ and zikv+, chat+ neurons in the ventral horns were counted manually using the cell counter in imagej. because there were occasionally zikv+ cells in the ventral horns with neuronal morphology that had low or undetectable chat levels ( fig. (b -b )), these cells were also counted as ventral motor neurons. for analysis of ly g (neutrophils), cd (t cells), and iba (microglia/macrophages) signals, one section per animal was analyzed. for the nerve cross sections, a stereological counting grid containing × μm boxes where each box was spaced μm from the boxes above and below it was placed over the nerve to obtain counts from a sample of the nerve, representing approximately one fourth of the total area. an roi was placed around the whole nerve, and the counting grid was cut to eliminate all areas of the grid outside of the nerve (see fig. a ). stereological counting rules were used to count the number of myelinated axons, unmyelinated axons, and empty myelin sheaths within the boxes of the cut grid. images were converted to composite images, so the neurofilament and myelin basic protein (mbp) channels could be toggled on and off during counting. all counts were done by a researcher who was blind to the status of each animal. the area of the cut grid as well as the area of the whole roi were calculated and used to extrapolate the number of myelinated axons, unmyelinated axons, and empty myelin sheaths within the whole area. three sections per animal were analyzed. for nmj analysis, two sections per animal were analyzed. confocal z-stacks (~ -μm step size) of two optimal fields of view containing nmj endplates (α-btx+) and nerve fibers (nf-h+) were analyzed per section for a total of four fields of view per animal. the number of endplates and the number of innervated endplates (co-localized with some neurofilament signal) were counted manually with the cell counter in imagej. images were converted to composite images, so the nf-h and α-btx channels could be toggled on and off during counting. total numbers of innervated nmjs (nf-h+, α-btx+) and all nmjs (α-btx+) from all four fields analyzed were used to calculate the percentage of nmjs that were innervated per animal. cmap measurements were performed by a researcher who was blind to the infection status and vps score of each mouse. animals were anesthetized with isoflurane and maintained at °c body temperature with a rectal probe and heating pad, and their hindlimbs were shaved. monopolar needle electrodes (tai-chi brand, acupuncture needles) were inserted into the sciatic notch or epidural to the lumbosacral spinal cord (after surgical exposure) to stimulate the gastrocnemius muscle with . -ms pulses of current using a stimulus isolator (wpi isostim a ). for lumbosacral spinal cord stimulation, monopolar needle electrodes (biopac systems, el ) were used, and the anode was inserted between the t and l vertebrae, and the cathode was inserted between the l and l vertebrae (basoglu et al. ; harrison et al. ) . no laminectomy was necessary. muscle responses were recorded with -mm-diameter shielded ag/agcl surface recording electrodes (el s, biopac system) filled with electrode gel. electrodes were placed on the skin at the belly of the muscle and at the anterior aspect of the ankle and connected to a differential amplifier (dam , wpi) with a gain of × and filtered with a - k hz band pass filter. data were acquired at a k/s sampling rate with powerlab / and labchart software (adinstruments). responses to five pulses at hz were averaged, and the current was increased incrementally until a maximum amplitude was reached. maximal cmap amplitudes were measured from peak to peak of the m wave. for experiments with α-btx, uninfected, male ag mice, about . months of age, were used. after finding the stimulation current that gave the maximal cmap response, μl of . , . , or . μm α-btx (biotium, inc., # - ) was injected into the gastrocnemius muscle (with the gel pads kept in place) with a g needle (nakanishi et al. ) . cmap responses after maximal stimulation current were recorded at intervals for up to h after α-btx injection. dilutions of α-btx were made with saline, and thus, a μl saline injection was used in the control. data were graphed and analyzed with prism (graphpad software, inc.) for statistical significance using t tests or two-way anovas with post hoc t tests. linear regression was used for correlation analyses. experiment no. disease and behavioral motor deficits zikv-infected animals began losing weight by dpi and started succumbing to the disease by dpi (fig. ) . by dpi, all zikv-infected animals had died or been humanely euthanized. the onset of hindlimb motor deficits in zikv-infected mice ranged from to dpi (fig. a, b) . initial symptoms included tail weakness (tail not in the up position during walking) and subtle hindlimb lateral skidding and weakness (as indicated by a limp or wobble). disease progressed quickly after the onset of motor deficits and morbidity occurred within - days. the severity of motor deficits generally increased rapidly, with vps scores reaching as high as or before death (hindlimb dragging with little to no joint movement). mice were grouped into early ( - dpi) and late ( - dpi) onset groups in fig. c , d so that scores from later onset mice would not mask the progression of disease in the early onset mice. deficits were more severe on the right side compared with the left, corresponding to the side of virus inoculation. forelimbs were rarely affected. seizure-like activity was also observed in zikv-infected mice (fig. ) . six of zikv-infected mice had some seizurelike activity, and mice were scored as severe (fig. a) . seizure-like activity did not correlate with vps score (fig. b) . half of the mice in which severe seizure-like activity was noted had no to only subtle hindlimb deficits after seizure activity had subsided. additionally, there were mice with complete paralysis (vps score of ) that were never observed to have seizure-like activity. two other disease phenotypes, walking in circles and extreme mobile activity (not shown), were also observed (data not shown). interestingly, the severity of hindlimb paralysis and seizure-like activity did not correlate well with changes in body weight ( supplementary fig. s ), suggesting that mechanisms affecting neuroinvasion might operate independently from those affecting systemic infection and body weight changes. initial histopathologic analysis of our relatively thick, frozen sections with hematoxylin and eosin staining was considered to be preliminary. nevertheless, the following preliminary results were evidence of mild to moderate, multifocal encephalitis in the brain; moderate to severe, multifocal myelitis in the spinal cord; and no lesions in the gastrocnemius muscle or sciatic nerve (data not shown). to further identify anatomical features that might be associated with motor deficits, we performed immunohistofluorescent analyses of the motor cortex, lumbosacral spinal cord, sciatic nerve, and gastrocnemius muscle. the kidney, liver, and pancreas were collected to assess systemic infection level. using a polyclonal antibody against zikv envelope glycoprotein, zikv immunoreactive (ir) was seen in all parts of the adult ag mouse brain, including motor cortex, cerebellum, hippocampus, and brainstem ( fig. (a) ). in the motor cortex, zikv infects both neurons and astrocytes (arrows in fig. (e -e ) ), and many of the neurons have the morphology and location of layer v pyramidal neurons which are upper motor neurons that project to the spinal cord ( fig. (e and e -e ) ). quantification of a region of the motor cortex containing layer v (shown in box in fig. (d)) revealed that - % and up to - % of the area were zikv ir in zikv-infected mice (fig. (l) ). additionally, - % of astrocytes in this area were zikv ir ( fig. (m)), as measured by pixel-based quantification. there were also significant increases in astrogliosis ( fig. (e, e -e , and p)), t cell infiltration ( fig. (g, g , and q)), and neutrophil infiltration ( fig. (i, i , and r)) in the zikvinfected motor cortex compared to sham infected ( fig. (d, f, and h) ). interestingly, microglia and macrophages were virtually undetectable in the zikv-infected ag mouse brain (fig. (k and s) ), though resting microglia were apparent in sham-infected brains (fig. (j and j ) . the pyramidal cell layer of the hippocampus was also strikingly zikv ir in many zikv-infected mice ( fig. (c) ). quantification of this region (outlined in fig. (b) ) demonstrated that - % of this layer was zikv ir in three of eight mice (fig. (n) ). because of the association between the hippocampus and seizure activity, the severity of seizure-like activity was plotted against hippocampus pyramidal cell layer zikv infection levels, but no correlation was found (fig. (o) ). likewise, no correlation was found between motor cortex infection level and seizure-like activity (data not shown). zikv ir was also abundant in the lumbosacral spinal cord which contains lower motor neurons that innervate the hindlimbs (fig. (a-d) ). overall, - % of the crosssectional area was zikv ir (fig. (k) ). as with the brain, astrocytes (arrowheads in fig. (d -d and m) ) and neurons were infected. using a chat antibody to label ventral motor neurons, many were found to be zikv ir (arrows in fig. (b -b ) ) and a few ventral cells with clear neuronal morphology were strongly zikv ir, but only weakly chat ir (asterisks in fig. (b -b ) ). as many as - % of ventral motor neurons were zikv ir (fig. (l) ), which suggests that zikv can infect and replicate in lower motor neurons in the spinal cord. the number of motor neurons at the l -l and s levels was also diminished in many zikv-infected mice (fig. (n and o) ), but the difference from sham-infected mice did not reach statistical significance, likely because the disease progressed so rapidly. as in the brain, astrogliosis (fig. (c, d, and p) ), t cell infiltration (fig. (e, f, f , and q) , and neutrophil infiltration (fig. (g, h, h , and r) were markedly increased in zikvinfected mice compared to sham-injected mice. conversely, microglia and macrophages were markedly reduced or absent (fig. (j and s) ) in these zikv-infected ag mice, as observed in the brain, though resting microglia were evident in sham-infected spinal cords (fig. (i and i ) . zikv ir reactivity was detected in cross sections of the sciatic nerve ( fig. c-e) ; however, this did not appear to have a statistically significant effect on the number of myelinated axons (fig. a , b, f), unmyelinated axons (fig. g) , or empty myelin sheaths (fig. h) . gastrocnemius muscle, kidney, liver, and pancreas zikv ir and neutrophil infiltration were not detected in the gastrocnemius muscles of zikv-infected mice (data not shown). infection of lower motor neurons in the spinal cord and their axons in the sciatic nerve did not appear to lead to a statistically significant loss of nmj number or innervation (fig. ) . no zikv ir was detected in the kidney, liver, or pancreas of zikv-infected mice (data not shown). to determine which anatomical features might be correlated with hindlimb motor deficits, we used our data to make preliminary correlations between histological parameters of the spinal cord (fig. a-f ), motor cortex ( fig. g-k) , nmj (fig. l) , and sciatic nerve (fig. m) and vps scores. statistically significant correlations were only found with spinal cord parameters. increased numbers of zikv-infected ventral motor neurons in the lumbosacral spinal cord are associated with higher vps scores (fig. a , p < . ), and higher vps scores are associated with decreased survival of spinal motor neurons (fig. c, p < . ) . these data suggest that zikv infection of lower motor neurons and lower motor death adversely affects hindlimb function. interestingly, the level of neutrophil infiltration was inversely proportional to vps score (fig. f , p < . ), which suggests that the presence of neutrophils might mitigate motor deficits. to get a preliminary indication of how neutrophil invasion and spinal motor neuron viral infection are related to spinal motor neuron survival (as opposed to vps score), correlation graphs with these parameters were prepared as well and similar trends were observed (supplemental fig. s ). vps scores did not appear to correlate with virus levels in the motor cortex (fig. g , h) or with sciatic nerve infection levels (data not shown). overall, these results suggest that hindlimb motor deficits in these ag mice during the acute phase of zikv infection are likely caused primarily by spinal cord myelitis, though upper motor neuron disease and peripheral neuropathy may contribute. experiment no. reproduced the disease and motor phenotypes seen in experiment no. . zikv-infected animals began losing weight by dpi and started succumbing to the disease by dpi (supplemental fig. s ). all zikv-infected mice had died or been humanely euthanized by instead of dpi. vps assessments were done less frequently, but symptoms appeared at a similar timing and progressed at a similar rate (supplemental fig. s ). when mice in experiment no. had vps scores ≥ , gastrocnemius muscle cmaps were recorded in response to stimulation at the sciatic notch, followed by stimulation of the lumbosacral spinal cord to further delineate the cause of the hindlimb motor deficits. we reasoned that since motor deficits progress quickly after symptom onset and many motor neurons, though infected, have not died by the time severe deficits occur, axons and nmjs may still be healthy even if the neuronal cell body is sick. thus, cmap amplitudes may be normal in response to sciatic notch stimulation, but decreased upon lumbosacral spinal cord stimulation. responses to stimulation at these two sites, therefore, might distinguish between myositis/neuritis and myelitis. because cmap measurement in response to lumbar spinal cord stimulation was an invasive procedure, pre-infection baselines were only obtained in response to sciatic notch stimulation. figure a shows the cmap amplitudes for each animal pre-infection and post-infection in response to stimulation at the two sites. there are no statistically significant differences between groups when these babsolute^values are used. the use of relative values, however, are more compelling, and may be necessary because individual differences in the thickness and composition of tissue between the surface of the skin and the muscle can affect cmap amplitudes (nordander et al. ) . when post-infection cmap amplitude was expressed relative to pre-infection amplitude (both in response to sciatic notch stimulation), there was no statistically significant difference between the zikv-infected and sham-infected groups (fig. b , bpre minus post, at notch^), suggesting that the axons and nmjs are healthy after infection and motor deficits are not due to myositis or neuritis. when post-infection cmap amplitude in response to stimulation of the spinal cord was expressed relative to the amplitude in response to stimulation at the sciatic notch, however, there was a statistically significant relative decrease in amplitude in the zikv-infected group (fig. b , bpost, at s. cord minus notch,^p < . ). this suggests that, after infection and at the time motor deficits are observed, the axons and nmjs are healthy enough to generate a normal cmap response when axons are stimulated at the sciatic notch, but the neuronal cell bodies in the spinal cord are impaired and less able to generate normal cmap responses when stimulated at the lumbosacral spinal cord. this indicates that myelitis, rather than myositis or neuritis, contributes to the motor deficits. the fact that vps score did not correlate with the relative cmap amplitude in response to spinal cord stimulation (fig. c) , however, suggests that other factors also contribute to hindlimb motor deficits. to validate that the cmap indeed reflected muscle activity, and not just nerve activity, an inhibitor of the nmj nicotinic acetylcholine receptor, α-btx, was injected into the gastrocnemius muscles of uninfected adult ag mice, and cmaps were recorded for up to h after injection. cmap amplitudes decreased in a dose-dependent manner over the course of the h after α-btx injection, whereas cmap amplitude in the saline-injected animal was relatively stable (fig. ) . these data suggest that the cmap reflects mostly muscle rather than nerve activity, and thus, cmap measured the health status of all structures between the point of stimulation and the muscle (including the nerve and nmj). this study demonstrates that a contemporary strain of zikv (prvabc ) can widely infect astrocytes and neurons in the brain and spinal cord of adult ag mice and cause rapidly progressing hindlimb paralysis, as well as severe seizure activity, during the acute phase of disease. motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or gbs-like syndrome. this is an important new finding because the most severe histopathologies previously reported in ag mice were in the brain and muscle (aliota et al. ) . the finding that myelitis is likely the primary cause of zikv-induced paralysis is also in alignment with what has been seen with other flavivirus-induced motor deficits (sejvar et al. ; solomon et al. ). this conclusion is also supported by our data suggesting that muscle and peripheral nerve functions are normal. electrophysiological cmap amplitude measurements of sciatic nerve and sural muscles were normal in response to stimulation at the sciatic notch. additionally, the anatomy of the sciatic nerve and gastrocnemius muscle, as revealed by neurofilament/mbp and neurofilament/nmj staining, respectively, was minimally disrupted. in contrast, zikv ir was high, intense, and obvious in the brain and spinal cord. the severity of hindlimb motor deficits correlated with increased numbers of zikv-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. relative cmap amplitudes upon stimulation at the lumbar spinal cord were also reduced when obvious motor deficits were present (vps score ≥ ). zikv infections in the brain and spinal cord were also associated with astrogliosis as well as t cell and neutrophil infiltration. thus, acute zikv infection of adult ag mice may be a useful model for studying the mechanisms of zikv-induced myelitis (anaya et al. ; dirlikov et al. ) , encephalitis, and seizure activity. the results of this study may be clinically relevant, because there have been several reports of myelitis during recent zikv outbreaks. in the february outbreak in guadeloupe, a -year-old girl developed hemiparalysis that was associated with detection of zikv in the cerebrospinal fluid. magnetic resonance imaging revealed extensive lesions in the cervical and thoracic spinal cord (mecharles et al. ) . investigators reporting on zikv-associated gbs in puerto rico noted cases of neurologic disorders other than gbs, of which were (dirlikov et al. ) . in a case-controlled study in colombia from a national population-based surveillance system (anaya et al. ) , patients with zikv-associated gbs were compared to matched controls. thirteen of these patients were reported to have other neurological syndromes: three with myelitis, three with peripheral facial palsy, six with transverse myelitis, and one with thoraco-lumbosacral myelopathy. the observation that zikv can infect and kill spinal motor neurons and that this correlates with hindlimb motor deficits is a unique finding of this report and may be clinically important. pathology reports of human zikv infection are limited, but do reveal neuronophagia in the brain , which suggests that zikv may be capable of eliciting neuron death in human neurological tissues as well as in the ag mouse. zikv infection has previously been reported in the spinal cords of ag mice (julander et al. ; zmurko et al. ) , and zikv-induced histopathology or myelitis has been observed in ifnar −/− (lazear et al. ) and swiss webster mice infected as newborns (fernandes et al. ) . the zikv-induced motor deficits in ag mice are reminiscent of motor deficits caused by other flaviviruses, such as wnv, in immunocompetent mice and hamsters (morrey et al. ; morrey et al. ; shrestha et al. ; siddharthan et al. ; xiao et al. ; zukor et al. ) . these flavivirus models also demonstrate virus infection and killing of spinal motor neurons together with mild to severe signs of motor impairment (zukor et al. ) . the vast majority of adults infected with zikv do not develop severe neurological disease such as encephalitis, yet the ag mice of this study are immunodeficient and develop encephalitis and myelitis. as such, the ag mouse model is probably more relevant to infection of immunocompromised patients. some clinical reports of infected immunocompromised patients describe severe neurological complications (henry et al. ; schwartzmann et al. ) . for example, an immunosuppressed heart transplant patient developed acute neurological impairment and mental deterioration after zikv infection, which culminated in death. autopsy revealed zikv infection of the brain and cerebral spinal fluid by rt-pcr, immunohistochemistry, and electron microscopy. the histopathology of the brain was consistent with meningoencephalitis (schwartzmann et al. ) . the fact that relative cmap amplitude after spinal cord stimulation did not statistically correlate with vps score suggests that damage upstream of spinal motor neurons may contribute to the hindlimb deficits. for example, cmap amplitude was not reduced in two infected mice with overt paralysis (vps score - , fig. c ). paralysis in these animals could be caused by motor cortex, cerebellum, or brainstem dysfunction, as extensive infection was observed in these areas of the brain. while upper motor neuron disease is associated with rigid, rather than flaccid paralysis, we occasionally saw symptoms that could be interpreted as rigidity, such as walking with high haunches and extended/stiff limbs, but it was difficult to reliably assess this with the vps test. another possible explanation for the paralysis in animals with normal relative cmap amplitudes is that spinal motor neuron infection damages dendrites and post-synaptic densities such that the neurons are not able to receive signals physiologically, but when stimulated externally with an electrode, the axon hillock and axon are healthy enough to propagate an action potential to the muscle. the observation of seizure-like activity in infected ag mice in this study and in immunocompetent mice infected as neonates in another study (manangeeswaran et al. ) may have clinical relevance since recent zikv infections have been associated with seizures in humans (asadi-pooya ; pastula et al. ) . notably, the seizure-like activity in this study was not correlated with the vps score, which suggests that the seizure activity was not simply a manifestation of motor deficits. to further investigate the relevance, seizures would need to be confirmed in zikv-infected mice with more detailed analyses. motor deficits seen in this study are not likely to be the result of peripheral neuropathy. while zikv ir was observed in the sciatic nerve, axon and myelin morphology did not appear to be altered. zikv also did not appear to alter the function of the sciatic nerve, which is supported by the observation that gastrocnemius cmap amplitudes in response to sciatic notch stimulation were not affected. the observation of viral antigen in axons is consistent with prior findings that four families of viruses, including flaviviruses, can undergo axonal transport and spread in the nervous system (samuel et al. ; taylor and enquist ; wang et al. ), as well as the finding that zikv appears to be able to travel in axons (van den pol et al. ). we were not able to determine what effect zikv might have on the development of peripheral neuropathies, such as gbs, in this study because the mice die during the acute infection and peripheral neuropathies tend to develop in chronic stages. in the current study, inflammatory cellular responses, such as astrogliosis, neutrophil infiltration, and t cell infiltration, were present in the brain and spinal cord. the possible influence of each of these responses to disease severity, whether causative or protective, is explored below. future work should comprehensively establish correlation with larger sample sizes and then determine which relationships are causative or protective, so that the cellular mechanisms underlying zikv-induced motor deficits can be understood. results from this study suggest that neutrophils play a role in mitigating or protecting zikv-infected ag mice from motor deficits, probably by enhancing immune responses that reduce viral load. increased numbers of neutrophils, as detected by the ly g antibody, were inversely correlated with vps score (p < . ). studies with wnv demonstrate that neutrophils can play complex roles during infection. they appear to be protective early in wnv infection, but detrimental later. when neutrophils were depleted before wnv challenge in c h/hej mice, mortality increased, but when they were depleted after wnv challenge, mortality decreased (bai et al. ) . another level of complexity regarding the role of neutrophils involves mosquito bites. when neutrophils are depleted and inflammasome activity is blocked, the ability of mosquito bites to promote infection and inflammation is abrogated (pingen et al. ) . therefore, the role of neutrophils in zikv-induced disease should be further investigated. the drastic reduction in iba ir (a marker for microglia and macrophages) in the brains and spinal cords of ag mice infected with zikv was a unique finding. it could mean that microglia and macrophages are present, but the iba marker is strongly downregulated, or it could mean that microglia are killed and macrophage infiltration is inhibited. after wnv infection in c bl mice, microglia and macrophages are strongly activated (zukor et al. ) . they are also strongly activated in wild-type c bl or s /svimj mice infected with zikv intracerebrally at e . (shao et al. ) . thus, the decrease in iba ir after zikv infection in our study might be unique to the ag mouse strain and could indicate a role for microglia and macrophages in protecting against lethal zikv infection. for example, m macrophages elicit proinflammatory responses and enable phagocytosis and killing of pathogens, and m macrophages function in protective and homeostasis of the cns (kabba et al. ) . a certain balance of these functions could be overall protective. to further elucidate immunological cellular responses, decreased iba ir cells and modulation of other immune cells should be validated with flow cytometric analysis in future studies. t cell infiltration was associated with zikv infection in the brain and spinal cord, though there was no correlation between the level of infiltration and the severity of motor deficits. this could indicate that t cells play both beneficial and detrimental roles (ousman and kubes ; prinz and priller ) . for example, cd (+) t cells contribute to resolution of wnv neurological infection by helping to clear wnv from neurons (mccandless et al. ; shrestha et al. ) . t cells also play beneficial roles in la crosse virus infection (winkler et al. ) . t cells, however, are the cause of fatal meningoencephalitis after tacaribe arenavirus infection (ireland et al. ) . future studies will delineate the role of t cells in the development of motor deficits in zikv-infected ag mice. astrogliosis in the spinal cord was strongly associated with zikv infection, and many astrocytes were infected by the virus. as astrocytes regulate synapse formation, function, and elimination (chung et al. ) and control glutamate excitotoxicity (murphy-royal et al. ) , this likely had a significant effect on motor neuron health and function. in a model of human coronavirus, expression of the primary glutamate transporter in the adult cns, glt- , was decreased in astrocytes. this was associated with flaccid hindlimb paralysis even in the absence of significant neuronal death. blockade of -amino- -( -methyl- -oxo- , -oxazol- -yl) propranoic acid (ampa) receptors with a non-competitive antagonist improved hindlimb function (brison et al. ) . in mouse models of experimental autoimmune encephalomyelitis, astrocytes are implicated in the temporary stripping of excitatory synapses from motor neurons in the spinal cord, leading to severe flaccid paralysis in the absence of neuronal cell death (blakely et al. ; marques et al. ). astrogliosis may not have correlated with vps scores in our studies because it may precede and be required for the onset of motor deficits (blakely et al. ) . in two independent experiments, female mice were found to die slightly earlier than male mice, though this was not statistically significant. other zikv studies with ag mice at our institute, however, suggest that males and females are equally susceptible (data not shown). additionally, experiment no. appeared to have two distinct groups of mice with an 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disease progression in a robust mouse infection model fluorescent whole-mount method for visualizing three-dimensional relationships in intact and regenerating adult newt spinal cords phrenic nerve deficits and neurological immunopathology associated with acute west nile virus infection in mice and hamsters acknowledgements we thank the following individuals from the institute for antiviral research at utah state university for their excellent technical assistance: mitchell stevens for help with vps scoring; brent hunter for help with immunohistofluorescent staining; christian morrill for help with image quantification; heidi julander for help with ag colony management; rich albrechtsen for help with setting up the cmap assays; skot neilson for overseeing animal studies; jared bennet, jason fairbourn, john mcclatchy, devin pfister, jean marie maxwell, taylor redding, rachelle stanton, and trevor truex for assisting with animal studies; and neil motter for maintenance and design of electrophysiological instruments. we also thank arnaud van wettere, dvm, ms, phd, dacvp at utah state university's veterinary diagnostic laboratory for histopathologic analysis of tissues. the authors declare that they have no conflict of interest.open access this article is distributed under the terms of the creative comm ons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -ypefqm authors: roberts, christine c.; maslow, joel n. title: assay challenges for emerging infectious diseases: the zika experience date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: ypefqm from the perspective of vaccine development, it is imperative to accurately diagnose target infections in order to exclude subjects with prior exposure from evaluations of vaccine effectiveness, to track incident infection during the course of a clinical trial and to differentiate immune reactions due to natural infections from responses that are vaccine related. when vaccine development is accelerated to a rapid pace in response to emerging infectious disease threats, the challenges to develop such diagnostic tools is even greater. this was observed through the recent expansion of zika virus infections into the western hemisphere in – . when initial zika vaccine clinical trials were being designed and launched in response to the outbreak, there were no standardized sets of viral and immunological assays, and no approved diagnostic tests for zika virus infection. the diagnosis of zika virus infection is still an area of active research and development on many fronts. here we review emerging infectious disease vaccine clinical assay development and trial execution with a special focus on the state of zika virus clinical assays and diagnostics. the declaration by the world health organization (who) that zika is a public health emergency of international concern in february led to a global effort to support vaccine development and control the spread of zika virus (zikv). our collaborative dna vaccine consortium focused and accelerated pre-clinical, manufacturing and early clinical development efforts to bring forward the first zika vaccine, gls- , into human clinical trials [ ] [ ] [ ] . at the outset, it was clear that gaps would need to be filled as the public health and science communities learned and shared new information on zika. one of the clear gaps affecting both public health efforts and vaccine development programs was a lack of standardized reagents and methods to test for evidence of current or prior zika infection. the need for fast and accurate diagnostic tests of infection in an outbreak situation is obvious: identify the source or epicenter so that appropriate healthcare measures can be quickly instituted. expanding that concept to the public health scale and attaining accurate infectious disease diagnoses allows for better understanding of the course and severity of an outbreak and aids decision-making for population-level countermeasure implementation. the clinical assays with which the immune response and pathogen presence are measured in vaccine trials become part of the basis for licensure for all vaccine products [ ] . because vaccines are tested in healthy populations through all phases of clinical development for immune response and/or pathogen presence whereas drugs/biologics (post-phase i) are most often tested in a population with specific disease to demonstrate improvement, the selected methods to measure vaccine responses and endpoints are of the utmost importance. the identification of an immune correlate of protection for each vaccine is highly desirable, though not always attainable [ ] . here we will use the recent experience with the zikv outbreak and ensuing public health countermeasures for containment and vaccine development as an example of challenges faced during emerging infectious disease emergencies. zika virus was discovered in during a survey to map the extent of yellow fever in the entebbe region of uganda. the virus was cultured from the serum of a sentinel macaque placed in the zika forest that developed fever but was otherwise well. initially restricted to equatorial regions of africa and asia, zikv started to spread eastward across the pacific ocean with an outbreak on yap island, micronesia in [ ] , in french polynesia in [ ] , and reaching brazil in late or early [ ] . zikv infection typically causes a self-limited illness that is minimally symptomatic for most individuals. zika infection presents similarly to dengue or chikungunya with fever in most, rash, malaise, myalgia, conjunctivitis and retro-orbital pain but may present with few, if any, discernable symptoms [ ] . however, documentation in brazil of severe neurologic complications associated with zikv infection beginning around october raised worldwide alert. the most publicized and dramatic complications are those that occur during fetal development. they include microcephaly, intra-uterine growth retardation, cerebral calcifications, ocular calcifications and other ocular abnormalities-with an attack rate estimated at - % of women who become infected during pregnancy [ ] . zikv can also directly infect the placenta and can result in spontaneous miscarriage with an unknown prevalence [ ] [ ] [ ] [ ] . in adults, the most common complication of zikv infection is guillain-barré syndrome occurring at an estimated rate of in cases [ ] . zika has also resulted in deaths among adults with and without other complicating factors [ , ] . there are no approved therapies or vaccines for zikv infection. there have been a number of vaccines developed for other flavivirus infections. live virus vaccines utilizing chimeric viral constructs have been approved for use to prevent yellow fever and dengue. dna vaccines have been developed and published for dengue [ , ] , west nile virus [ ] [ ] [ ] [ ] [ ] , and japanese encephalitis virus [ ] . notably, dna vaccines targeting west nile virus [ , ] and dengue virus [ ] have been tested as part of phase i clinical trials without vaccine associated toxicity. currently, as recently reviewed elsewhere, a number of vaccine modalities targeting zikv have reached early stage clinical trials and even more are in preclinical development [ ] [ ] [ ] . zikv is spread primarily through aedes species mosquitoes, mainly aedes aegypti, but can be carried by other mosquito species [ ] and does not appear to be transmission competent except for aedes species [ ] [ ] [ ] [ ] [ ] [ ] . aedes mosquitoes also transmit other arboviruses such as dengue and chikungunya [ , ] . in fact, these infections are co-endemic in most regions and may cause concurrent infections [ ] [ ] [ ] [ ] . alternative nonmosquito-borne routes of zikv spread include: blood transfusions, breast milk [ ] , sexual transmission [ ] [ ] [ ] , and may include urine and saliva [ ] [ ] [ ] [ ] . the additional potential routes of zikv transmission have increased the need for definitive monitoring and a variety of surveillance strategies to prevent disease spread [ ] [ ] [ ] [ ] [ ] . in an outbreak situation, such as with zika, it is important to have the ability to quickly develop both diagnostic kits for public health purposes and vaccine clinical assays to support pre-clinical studies and early stage clinical trials. both were largely unavailable on a commercial scale or for widespread use at the outset of the zika outbreak, though development ensued at a rapid pace upon the declaration of a worldwide public health emergency. because there is significant homology between zikv and other cocirculating flaviviruses, detection and diagnosis has had the extra challenge of avoiding cross-reactivity without sacrificing sensitivity. while the avoidance of cross-reactivity is more easily engineered into molecular tests of virus rna because primers or probes can be designed to be virus-, antigen-and serotype-specific [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is not as easily achieved for immunoassays [ ] [ ] [ ] [ ] [ ] [ ] . diagnostic assays are often the same style tests as those used in vaccine development, but are for the intended purpose of identifying the source of a patient's illness to enable the initiation of appropriate treatments by healthcare professionals. the need for sensitivity and specificity as it relates to clinical disease identification for patient treatment is of the utmost concern. early in the zika outbreak, the us centers for disease control and some academic laboratories studying flaviviruses had assays developed for zikv that generally supported their own research interests [ ] [ ] [ ] [ ] [ ] [ ] [ ] . making sufficient quantities of these tests available for public health use while ensuring consistency and quality was a significant challenge. additionally, cross-reactivity in a number of immunological assays and the short time frame in which viremia can be detected in bodily fluids necessitated the institution of an algorithm to confirm zikv infection that was based on a combination of risk factors, clinical symptoms and diagnostic test results [ ] . the centers for disease control (cdc) issued testing guidance for healthcare providers using algorithms that included use of molecular testing for pregnant or non-pregnant and symptomatic or non-symptomatic individuals, the types of specimens and timing of collection of specimens that would provide the most reliable results [ ] . in addition, guidance algorithms were provided for the use and interpretation of zikv igm assays to indicate recent exposure with or without accompanying molecular test results [ ] . in july of , united states (us) health and human services (hhs) sponsored a hhs summit to accelerate zika diagnostics development, recognizing the important need. at that point in time, very few tests had gained emergency use authorization (eua) from the food and drug administration (fda). the five serological zika diagnostic kits currently authorized by the us fda are shown in table and fourteen viral diagnostic kits in table [ ] . at the fda medical countermeasures webpage, links can also be found for each eua approved molecular assay's key [ ] and performance [ ] characteristics including instruments approved for use and limits of detection. although no zika diagnostic kit, serological or viral, has been fully approved by the fda to date, the fda has worked collaboratively with developers to accelerate both eua approvals and the transition to formal full approvals of zika diagnostic kits using standard review timelines [ ] . a number of studies have evaluated the various eua tests both for independent sensitivity and specificity [ , , , , , , , ] assessments of assay performance and as part of comparative studies [ , [ ] [ ] [ ] [ ] to determine those best to use for the diagnostic or epidemiological surveillance need. a large number of companies have also developed tests that remain classified as "research use only" (ruo) for which emergency authorizations have not been granted. other authorizations for emergency use have been granted to different diagnostic kits by other agencies, such as who's emergency use assessment and listing procedures (euals) [ ] . while many diagnostic kits use methods similar or identical to those that may be used to support a vaccine clinical trial, often they are focused more toward the support of a clinical patient diagnosis and may not be sensitive enough for use to support vaccine development. often there is not enough data at the outset of a vaccine program, but especially in the case of emerging infectious diseases, to understand what assays will be most useful or informative or will perhaps even provide a correlate of protection for the vaccine into the future. a few general assumptions can be made at the outset about which types of assays will be needed to detect vaccine-induced humoral and cellular immune responses and these will evolve over time. the typical go-to methods used for vaccine clinical assays are: antibody binding-enzyme-linked immunosorbent assays (elisa); functional-virus neutralization or bactericidal; cellular-interferon gamma enzyme-linked immunospot assay (ifnγ-elispot) using the target antigen or antigen-derived peptide pools; and molecular or culture methods to detect the pathogen. the technologies used to develop and run these assays have improved over the years to allow for higher throughput, multiplexing, reduced sample volumes, and automation. often, more tests of a larger variety are done early in a program and are then whittled down based on the usefulness of the data generated so that just the relevant few remain to support large trials and licensure [ ] . when the first zikv vaccine trials commenced in late summer of , even though a few zika diagnostics had achieved eua status, none were commercially available and were initially restricted to public health use only. challenges to vaccine development centered around the determination of prior zikv exposure and immunity and determination of incident infection among study participants. each of the serological assays listed in table detect igm reflective of recent infection, however many, such as the mac-elisa and the inbios assay, will detect igm against the zikv envelope which is the target antigen for many vaccines. igg-based assays have not yet been validated primarily due to cross-reactivity between zikv and other flaviviruses, principally dengue. igm immunoassays targeted to the ns antigen are generally more specific than those directed to the viral envelope [ ] . in september , we initiated the first clinical trial of the gls- zikv dna vaccine in an endemic region, puerto rico, an area also known to have high rates of exposure to dengue. because no widely accepted standardized assays nor any international reference standards or reagents existed at the time of trial initiation, individual vaccine projects needed to rely on internally developed clinical assays to understand prior exposure and vaccine related immune responses. similar to patient diagnostic tests for zikv and as mentioned above, there was no accepted "gold standard" for any of the immunoassays one might choose to develop or use in a vaccine program. the extensive experience of our collaborative dna vaccine team allowed us to develop zikv-specific tests such as elisa, virus microneutralization and elispot around the development and pre-clinical testing of our zikv plasmid dna vaccine constructs [ , , , ] . these assays performed consistently on a pre-clinical scale and we were able to quickly expand their use to support our two phase gls- vaccine clinical trials. a concern always remains, however, that the lack of highly-characterized reagents and controls in early versions of vaccine clinical assays will result in difficulties in the maintenance of the assays through its full life cycle. sourcing, batch-to-batch variability and overall quality of critical reagents, standards and controls can become an issue over time. lack of standardization across the scientific field can be confounding in that interpretation of results from multiple "home brew" assays across labs are not directly comparable in the absence of an accepted international standard or a proficiency panel of samples [ ] . the main methodologies used to detect incident zikv infection are currently molecular-based, mainly reverse transcriptase polymerase chain reaction (rt-pcr) [ , , , , , , , , ] or varieties thereof [ , , ] , since culture methods can be both difficult and laborious [ ] [ ] [ ] . those viral detection systems with eua approval are shown in table . in clinical trials, identification of newly infected subjects over the treatment and follow-up periods are necessary to determine vaccine efficacy. as discussed earlier, for most individuals zikv infection is minimally symptomatic such that few present to clinical care. zikv is detectable in serum by rt-pcr for only a short interval following the onset of symptoms, typically for only seven days to a maximum of days [ , ] , though longer periods of rt-pcr detection (up to days) have been observed in serum of pregnant women [ ] , which may contribute to the incidence of zikv-related birth defects. zikv is excreted into the urine for approximately four weeks in most individuals, though this observation was not documented until over a year into the epidemic [ , , , ] . because of this, rt-pcr-based diagnosis of incident infections in a vaccine clinical trial would require very frequent sampling. other sample types have been evaluated for rt-pcr detection including saliva, whole blood, plasma, brain tissue, amniotic fluid and vaginal secretions [ , , , , , ] . a key concern from developers of both diagnostics and of vaccines or therapeutics for zikv highlighted at the hhs summit for diagnostics in july was the lack of well-characterized human zikv specimens which groups could use to fully understand the performance characteristics of the assays being developed and, eventually, work toward some standardization across the field. the who has initiated in, july , a collaborative study effort for the development of nucleic acid testing international standard for zika [ ] . additionally, in july , a plasma sample panel became available through the us fda for use in evaluating zikv immunoassay performance [ ] . reagents for newly emergent infectious diseases like zikv were not readily available from commercial vendors that had consistent production methods and quality controls in place, thus many reagents were not well characterized early on in the development of zikv clinical assays. because validated assays supporting vaccine efficacy endpoints need to support clinical and regulatory expectations over the life of the product, it is imperative that a line-of-sight is maintained such that reliable and qualified materials in appropriate quantity are available for resolving issues that arise. assays typically require some troubleshooting over time, changes to reagent lots or instruments, potential for multiplexing, platform changes to increase testing throughput, or a desire to bridge to a new technology [ ] . the development, qualification, validation and maintenance of vaccine clinical assays should be done in close consultation with biostatisticians and bench scientists to ensure the optimal assay design, control parameters and performance characteristics for the needs of the vaccine program from beginning to end. it should be noted that vaccine assay quality is highly dependent upon clinical study execution from collection through final data reporting. sample collection & handling are critical to the quality of the specimen and its ability to be used in an assay. implementing methods to assure the following are keys to successful vaccine clinical trials: proper sample storage and shipping conditions, processing and aliquotting with methods for contamination control, proper training of site and clinical research organization lab personnel, chain of custody verification of samples from collection to final valid test result, quality control checks and good data management. in the execution of vaccine clinical trials, there is the need for testing methodology to be reliable, reproducible (accuracy, specificity, robustness), and occasionally to provide rapid diagnosis (on-site or point-of-care testing, if needed), which contributes to patient care as well as to the understanding of vaccine efficacy or disease epidemiology. the diagnostic assay development response to zikv was quite rapid with the eua approval of different molecular detection assays and five serological assays in roughly months' time (tables and ) and the initiation of efforts to build international reference standards for both assay types. however, at the time of writing, there are still no fully approved diagnostics, no established "gold standard" detection methods nor any fully characterized and accepted international reference standards for zikv. while our understanding of the immune response to zikv has greatly expanded since the start of the most recent outbreak and leverages the years of vaccine research for other flaviviruses, such as dengue, there is still no established immune correlate of protection. other flavivirus vaccines have established immune correlates that are based on neutralization titers and it has been assumed that zikv will, as well [ ] . post-vaccination serum from participants enrolled in our group's gls- zika dna vaccine phase trial protected % of interferon α/β receptor knockout mice (ifnar) in a lethal-challenge model of zikv infection, however this protection was not dependent upon neutralizing antibody titers. other vaccines in clinical development have achieved neutralizing antibody titers in humans which were similar to the titers conferring protection in pre-clinical studies of the vaccines [ , ] . serum from participants in a phase clinical study receiving a purified inactivated zikv vaccine was also able to protect mice in a passive transfer mouse model [ ] . the development and validation of sensitive, specific and standardized zikv immunoassays to better characterize the immune response to vaccination are necessary to work toward the establishment of an immune correlate of protection in humans. efforts are being made through the work of the coalition for epidemic preparedness innovations (cepi) and others to ensure that rapid response mechanisms are in place to address emerging infectious diseases. well-seasoned development teams have accepted the challenges and funding to support building the vaccine design, manufacture and clinical assessment infrastructure needed to save lives in outbreak situations. scientific and quality principles must still apply although speed may be required when developing vaccine or diagnostic assays during an outbreak of a new emerging infectious disease. eid public health and countermeasure programs often have unique challenges for diagnostic and vaccine clinical assay development purposes including: . incomplete understanding of biology or epidemiology for appropriate target selection. lack of available reagent sources; inconsistency in quantity and/or quality of those available. difficulty in obtaining relevant human sample panels for the evaluation of test methods. challenges to produce clear line of sight sourcing and quality from early vaccine development through licensure. although the speed at which the diagnostic or vaccine development field needs to move will be dependent upon the urgency of the emerging infectious pathogen and its impact on human life, biological assay standardization is a critical component and requires three separate activities: • development of relevant, sensitive, specific and preferably quantitative biological assays. use of biostatistics to analyze assay performance data from development to validation to life cycle performance management. • ability to prepare stable and reproducible results over time to support diagnostic or vaccine program needs. if assay performance consistency and quality is not demonstrated, the validity of clinical study results may be questioned. assay standardization for any newly emergent infectious disease will be challenging and will: (i) take time to develop, collect and characterize quality reagents, (ii) to achieve sufficient sources of confirmed positive samples for establishment of serological standards, and (iii) to confirm new molecular standards, though this is more straightforward for molecular assays than serology. any efforts to prepare reagents, 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immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - qvyhjlp authors: roy, amrita; lim, liangzhong; srivastava, shagun; lu, yimei; song, jianxing title: solution conformations of zika ns b-ns pro and its inhibition by natural products from edible plants date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qvyhjlp the recent zika viral (zikv) epidemic has been associated with severe neurological pathologies such as neonatal microcephaly and guillain-barre syndrome but unfortunately no vaccine or medication is effectively available yet. zika ns b-ns pro is essential for the proteolysis of the viral polyprotein and thereby viral replication. thus ns b-ns pro represents an attractive target for anti-zika drug discovery/design. here, we have characterized the solution conformations and catalytic parameters of both linked and unlinked zika ns b-ns pro complexes and found that the unlinked complex manifested well-dispersed nmr spectra. subsequently with selective isotope-labeling using nmr spectroscopy, we demonstrated that c-terminal residues (r -k ) of ns b is highly disordered without any stable tertiary and secondary structures in the zika ns b-ns pro complex in the free state. upon binding to the well-characterized serine protease inhibitor, bovine pancreatic trypsin inhibitor (bpti), only the extreme c-terminal residues (l -k ) remain disordered. additionally, we have identified five flavonoids and one natural phenol rich in edible plants including fruits and vegetables, which inhibit zika ns b-ns pro in a non-competitive mode, with ki ranging from nm for myricetin to . μm for apigenin. molecular docking showed that they all bind to a pocket on the back of the active site and their structure-activity relationship was elucidated. our study provides valuable insights into the solution conformation of zika ns b-ns pro and further deciphers its susceptibility towards allosteric inhibition by natural products. as these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in terms of both inhibitory mode and chemical scaffold, our finding might open a new avenue for development of better allosteric inhibitors to fight zikv infection. zika virus (zikv) was a neglected, mosquito-borne flavivirus because of its assumed small geographical spread and mild clinical symptoms [ ] such as fever, headache, rashes and etc [ ] . the first biological zikv sample was isolated from a sentinel rhesus monkey in the zika plos one | https://doi.org/ . /journal.pone. july , / a a a a a forest of uganda in [ ] , and it was later found zikv is transmitted to humans by aedes mosquitoes. however since , large epidemics of asian genotype zikv have been reported around the world [ , ] . recently it is estimated that one-third of the world population might be at risk of infection [ ] . the rapid rise in zika infection is compounded by the ease of vertical [ ] and sexual human-to-human transmissions [ ] . recent studies have associated zikv infection with other diseases: guillain-barré syndrome and microcephaly in newborn infants of mothers infected with zikv during pregnancy [ , [ ] [ ] [ ] , thrombocytopenia [ ] , multipleorgan failures [ ] , and possibly male infertility [ ] . consequently who has declared a public health emergency for zikv infection [ ] . zikv represents a significant challenge to the public health of the whole world but unfortunately there is no available effective vaccine or therapy so far. zikv has a single-stranded positive sense rna genome of . kb, belongs to the flavivirus genus which also contains viruses causing dengue, yellow fever, west nile, japanese encephalitis and tick-borne encephalitis [ , ] . zikv shares a high degree of sequence and structural homology with other flaviviruses particularly dengue virus, thus resulting in immunological cross-reactivity [ ] . current zika outbreaks are largely localized within dengue-endemic areas, it is thus proposed that preexisting dengue-induced antibodies may enhance zika infection by antibody-dependent enhancement (ade), a factor that makes the vaccine approaches extremely challenging [ ] . while several recent studies focused on the possibility of developing neutralizing antibodies against zikv [ ] [ ] [ ] , it may take quite a while before such neutralizing antibodies for zikv enter clinical trials. zikv genome is translated into a single~ , -residue polyprotein, which is further cleaved into structural proteins and non-structural proteins [ ] . the correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and a viral ns b-ns protease (ns b-ns pro) and this property makes the flaviviral ns b-ns pro a well-established target for developing antiviral drugs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while there are studies testing the inhibition effects of certain drugs on ns b-ns pro such as bromocriptine [ ] , boronate [ ] , or small compounds inhibitors [ ] , we are interested in new chemical scaffolds that are found in the edible foods. our focus here is on whether natural edible products contain small molecules that have some inhibitory effects on the enzymatic activities of zikv ns b-ns pro. thus in the present study, we have characterized the solution conformations and catalysis of two enzymatically active zika ns b-ns pro: one with ns b and ns pro linked by extensively used (gly) -ser-(gly) sequence; and another with ns b and ns pro unlinked. subsequently, we identified several small molecular inhibitors from edible plants: five flavonoids and one natural phenol which were shown to inhibit zika ns b-ns pro in a non-competitive mode. further molecular docking suggests that these molecules inhibit zika ns b-ns pro by binding to a pocket on the back of the active site and allosterically affect the structure-activity property of zika ns b-ns pro. as such, identification of these natural product inhibitors here night provide new avenues for development of allosteric inhibitors against zikv infection. based on the sequence alignment with ns b and ns pro of the dengue serotype we previously characterized [ ] , the corresponding zika sequences were identified for the ns b (s a we also constructed a zika protease with ns b and ns pro linked by a (gly) -ser-(gly) sequence which was extensively used for functional and structural characterization of flaviviral ns b-ns pro complexes [ ] [ ] [ ] [ ] [ ] . the linked ns b-ns pro protein was detected in the pellet of e. coli cells with induction of mm isopropyl β-d-thiogalactopyranoside (iptg) for four hours at ˚c, while a portion of recombinant protein was found to be in supernatant with induction of . mm iptg overnight at ˚c. consequently, we purified the linked ns b-ns pro by ni + -affinity column under two conditions: the soluble form directly from the supernatant under native condition, but the insoluble form from inclusion body under denaturing condition, which was easily refolded by overnight dialysis against pbs buffer (ph . ) with mm β-mercaptoethanol (column of s c fig) . the linked complexes without his-tag were successfully obtained by cleavage with thrombin covalently linked to beads, followed by fplc purifications on a gel filtration column (hiload / superdex ) (column of s c fig) . nevertheless, the linked zika ns b-ns pro complexes purified directly from supernatant and from the refolding were indistinguishable as judged from both enzymatic activity and biophysical characterizations by cd, fluorescence and nmr. on the other hand, the wild-type ns b and ns pro domain are not covalently linked. furthermore, it has been previously demonstrated that only the unlinked dengue ns b-ns pro manifested well-dispersed nmr spectra [ , ] . therefore, we went on further to express the isolated zika ns b and ns pro. while the ns pro protein was found to be in inclusion body, the ns b was detected in supernatant. so we purified them by ni + -affinity column under denaturing condition for ns pro and under native condition for ns b. we first attempted to refold ns pro alone without ns b by dialyzing ns pro overnight against pbs buffer (ph . ) with mm β-mercaptoethanol, but all ns pro protein precipitated during dialysis and no enzymatic activity could be detected, suggesting that zika ns pro domain also needs ns b to fold correctly, as previously observed on all other flaviviral ns b-ns pro [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, using the same protocol, the mixture of ns b and ns pro was easily refolded into the soluble complex (column of s d fig) , was subjected to further cleavage of his-tag and the final fplc purification (column of s d fig). as small peptides diffuse significantly and thus usually cannot be seen in the sds-page system we used here, we checked the presence of the ns b peptide in the finally purified unlinked ns b-ns pro complex by the reverse-phase (rp) high pressure liquid chromatography (hplc) with an analytic c column. the hplc profile clearly showed that two peaks exist: one with the shorter retention time is for ns b while another with the longer retention time is for ns pro (s f fig) . first we acquired h nmr one-dimensional spectra for both linked and unlinked ns b-ns pro ( fig a) . both spectra have similar up-field peaks, which can only be observed on a well-folded protein with the tight tertiary packing and will disappear even upon a slight disruption to its tight tertiary packing [ ] . fig a clearly indicates both linked and unlinked complexes are well folded. an interesting note here is the peaks of linked complex are broader than those of the unlinked complex, which implies the linkage between ns b and ns pro introduced additional μs-ms conformational dynamics; this phenomenon was observed for the linked dengue ns b-ns pro [ , , ] . while this linkage significantly facilitated the crystallization of the linked flavi-viral ns b-ns pro complexes [ ] [ ] [ ] , this linkage significantly broadened nmr signals of linked ns b-ns pro complexes [ , , ] . thus, high-resolution nmr can be done on the unlinked form of dengue ns b-ns pro which was found to manifest well-dispersed nmr spectra [ , ] . similarly here, the linked zika ns b-ns pro also had broad nmr signals and consequently only a very small portion of hsqc peaks could be detected ( fig b) . by contrast, the unlinked complex had sharper d up-field peaks ( fig a) and a well-dispersed hsqc spectrum (fig b) . and unlinked (red) zika ns b-ns pro, as well as ns b ( - )-ns pro (green) complexes at a protein concentration of μm. cyan arrows are used to indicate the very up-field peaks. (b) superimposition of h- n hsqc spectra of n-labeled unlinked (blue) and linked (red) ns b-ns pro complexes at a protein concentration of μm. nmr spectra were acquired at ˚c in mm sodium phosphate buffer at ph . . pink arrows are used to indicate five hsqc peaks for ns b-trp , as well as ns pro-trp , ns pro-trp , ns pro-trp and ns pro-trp side chains only observed for the unlinked complex. (c) far-uv cd spectra of linked (blue) and unlinked (red) zika ns b-ns pro, as well as ns b ( - )-ns pro (green) complexes at a protein concentration of μm, together with that of dengue- ns b-ns pro complex previously obtained (black) (ref. we further characterized zika ns b-ns pro complexes with one being labeled and the other unlabeled. as seen in s a fig, the [ , , ] which unambiguously indicates dengue ns b has a tight tertiary packing unlike the zika one. therefore, our results suggest that in the zika ns b-ns pro complex, ns b has a portion of residues undergo μs-ms dynamics which made their nmr peaks too broad to be detectable; while the rest of ns b is highly disordered and lacks tight tertiary packing, which results in a narrowly-dispersed hsqc spectrum (s b fig) . both linked and unlinked zika ns b-ns pro complexes have far-uv cd spectra with the maximal negative signal at the wavelength of~ nm and lacks any positive signal below nm, which is different from that of the unlinked dengue complex of the same length [ ] which has the maximal negative signal at nm and large positive signal at nm ( fig c) . cd studies provide another piece of evidence that zika ns b-ns pro complexes contain more disordered regions than the dengue one. we have also collected spectra of intrinsic uv fluorescence from five trp residues in both linked and unlinked ns b-ns pro complexes ( fig d) , and both complexes have similar spectra with the emission maxima ranging from to nm, very similar to what were observed on the linked ns b-ns pro complexes of all four dengue serotypes ( nm) [ ] , which suggests that in zika ns b-ns pro, all five trp residues are similarly buried as dengue ones, as trp residue in the unfolded proteins has an emission maximum wavelength > nm [ ] . to screen inhibitors from natural products which are largely insoluble in aqueous buffers, we introduced organic solvents such as dimethyl sulfoxide (dmso) and glycerol into the activity assay buffers. we assessed their effects on the conformations of zika ns b-ns pro by monitoring intrinsic uv fluorescence. the results indicate that dmso induced significant changes in the intrinsic uv fluorescence spectra of zikv's ns b-ns pro (fig e) , while glycerol has no significant effect even with concentration up to % (fig f) . as the isolated zika ns b ( - ) was soluble in buffers, we acquired triple-resonance experiments hncacb, cbca(co)nh on a double labeled sample, and achieved the sequential assignment for all residues of ns b ( - ) except for pro , pro and pro which have no amide protons. fig a presents its (Δcα-Δcβ) chemical shifts, which represent a sensitive indicator of the residual secondary structures in disordered proteins [ , ] . the small absolute values of (Δcα-Δcβ) chemical shifts over the whole sequence clearly indicate that the isolated zika ns b ( - ) lacks stable secondary structure. we used ssp program [ ] to gain quantitative insights into the populations of different secondary structures and found residues arg -lys all have small but negative ssp score ( fig b) which implies these residues are in extended conformations that are weakly populated [ , ] . strikingly, superimposing the hsqc spectra of the n-labeled ns b in the isolated state against its complex form with unlabeled ns pro revealed that c-terminal residues arg -lys were the detectable hsqc peaks of ns b in complexed form, while n-terminal residues ser -ser were the disappeared hsqc peaks (fig c) . furthermore, we titrated the n-labeled ns b in complex with unlabeled ns pro by adding unlabeled bpti, which is a tight inhibitor and converts open conformation of flaviviral ns b-ns pro complexes to the closed form by both nmr [ ] and crystallography [ ] . here, upon adding bpti, the hsqc peaks of arg -ser disappeared and only very weak hsqc peaks of leu -lys were detectable ( fig d) . previously, we have characterized many binding interactions by nmr and found that the binding-induced disappearance of hsqc peaks was due to intermediate binding affinity (with kd values between nm and μm), or/and enhancement of μs-ms dynamics triggered by binding and binding interactions between two well-folded proteins [ ] and between disordered peptides and a well-folded protein [ ] . our nmr results here indicated that only c-terminal residues of zikv ns b remained disordered both in the complex form (arg -lys ; fig e) and in presence of bpti (leu -lys ; fig f) , and the conformation of these c-terminal residues in zikv ns b in these two forms are similar to the uncomplexed form as their hsqc peaks are superimposable ( fig c) . our observations are also supported secondary structure score obtained by analyzing chemical shifts with the ssp program. a score of + is for the well-formed helix while a score of - for the well-formed extended strand. (c) superimposition of h- n hsqc spectra of n-labeled ns b alone (blue) and n-labeled ns b in complex with unlabeled ns pro (red). the green is used to highlight two residues gly and gly , which could only be detected in the hsqc spectrum of ns b in the free state. (d) superimposition of n-labeled ns b alone (blue) and h- n hsqc spectra of n-labeled ns b in complex with unlabeled ns pro in the presence of unlabelled bovine pancreatic trypsin inhibitor (bpti) at a molar ratio of : (red). nmr spectra were acquired at ˚c in mm sodium phosphate buffer at ph . . a proposed diagram showing the conformations of ns b in the open conformation (e) and in the close conformation (f) triggered by complexing with bpti. blue arrows are used for indicating β-strands formed over ns b, while purple dashed lines are for flexible regions of ns b, whose hsqc peaks could be detected. https://doi.org/ . /journal.pone. .g by two independent observations: the c-terminal residues of ns b beyond val are completely invisible in the crystal structure of zika ns b-ns pro in the closed conformation [ ] ; a report on the apo/open-form of zika ns b-ns pro [ ] stated ". . . c-terminus of ns b (residues - ) is largely unseen, highlighting its high intrinsic flexibility." in sharp contrast, the crystal structure of apo denv ns b-ns pro complex showed the c-terminal loop of ns b adopts a defined conformation despite some discontinuous electron densities beyond residue [ ] . so why do zika and dengue ns b have such radical differences in conformations and dynamics? examination of ns b sequences reveals that the zika ns b region corresponding to the dengue ns b glu -thr shows no sequence homology at all (s fig) , thus implying sequence variations over this region may at least partly account for the loss of the contacts between the zika ns b and ns pro corresponding to the dengue ns b gln -leu and ns pro leu -ser , thus leading to the high dynamics of zika ns b c-half. thus we cloned and expressed a his-tagged zika ns b ( - ) with the c-half deleted. despite its low expression level and insolubility, we managed to purify a sufficient amount of ns b ( - ) for refolding with ns pro with the same protocol described above. interestingly, ns b ( - ) is sufficient to form a soluble complex with ns pro (s e fig) . however, its nmr peaks are broader than those of the linked ns b-ns pro (fig a) , which is likely due to μs-ms conformational dynamics or/and dynamic aggregation. on the other hand, its intrinsic uv fluorescence spectrum indicates that its four trp residues are similarly buried as linked and unlinked zika complexes with the full-length soluble domain of ns b ( fig d) . most interestingly, this complex with the c-half of ns b deleted contains less amount of disordered region than both linked and unlinked zika complexes with the full-length ns b, as seen by its cd spectrum having maximal negative signal shifted to nm and has positive elliptical signal at nm ( fig c) , despite being less disordered, this complex (with the later c-half of ns b deleted) showed no detectable enzymatic activity even with the protease concentration up to μm, which suggests this complex is enzymatically inactive. previously, we generated a truncated dengue ns b with the same c-half deleted but the shorter ns b is unable to form a soluble complex with its ns pro [ ] . however, we also generated another truncated dengue ns b with only residues - deleted, which was designated as ns b ( - ; Δ - ). interestingly, ns b ( - ; Δ - ) was able to form a soluble but inactive complex with its ns pro, which appeared to be highly disordered as reflected by its cd spectrum, and highly dynamic as judged by its nmr spectrum [ ] . together with recent reports on the crystal structures of zika ns b-ns pro complexes in both open and closed conformations [ , ] , our current results reveal that in solution the ns b residues over arg -lys are highly disordered in the open conformation. however, upon conversion into closed conformation such as triggered by bpti binding, the ns b residues arg -ser become further bound to the ns pro domain. on the other hand, our results suggest that despite being intrinsically disordered [ ] , the c-half of zika ns b is absolutely required for implementing the catalytic actions, thus implying that the closed conformation might be enzymatically-active, which was also previously speculated [ ] [ ] [ ] [ ] , ] . additionally, being disordered for the ns b c-half might have other functional advantages offered by intrinsically disordered proteins [ ] , such as to allow the formation of dynamic replication complex observed in hcv replication [ , ] . we have extensively characterized the catalytic properties of both linked and unlinked zika ns b-ns pro complexes in different buffer conditions. to allow comparison with the previously published results on the (gly) -ser-(gly) linked ns b-ns proteases of all four dengue serotypes [ ] , we have selected the same three substrates. these substrates, namely bz-nkrr-amc, boc-grr-amc and boc-gkr-amc, were extensively used for characterization of other flaviviral ns b-ns pro. moreover, we have used the same buffer as previously published [ ] , for the measurement of proteolytic activities. furthermore, the effects of salts and organic solvents on the zika protease activity were assessed. interestingly, as shown in s a fig, the zika ns b-ns pro efficiently cleaved bz-nkrr-amc, but unexpectedly showed weak activity on other two substrates. these results imply that besides requiring dibasic residues at the p and p sites (which is characteristic of the flaviviral ns proteases), zika ns b-ns pro appears to have a higher need for a basic residue at p site than dengue complexes [ ] ; as our current focus was not on profiling substrate specificity, we did not test this hypothesis on more substrates but instead measured all kinetic constants of zika complexes with bz-nkrr-amc. we found enzymatic activities of both linked and unlinked zikv complexes showed similar dependence on the ph values with the optimal ph at~ . (s b fig), which were similar to other flaviviral ns b-ns pro linked by (gly )-ser-(gly ) [ ] [ ] [ ] [ ] , different from dengue- ns b-ns pro, for which the optimal ph significantly switched from basic ph to neutral ph upon separating ns b from ns pro [ ] . secondly, like other flaviviral ns b-ns pro, the catalytic activity of zika ns b-ns pro also reduced significantly upon increasing the concentrations of nacl (s c fig). in the presence of mm nacl, the catalytic activity is only . % and . % of the linked and unlinked enzymes respectively in mm tris buffer at ph . . thirdly, glycerol concentrations also largely affect the enzymatic activity. the linked zika complex showed the highest activity in the presence of % glycerol while the unlinked had the highest activity in the presence of % glycerol (s d fig) . hence, we measured the kinetic parameters for the unlinked complex in the same buffers but with varying concentrations of organic solvents (s e fig) and the results were presented in table . the linked and unlinked zika complexes only have slightly differences for their kinetic constants in the buffer without any organic solvent. recently, the kinetic constants were just published for a zika ns b-ns pro which has ns b ( - ) and ns ( - ) also linked by the same (gly) -ser-(gly) linker [ ] . it has a km of . μm and kcat of . s - respectively. while the kcat values are almost identical, the km value is~ . -fold less than our current one. this small difference is likely due to: ) the difference of substrate as bz-nkkr-amc was used in the study [ ] ; or/and ) the slight differences of ns b and ns pro lengths, ) or/and the difference of the buffers: our buffer is mm tris ph . while the buffer in the publication [ ] is only mm tris ph . with % glycerol and mm chaps. it is extensively demonstrated that high salt concentrations has decreased, while glycerol at low concentrations has increased the activity of the flaviviral proteases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, we measured the activity of our linked zika protease in mm tris ph . , and the catalytic activity is . -time higher. due to the urgency to fight zikv infection, we attempted to screen the inhibitors of zika ns b-ns pro from natural products rich in edible plants. to better reflect the situation in vivo, here we selected the unlinked zika ns b-ns pro complex for screening. however as most natural products are largely insoluble in aqueous buffers, we have assessed effects of dmso and glycerol on the conformations (fig e and f ) as well as on catalytic parameters (table ) . consequently, mm tris buffer at ph . + % glycerol was utilized as the screening buffer as it could dissolve all natural products in the present study but has no significant effect on the conformation of zika ns b-ns pro (fig f) . remarkably, we have identified six compounds to have significant inhibitory effects, which belong to flavonoid and natural phenol ( fig a) . subsequently, we have determined their values of ic (s fig and table ); and inhibitory constant ki (fig c; table ). noticeably, despite sharing the same scaffold, five flavonoids have very distinctive inhibitory effects, with myricetin being the highest (ic of . μm and ki of . μm) and apigenin the weakest (ic of . μm and ki of . μm). on the contrary, no inhibitory activity was detected even at a compound concentration up to μm for daidzein, an isoflavone; as well as catechine that has a chemical structure identical to that of quercetin, except that the c-ring of catechin is a dihydropyran heterocycle (fig b) . noticeably, isorhamnetin, also called '-methylquercetin, has a much weaker inhibitory activity (ic of . μm and ki of . μm) than quercetin (ic of . μm and ki of . μm), although isorhamnetin is a derivative of quercetin only with proton of the hydroxyl group at ' position of phenyl ring replaced by a methyl group (fig a) . by contrast, quercetin and luteolin have almost the same inhibitory activity (table ) , although a hydroxyl group at position of benzopyran ring is absent in luteolin (fig a) . furthermore, a significant inhibitory effect (ic of . μm and ki of . μm) was detected for curcumin, a natural phenol with two aromatic rings linked by heptadiene group (fig a) , while no inhibitory effect was detected for resveratrol, also a natural phenol with two aromatic rings but linked by unsaturated ethene group (fig b) . these results suggest that these natural products inhibit zika ns b-ns pro specifically. another significant finding is that the six compounds inhibit zika ns b-ns pro by changing vmax but not km (fig c) . this indicates that these compounds inhibit zika ns b-ns pro in a non-competitive mode. in other words, six compounds are most likely to allosterically inhibit zika ns b-ns pro with their binding sites having no overlap with that for the substrate. indeed, previously myricetin and quercetin have been characterized to allosterically inhibit dengue- ns b-ns pro with ki values of . and . μm respectively, which, however, are much weaker than those for zika ns b-ns pro here. unfortunately, nmr spectroscopy cannot be utilized to investigate the interaction between those compounds and zika ns b-ns pro as the presence of % glycerol significantly increased the rotational tumbling time of the protein which made nmr peaks too broad for detection. to facilitate a better understanding of the experimental results and elucidate structure-activity relationship of the compounds, we used autodock software [ ] to dock six small molecules to the crystal structure ( lc ) [ ] of zikv ns b-ns pro with the substrate-derived inhibitor cn- removed. strikingly, all six compounds bind to the pockets on the back of the active site of zika ns b-ns pro (fig a) , similar to flavonoids binding to dengue- ns b-ns pro such as myricetin and quercetin [ ] . interestingly in the complexes, the short β-sheet formed by ns b residue leu -leu and asp -leu has direct contacts with the active site inhibitor cn- on one side, and with the six compounds on another side (fig b and c ). as such the pocket for binding six compounds is constituted by the surfaces provided by both zika conformations and inhibition of zika ns b-ns pro ns b and ns pro (fig d and e) , whose inner surfaces are relatively polar and negatively charged. interestingly, five flavonoids are highly superimposable in the pocket with the phenyl ring contacting the inner wall formed by ns b, and with the benzopyran ring located in a pocket provided by ns pro (fig e) . amazingly, although one phenyl ring of curcumin also occupies the same pocket like flavonoids, another phenyl ring has established the binding to a new pocket of ns pro, which is not observed for five flavonoids (fig f and g) . previously a similar pocket has been extensively identified on the dengue- ns b-ns pro structure u i [ ] to bind flavonoids including myricetin and quercetin [ , ] . however, the binding affinities of myricetin and quercetin to bind dengue ns b-ns pro are much weaker than those for zika one. as seen in s fig, although the overall rmsd of all heavy atoms is only . Å between the crystal structures of zika [ ] and dengue- [ ] ns b-ns pro complexes, which are both bound with active-site inhibitors, the binding pockets have slightly-different local geometries but very distinctive electrostatic properties: the zika pocket is more polar and negatively charged, while the dengue- pocket is less polar and positively charged (s fig). we further analyzed the hydrogen bond networks formed between zika ns b-ns pro and six compounds. interestingly, myricetin, the strongest inhibitor, establishes six hydrogen bonds with four zika ns pro residues (fig a) . protons of ', '-hydroxyl groups on phenyl ring form two hydrogen bonds with the backbone oxygen atoms of lys of the zika ns pro domain, while oxygen atoms of ', '-hydroxyl groups on phenyl ring establishes two hydrogen bonds with the side chain nh atom of asn . furthermore, proton of -hydroxyl group on the benzopyran ring forms a hydrogen bond with the side chain oxygen atom of gln , while proton of -hydroxyl group on benzopyran ring forms a hydrogen bond with the backbone nitrogen atom of gly . due to the absence of '-hydroxyl group on phenyl ring in quercetin, one hydrogen bond is missing between oxygen atom of '-hydroxyl groups and the side chain nh atom of asn (fig b) , which may at least partly account for its slightly weak affinity than that of myricetin. strikingly, due to the replacement of proton of '-hydroxyl group of quercetin by a methyl group in isorhamnetin, the hydrogen bond between proton of '-hydroxyl group and backbone oxygen of lys is lost (fig c) . this hydrogen bond appears to significantly contribute to the inhibitory activity as the inhibitory constant ki of isorhamnetin increases by~ times as compared to quercetin (table ) . on the other hand, luteolin establishes the same hydrogen bond network with zika ns b-ns pro residues as quercetin except for the loss of the hydrogen bond with gln due to the absence of -hydroxyl group in luteolin (fig d) . however, this hydrogen bond appears to be not very important as only a very slight reduction of the inhibitory activity was observed for luteolin as compared to quercetin (table ). however, the further absence of '-hydroxyl group on phenyl ring in apigenin results in loss of a hydrogen bond between '-hydroxyl group on phenyl ring and backbone oxygen atom of lys (fig e) . this hydrogen bond appears to be critical for its inhibitory activity as apigenin has a ki increased by~ times as compared to luteolin (table ) . amazingly, resveratrol has no detectable inhibitory activity but curcumin shows strong inhibitory activity comparable to quercetin. in fact, resveratrol and curcumin have similar structures but the linker between two phenyl rings of resveratrol is -carbon shorter than that of curcumin. the complex model between zika ns b-ns pro and curcumin (fig f) provides an explanation to the experimental result. with a longer linker, one phenyl ring of curcumin occupies the pocket identical to five flavonoids with the formation of hydrogen bonds with gln and gly , while another phenyl ring has additional contacts with a new pocket with unique hydrogen bonds with asp and ile . as such, although curcumin and isorhamnetin have the same '-methoxy and '-hydroxyl groups on the phenyl rings, likely by having bivalent binding sites, curcumin gains an inhibitory affinity which is much higher than that of isorhamnetin (table ) . a high affinity, which is achieved by establishing bivalent or multivalent binding sites, has been extensively found, such as on bivalent thrombin-inhibitor interactions [ ] . knowledge of catalysis, structures and dynamics of all structural states is beneficial for design of inhibitors with high affinity and specificity towards enzymes including viral proteases [ ] [ ] [ ] [ ] [ ] . this knowledge is particularly relevant to the flaviviral ns b-ns pro complexes as it is proposed that their catalytic activities require a transition from the open (inactive) to closed (active) conformation [ , , , ] . interestingly, regardless of being in the open or closed conformations, ns pro domains of different flaviviral ns b-ns pro complexes universally adopt the same chymotrypsin fold. by contrast, while the n-half of ns b assumes a similar βstrand packed to the ns pro domain in both open and closed conformations, the c-half of ns b shows a significant structural diversity in different structures determined so far. in the closed conformation, ns b structures of flaviviral ns b-ns pro complexes show a similar a short β-hairpin formation over the c-half of ns b and is tightly bound to the ns pro chymotrypsin fold (fig a) . however in the open conformation, the structural properties of the ns b c-half have been shown to be quite diverse. for the well-studied dengue- ns b-ns pro in the open conformation, most ns b residues are tightly packed with the ns pro domain as revealed by the crystal structure [ ] , and evident from its well-dispersed hsqc spectrum (s c fig) reconstructed from a previous report [ ] . in the present study, we first constructed and characterized the zika ns b-ns pro complex with ns b and ns pro linked by an artificial (gly) -(ser)-(gly) sequence which has been found to dramatically facilitate the crystallization of flaviviral ns b-ns pro complexes [ , , , ] . despite slight differences in sequence length, the catalytic parameters (table ) of our linked zika ns b-ns pro complex have no significant difference from those recently published [ ] . unfortunately, as previously observed on dengue- ns b-ns pro complexes [ , , ] , our linked zika complex also underwent significant μs-ms dynamics, thus making its nmr signals too broad to be detected (fig a and b) . as a consequence, we devoted efforts to generate and characterize an unlinked zika ns b-ns pro complex by using a protocol we previously established for the dengue- ns b-ns pro complex [ ] . this approach is also required for the selective isotope-labeling of zika ns b or ns pro for high-resolution nmr studies. indeed, despite showing no significant difference of catalytic properties from the linked one (table ) , the unlinked zika ns b-ns pro complex suddenly manifested a well-dispersed hsqc spectrum of the n-labeled ns pro domain in complex with unlabeled ns b with sharper nmr peaks (fig a and b) , which are consistent with previous nmr results on the unlinked dengue complexes [ , , ] . most importantly, this allowed us to selectively study the n-labeled ns b in complex with unlabeled ns pro. the results revealed that the zika ns b-ns pro complex, the c-terminal residues arg -lys of ns b remain highly disordered unlike the dengue- ns b-ns pro complex in the open conformation. binding to bpti appeared to trigger the conversion of zika ns b-ns pro complex into the closed conformation, in which the ns b c-terminal residues arg -ser become further bound to the ns pro domain. the intrinsic dynamics of the zika ns b c-half might be due to the significant sequence variations over ns b residues - (s fig). strikingly, this unique property for zika ns b-ns pro is not only observed in solution by our nmr investigation, but has been recently shown by the crystal structure of the apo/open-form of zika ns b-ns pro [ ] . in the future, it is of significant interest to explore what is the functional consequence of this unique property. one possibility might be that with the intrinsically disordered ns b c-half [ ] , the zika ns b-ns pro is more susceptible to the allosteric regulation [ ] [ ] [ ] . although many adults infected with zikv will have only mild or even no detectable symptoms, the zikv can be transmitted from a pregnant woman to her fetus, thus leading to birth defects such as microcephaly. this imposed a great challenge and urgency to fight zikv. therefore we attempted to screen inhibitors from natural products rich in edible plants for the unlinked zika ns b-ns pro, which represents a more realistic form in vivo. remarkably, we identified five flavonoids and one natural phenol to specifically inhibit zika ns b-ns pro with the strongest myricertin having a ki of nm. recently, lim et. al. has reported a ki value of . um for myricitin [ ] , the difference might be due to ) the linked zika ns b-ns pro which they have used for their enzyme inhibition assay whereas we have used an unlinked enzyme and ) the ph of the assay buffer they used is ph whereas we have used ph . . furthermore, analysis of structures and inhibitory activities reveals that for flavonoids, the presence of benzopyran ring and the position to connect to phenyl ring are critical for the inhibitory activity, as evident from the results that daidzein and catechin were inactive on inhibiting zika ns b-ns pro. furthermore, the number of hydroxyl groups on phenyl ring is a key determinant for the inhibitory activity. similar observation has been reported in a recent study where a number of polyphenols has been studied for inhibitory activity against linked zika ns b-ns pro [ ] . interestingly, our current molecular docking reveals that these compounds bind to a pocket on the back of the active site of the zika ns b-ns pro complex. while five flavonoids bind very similarly to the pocket constituted by both ns b and ns pro, curcumin can have an additional binding site outside of this pocket. amazingly, the pocket we observed on the zika ns b-ns pro to bind five flavonoids is very similar to that previously observed on the dengue ns b-ns pro [ , ] , but the additional binding site for curcumin represents a novel one. therefore, zika ns b-ns pro appears to be highly susceptible to allosteric inhibitions, as previously found on other flaviviral ns b-ns pro complexes [ , , , ] . noticeably, probably due to slight variations of the local geometry or/and significant difference in electrostatic potentials of the allosteric pockets of zika and dengue complexes, the same flavonoids inhibit the zika ns b-ns pro complex with higher activity [ ] . five flavonoids and curcumin are rich in various edible plants including caper, tea, red onion, celery, broccoli, green pepper and turmeric. additionally, they have been previously demonstrated to have beneficial effects on human health and thus extensively used as supplements [ , [ ] [ ] [ ] . as such, our finding might help the public to fight against zikv infection immediately. in the further, it is also of both fundamental and therapeutic interests to characterize the allosteric mechanisms by which these natural products inhibit zika ns b-ns pro by both experimental and computational approaches [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in both inhibitory mode and chemical scaffold [ , ] , our finding may also provide a promising foundation for further development of novel allosteric inhibitors of higher affinity and specificity to fight zikv infection. the identified genes encoding ns pro ( - ) and ns b ( - ) from the asian zika strain (genbank id: ku . ) were optimized and synthesized by genscript (piscataway, nj). with designed primers, the genes by genscript were used as templates for amplifying dna fragments encoding the isolated ns ( - ) and ns b ( - ) with the transmembrane regions removed, as well as ns b with the c-region further deleted. furthermore, dna fragments were designed for linking ns b ( - ) to ns ( - ) by (gly) -ser-(gly) using overlap pcr as previously described ( ) . amplified dna fragments were subsequently cloned into his-tagged pet a vector (novagen). dna sequences of all constructs were verified by automated dna sequencing. all pet a vectors containing different zika genes were transformed into escherichia coli bl (de ) star cell (thermo fisher scientific), which was cultured in luria-bertani broth containing μg/ml kanamycin at ˚c until the a reached . . subsequently, protein expressions of different constructs were induced with different conditions. to obtain soluble form of the linked ns b-ns pro complex, protein expression was induced with . mm isopropyl β-d-thiogalactopyranoside (iptg) overnight at ˚c. however, the expression level of the soluble form was low and consequently the linked complex was also prompted into inclusion body with induction of mm iptg for hours at ˚c, which yielded to a much higher expression level. for isolated ns b and ns , protein expressions were induced with mm iptg for hours at ˚c. for the linked ns b-ns pro complex, the soluble form was purified by ni + -affinity chromatography under native condition. subsequently, the his-tag was removed by the cleavage with thrombin-agarose beads from thrombin cleancleave™ kit (sigma-aldrich, st. louis, mo), followed by binding to an excess amount of ni-nta beads to remove his-tag and uncleaved fusion protein, as well as a final fplc purification on a gel filtration column (hiload / superdex ). to obtain the linked complex in inclusion body, the cell pellets were re-suspended in cold pbs buffer at ph . , containing mm β-mercaptoethanol and m urea and the supernatant containing the recombinant proteins were purified by ni-nta affinity column under denaturing condition. eluted fractions were subjected to dialysis against pbs buffer (ph . ) with mm β-mercaptoethanol at ˚c overnight to allow the refolding of the complex. the refolded complex was subjected to the cleavage of his-tag and final fplc purification as described above. the isolated ns b ( - ) and ns were purified by ni-nta affinity column under denaturing condition, while ns b ( - ) was purified under native condition. subsequently, the purified ns b ( - )/ns b and ns were mixed up with an excess amount of ns . the mixture was subjected to refolding by dialysis against pbs buffer (ph . ) with mm β-mercaptoethanol at ˚c overnight as we previously established for dengue ns b-ns pro complex ( ) . the refolded complex was subjected to the cleavage of his-tag and further fplc purification as described above. the generation of the isotope-labeled proteins for nmr studies followed a similar procedure except that the bacteria were grown in m medium with the addition of ( nh ) so for n labeling and ( nh ) so /[ c]-glucose for double labeling [ ] . all recombinant protease samples were checked by tris-glycine sds page. however, for this sds-page system, small peptides diffuse significantly and thus usually cannot be seen. therefore, reverse-phase (rp) high pressure liquid chromatography (hplc) with an analytic c column was used to check the presence of the isolated ns b peptides with a molecular weights less than kda. molecular weight verification and protein sequencing were conducted with time-of-flightmass spectrometer (applied biosystems). protein concentration was determined by the uv spectroscopic method with m urea [ ] . intrinsic uv fluorescence spectra were measured with a cary eclipse fluorescence spectrophotometer as we previously described [ ] with the excitation wavelength at nm. circular dichroism (cd) experiments were performed on a jasco j- spectropolarimeter and data from five independent scans were added and averaged [ ] . to assess the effects of dmso and glycerol on the conformation of zika ns b-ns pro, we monitored the change of its intrinsic uv fluorescence instead of circular dichroism (cd), because organic solvents were found to provoke very high non-specific noises. all nmr experiments were acquired on an mhz bruker avance spectrometer equipped with pulse field gradient units as described previously [ ] . to achieve sequential assignment, n-/ c-double labeled zika ns b sample was prepared at a protein concentration of μm in mm phosphate buffer. a pair of triple-resonance experiments hncacb, cbca(co) nh were acquired [ ] . to investigate the binding interaction between zika ns b-ns pro and bpti, hsqc spectra of zika ns b-ns pro only with ns b n-labeled were acquired in the absence and in the presence of bpti (sigma-aldrich) at different ratios. to allow comparison with the ns b-ns pro complexes of four dengue serotypes ( ), we selected three fluorophore-tagged substrates previously used ( ) : namely bz-nle-lys-arg-arg-amc, boc-gly-arg-arg-amc and boc-gly-lys-arg-amc (bachem ag, bubendorf), which were dissolved in dimethyl sulfoxide for preparing stock solutions ( mm). all enzymatic experiments were performed in triplicate and data are presented as mean ± sd, while ic , km and ki were obtained by fitting with graphpad prism . [ ] . the ph dependence was measured with a protease concentration of nm and substrate (bz-nkrr-amc) concentration of μm at . ph intervals using the following buffers: mm citrate-phosphate buffer for ph - , mm phosphate buffer for ph . - , mm tris-hcl buffer for ph . - . , and mm na-bicarbonate buffer for ph - . . for steady state kinetics, we used the exactly the same buffer as a previous one on profiling substrate specificity for the ns b-ns pro complexes of all four dengue serotypes ): mm tris-hcl at ph . . to screen natural product inhibitors of zika ns b-ns pro, we also measured the km values of zika ns b-ns pro in the presence of dmso and glycerol which allow the solubilization of these compounds in the assay buffer. briefly, zika protease at nm was incubated with substrates ranging from to μm in μl assay buffer at ˚c. progression of enzymatic reaction was monitored as an increase in fluorescence at λ ex of nm and λ em of nm. fluorescence intensity is reported in arbitrary units. initial fluorescence or absorbance velocities (relative fluorescence units per minute or relative absorbance units per minute) were converted to ms - from a standard acmc calibration curve. subsequently, the curves were fitted to the michaelis-menten equation by nonlinear regression. all natural products were purchased from sigma-aldrich, which are all hplc purified. after optimization of buffer conditions, here, we selected mm tris-hcl buffer at ph . in the presence of % glycerol which could dissolve all natural products. briefly, for the initial screening, the zika protease at nm was preincubated for min with different compounds at final concentrations of and μm dissolved in μl dmso, followed by adding bz-nkrr-amc to μm to initiate reaction. only the compounds showing significant inhibitions at both concentrations were subjected to further determination of ic and ki. for ic determination, the zika protease at nm was preincubated at ˚c for min with natural products at various final concentrations in μl dmso; and subsequently the reaction was initiated by adding bz-nkrr-amc to μm. for ki determination, the assay was performed with different final concentrations of the inhibitors and substrate. briefly, the zika protease at nm was preincubated with the inhibitor at different concentrations for min at ˚c. subsequently, the reaction was initiated by addition of the corresponding concentration series of the substrate. all measurements were performed in triplicate and data are presented as mean ± sd. the ki was obtained by fitting in the non-competitive inhibition mode with graphpad prism . , with an equation: vmaxinh = vmax/( +i/ki), while i is the concentration of inhibitor [ ] . to gain insight into structural details of the binding pocket, we docked all six active natural products to the crystal structure (pdb code of lc ) of zika ns b-ns pro in complex with an active site inhibitor cn- [ ] . the chemical structures of the compounds were downloaded from zinc (http://zinc.docking.org) and chemicalbook database (http://www. chemicalbook.com) respectively. subsequently the structures were geometrically optimized with avogadro [ ] . the partial charges of all atoms in small compounds and zika ns b-ns pro were assigned with gasteiger-marsili charges, and non-polar hydrogen atoms were merged into the appropriate heavy atoms with autodocktools [ ] . autodock software (version . ) was utilized to dock six compounds to the crystal structure of zika ns b-ns pro. the grid box was set with × × (x,y,z axis) with the default . Å spacing. the initial population size was set to , and the number of energy evaluations was set to , , , and number of docking runs was set as . the results were clustered with each cluster having a tolerance of Å. the complexes with the lowest energy were selected for analysis and display. zika fever and congenital zika syndrome: an unexpected emerging arboviral disease zika virus: an update on epidemiology, pathology, molecular biology, and animal model isolations and serological specificity zika virus: history of a newly emerging arbovirus complete coding sequence of zika virus from a french polynesia outbreak in potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study zika virus associated with microcephaly evidence of sexual transmission of zika virus zika virus disrupts neural progenitor development and leads to microcephaly in mice the brazilian zika virus strain causes birth defects in 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critical role of the extra domain in dimerization of the enzyme defining the extra domain as a new target for design of highly specific protease inhibitors fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting avogadro: an advanced semantic chemical editor, visualization, and analysis platform key: cord- -v r d a authors: chan, jasper fuk-woo; zhang, anna jinxia; chan, chris chung-sing; yip, cyril chik-yan; mak, winger wing-nga; zhu, houshun; poon, vincent kwok-man; tee, kah-meng; zhu, zheng; cai, jian-piao; tsang, jessica oi-ling; chik, kenn ka-heng; yin, feifei; chan, kwok-hung; kok, kin-hang; jin, dong-yan; au-yeung, rex kwok-him; yuen, kwok-yung title: zika virus infection in dexamethasone-immunosuppressed mice demonstrating disseminated infection with multi-organ involvement including orchitis effectively treated by recombinant type i interferons date: - - journal: ebiomedicine doi: . /j.ebiom. . . sha: doc_id: cord_uid: v r d a background: disseminated or fatal zika virus (zikv) infections were reported in immunosuppressed patients. existing interferon-signaling/receptor-deficient mouse models may not be suitable for evaluating treatment effects of recombinant interferons. methods: we developed a novel mouse model for zikv infection by immunosuppressing balb/c mice with dexamethasone. results: dexamethasone-immunosuppressed male mice ( – weeks) developed disseminated infection as evidenced by the detection of zikv-ns protein expression and high viral loads in multiple organs. they had ≥ % weight loss and high clinical scores soon after dexamethasone withdrawal ( dpi), which warranted euthanasia at dpi. viral loads in blood and most tissues at dpi were significantly higher than those at dpi (p < . ). histological examination revealed prominent inflammatory infiltrates in multiple organs, and cd + and cd + inflammatory cells were seen in the testis. these findings suggested that clinical deterioration occurred during viral clearance by host immune response. type i interferon treatments improved clinical outcome of mice ( % vs % survival). conclusions: besides virus dissemination, inflammation of various tissues, especially orchitis, may be potential complications of zikv infection with significant implications on disease transmission and male fertility. interferon treatment should be considered in patients at high risks for zikv-associated complications when the potential benefits outweigh the side effects of treatment. zika virus (zikv) is an emerging flavivirus that has been largely neglected for n years after its discovery due to its restricted geographical distribution and its presumed low clinical significance (chan et al., a) . since , large-scale outbreaks of zikv infection have occurred in the pacific islands, latin america, and most recently, usa and southeast asia (duffy et al., ; musso and gubler, ; zhu et al., ) . as of october , n countries/territories have ebiomedicine ( ) - reported continuing mosquito-borne transmission of zikv (world health organization. zika situation report. october , ) . in addition to mosquito-borne transmission, sexual and transplacental transmissions of zikv have also been reported (chan et al., a; musso et al., a; foy et al., ; calvet et al., ) . these non-vectorborne transmission routes render the control of the continuing epidemic more complicated. zikv was not considered as an important human pathogen in the past as most infected adult patients were asymptomatic or developed a self-limiting acute febrile illness which resolved within - weeks (chan et al., a; duffy et al., ). however, it has been recently recognized that infected mothers may transmit the virus transplacentally to developing fetuses, leading to congenital malformations, including microcephaly, cerebral malformations, ophthalmological and hearing defects, and arthrogryposis (chan et al., a; mlakar et al., ; de paula et al., ; leal et al., ) . some infected adults may also develop severe neurological complications, such as guillain-barré syndrome, meningoencephalitis, and myelitis (cao-lormeau et al., ; carteaux et al., ; mecharles et al., ) . moreover, zikv-related fatalities have been increasingly recognized. most of the patients with fatal infection had underlying medical conditions and some were markedly immunosuppressed, including a patient with systemic lupus erythematosus and rheumatoid arthritis who was on corticosteroid therapy and died of disseminated infection with detectable zikv rna in blood, brain, spleen, liver, kidney, lung, and heart obtained at postmortem examination (pan american health organization/world health oganization (paho/who), ; sarmiento-ospina et al., ; arzuza-ortega et al., ) . a number of animal models have been developed for studying the pathogenesis and evaluating countermeasures for zikv infection. rhesus macaques with subcutaneous zikv inoculation develop mild clinical signs that resemble the self-limiting illness in most infected immunocompetent adults (dudley et al., ) . this non-human primate model provides a robust platform for the evaluation of vaccines and host immune response (abbink et al., ) . however, the mild clinical disease in these primates is suboptimal for antiviral treatment evaluation. moreover, expertise and facilities for working with non-human primates are not available in most research laboratories. wild-type adult balb/c mice are not susceptible to intraperitoneal zikv inoculation (dick, ) . suckling and young mice with intracerebral zikv inoculation develop disease that is localized to the central nervous system (dick, ; way et al., ; weinbren and williams, ; bell et al., ) . pregnant mice and fetal mice with partially intact type i interferon signaling response (fetuses of female mice deficienct in type i interferon signaling response crossed to wild-type male mice) were used to study pathogenesis in pregnancy and maternalfetal transmission, but these models are technically more demanding (miner et al., ; cugola et al., ) . type i/ii interferon-signaling-/ receptor-deficient mice with intraperitoneal or subcutaneous zikv inoculation develop fatal, disseminated infection (lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ) . these models are useful for the evaluation of countermeasures for zikv infection as the protective effects of antivirals drugs and vaccines are more easily observed in treated mice. however, such models have complete/nearcomplete deficiency in interferon response and do not resemble the real clinical situation in immunosuppressed humans. moreover, these mice are suboptimal for the study of host immune response and may be too expensive for laboratories in resource-limited areas. because of these limitations and knowledge gaps, we developed and characterized a more readily available mouse model which resembles immunosuppressed hosts with disseminated infection. we showed that these mice developed inflammation in multiple organs, including the testes, which may have important implications on zikv's longterm outcome and effects on fertility. we also utilized this novel animal model to show that early treatment with clinically approved recombinant type i interferons improved the clinical outcome of these mice. a clinical isolate of zikv (puerto rico strain prvabc ) was kindly provided by brandy russell and barbara johnson, centers for disease control and prevention, usa. the virus was amplified by three additional passages in vero cells (atcc) in minimum essential medium (mem) supplemented with % fetal calf serum and units/ml penicillin plus μg/ml streptomycin to make working stocks of the virus. for virus titration, aliquots of zikv were applied on confluent vero cells in well plates for % tissue culture infectious dose (tcid ) assay as we previously described with slight modifications (zhou et al., ) . briefly, serial -fold dilutions of zikv were inoculated in a vero cell monolayer in quadruplicate and cultured in penicillin/streptomycinsupplemented mem. the plates were observed for cytopathic effect for days. viral titer was calculated with the reed and münch endpoint method. one tcid was interpreted as the amount of virus that causes cytopathic effect in % of inoculated wells. approval was obtained from the committee on the use of live animals in teaching and research of the university of hong kong. male and female balb/c mice, - weeks old, were obtained from the laboratory animal unit of the university of hong kong. the mice were kept in biosafety level- housing and given access to standard pellet feed and water ad libitum. virus inoculation experiments were performed in a biosafety level- animal facility according to the standard operating procedures approved by the committee on the use of live animals in teaching and research of the university of hong kong as we described previously (zhang et al., ) . the mice were randomly divided into groups and given different regimens of virus inoculation, dexamethasone, and recombinant interferon treatment (table ) . phosphate-buffered saline (pbs) was used to dilute the virus stocks to the desired concentration, and inocula were back-titrated to verify the dose given. on the day of virus inoculation, a dose of the virus equivalent to × tcid ( . × plaque forming units) in μl of pbs was inoculated via the intraperitoneal route into mice under ketamine ( mg/kg) and xylazine ( mg/kg) anesthesia. mice in the negative-control groups (groups to ) were injected with the same volume of pbs. mice were monitored three times each day for clinical signs of disease and a numerical score was assigned at each observation as previously described (dowall et al., ; graham et al., ) . their body weight and survival were monitored for days post-inoculation (dpi) or until euthanasia. three mice in each group (except groups and which included mock-infected control mice without dexamethasone immunosuppression and group which included zikv-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal) were sacrificed at dpi for virological, histological, and immunohistochemistry analyses. the remaining mice were sacrificed at dpi or euthanized when there was a % weight loss or % weight loss with ≥ clinical sign (dowall et al., ) . samples of brain, testis/ epididymis (male), prostate (male), ovary/uterus (female), kidney, urinary bladder, spleen, liver, pancreas, intestine, heart, lung, and salivary gland were collected at necropsy. the specimens were separated into two parts, one immediately fixed in % pbs-buffered formalin, the other immediately frozen at − °c until further experiments. blood samples were also collected for rna extraction and real-time pcr analysis. paraffin-embedded tissues were cut into - μm sections, mounted on slides, and stained with hematoxylin and eosin (h&e) for light microscopy examination as we previously described (zheng et al., ) . for immunohistochemical staining of zikv-ns antigen, mouse antiserum against zikv-ns protein prepared as we previously described was used as primary antibody (chan et al., b) . de-paraffinized and rehydrated tissue sections were treated with antigen unmasking solution according to manufacturer's instructions (vector laboratories inc., burlingame, ca, usa) and then stained with mouse on mouse polymer ihc kit (abcam, cambridge, united kingdom). the primary antibody mouse anti-zikv-ns antiserum ( : dilution with % bsa/pbs) was incubated at °c overnight. this was followed by mouse on mouse hrp polymer kit (abcam) with horseradish peroxidase-conjugated secondary antibody for min. color development was performed using , ′-diaminobenzidine (dab) (vector laboratories, burlingame, ca, usa). for immunohistochemical staining of cd and cd , the sections were incubated at °c for overnight with primary antibody (rabbit anti-mouse cd , or rat anti-mouse cd α (abcam) after antigen unmasking and blocking. this was then followed by incubation with biotin-conjugated goat anti-rabbit igg or goat anti-rat igg (calbiochem, darmstadt, germany) for min at room temperature. streptavidin/peroxidase complex reagent (vector laboratories) was then added and incubated at room temperature for min. color development was done with dab (vector laboratories). all tissue sections were examined microscopically by two pathologists in an operatorblinded manner. images were captured with nikon i imaging system equipped with spot-advance computer software. total nucleic acid (tna) was extracted from the blood and necropsied tissues using ez virus mini kit v . and qiasymphony dsp virus/pathogen mini kit (qiagen, hilden, germany), respectively, as we previously described (zheng et al., ; chan et al., b; chan et al., a) . zikv envelope gene was measured by using quantinova probe rt-pcr kit (qiagen) in lightcycler real-time pcr system (roche diagnostics, basel, switzerland). μl of purified tna was amplified in a μl-reaction containing μl of × quantinova probe rt-pcr master mix, . μl qn probe rt-mix, . μm forward primer, . μm reverse primer, and nm probe. forward primer ( ′-cgytgcccaacacaagg- ′), reverse primer ( ′-ccacyaaygttcttttgcabaca- ′), and probe ( ′-hex-agcctaccttgayaagcartcagacactc-iabkfq- ′) targeting the zikv envelope gene as we previously described were used (chan et al., b) . reactions were incubated at °c for min, followed by °c for min, and then thermal cycled for cycles ( °c for s, °c for s). internal control β-actin gene was measured by using quantinova sybr green rt-pcr kit (qiagen) in lightcycler real-time pcr system. μl of purified tna was amplified in a μl-reaction containing μl of × quantinova sybr green rt-pcr master mix, . μl qn sybr green rt-mix, . μm forward primer ( ′-acggccaggtcatcactattg- ′) and . μm reverse primer ( ′-caagaaggaaggctggaaaag- ′) for the β-actin gene. reactions were incubated at °c for min, followed by °c for min, and then thermal cycled for cycles ( °c for s, °c for s). a series of -fold dilutions equivalent to × to × copies/reaction mixture were prepared to generate standard curves and run in parallel with the test samples. all data were analyzed with graphpad prism software (graphpad software, inc). kaplan-meier survival curves were analyzed by the log rank test, and weight losses were compared using two-way anova. student's t-test was used to determine significant differences in virus titers, and tukey-kramer post hoc tests were used to discern differences among individual treatment groups as previously reported rossi et al., ) . p-values b . were considered statistically significant. to establish a novel mouse model for zikv infection, we compared the clinical, histological, and virological findings of male (group ) and female (group ) mice with dexamethasone immunosuppression and zikv inoculation with those of the appropriate controls (groups to ) (table ). in terms of the clinical parameters, the dexamethasone-immunosuppressed mice developed mild (~ %) weight loss (fig. a) and no mortality at dpi ( fig. b and c) . the weight losses of the dexamethasone-immunosuppressed mice with zikv inoculation (groups and ) table eleven groups of mice receiving different regimens of virus inoculation, dexamethasone, and antiviral treatment in this study a . group gender routes and inoculum of zikv ( dpi) dexamethasone antiviral treatment b date of sacrifice/euthanasia m ip × tcid mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi c f ip × tcid mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi m ip × tcid no no (n = ) and (n = ) dpi f ip × tcid no no (n = ) and (n = ) dpi m no mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi f no mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi no (n = ) dpi m ip × tcid mg/kg q h ip, from days before to dpi inclusively no (n = ) dpi m ip × tcid mg/kg q h ip, from days before to dpi inclusively pegylated ifn-α b iu/dose q h sc at , , and dpi (n = ) and (n = ) dpi m ip × tcid mg/kg q h ip, from days before to dpi inclusively ifn-β b , iu/dose q h ip at , , , , and dpi (n = ) and (n = ) dpi abbreviations: dpi, days post-inoculation; f, female; ifn, interferon; ip, intraperitoneal; m, male; sc, subcutaneous. a - week-old balb/c mice were used in all the groups. b the preparations of pegylated ifn-α b and ifn-β b used in this study were pegintron (merck & co., inc., whitehouse station, nj, usa) and betaferon (bayer schering pharma ag, berlin, germany), respectively. c the mice in group were euthanized at dpi as they had ≥ % weight loss and ≥ clinical symptom. were consistently more significant than those of their comparators, including the zikv-inoculated mice without dexamethasone immunosuppression (groups and ) and mock-infected mice without dexamethasone immunosuppression (groups and ) starting at dpi (p b . ). minimal histological changes and inflammatory infiltrates were seen in the tissues of the male and female mice with dexamethasone immunosuppression and zikv inoculation (groups and ). on the other hand, zikv-ns protein expression was detected by immunohistochemical staining in most tissues of these mice, but not in dexamethasone-immunosuppressed mice with mock infection (groups and ), suggesting that the viral protein expression was specific and not related to dexamethasone effects (fig. ) . the dexamethasone-immunosuppressed mice with zikv inoculation (groups and ) also had high mean viral loads in blood and most tissues at dpi, especially in the testis/epididymis, ovary/uterus, prostate, spleen, and pancreas (figs. a and b and s a and b). these findings at dpi were suggestive of disseminated but non-lethal zikv infection involving different organs with minimal inflammatory response due to dexamethasone immunosuppression. to investigate the possible effects of immune reconstitution in the dexamethasone-immunosuppressed mice, dexamethasone was stopped after dpi. this led to prominent weight loss and increased symptoms in the dexamethasone-immunosuppressed mice (groups and ). the most prominent body weight loss was observed in the male mice with dexamethasone immunosuppression and zikv inoculation (group ), with all mice having weight loss of ≥ % at dpi (fig. a) . all of the female mice with dexamethasone immunosuppression and zikv inoculation (group ) also had progressive weight loss and / ( . %) of them had ≥ % weight loss at dpi. in contrast, none of the mice with dexamethasone immunosuppression from days before to days post-infection (group ) developed abrupt weight loss between dpi and dpi (fig. a) . the weight loss of mice in groups and became consistently more than those of their comparators in the other control groups (groups to ), including those in the dexamethasone-immunosuppressed mice with mock infection (groups and ) since dpi (p b . ). together, these findings suggested that the combination of immune reconstitution after dexamethasone withdrawal and disseminated virus infection were responsible for the abrupt clinical deterioration. all of the mice in groups and developed rapid breathing, lethargy, and/or ruffled fur since dpi (group ) or dpi (group ), shortly after dexamethasone was stopped (fig. b) . the reasons for the earlier onset of weight loss and symptoms in the male mice were not fully understood, but might be related to the higher cumulative dose of dexamethasone because of their higher baseline body weights and/or possible effects of androgen on virus replication (tian et al., ) . based on the predefined criteria, all ( %) male and / ( . %) female mice were euthanized at dpi and dpi, respectively (fig. c) . comparatively, all the mice in the other control groups (groups to ) had either gained weight or had b % weight loss with spontaneous recovery at dpi (fig. a) , remained asymptomatic (fig. b) , and survived through the study period (fig. c) . at euthanasia ( - dpi), h&e staining of the necropsied tissues of these mice showed prominent acute inflammatory reactions with predominantly lymphocytic infiltrates. the most prominent inflammatory changes were seen in the brain (cortical parenchymal and perivascular lymphocytic infiltrates) (fig. a to c), kidney (acute tubulitis and interstitial inflammation) (fig. d to f), and testis (necrotic and hemorrhagic seminiferous tubules with marked lymphocytic infiltration in the perimeter of the tubules and the interstitium) (fig. a to d) . zikv-ns protein expression was still visible, but to a lesser degree, in the immunohistochemical staining of the testis/epididymis, ovary/uterus, kidney, spleen, small intestine, pancreas, and salivary gland of the dexamethasone-immunosuppressed mice with zikv inoculation at - dpi compared with dpi. in contrast, no inflammatory reaction and viral protein expression were seen in any organ of the control mice with zikv inoculation alone (groups and ) or dexamethasone immunosuppression alone (groups and ) ( fig. c and d) . these findings confirmed that mice with zikv inoculation but no dexamethasone immunosuppression were not susceptible to infection as previously reported, and that the histological changes in the model mice (groups and ) were unrelated to dexamethasone-induced effects such as drug-induced testicular toxicity (lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ; khorsandi et al., ) . the absence of inflammatory infiltrates in zikv-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal (group ) supported the role of the host immune response in eliciting the clinical and histological changes in the zikv-inoculated mice with dexamethasone withdrawal (groups and ). to further confirm the presence of inflammatory infiltrates and characterize the cell types involved in the host immune response, we stained the necropsied testis of the dexamethasone-immunosuppressed mice with zikv inoculation and those of the dexamethasoneimmunosuppressed mock-infected control mice with cd (pan-leukocyte) and cd (cytotoxic t lymphocyte) antibodies. corroborative to the histological findings, only the testis of the dexamethasone-immunosuppressed mice with zikv inoculation, but not those of the control mice, stained positive for cd ( fig. e and f) and cd antibodies ( fig. g and h). these findings confirmed the presence of inflammatory infiltrates and especially cd + t lymphocytes in the testis of the zikv-infected mice. the dexamethasone-immunosuppressed mice with zikv inoculation (groups and ) also had significantly lower mean viral loads in blood and most tissues at euthanasia at - dpi as compared with those collected at dpi (↓ - log copies/ β-actin at - dpi) (figs. a and b and s a and b). at euthanasia ( - dpi), viral rna was still detectable in most tissues of the male mice (up to log copies/ β-actin), but viremia was absent. the control mice with zikv inoculation but no dexamethasone immunosuppression had undetectable viral rna in blood and most tissues, which was consistent with previous reports (lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ) . overall, these findings were suggestive of multi-organ inflammation upon immune reconstitution with partial viral clearance in the mice after withdrawal of dexamethasone immunosuppression. we next evaluated the effects of recombinant type i interferon treatment in our mouse model. we used male mice as they had earlier onset of weight loss and clinical symptoms requiring necropsy at dpi. the mice were treated with pegylated interferon-α b (pegintron®, merck & co., inc., whitehouse station, nj, usa) iu/dose every h subcutaneously at dpi, dpi, and dpi (group ) or interferon-β b (betaferon®, bayer schering pharma ag, berlin, germany) , iu/dose every h intraperitoneally at dpi, dpi, dpi, dpi, and dpi (group ). as shown in fig. a , the mice treated with pegylated interferon-α b (group ) or interferon-β b (group ) had b % weight loss with spontaneous recovery at dpi. the weight loss of the untreated group became significantly more than those of the mice treated with either interferon-α or interferon-β b starting at dpi (p b . ). all of these mice remained asymptomatic and survived through the study period ( fig. b and c) . none of their tissues showed prominent inflammatory reactions in h&e staining at dpi or dpi. zikv-ns protein expression was only rarely seen in the immunohistochemical staining of the testis/epididymis, kidney, spleen, small intestine, lung, and pancreas collected at dpi, and testis, epididymis, kidney, and spleen at dpi. they had reduced mean viral loads in blood and all the tissues (↓ - log copies/ β-actin) as compared with those of the untreated mice at dpi and dpi ( fig. a and b) . the reductions were most significant in the tissues with high viral loads, such as the spleen, testis, pancreas, and prostate (p b . ). overall, these findings suggested that early use of systemic recombinant type i interferons improved the clinical, histological, and virological parameters of mice with disseminated zikv infection. the full spectrum of clinical manifestations and complications of zikv infection remains incompletely understood as of today. the previous assumption that zikv infection is an entirely self-limiting disease without severe or long-lasting sequelae has been overturned by the increasing recognition of congenital malformations, neurological complications, immune-mediated thrombocytopenia, and even fatality in some immunosuppressed patients (pan american health organization/ a b fig. . viral loads in the blood and major organs of dexamethasone-immunosuppressed balb/c male and female mice with zikv inoculation. mice ((a) male ( dpi, n = from two independent experiments; dpi, n = from two independent experiments) and (b) female ( dpi, n = from two independent experiments; dpi, n = from two independent experiments)) with dexamethasone immunosuppression had higher blood and tissue viral loads at dpi while they were on dexamethasone than at euthanasia ( dpi for male mice and dpi for female mice) after dexamethasone was stopped ( dpi). zikv rna copies in the blood and tissues of the mice were determined by real-time rt-pcr and normalized by β-actin as described in the text. * denotes p-values of b . . *** denotes p-values b . . error bars represent standard error of the mean. world health oganization (paho/who), ; sarmiento-ospina et al., ; arzuza-ortega et al., ; duijster et al., ) . notably, patients with severe non-pregnancy-related complications of zikv often deteriorated suddenly after an initially mild disease phase as the viral load began to decrease (cao-lormeau et al., ; mecharles et al., ; sarmiento-ospina et al., ) . this has led us to hypothesize that, like many other flavivirus infections, including yellow fever, dengue, and west nile virus infection, the host immune response may also play a role in these zikv-associated complications, especially during the viral clearance phase by the host immune system (quaresma et al., ; screaton et al., ; wang et al., ) . in this study, we characterized a novel and readily available mouse model for severe zikv infection which attempts to provide an alternative venue for studying the host immune response of and evaluating countermeasures for zikv infection. the findings in our study have important implications on the pathogenesis, potential complications, and treatment of zikv infection. the dexamethasone-immunosuppressed mice with zikv inoculation in our study developed disseminated infection with viremia and multi-organ involvement, including the brain, urogenital tract, intestine, liver, spleen, pancreas, heart, lung, and salivary gland as evident by zikv-ns protein expression on immunohistochemical staining and/or detectable viral load in these tissues. immunohistochemistry staining of the testis confirmed the presence of inflammatory cell infiltrate (pan-leukocyte marker cd +) with predominantly cd + t lymphocytes. clinically, the male mice developed earlier onset of disease than the female mice, with ≥ % weight loss and ≥ clinical sign, which warranted euthanasia at dpi. their weight loss, clinical scores, and histological evidence of inflammatory reactions were most severe soon after dexamethasone withdrawal, when viral loads had already decreased by about - log copies/ β-actin. overall, these findings suggested that, like the other related flaviviruses, the host immune response might have led to marked clinical deterioration in the face of disseminated zikv infection at the time when immune-mediated clearance of the virus began. our findings provided an additional explanation for the pathogenesis of fatal zikv infection, which has been proposed to be related to uncontrolled virus dissemination in previously described mouse models utilizing types i/ii interferon-signaling-/receptor-deficient mice that were unable to mount a robust host innate immune response. our mouse model is also useful for studying zikv's tissue tropism and potential complications of severe zikv infection. in addition to the reported findings of detectable virus particles and/or rna in the brain, spinal cord, kidney, spleen, liver, testis, ovary, heart, lung, muscle, and blood of types i/ii interferon-signaling-/receptor-deficient mice with zikv infection, our study identified intestine, pancreas, and salivary gland as other possible tissues and anatomical sites for virus infection (dick, ; lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ) . this tissue tropism of zikv in our mouse model concurs with the in-vitro observation that zikv efficiently replicates in diverse cell types of neuronal, testicular, prostatic, renal, intestinal, hepatic, and placental origin (chan et al., b; brault et al., ; hughes et al., ) . such degree of virus dissemination and multiorgan involvement is also compatible with the clinical findings in patients with severe and/or fatal zikv infection, in whom viral particles and/or rna were detected in multiple organs at post-mortem examination (pan american health organization/world health oganization (paho/who), ; sarmiento-ospina et al., ; arzuza-ortega et al., ) . while inflammatory neurological complications, such as guillain-barré syndrome, meningoencephalitis, and myelitis, have been recently reported in patients with zikv infection, inflammatory disorders of the other non-neuronal tissues were not well recognized (cao-lormeau et al., ; carteaux et al., ; mecharles et al., ) . our findings showed that inflammation could be observed in multiple organs including the testis, kidney, spleen, liver, intestine, pancreas, lung, and salivary gland outside the nervous system. among these non-neuronal tissues, the inflammatory reactions were most prominent in the testis of our model mice. some patients with zikv infection reported hematospermia, pelvic pain, and dysuria with detectable viral particles and/or rna in their semen (chan et al., a; musso et al., a; foy et al., ) . histological evidence of orchitis has not been reported due to the difficulty in obtaining the patients' testicular tissues for histological examination. previous mouse fig. . representative histological findings in the brain and kidney of dexamethasone-immunosuppressed balb/c mice with zikv inoculation and dexamethasone-immunosuppressed mock-infected control mice. the brain and kidneys of all sacrificed mice were examined. each organ was entirely embedded in one paraffin block, and one full-face paraffin section at the maximum diameter of each organ was examined per block. sections of the brain ( dpi) of a dexamethasone-immunosuppressed mouse with zikv inoculation showing (a) moderate degree of perivascular lymphocytic infiltrate and (b) marked lymphocytic infiltration in the cortical parenchyma (h&e, original magnification × ). section of the brain ( dpi) of dexamethasone-immunosuppressed mock-infected mouse showing normal architecture in the parenchyma (c) (h&e, original magnification × ). sections of the kidney ( dpi) showing (d) acute tubulitis with a large amount of inflammatory exudation in tubular lumens and (e) moderate degree of interstitial inflammation (h&e, original magnification × ). (f) section of the kidney ( dpi) of a dexamethasone-immunosuppressed mock-infected mouse showing normal architecture (h&e, original magnification × ). models for zikv infection utilizing types i/ii interferon-signaling-/receptor-deficient mice have also showed that viral particles and rna could be detected in the mice's testes, but histological analysis were not reported (lazear et al., ) . the markedly necrotic and hemorrhagic seminiferous tubules observed in our mice are highly alarming as orchitis may have long-term effects on fertility. these changes were not accountable by dexamethasone-induced testicular toxicity, as they were morphologically different from the latter, and were not present in any of the testes of the control mice with dexamethasone treatment and mock infection (khorsandi et al., ) . during revision of this work, similar findings were reported in the testes of c bl/ mice treated with anti-ifna blocking monoclonal antibody and inoculated with zikv . clinical studies to confirm the presence of orchitis and to assess the fertility of convalescent male patients should be conducted to ascertain the long-term consequences of zikv infection regarding reproductive and hormonal derangements. inflammation of other organs, such as acute tubulitis, interstitial nephritis, sialadenitis, hepatitis, enteritis, and acute pancreatitis have been reported in patients with zikv or other flavivirus infections (chan et al., a; sarmiento-ospina et al., ; arzuza-ortega et al., ; duijster et al., ; bonaldo et al., ; gourinat et al., ; musso et al., b; mercado et al., ; bhagat et al., ; torres et al., ; macnamara, ; chatterjee et al., ) . these potential complications may be increasingly recognized as the zikv epidemic continues to expand into developed countries with a large ageing and immunosuppressed population. finally, our mouse model also provided a novel avenue for the evaluation of anti-zikv treatment. type i interferons have broad-spectrum antiviral activities including those against zikv, but type i interferonsignaling-/receptor-deficient mice were not suitable for evaluation of the effects of recombinant type i interferons. we therefore evaluated the antiviral effects of two commercially available preparations of recombinant type i interferons in this new mouse model (hamel et al., ; zumla et al., ; chan et al., ; chan et al., b) . we showed that the early use of either drug was associated with improved clinical outcome with no fatality ( % fatality in untreated mice), markedly decreased inflammatory response after dexamethasone withdrawal, and reduced viral loads in various tissues of the mice as compared to those of the untreated mice. the viral load reductions were especially significant in the early phase of the disease ( dpi), when the mice were on dexamethasone. these findings suggested that the early use of recombinant interferons might help to control viral replication during the initial phase of infection, and prevent the subsequent development of severe complications related to an exaggerated immune response in the presence of high viral loads as seen in the untreated mice. it is important to further confirm these results in our mouse model using different zikv strains and in clinical trials because zikv antagonizes mouse stat less efficiently than human stat , and thus may be more susceptible to type i interferons in mice (grant et al., ) . while most patients with mild zikv infection may not require systemic interferon treatment, clinical trials should be considered to evaluate the benefits of the early use of interferon treatment in patients at risk of developing severe zikv-associated complications, such as those with underlying comorbidities (sarmiento-ospina et al., ) . the increased risk of fetal loss and low birth weight associated with interferon therapy in the first trimester of pregnancy may be outweighed by the risk of congenital malformations due to zikv infection. the optimal timing of treatment commencement should be further investigated as late commencement of interferon treatment may be useless or deleterious and should be avoided (solomon et al., ) . in summary, this novel mouse model is useful for investigating host immune response-associated damage of and evaluating countermeasures for zikv infection. inflammation of different visceral organs may be important complications of zikv that should be further studied in infected humans. long-term monitoring of the testicular function of zikvinfected male patients should be considered. clinical trials should be , inc., whitehouse station, nj, usa) iu/dose every h subcutaneously at dpi, dpi, and dpi or interferon-β b (betaferon®, bayer schering pharma ag, berlin, germany) , iu/dose every h intraperitoneally at dpi, dpi, dpi, dpi, and dpi. no interferon treatment group (n = ), interferon-α b treatment group (n = ), and interferon-β b treatment group (n = ). results were combined from two independent experiments. p-values b . are indicated by (no interferon treatment versus interferon-α b treatment) and * (no interferon treatment versus interferon-β b treatment). abbreviation: ifn, interferon. clinical scores: normal = ; ruffled fur = ; lethargy, pinched, hunched, wasp waisted = ; labored breathing, rapid breathing, inactive, neurological = ; and immobile = . considered for evaluating the effects of recombinant interferon treatments in patients at high risk for zikv-associated complications when the potential benefits may outweight the side effects of treatment. supplementary data to this article can be found online at http://dx. doi.org/ . /j.ebiom. . . . jfwc, ajz, ccsc, and kyy designed the study. ajz, ccsc, hz, and vkmp performed infections. ccyy, jolt, kkhc, and khc prepared virus stocks and viral titrations. ajz, wwnm, vkmp, and rkhay prepared histology and immunohistochemistry slides. ccyy, kmt, zz, and jpc performed viral load studies. jfwc, ajz, rkhay, vkmp, and kyy acquired images at the microscope, analyzed, and quantified the data. fy, khk, dyj provided technical assistance and edited the manuscript. jfw, ajz, and kyy wrote the manuscript. jasper f.w. chan has received travel grants from pfizer corporation hong kong and astellas pharma hong kong corporation limited, and was an invited speaker for gilead sciences hong kong limited. the other authors declared no conflict of interests. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. we are grateful to can li, andrew chak-yiu lee, and shuofeng yuan for their facilitation of the study. this work was partly supported by the donations of larry chi-kin yung, and hui hoy and chow sin lan charity fund limited; and funding from the consultancy service for enhancing α β α β a b fig. . viral loads in the blood and major organs of dexamethasone-immunosuppressed balb/c mice with zikv inoculation with or without recombinant type i interferon treatment. male mice with interferon-α b or -β b treatment had reduced zikv blood and tissue viral loads as compared to untreated mice at (a) dpi (n = per group from two independent experiments) and (b) dpi (n = - per group from two independent experiments). zikv rna copies in the blood and tissues of the mice were determined by real-time rt-pcr and normalized by β-actin as described in the text. * denotes p-values of b . . error bars represent standard error of the mean. protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys characterization of lethal zika virus infection in ag mice fatal sickle cell disease and zika virus infection in girl from colombia zika virus infection of the central nervous system of mice acute glomerulonephritis in dengue haemorrhagic fever in the absence of shock, sepsis, haemolysis or 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plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/ h n virus active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic coronaviruses -drug discovery and therapeutic options key: cord- -azrqz hf authors: ganasegeran, kurubaran; abdulrahman, surajudeen abiola title: artificial intelligence applications in tracking health behaviors during disease epidemics date: - - journal: human behaviour analysis using intelligent systems doi: . / - - - - _ sha: doc_id: cord_uid: azrqz hf the threat of emerging and re-emerging infectious diseases to global population health remains significantly enormous, and the pandemic preparedness capabilities necessary to confront such threats must be of greater potency. artificial intelligence (ai) offers new hope in not only effectively pre-empting, preventing and combating the threats of infectious disease epidemics, but also facilitating the understanding of health-seeking behaviors and public emotions during epidemics. from a systems-thinking perspective, and in today’s world of seamless boundaries and global interconnectivity, ai offers enormous potential for public health practitioners and policy makers to revolutionize healthcare and population health through focussed, context-specific interventions that promote cost-savings on therapeutic care, expand access to health information and services, and enhance individual responsibility for their health and well-being. this chapter systematically appraises the dawn of ai technology towards empowering population health to combat the rise of infectious disease epidemics. infectious diseases disrespect national and international borders. they pose substantial threats and serious repercussions to global public health security. while the asia-pacific region was generally regarded as the main epicenter of emerging infectious diseases, with outbreaks of avian flu, asian flu and severe acute respiratory syndrome (sars) [ ] , the recent and unexpected emergence of zika pandemic spurred global concerns about pandemic preparedness capabilities particularly as it relates to training and deployment of healthcare workforce at a massive level, worldwide. despite coordinated global efforts, containing the "red alert" pandemic of zika remained a challenge, as both healthcare workers and public health advocates were uncertain about such disastrous contagion causing serious complications including congenital microcephaly in newborns and neurological deficits in adults [ , ] . control measures were obtunded as public health advocates were initially speculative about the potential transmission route of zika, while clinicians in hospitals were irresolute, instituting multiple levels of care and management to tackle the complications of zika. this debacle gave rise to an urgent need to debate the circumstances under which the zika epidemic has challenged human intelligence behavior and capacity to battle the threat effectively and efficiently. as population explosions and uncontrolled human mobility across nations catalyzes rapid disease propagation, our next question is, what else above human intelligence could help resolve such unprecedented epidemic crisis? scientists believe that the time has come to institute analytic technologies-such as artificial intelligence (ai)-in healthcare to help prevent and resolve such large disease epidemics [ , ] . adaptive ai applications could mould human behavior to practice preventive behaviors and disease control strategies [ ] , thereby improving global health. this chapter will systematically discuss the dawn of ai technology in healthcare that could potentially empower the human population to tackle unprecedented infectious disease epidemics. the human population has witnessed four major revolutions till date (fig. ) ; the foremost being the first industrial revolution that introduced steam engine to the world [ ] . this was followed by the second industrial revolution that introduced electrical-energy based productions. the first information revolution was conceptualized during the third industrial revolution in the late th century. it was during this time that computers and internet-based knowledge began and has since then shaped human interactions. in early st century, the fourth industrial revolution accelerated the second information revolution. the entire phase of human daily functions transformed with the debut of ai, bringing together massive information flow from different specialties. these culminated in the rise of big data with systems integration across the internet of things (iot) and cloud computing systems. current revolutionary era is based on extreme automation for global connectivity, in which ai would definitely play an imperative role as a resource to utilize. at the peak of emergent multi-function contexts of ai and the rise of big data analytics, the united nations (un) in unified global experts to galvanize a dynamic consensus on the adoption and expansion of ai use in delivering good public care services [ ] . succinctly, various stakeholders were assembled together in another un meeting to assess the role of ai towards achieving sustainable developmental goals (sdgs) [ ] . from the healthcare perspective, massive data have been obtained from public health surveillance efforts with the advancement of ai. one major public health field that gained momentum to develop various ai applications for disease prevention was the infectious disease domain [ ] . the human population is currently able to access potentially useful massive data sources of infectious disease spread through sentinel reporting systems, national surveillance systems (usually operated by national or regional disease centers such as the center for disease control (cdc)), genome databases, internet search queries (also called infodemiology and infoveillance studies) [ ] [ ] [ ] , twitter data analysis [ , ] , outbreak investigation reports, transportation dynamics [ ] , vaccine reports [ ] and human dynamics information [ ] . with the influx of massive data volume, effective data integration, management and knowledge extraction systems are required [ ] . epidemic modeling and disease-spread simulations form new horizons to understand the effects of citizen behaviors or government health policy measures [ ] . a simple integrated effect of disease knowledge discovery is exhibited in fig. . as humans, we are able to perform simple essential tasks such as object detection, visual interpretation and speech recognition. our interpretation is instantaneous when we look at an object or image, or when we hear voices or noises surrounding us. our next question is-could ai perform these essential intellectual tasks as well? the answer is absolutely yes, but in a different mode of function. while human interpretation is solely dependent on cognitive functions, ai requires mathematical algorithms to automate machines for execution of such functions [ ] . machines here refer to programmable computers! an example is to visualize the cause of an outbreak; dengue, chikungunya or zika, of which these diseases are commonly caused by the vector mosquito. in massive epidemics, elimination of the vector is important, and human cognitive functions can never detect all mosquitos in an outbreak investigation area! however, this can be easily detected through deployment of ai in areas which have loads of mosquito vectors to facilitate control measures. figure exhibits how human and ai technology interpret the vector differently. while human interpretation is instantaneous, ai evaluates the same image as humans do, but translated into codes [ ] , facilitating massive detection. while ai aims to mimic human cognitive functions, it lacks intuitive behaviors. scientists postulate that such synthetic intelligence which could be on par with human intelligence can be called "computational intelligence." however, the primary goal [ ] of ai was to create a system programming that is capable to think and act rationally like humans, although such machines may lack intuitive or emotional capabilities. as such, ai has been appropriately defined in simple and straightforward terms, as "a branch of computer science that deals with simulation of intelligent behaviors as humans using computers [ ] ". in principle, there are three types of ai. if a machine is able to think as humans do and perform a task similar to human intellectual capabilities, then that machine functionality is referred to as artificial general intelligence [ ] . if a machine performs a single task extremely well, this is known as artificial narrow intelligence [ ] . if the same machine out-smart the best humans in all fields from scientific creativity to general wisdom or social skills, this is referred to as artificial super intelligence [ ] . at present, virtually all contemporary ai application systems utilize artificial narrow intelligence. there are numerous concepts to function underlying ai applications in healthcare. based on the required functions, these concepts are clumped together to automate a single application-such as tracking infectious disease health seeking behavior. the following sub-sections summarize key concepts of different ai subsets adopted in emerging literature of infectious diseases. machine learning (ml). ml is a subfield of ai that implies learning from previous experiences (fig. ) . the system finds solution to a problem by extracting fig. interpreting the vector from the human and ai perspective. source da silva motta et al. [ ] previous relevant data, learn from this data and predicts new outcomes [ ] . ml applications are sub-divided into three categories: i. supervised learning: uses patterns of identified data (e.g. training data) ii. unsupervised learning: finds and learns from patterns of data (e.g. data-mining that involves identification of patterns in large datasets) iii. reinforcement learning: an extension of supervised learning that "rewards" and "punishes" when an application interacts with the environment. table illustrates some common examples of supervised and unsupervised ml methods that are currently adopted and utilized to track health seeking behaviors during infectious disease epidemics [ ] . deep learning (dl). dl is a specific subset of ml that uses neural networks (fig. ) . in short, it is basically a synthetic replica of the human brain structure and functionality [ ] . dl can execute multiple functions like image recognition and natural language processing (nlp). the system is capable of handling large datasets of information flow. image recognition. ai has the capability to process large amount of data about characteristics of a particular phenomenon in the form of images or signals [ ] . motion images and sounds are examples of signals that could be analyzed using artificial neural networks (anns) [ ] . recently, researchers from the usa proposed a system that could rapidly identify potential arbovirus outbreaks (mosquito, ticks or other arthropod borne viruses) [ ] . the system identifies images of mosquito larvae captured and delivered by a group of citizen scientists. not only did the developed prototype facilitate collection of images, it also facilitated training of image classifiers for the recognition of a particular specimen. this sets a base for execution of expert validation process and data analytics. it was found that recognition of specimen in images provided by citizen scientists was useful to generate visualizations of susceptible geographical regions of arboviruses threat (fig. ) . the system was capable of identifying mosquito larvae with great accuracy. the rapid identification of potential outbreak to a susceptible community could alert preventive behaviors and policy drafting in the quest to control potential epidemics. natural language processing (nlp). nlp bridges the gap between languages that humans and machines use to operate. algorithms are built to allow machines to identify keywords and phrases in an unstructured written text. ai applications then interpret the meaning of these texts for actionable knowledge [ ] . expert systems (es). es incorporates expert-level competence to resolve a particular problem [ ] . the system is constituted of two main components, namely knowledge base and a reasoning engine. it solves complex problems through reasoning a set of incomplete or uncertain information through a series of complex rules. in recent years, fuzzy logic, a set of mathematical principles for knowledge representation was crafted to accelerate the evolution of es. such strategy was utilized by a team of researchers from south africa to improve predictions of cholera outbreaks [ ] . public reaction and behavior towards disease outbreaks could be difficult to predict. with the rise of big data analytics and a pool of ai applications in place, public health researchers were able to correlate population's behavior during an outbreak [ ] . the following examples illustrate real life applications of ai during disease epidemics: twitter, a free social-networking micro-blogging service has enabled loads of users to send and read each other's "tweets (short, -character messages)." as important information and geo-political events are embedded within the twitter stream, researchers now postulate that twitter users' reactions may be useful for tracking and forecasting behavior during disease epidemics. the zika pandemic. most of the world's populations are living in endemic areas for common mosquito-borne diseases. the zika pandemic between the years of and marked the largest known outbreak, reaching a "red-alert" warning of multiple complications requiring global public health interventions. in such exigencies, population health behaviors are important for potential control measures. daughton and paul postulated that internet data has been effective to track human health seeking behaviors during disease outbreaks [ ] . they used twitter data between and respectively to identify and describe self-disclosures of an individual's behavior change during disease spread. they combined keyword filtering and ml classifications to identify first-person reactions to zika. a total of , english tweets were analyzed. keywords include "travel," "travelling intentions," and "cancellations." individual demographic characteristics, users' networking and linguistic patterns were compared with controls. the study found variations between individual characteristics, users' social network attributes and language styles in twitter users. these users changed or considered to change their travel behaviors in response to zika. significant differences were observed between geographic areas in the usa, with higher discussion among women than men and some differences in levels of exposure to zika-related information. this finding concludes that applying ai concepts could contribute to better understanding on how public perceives and reacts towards the risks of infectious disease outbreak. the influenza a h n pandemic. signorini and colleagues in analyzed twitter embedded data for tracking rapid evolvement of public concerns with respect to h n or swine flu, while concurrently measuring actual disease activity [ ] . the researchers explored public concerns by collecting tweets using pre-specified search terms related to h n activity with additional keywords related to disease transmission, disease countermeasures and food consumption within the united states. they utilized influenza-like illness surveillance data and predicted an estimation model using supervised learning method in machine learning. the results showed that twitter was useful to measure public interest or concern about health-related events associated with h n . these include an observed periodical spikes related to user twitter activity that were linked to preventive measures (hand-hygiene practices and usage of masks), travel and food consumption behaviors, drug related tweets about specific anti-viral and vaccine uptake. they concluded that twitter accurately estimated influenza outbreak through ai applications [ ] . the integration of internet data into public health informatics has been regarded as a powerful tool to explore real-time human health-seeking behaviors during disease epidemics. one such popular tool widely utilized is google trends, an open tool that provides traffic information regarding trends, patterns and variations of online interests using user-specified keywords and topics over time [ ] . such adaptations formed two conceptualizations: the first was "infodemiology," defined as "the science of distribution and determinants of information in an electronic medium, specifically the internet, or in a population, with the ultimate aim to inform public health and public policy [ ] ;" the second was "infoveillance," defined as "the longitudinal tracking of infodemiology metrics for surveillance and trend analysis [ ] ." examining health-behavior patterns during dengue outbreaks. dengue is highly endemic across the south-east asian countries. recently, a group of researchers from the philippines conducted an infodemiology and infoveillance study by using spatio-temporal concepts to explore relationships of weekly google dengue trends (gdt) data from the internet and dengue incidence data from manila city between and [ ] . they subsequently examined health-seeking behaviors using dengue-related search queries from the population. their findings suggested that weekly temporal gdt patterns were nearly similar to weekly dengue incidence reports. themes retrieved from dengue-related search queries include: "dengue," "symptoms and signs of dengue," "treatment and prevention of dengue," "mosquito," and "other diseases." most search queries were directed towards manifestations of dengue. the researchers concluded that gdt is a useful component to complement conventional disease surveillance methods. this concept could assists towards identifying dengue hotspots to facilitate appropriate and timely public health decisions and preventive strategies [ ] . health-seeking behavior of ebola outbreak. an unprecedented ebola contagion that plagued most west african countries in marked the rise of global public health interest in pandemic preparedness interventions. millions of ebola-related internet hit searches were retrieved. with such high fluxes of health-seeking behavior using computers, a group of italian researchers' evaluated google trends search queries for terms related to "ebola" outbreak at the global level and across countries where primary cases of ebola were reported [ ] . the researchers subsequently explored correlations between overall and weekly web hit searches of terms in relation to the total number and weekly new cases of ebola incidence. the highest search volumes that generated ebola related queries were captured across the west african countries, mainly affected by the ebola epidemic. web searches were concentrated across state capitals. however, in western countries, the distribution of web searches remained fixed across national territories. correlations between the total number of new weekly cases of ebola and the weekly google trends index varied from weak to moderate among the african countries afflicted by ebola. correlations between the total number of ebola cases registered in all countries and the google trends index was relatively high. the researchers concluded that google trends data strongly correlated with global epidemiological data. global agencies could utilize such information to correctly identify outbreaks, and craft appropriate actionable interventions for disease prevention urgently [ ] . public reactions toward chikungunya outbreaks. the italian outbreak of chikungunya posed substantial public health concerns, catalyzing public interests in terms of internet searches and social media interactions. a group of researchers were determined to investigate chikungunya-related digital health-seeking behaviors, and subsequently explored probable associations between epidemiological data and internet traffic sources [ ] . public reactions from italy toward chikungunya outbreaks were mined from google trends, google news, twitter traffics, wikipedia visits and edits, and pubmed articles to yield a structural equation model. the relationships between overall chikungunya cases, as well as autochthonous cases and tweet productions were mediated by chikungunya-related web searches. but in the allochthonous case model, tweet productions were not significantly mediated by epidemiological figures, instead, web searches posed significant mediating tweets. inconsistent associations were detected in mediation models involving wikipedia usage. the effects between news consumption and tweets production were suppressed in this regard. subsequently, inconsistent mediation effects were found between wikipedia usage and tweets production, with web searches as a mediator. after adjustment of internet penetration index, similar findings were retrieved with the adjusted model showing relationship between google news and twitter to be partially mediated by wikipedia usage. the link between wikipedia usage and pubmed/medline was fully mediated by google news, and differed from the unadjusted model. the researchers found significant public reactions to the chikungunya outbreak. they concluded that health authorities could be made aware immediately of such phenomenon with the aid of new technologies for collecting public concerns, disseminating awareness and avoiding misleading information [ ] . expert systems are built upon the basis to act as a diagnostic tool to accelerate detection of infectious disease epidemics, determining the intensity or concentration of vector-agents within the triads of infectious disease dynamics. the malaria control strategy using expert systems. malaria constitutes a "red-alert" health threat to the african communities. a group of researchers from nigeria built an expert system for malaria environmental diagnosis with the aim of providing a decisional support tool for researchers and health policy-makers [ ] . as prevailing malaria control measures were deemed insufficient, this group of researchers developed a prototype that constituted components of "knowledge," "applications," "system database," "user graphics interface," and "user components." the user component utilized java, while the application component used java expert system shell (jess) and the java ide of netbeans. the database component used sql server. the system was able to act as a diagnostic tool to determine the intensity of malarial parasites in designated geographical areas across africa. the proposed prototype proved useful and cost-effective in curbing malaria spread [ ] . whereas ai is gaining increasing popularity and acceptance as a quick fix to the myriad of challenges faced with pandemic preparedness using traditional population-based approaches, it is not without its own limitations. even in resource-rich settings, there are challenges associated with building and updating the knowledge base of expert systems [ ] , providing high-quality datasets upon which machine learning algorithms can be premised, and ethical issues associated with data ownership and management [ ] . additionally, resource-limited settings are further plagued with constraints of poorly organized and integrated health systems, poor it and communication infrastructure, and socio-economic and cultural contexts [ , ] that significantly impact successful implementation of ai systems. beyond these, the dynamics of human behavior and other environmental covariates (such as mass/social media, public emotions, public policy etc.) may not only influence the accuracy of epidemic disease modeling frameworks but also impact health seeking behavior during epidemics [ ] . more than ever before, public health experts, it developers and other stakeholders must work together to address concerns related to scalability of ai for healthcare, data integration and interoperability, security, privacy and ethics of aggregated digital data. finally, the transparency of predictive ai algorithms have been called to question, particularly given their 'black box' nature which makes them prone to biases in settings of significant inequalities [ ] . perhaps, it may be premature to describe ai as the future of healthcare given it is still in its infancy, however, it has become increasingly difficult to not acknowledge the substantial contributions of ai systems to the field of public health medicine. notwithstanding current challenges with the widespread adoption of ai particularly in resource-limited settings, the use of ai in providing in-depth knowledge on individuals' health, predicting population health risks and improving pandemic preparedness capabilities is likely to increase substantially in the near future [ ] . further, the rapidly expanding mobile phone penetrance, developments in cloud computing, substantial investments in health informatics, electronic medical records (emrs) and mobile health (mhealth) applications, even in resource-constrained settings, holds significant promise for increasing use and scalability of ai applications in improving public health outcomes [ ] . public health policy, practice and research will continue to benefit from the expanding framework of infodemiology and infoveillance in analyzing health information search, communication and publication behavior on the internet [ , ] . advances in cryptographic technologies-including block chain is likely to allay fears and concerns with security, privacy and confidentiality of public digital data/information [ ] . there is no doubt that ai is and will continue to revolutionize healthcare and population health. from prevention and health promotion to diagnosis and treatment, ai is increasingly being deployed to improve clinical decision-making, enhance personalized care and public health outcomes. in particular, ai offers enormous potential for cost-savings on therapeutic care given its predictive accuracy of potential outbreaks and epidemics and ability to enhance positive health seeking behaviors (at individual and population levels) during epidemics predicated upon robust infodemiology and infoveillance frameworks supported by expert systems, machine learning algorithms and mobile applications. amazing as the future of ai in healthcare seems, there are significant legal and ethical concerns that need to be addressed in order to pave way for robust implementation and scalability across a variety of socio-cultural, epidemiological, health system and political contexts. tracking infectious disease spread for global pandemic containment an update on zika virus infection neurologic complications associated with the zika virus in brazilian adults will artificial intelligence solve the human crisis in healthcare? bmc artificial intelligence for infectious disease big data analytics the human behavior-change project: harnessing the power of artificial intelligence and machine learning for evidence synthesis and interpretation recommendations for implementing the strategic initiative united nations: looking to future un to consider how artificial intelligence could help achieve economic growth and reduce inequalities detecting influenza epidemics using search engine query data google trends in 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building a computer-based expert system for malaria environmental diagnosis: an alternative malaria control strategy developing and using expert systems and neural networks in medicine: a review on benefits and challenges semantics derived automatically from language corpora contain human-like biases machine bias accounting for healthcare-seeking behaviors and testing practices in real-time influenza forecasts algorithmic transparency via quantitative input influence: theory and experiments with learning systems. security and privacy (sp) health intelligence: how artificial intelligence transforms population and personalized health tracking health seeking behavior during an ebola outbreak via mobile phones and sms adopting m-health in clinical practice: a boon or a bane? acknowledgements we thank the ministry of health malaysia for the support to publish this chapter. key: cord- -ycdaw vh authors: maslow, joel n. title: zika vaccine development—current progress and challenges for the future date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: ycdaw vh zika virus is an emergent pathogen that gained significant importance during the epidemic in south and central america as unusual and alarming complications of infection were recognized. although initially considered a self-limited benign infection, a panoply of neurologic complications were recognized including a guillain–barré-like syndrome and in-utero fetal infection causing microcephaly, blindness, and other congenital neurologic complications. numerous zika virus vaccines were developed, with nine different vaccines representing five different platforms entered into clinical trials, one progressing to phase ii. here we review the current landscape and challenges confronting zika virus vaccine development. the zika virus, discovered in uganda in [ ] , was shown to be endemic through sub-saharan africa and tropical areas of southeastern asia in studies through the second half of the th century [ ] . isolated outbreaks occurred in yap island in [ ] and on french polynesia in [ ] . starting in mid- , zika virus infection achieved epidemic status, spreading rapidly through south america, central america, and the caribbean islands [ ] . it was soon recognized that zika virus infection occurring during pregnancy caused microcephaly and other congenital disorders in the developing fetus, the latter being the primary reason for the world health organization (who) labelling zika as an international threat in early . beginning in late , numerous academic labs and pharmaceutical companies initiated work to develop a vaccine against zika, however, by the time the first vaccines entered clinical trials, the zika epidemic had started to wane creating significant challenges to vaccine assessment [ ] that has engendered discussion of other regulatory pathways to licensure [ ] . in this paper, we provide a brief update of current progress in zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure. zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. the frequency and degree of symptomatology has, however, varied between studies. a retrospective study immediately following the outbreak on yap island found that only % of survey participants reported symptoms consistent with zika virus infection [ ] , a figure that has been cited to suggest that zika virus infection is asymptomatic in as many as % of individuals. however, of individuals who provided blood samples, representing % of households, % were symptomatic [ ] . a second retrospective study of the larger outbreak in french polynesia [ , ] , similarly appeared to have a low rate of symptomatology based on a sample of blood donors [ ] . however, a subsequent seroprevalence study found that % of those with evidence of prior infection had symptoms consistent with zika virus infection [ ] . a more recent meta-analysis of studies noted that between and % of cases were reported as asymptomatic, although as the authors note, assessment of the prevalence of symptoms was not the goal of many studies [ ] . in adults, the most common reported complication of zika virus infection is a guillain-barré-like illness that had a prevalence of . cases per cases of infection in the french polynesian epidemic [ ] . of interest, a recent meta-analysis has questioned this causal association [ ] . other less common reported neurologic complications in adults include meningoencephalitis [ ] and an adem illness. in contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as % children born to mothers infected during pregnancy [ ] [ ] [ ] [ ] [ ] [ ] . additionally, zika can directly invade the placenta and has been associated with prolonged maternal viremia [ ] [ ] [ ] . zika virus can also be transmitted through sexual contact, first reported for a researcher returning from senegal in [ ] . numerous subsequent case reports were published among travelers as part of the epidemic affecting the americas [ ] . the frequency of sexual transmission in endemic regions is unknown and could not be differentiated from mosquito-borne transmission. zika virus carriage in the male urogenital tract is common through days post infection and may persist in some to months [ ] . in mice, zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates [ ] [ ] [ ] . in one study of a zika virus dna vaccine, the adverse effects in the male reproductive system were prevented by vaccination [ ] . the question of whether zika virus can adversely affect human reproductive potential and, if so, whether such effects would be age-related is unknown. females may also excrete zika virus rna for extended times. a prospective study of five women showed that zika rna was detected in vaginal fluid for as long as months and a month or more in three of the five [ ] . murine studies showed that zika virus caused infection of the ovaries of non-immunosuppressed c bl/ mice and induced a t-cell inflammatory reaction, but without affecting reproductive potential [ ] . in contrast to the above human study, female macaques rapidly cleared virus from the genital tract [ ] . thus, while zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. however, vaccination of females at or entering reproductive age and their male partners is prudent. as discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety [ ] . the one unknown aspect is whether vaccination of males of any age may be beneficial to protect against testicular complications. calls for generalized vaccination programs have, however, been put on hold due to the decreasing incidence of zika infection rates, as discussed in the next section. in late and through mid- , global concern for this spreading epidemic was increasing, however, the experience in the two prior epidemics was predictive of the subsequent epidemiology in the americas. the zika virus outbreaks in yap island and french polynesia were characterized by rapid onset and rapid resolution over a span of - months [ , ] . the zika epidemic in the americas was characterized by a longer time to reach peak incidence, taking approximately one year for brazil [ ] presumably related to the larger and more varied geography, with attack rates decreasing greater than -fold the next year [ ] . data published by the pan american health organization (paho) found similar patterns through south and central america (https://www.paho.org/). the almost complete disappearance of zika has created significant barriers to ongoing vaccine studies as attack rates have fallen below those need to meet reasonable sample sizes [ ] . the who removed the designation of zika a virus of global concern in as case rates have fallen. in march , the who and the national institutes of allergy and infectious diseases (niaid) convened a meeting of academics, representatives from industry, and regulators to discuss the approach to zika virus vaccine development in an era of waning incidence [ ] . although zika virus remains endemic in asia, africa, and central and south america, current transmission rates, coupled with high background herd immunity, would require an extremely large and logistically difficult study. as population immunity wanes, future outbreaks are likely. the ability to conduct a meaningful future clinical trial may be dependent upon having pre-existing site and regulatory approvals with an established vaccine network to enable rapid response to new reports suggesting increased disease activity. vaccine development against the zika virus began in earnest in late following the reports of microcephaly in fetuses and infants in brazil. of note, the first demonstration of immunoprotection was as part of a study to define the ultrastructural characteristics of zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection [ ] . poland et al. provide a comprehensive listing of almost zika virus vaccines in development as of [ ] . diamond et al. discuss potential immunoreactive epitopes on the zika envelope and provide further information on those vaccines that progressed into clinical trials [ ] . the large number of delivery platforms include live attenuated and inactivated whole-viruses; viral-vectored vaccines utilizing adeno-associated virus, vesicular stomatitis virus, measles virus, and dengue or yellow fever chimeric vaccines; dna and mrna vaccines; and peptide and protein subunit vaccines. most have been assessed in animal models utilizing non-human primates and/or lethal challenge experiments involving immunosuppressed mice [ ] . as of , six vaccines had advanced to phase i studies [ , , ] . since that time, two additional vaccines have entered into clinical trials (table ) . a total of three dna vaccines have entered into human testing [ , ] , including one that has advanced to phase ii (table ) . gls- , a dna vaccine encoding for the zika virus prme genes designed as a consensus based on available zika virus sequences through december [ ] , was the first to enter into clinical trials. in pre-clinical studies, vaccinated mice and non-human primates were shown to develop b and t-cell immune responses against the zika virus envelope and protected against development of neurologic disease and death in immunosuppressed, interferon α, β receptor deficient (ifnar) mice [ ] . moreover, histologic sections of brain tissue showed that vaccinated mice were without inflammatory infiltrates evident in non-immunized mice [ ] . subsequent studies showed that the vaccine protected against testicular damage, testicular atrophy, spermatozoal damage, and infertility in mice [ , ] . a phase i study evaluated gls- administered via intradermal injection (id), followed by electroporation (ep) at doses of either or mg per vaccinations followed immediately by electroporation at baseline, weeks and weeks [ ] . there were no serious adverse events (sae) reported as part of the study, with the most frequent adverse events related to discomfort, pain, or swelling at the injection site. seroconversion was observed in % of individuals after two vaccinations and % after three vaccinations with gls- [ ] . neutralizing antibodies were detected for % of participants in a vero-cell (monkey kidney cell) assay, however, % of participants demonstrated the ability to neutralize infection of u mg neuroblastoma cells [ ] . vaccine responses were maintained through a year of follow-up. there was no difference in responses based on dose level. notably, passive transfer of immune serum from vaccinated participants was able to protect % of ifnar mice against lethal infection independent of the presence of vero cell neutralizing antibodies [ ] . gls- is being evaluated as part of a second double-blind, placebo-controlled clinical trial (nct ) performed in puerto rico. analysis of the latter study is in progress. two additional dna vaccines, based on the french polynesian h/pf/ strain were developed as chimeras that included the jev prm signal sequence followed by the zika envelope (e) gene (vrc ) or a similar construct with the terminal amino acids of e, representing the stem and transmembrane regions, exchanged for the analogous jev sequence (vrc ) [ ] . both vaccines were immunogenic for mice and nhps and protected > % of nhps against viremia at a dose of either or mg given twice [ ] . both vaccine candidates were advanced into clinical trials with mg of administered intramuscularly at weeks , and with vaccine vrc administered by needle and syringe while vrc was administered either as a single dose or split dose by needle and syringe or as a split dose given by the pharmajet needle-free device [ ] . seroconversion was % in the group administered vaccine with the needle-free device, less for vaccine administered as split-dose by needle and syringe, and lowest for vaccine given as a single injection with needle and syringe. the vrc vaccine was advanced into phase ii studies in the americas that utilized clinical sites with clinical site selection guided by epidemiologic modeling [ ] . long-term follow-up has not been reported. three inactivated vaccines have entered into clinical studies, of which clinical data for only zpiv vaccine has been published [ ] . zpiv is a whole inactivated virus vaccine of puerto rican strain prvabc [ ] . studies in mice showed that a single vaccination given intramuscularly with alum generated antibody titers of approximately . log and fully protected balb/c immunocompetent mice from viremia, whereas unvaccinated mice were unprotected and subcutaneously vaccinated mice were only partially protected against the zika brazil strain [ ] . a subsequent study in non-human primates vaccinated twice at four-week intervals with alum generated binding and microneutralization antibody titers of . and . log , respectively, and complete protection against viremia and viruria following challenge with either brazilian or puerto rican strains of zika virus [ ] . zpiv safety and immunogenicity was tested in three clinical trials to assess zika vaccine responses relative to dose level, vaccination schedule, or following vaccination with either the yellow fever yf-vax or japanese encephalitis virus (jev) ixaro vaccines to assess responses in a flavivirus-exposed population [ ] . there were no vaccine-associated saes reported. the most common adverse events were pain and tenderness at the injection site; no neurologic events were reported. seroconversion was % using a cutoff for peak geometric mean titer of : and % using a titer of : . response rates after a single immunization were % and . % using cutoffs of : or : , respectively. vaccine responses were observed through day . passive transfer of purified igg derived from zika immunized participants to groups of balb/c mice per participant provided sterilizing immunity against viremia for % ( of ) of mice overall, with viremia observed for one or more mice per group inoculated with sera from five ( %) individuals [ ] . clinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. an mrna vaccine that incorporates the prm-e genes of a micronesian strain of zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the dii region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection [ ] . immunization of ag mice with un-modified lipid nanoparticle-encapsulated vaccines mrna was immunogenic and protective against lethal infection; immunization of c bl/ immunocompetent mice followed by treatment with anti-ifnar blocking antibody showed protection against viremia in approximately % of animals [ ] . pizv, an inactivated vaccine derived from puerto rican strain prvabc selected as without passage-related mutations, protected against lethal zika virus challenge in ag mice when administered with alum adjuvant [ ] . a measles-vectored vaccine encoding the zika virus prme was shown to lessen viremia in pregnant ifnar mice and prevented clinical disease in mouse pups [ ] . preclinical data for vla- inactivated viral vaccines and the rzikv/d ∆ - live virus vaccine has not yet been reported. a number of animal models have been assessed for vaccine development and have been reviewed elsewhere [ ] . immunocompetent mice can develop transient viremia following infection and may represent models to test sterilizing immunity. interferon α/β receptor knockout mice (ag or ifnar -/-) develop lethal infection following zika virus infection and have been used as a more stringent measure of protection. non-human primates develop a self-limited mild illness associated with viremia and can transmit virus in utero to primate fetus [ ] . the logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (chim). the conduct and planning for human challenge trials raises unique medical and ethical considerations. for zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks. as reviewed above, published studies provide estimates that zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. in adults, two sets of complications warrant consideration for a proposed chim study. as reviewed above, a guillain-barre-like syndrome occurs in approximately of zika virus infections [ ] , whereas other neurologic complications such as meningoencephalitis, myelitis [ , , ] , and acute disseminated encephalomyelitis [ ] are rare. second, and perhaps more important, is the risk of transmission to a sexual partner and the potential for infection during pregnancy. zika virus commonly persists in the male urogenital tract for months, and may persist in some individuals for up to months [ ] . some have considered limiting studies to non-pregnant females as zika virus colonization of the female genital tract may be temporally limited. the fact that zika virus is known to cause testicular atrophy in mice [ ] [ ] [ ] , raises yet another as yet theoretical concern for humans. these questions as well as the theoretical potential for vector-borne transmission were debated in detail in late with the conclusion that the benefits of a human challenge infection did not outweigh the risks [ ] , however, this analysis was performed just as the initial epidemic wave in the americas was ending. the group published a follow-up in [ ] . despite the recognition that conducting a placebo-controlled vaccine trial had become significantly more difficult due to declining case rates, the group's conclusion was essentially unchanged. to address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions [ ] with a goal to prevent viremia. whether the attenuated viral strain (rzikv/d ∆ - ) being tested in a phase i study will serve as putative challenge strain is as yet undetermined. in summary, zika vaccine development continues with multiple candidate vaccines in clinical trials. because of the significant decline in incidence, evaluation of vaccine efficacy is increasingly difficult. there has been renewed interest in animal model and human infection models of infection. the author is an employee of geneone life science, inc., a developer of dna-based vaccines including a vaccine against the zika virus. walter reed army institute of research niaid national institutes of allergy and infectious disease vrc vaccine research center zika virus: (i). isolations and serological specificity zika virus: following the path of dengue and chikungunya? lancet zika virus outbreak on yap island, federated states of micronesia european centre for disease prevention and control. rapid risk assessment: zika virus infection outbreak, french polynesia zika virus in the americas-yet another arbovirus threat steep drop in 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immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials safety and immunogenicity of an anti-zika virus dna vaccine-preliminary report in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunization with a prmenv dna vaccine rapid development of a dna vaccine for zika virus preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials vaccine protection against zika virus from brazil protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys modified mrna vaccines protect against zika virus infection purified inactivated zika vaccine candidates afford protection against lethal challenge in mice a measles virus-based vaccine candidate mediates protection against zika virus in an allogeneic mouse pregnancy model animal models of zika virus infection, pathogenesis, and immunity zika virus infection during pregnancy in mice causes placental damage and fetal demise increased hospitalizations for neuropathies as indicators of zika virus infection, according to health information system data guillain-barré syndrome, acute disseminated encephalomyelitis and encephalitis associated with zika virus infection in brazil: detection of viral rna and isolation of virus during late infection ethical considerations for zika virus human challenge trials; national institutes of health bystander risk, social value, and ethics of human research zika vaccines: role for controlled human infection key: cord- -f uvohf authors: malmlov, ashley; bantle, collin; aboellail, tawfik; wagner, kaitlyn; campbell, corey l.; eckley, miles; chotiwan, nunya; gullberg, rebekah c.; perera, rushika; tjalkens, ronald; schountz, tony title: experimental zika virus infection of jamaican fruit bats (artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: f uvohf the emergence of zika virus (zikv) in the new world has led to more than , human infections. perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with guillain-barré syndrome (gbs). zikv is transmitted to humans by aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal aedes sp. mosquitos and non-human primates. in the s and ‘ s, several bat species were shown to be naturally and experimentally susceptible to zikv with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. because of zikv emergence in the americas, we sought to determine susceptibility of jamaican fruit bats (artibeus jamaicensis), one of the most common bats in the new world. bats were inoculated with zikv prvabc but did not show signs of disease. bats held to days post-inoculation (pi) had detectable antibody by elisa and viral rna was detected by qrt-pcr in the brain, saliva and urine in some of the bats. immunoreactivity using polyclonal anti-zikv antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular leydig cells. the virus likely localized to the brain via infection of iba (+) macrophage/microglial cells. jamaican fruit bats, therefore, may be a useful animal model for the study of zikv infection. this work also raises the possibility that bats may have a role in zika virus ecology in endemic regions, and that zikv may pose a wildlife disease threat to bat populations. introduction zika virus (zikv) was first isolated from a sentinel rhesus macaque in uganda in and subsequently from aedes africanus mosquitoes in the same location [ ] . the first human cases were identified in in nigeria and serosurveys found evidence of a broad geographic distribution for zikv throughout africa and asia with sporadic cases in humans [ , ] . the first recognized zikv epidemic occurred in yap state, federated state of micronesia in . an estimated % of residents were infected, and of those % presented with clinical disease [ ] . in , a second epidemic occurred in french polynesia with , cases reported. during the latter outbreak, the incidence rate of guillain-barré syndrome (gbs) increased -fold and first indication of a connection between zikv infection and gbs was established [ ] . the virus spread to brazil in [ , ] and has since disseminated throughout much of tropical south america, central america, the caribbean, and the southern united states, with more than , confirmed cases [ ] . zikv can also cause congenital zika syndrome (czs) in naïve populations and is therefore a virus of high concern [ ] . zika virus is maintained in an urban cycle, transmitted between an aedes mosquito vector and humans thereby maintaining endemicity [ ] . it is generally accepted that the virus transmits between non-human primates and vectors in a sylvatic cycle; however, the sylvatic cycle has not been well characterized in the old world and little is known about a new world sylvatic cycle [ , ] . molecular analysis of zikv to better understand viral phylogenetics suggests that animal hosts affected viral evolution and therefore may play an important role in viral ecology [ ] . in the s and ' s, the susceptibility of bats to zikv was investigated. shepherd and williams [ ] screened wild bats from different species in uganda for antibodies against zikv and found / little free-tail bats (tadarida pumila) and / angolan freetail bats (t. condylura) were seropositive by hemagglutination inhibition assay. additionally, two angolan free-tail bats were experimentally inoculated with zikv and serially bled to test for viremia. both animals were viremic on days , and as determined by paralysis in mice inoculated with the sera from those two bats [ ] . simpson and o'sullivan [ ] experimentally inoculated three straw-colored fruit bats (eidolon helvum), three egyptian fruit bats (rousettus aegyptiacusi), and five angolan free-tail bats. two of the straw-colored fruit bats were viremic and had seroconverted. one of the egyptian fruit bats was viremic and two had seroconverted. the angolan free-tail bats were euthanized on days , , , and days post inoculation and screened for viral tropism. at one day post infection, a kidney was trace positive [ ] . finally, reagan et al. [ ] inoculated new world little brown bats (myotis lucifigus) by different routes: intracranial, intraperitoneal, intradermal, intrarectal and intranasal. bats in all groups, with the exception of the intranasal group, developed fatal neurological disease - days post inoculation. brain tissue was virus-positive in all animals with clinical disease, determined by inoculation of mice with brain homogenate suspension [ ] . considering the evidence that african bats are naturally susceptible to zikv and that little brown bats develop disease, the question emerged: could bats serve as a natural reservoir host for zikv in the new world? to test this hypothesis, we inoculated jamaican fruit bats (artibeus jamaicensis), among the most abundant bats in the caribbean, central america and mexico, with zikv to examine virology, immunology and pathology of the infection. although virus was detected in several organs, including the testes and brains, no overt clinical signs were detected, and substantial viremia or viruria was not evident. these results suggest that jamaican fruit bats are unlikely to serve as amplification hosts but that zikv infection may constitute a wildlife disease threat to bats. bats for this project were obtained from the colorado state university breeding colony approved by the institutional animal care and use committee (protocol - a). two experimental infections were conducted; a pilot study and a time course study. in the pilotstudy, three male bats (aj-z , aj-z , aj-z ) were intradermally inoculated with . x plaque forming units (pfu) zikv, strain prvabc ; a high dose to assess susceptibility. no signs of disease were apparent during this day experiment; however, all three bats had antibody titers of on day (table ) . after demonstration of susceptibility in the pilot study, a time course study was conducted. six male bats (aj-z through aj-z ) were identically inoculated and two were euthanized at , and days post inoculation (dpi). no conspicuous signs of disease were observed in any of the inoculated bats. necropsies immediately followed euthanasia and no significant gross pathology was evident. quantitative probe-based reverse transcription pcr (qrt-pcr) was performed on seruminoculated vero cell supernatants, serum, brain, lung, liver, spleen, kidney, urinary bladder, prostate and testes from bats from both studies. in addition, urine collected during the time course study was similarly assayed. urine from bats aj-z at dpi and aj-z at dpi had low levels of vrna whereas bat aj-z , euthanized at dpi, had low levels of vrna in its brain ( fig ) . all other samples were negative. sera from aj-z at dpi, and aj-z and aj-z at dpi were negative by elisa. sera were blind passaged on vero e cells in an attempt to isolate zikv and all were negative for cpe and pcr. hematoxylin and eosin stain (h&e). heart, lung, liver, kidney, testes, prostate, urinary bladder, and brain were collected from all animals as well as salivary glands from / bats. all samples were blindly read by one pathologist. a summary of the consistent histopathology findings is listed in table . for the time course study, aj-z at dpi showed mild pulmonary congestion with multifocal areas of interstitial pneumonia, mild intra-alveolar hemorrhage and mild atelectasis. terminal airways had slightly increased amounts of mucus. kidneys had multifocal interstitial infiltrates of small numbers of lymphocytes. all other tissues were within normal limits. in aj- z at dpi, lungs showed milder pathology than aj-z with minimal interstitial to perivascular infiltrates predominately lymphocytes and macrophages with a band of collapsed air spaces subjacent to the pleural surface. there were focal lesions in the left ventricle of the heart where there was individual cell loss or else fragmentation of the sarcoplasm of scattered cardiomyocytes. degenerate/necrotic cardiomyocytes were accompanied by infiltrations of small numbers of macrophages, lymphocytes and satellite cells. all other tissues were within normal limits. lungs from aj-z at dpi had minimal focal interstitial histiocytic pneumonia with atelectasis. kidneys showed multifocal chronic lymphohistiocytic pyelitis with a few degenerate and detached epithelial cells accumulating in the renal pelvis and infiltration of pelvic stroma by small numbers of mixed inflammatory cells. mandibular salivary gland showed focal moderate cellular infiltrates of periductular lymphocytes and macrophages. affected salivary ducts contained detached and degenerate epithelial cells and leukocytes. occasional ducts were encircled by granulation tissue and a few heterophils. rare apoptosis was evident in the lining epithelium of such ducts. all other tissues were within normal limits. aj-z at dpi had lungs with minimal alveolar septal infiltrates scattered within collapsed lung parenchyma along with multifocal microscopic hemorrhages. kidneys had multifocal areas of mineralization. in the outer medulla and at the cortico-medullary junction were rare perivascular infiltrates of lymphoplasmacytes. esophagus and lymphoid tissue associated with palatine salivary gland showed focal mild lymphoplasmacytic inflammation. moderate numbers of lymphocytes and plasma cells were arranged in columns parallel to the respiratory mucosal epithelium of the nasophayrnx. the lumen contained increased amounts of mucus and a few inflammatory cells, mainly heterophils and lymphocytes. in the testicles, there was focal testicular degeneration manifested by presence of giant spermatids in the lumina of affected seminiferous tubules and accumulation of a small numbers of interstitial lymphocytes and macrophages. all other tissues were within normal limits. lungs from aj-z at dpi had minimal interstitial to perivascular infiltrates with multifocal atelectasis and microscopic hemorrhages. the left papillary muscle of the heart showed rare multifocal cardiomyocyte necrosis characterized by rounding up of individual cardiomyocytes. necrotic cardiomyocytes appeared with hypereosinophilic cytoplasm, devoid of cross striations or fragmented and rarely vacuolated. minimal interstitial hypercellularity due to increased activity of satellite cells and infiltration of small numbers of lymphocytes was observed in the vicinity of degenerate/necrotic cardiac muscle fibers. kidneys had an area of focal lymphoplasmactyic pyelitis. additionally, there was a focal area of mineralization and inflammation in the inner medulla. all other tissues were within normal limits. aj-z at dpi had occasional focal inflammation and cardiomyocyte degeneration in the left ventricle and interventricular septum. area ca of the hippocampus in the brain showed focal pyrimidal neuronal necrosis with a focal area of mineralization around a vessel in the cerebral cortex along with focal gliosis and individual neuronal necrosis (fig ) . all other tissues were within normal limits. testicular, neural and salivary glands' lesions are believed to be associated with zikv infection as they were not seen with other viral infections. in the pilot study bats, aj-z at dpi had more prominent interstitial pneumonia with congestion of the lungs compared to earlier time points. the heart had minimal cardiomyocyte degeneration and necrosis with hypercellular interstitium and increased amounts of mature fibrous connective tissue. the kidney had focal interstitial infiltrates of the cortical and outer medullary interstitium. the brain showed degenerate neurons in area a of the hippocampus. all other tissues were within normal limits. aj-z had minimal focal testicular degeneration (fig ) . all other tissues were normal. aj-z had perivascular lymphocyte pulmonary infiltrates and atelectasis. heart demonstrated locally extensive lymphocytic and histiocytic pericarditis. kidneys showed multifocal interstitial lymphocytic infiltrates. brain had focal, perivascular infiltrates of small numbers of lymphocytes at the subfornical commissure. the reticular formation showed multifocal neuronal degeneration/necrosis. immunohistochemistry and immunofluorescence. tissues were stained with a polyclonal antibody for zikv (cdc, fort collins). ajz- at dpi with inflammation of the mandibular salivary gland had moderate immunoreactivity in the lumen of affected ducts (fig ) . aj-z at dpi had immunoreactive cells in the brain and mononuclear cell immunoreactivity in the testes ( fig ) . additionally, aj-z demonstrated immunoreactivity in purkinje cells of the cerebellum (fig a) . aj-z at dpi had immunoreactive cells around the pulmonary arteries in the lungs ( fig a) . aj-z also had immunoreactivity perivascullarly in the tunica albuginea of the testes (fig a) . scrotal skin had focal lymphocytic dermatitis with immunoreactive mononuclear cells ( fig d) . cell morphology consistently identified mononuclear cells compatible with macrophages and fibroblasts as the primary cell types showing immunoreactivity against zikv antigen. brain and testicular tissues stained with both goat polyclonal goat anti-iba (green) and monoclonal g- flavivirus e specific antibodies (red) showed co-localization (yellow) of zikv antigen in cytoplasm of activated microglial cells with their characteristic morphology in the cerebral cortex of infected bats dpi in the time course study and day dpi in the pilot study (fig ) . increased microgliosis was noted in the vicinity of co-localization sites. the gliosis was also prominent in the cerebellum and hippocampus especially around dead neurons. in the testicles, occasional macrophages showed similar co-localization similar to that noted in the brain in the testicular interstitium, inner layer of tunica albuginea and scrotum. cells consistent in morphology with leydig cells were similarly highlighted by zika viral antigen only showing strong immunoreactivity using polyclonal anti-zikv antibody. two bat infection experiments were conducted in this investigation; ) a pilot study to determine susceptibility of jamaican fruit bats to zikv infection, and ) a time course study to better understand pathophysiology and chronology of events pertaining to the dynamics of viremia, viral tropism, replication and shedding of the virus in a new world bat species. the goal was to determine whether bats can be used as an animal model for zikv pathogenesis and to assess the possible role of bats in zikv ecology in the new world. in the pilot experiment, no signs of disease were apparent during the -day study. sera collected at euthanasia indicated modest antibody titers of for each bat by elisa (table ) , whereas the human -convalescent control serum titer was � , . bats typically have low to modest antibody titers, perhaps due to limited somatic hypermutation and affinity maturation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . concerning viremia, cell-serum supernatants, blind passage supernatants, and neat serum results were all negative. although serum is routinely used for zikv diagnostics in humans, it may not be the most suitable sample [ ] [ ] [ ] [ ] [ ] . in one investigation zikv patient had negative serum sample for the duration of the study, whereas whole blood yielded positive qrt-pcr results from days to [ ] . one possible explanation for the phenomenon of negative serum in human patients is that the virus during acute infection disseminates via a cell-associated viremia or as novel findings suggest that the virus gets phagocytized in neutrophils and therefore whole blood is a more sensitive diagnostic sample than serum. viruria is commonly detected in zikv-infected humans [ ] ; therefore, urine may be an equally important diagnostic sample with higher viral load in early infection when compared to blood in humans and other primates [ ] [ ] [ ] [ ] . although urine collection from bats was challenging, we collected urine from some of the inoculated bats in the time course study. aj-z exhibited viruria only at dpi, and aj-z was equivocal only at dpi, corroborating the findings in other mammals that urine may be a route of viral shedding early in infection. urine from one human patient was positive from the first time point ( dpi) through dpi and again on day . similarly, saliva from that same patient was positive from day nine through day and again on day [ ] . another investigation compared diagnostic samples of infected patients and showed that urine was positive in of them, whereas serum was only positive in patients by qrt-pcr. the study concluded that viral loads in urine were tenfold higher compared to serum and that uremia lasted longer [ ] . these data corroborated the first study that identified zikv shed in urine in which there was a higher viral load in urine for longer duration compared to serum [ ] . zikv rna in plasma was detected in the bats by qrt-pcr between and dpi, but between to dpi in urine [ ] . the lack of detectable viremia in the serum of bats is congruent with some of the human and nhp investigations in that viremia is low and short-lived. detached renal pelvic urothelial cells and degenerate salivary gland ductular epithelium as seen in the current study will make urine and saliva equally important fluids to collect in order to maximize detection of zikv in the acute and established stages of infections. for this experiment, all male bats were used because female bats are prioritized for colony expansion. zikv exhibited tropism for the testes with strong immunoreactivity in reproductive organs (figs & ) . histologically, minimal focal testicular degeneration in two bats ( fig ) suggests viral related pathology may be minimal. in humans it has yet to be completely elucidated what reproductive organs harbor zikv, it has been determined that semen contains zikv both in both vasectomized and unvasectomized men [ , ] . this suggests that zikv is sequestered in the testes and/or accessory sex glands. mouse models have demonstrated zikv infection and associated pathology in the testes [ ] [ ] [ ] of humanized blt mouse model with infection primarily targeting macrophages and leydig cells [ ] . limited investigation has been done relating to infection of accessory sex glands in mouse models, but one study that assessed the prostate found no virus, possibly due to differential expression of the receptor candidate in the testes but not in the prostate [ ] . for this experiment the finding of viral antigen and viral rna in the testes but not in the prostate is consistent with published animal models and may suggest the potential for bats to serve as another animal model. three bats had histopathological alterations in the hippocampus at later time points and one bat had viral nucleic acid present in the brain as determined by qrt-pcr demonstrating tropism for the cns, a tissue predilection also documented in humans and animal models. zikv has a predilection for nervous tissue in animal studies and disease manifestation in humans. as a neurological teratogen, zikv has been detected in the brain mononuclear cells in human newborns with fatal microcephaly and fetal miscarriages. histological lesions are varied but may include parenchymal calcification, microglial nodules, gliosis, cell degeneration, mononuclear infiltration and necrosis [ ] [ ] [ ] . in non-human animal models, evidence for viral tropism has been found in brain and/or peripheral nervous tissue [ ] [ ] [ ] [ ] . in immunocompromised mouse models, the virus has a predilection for the brain but with the mice engineered for specific immune traits it is difficult to know to what extent this recapitulates natural zikv pathophysiology [ ] . in the bats used in this experiment, evidence of zikvinduced pathology in the brain is consistent with what has been seen in human newborns and fetuses. the novel finding of co-localizing zikv antigen in bat iba + microglial/macrophage cells lends support to the earlier evidence of microglial cell infection via axl ligand bridging zikv particles to glial cells [ ] . iba (aka, allograft inflammatory factor , aif ) is a microglia/macrophage-specific calcium-binding protein, which has actin-bundling activity that participates in membrane ruffling and phagocytic activity of activated microglia. activated microglial cells appeared with increased ability of cell migration and phagocytosis, which is controlled by remodeling of membrane cytoskeleton [ ] . the morphology of cells with co-localization in the brain of infected bats is consistent with activated microglia depicting prominent branched processes. recent primate models in rhesus and cynomolgus macaques demonstrated similar viral distribution of zikv antigen to that in bats, described herein. high-level of zikv was evident in cerebellar neurons and the same studies documented involvement of iba positive microglial cells in cns infections. in primate models there is increasing evidence that zikv antigen was detected in individuals with the highest peak plasma viremia, which in part implies that zikv may initially seed the cns by a passive spillover from circulating monocytes to resident microglial cells. this is further substantiated in all of human and animal studies, which did not show any evidence of disruption to bbb or viral distribution reminiscent of circumventricular distribution seen in alphavirus animal models [ ] . in addition to brain and testes immunoreactivity, scrotal skin and mandibular salivary gland also harbored viral antigen. distribution of viral antigen in bat tissues suggests that infection in this species recapitulates human infection, which is thought to start with infection of epidermal and dermal cells with subsequent dissemination to multiple organs including salivary glands as viral rna can be detected in human saliva [ , ] . the histopathology for aj- zika virus infects new world bats z , dpi showed sialoadenitis and the presence zikv antigen by ihc (fig ) . this suggests zikv may be shed in the saliva, although additional animal experiments need to be performed to confirm such a route of shedding. the results presented here suggest that jamaican fruit bats may be a suitable animal model for examining zikv infection to elucidate its pathogenesis. jamaican fruit bats may also serve as a model to ascertain sexual transmission, in utero transmission, teratogenesis and neurological pathophysiology. it may be that zikv is a wildlife disease threat for bats that could lead to infertility in some males, which could impact bat populations. zikv is thought to be maintained in two different distinct cycles: sylvatic-cycling between non-human primates (nhp) and mosquito species, and urban-cycling between humans and mosquito species [ ] . while there are limited data on what mosquito species feed on jamaican fruit bats, evidence for natural flavivirus infection has been identified in wild new world bats. dengue virus (denv) rna and antibodies to denv were detected in multiple species of bats, including jamaican fruit bats, in mexico [ ] . additionally, antibodies to denv were detected in multiple bat species including those of the artibeus genus in costa rica and ecuador [ , ] . these data indirectly provide evidence for mosquito-bat interactions in the wild; either through consumption of bat-blood meals taken by mosquitoes or bat consumption of infected mosquitoes. as it pertains to a wildlife reservoir, wild nhps have antibody to zikv including several monkey species trapped near ziika forest [ ] , and wild and semi-captive orangutans in borneo [ ] . not only have nhp been found to be seropositive, but also many other mammals, including rodents, horses, cows, and goats [ , ] . furthermore, experimental inoculation of various north american species resulted in seroconversion (cottontail rabbits, boar goats, pigs, and leopard frogs) and demonstrated viremia (nine-banded armadillo and leopard frogs) [ ] . molecular epidemiology suggests animals play an important role in an enzootic cycle [ ] . much about the enzootic cycle of zikv has yet to be understood but it stands to reason that bats may be capable of maintaining the virus in nature. jamaican fruit bats are found in northern south america, central america, and the caribbean-areas that now have zikv potentially exposing bat populations to the virus [ , ] . however, the data presented here suggest it is unlikely that jamaican fruit bats can serve as amplification hosts of zikv, unless virus sequesters in some as-yet unidentified way that could lead to periodic shedding of virus. it may also be that some bats become persistently infected and can transmit sexually to maintain virus within populations of bats. further experimental and field studies will be necessary to fully understand the ecological role of bats in zikv maintenance. all animal procedures were approved by the colorado state university (csu) institutional animal care and use committee (protocol - a) and were in compliance with u.s. animal welfare act. bats csu has a captive colony of jamaican fruit bats (artibeus jamaicensis), a neotropical fruit bat indigenous to much of south america, central america and the caribbean [ ] . colony bats are kept in a free flight room measuring 'w x 'l x 'h. roosting baskets are hung from the ceiling throughout the room and drapes of different cloth material are positioned for hanging and roosting. ambient temperature is maintained between ˚c and ˚c, with humidity between % and %, and a hour light/ hour dark light cycle via a computer-controlled system. diets consist of a combination of fruits (shamrock foods, fort collins, co), tekald primate diet (envigo, huntington, uk), molasses, nonfat dry milk and cherry gelatin that are placed in multiple feeding trays around the room once a day. fresh water is provided. in addition, fruit is hung around the room to stimulate foraging behavior and serve as enrichment. for infection experiments, bats were trapped using a butterfly net and placed in an "d x "w x "h cage for hours prior to inoculations to allow for acclimation. hanging clothes were provided for roosting and coverage. food and water are placed in open trays in the bottom of the cage and changed daily. tray liners were changed every two days, and cages and hanging clothes are changed every two weeks. due to the social nature of these bats, minimums of two bats were kept in cages at all times to mitigate potential stress. two sets of experiments were performed; a pilot study and a time course study. zika virus strain prvabc . prvabc was isolated in by centers for disease control and prevention (fort collins, co) from an infected individual who traveled to puerto rico (genbank accession no. hq ). the virus stock titer is x plaque forming units (pfu) per ml of media, and the fourth passage was used for both studies. for the pilot study, three male bats were anesthetized with % to % isoflurane to effect with an oxygen flow rate of . l/min, administered with a gas mask. animals were placed on a heating pad to maintain body temperature and respirations continuously monitored. the dorsum of each animal was disinfected with % ethanol and ul containing . x p.f.u of virus was administered subcutaneously (sc) at the level of the scapula with a sterile hypodermic gauge needle in a biosafety cabinet. when procedures were finished, bats were removed from isoflurane and placed back in the cage in ventral recumbency. respirations were monitored until animal was fully awake and ambulated normally. bats were identified as aj-z , aj-z and aj-z . animals were euthanized at days post-inoculation (dpi). for the time course study, six male bats were anesthetized under the same protocol as the pilot study. animals were placed in ventral recumbency. after disinfecting the dorsum of each animal with % ethanol, . mls of % lidocaine was administered sc at the level of the last rib with a gauge sterile hypodermic needle as a local anesthetic. iptt transponders (biomedic data systems, inc., seaford, de) were inserted sc at the level of the caudal edge of the scapula. twenty-five microliters containing . x p.f.u of virus was administered sc at the level of the cranial edge of the scapula. recovery followed the same protocol as for the pilot study bats. animals were identified as aj-z through aj-z . aj-z and aj-z were euthanized at two dpi. aj-z and aj-z were euthanized at dpi. aj-z and aj-z were euthanized at dpi. female bats were excluded from the study because they are prioritized for breeding to sustain and expand upon the colony. for the pilot study, bats were visually monitored twice daily for fourteen days, and then monitored once a day for an additional fourteen days. for the time course study, bats were monitored twice a day throughout the experiment. for both studies, energy levels, behavior, ability to ambulate, respirations, presence of oral or nasal discharge, and fecal consistency were all assessed. during the time course study urine was collected at , , and dpi from as many bats as possible. urine was collected by allowing bats to grasp screen cloth with their feet and then the bat was placed in a clear solo cup (dart container, lake forest, il) with the screen covering the top of the cup as a lid, and kept in place with a rubber band. this allowed the bats to hang in a clear container. bats were monitored for minutes. if they urinated, bats were removed from the collection contraption and placed back in the cage without disrupting the urine. urine collection was attempted on all remaining bats at each time point, but not all bats would urinate at each collection attempt. urine was successfully collected as follows: two dpi from aj-z and aj-z ; three dpi from aj-z , aj-z and aj-z ; five dpi from aj-z , aj-z , aj-z and aj-z ; and ten dpi from aj-z and aj-z . urine was pipetted off the surface of the cup with a sterile pipette tip and put in a . ml microcentrifuge tube and stored at - ˚c for future use. urine volume ranged between ul and ul. bats were deeply anesthetized and maintained with % isoflurane and an oxygen flow rate of . l/min. deep pain was assessed by firmly pinching skin and toes with forceps and assessed for any response. a thoracotomy was then performed with sterile standard scissors to puncture through the skin, muscle and diaphragm just caudal to the sternum and cut through the wall of the chest cavity caudally to cranially-removing and preventing negative pressure from building in the thorax. cardiac blood was collected with a gauge sterile needle inserted into the apex of the heart. a maximum blood volume of between and . mls is collected in a syringe and transferred to a red top tube (rtt). rtts sat at room temperature for one hour to allow a clot to form and then centrifuged at x g for min at room temperature. serum was removed from the clot, placed in a new microcentrifuge tube and stored at - ˚c. serum from bats at and dpi were used to assess for viremia. serum from dpi and the dpi pilot study bats were used to determine antibody titers. because blood draws yield a small volume of blood ( μl whole blood for a non-terminal blood draw, μl whole blood for terminal blood draw) it was necessary to prioritize samples to optimize data retrieved. in order to assay the serum for viral rna and perform serology, earlier time points were used to assess for viremia and later time points for seroconversion. along with sample partitioning for data maximization, the small blood volume led to concerns that there would be an undetectably small viral load. to circumvent this issue, neat serum and : diluted serum were inoculated onto vero cells to amplify any virus that may have been present at low levels. one blind passage on vero cells was done and cell supernatants assayed by qrt-pcr. the remaining serum from three of the four bats was assayed directly for zikv rna. necropsies were performed immediately after euthanasia. bats were assessed for gross pathology. the following tissues were collected for both experiments: heart, lung, liver, spleen, kidney, urinary bladder, prostate, testes, and brain. a portion of tissues were collected and kept at - ˚c for rna extraction, and a portion placed in % buffered formalin for histology at a : weight to volume ratio for histology. for a negative control animal a male bat was trapped from the colony and euthanized under the same protocol as the experimental infection bats. vero e cells (atcc) were propagated to % confluency in a -well tissue culture plate and infected with zikv strain prvabc at an m.o.i. of . . after a one hour incubation period, unbound virus was removed and replaced with % fbs-dmem and incubated for a maximum of three days. media was then replaced with % acetone for minutes at - ˚c to fix virusinfected cells to plate and serve as an antigen for enzyme-linked-immunosorbent assay (elisa). plates were stored at ˚c until use and used within two weeks. plates were washed x with . % tween -pbs and blocked with superblock t (tbs) blocking buffer (thermo fisher scientific, waltham, ma) for one hour at room temperature. serum from an uninfected bat was used for a negative control. a convalescent human serum sample (kindly provided by b. foy, csu) was used as a positive control. a two-fold serial dilution was used starting at : to : . diluted serum was placed in wells and incubated for two hours at room temperature. serum was removed and plates washed. hrp-conjugated protein a/g (thermo fisher scientific, waltham, ma) was added at a concentration of μg/ml to each well, and incubated for minutes at room temperature. hrp-conjugated protein a/g was used in place of a secondary antibody as it targets the fc portion of an antibody, which is highly conserved and therefore can be used for multiple animal species [ ] . plates were washed and μl of abts peroxidase substrate ( component) (kpl, gaithersburg, md) added according to manufacturers' instructions, incubated at room temperature for minutes, and then μl of abts peroxidase stop solution (kpl, gaithersburg, md) added. plates were read on an emax plus microplate reader (cambridge scientific, watertown, ma). absorbance was measured at nm and the limit of detectable response was set at three standard deviation values above mean negative control serum. trizol reagent was used for rna extraction from serum-cell supernatants, serum, urine and tissues according to ambion, life technologies protocol. for tissues, approximately mg of tissue was homogenized with one ml of trizol reagent. a mm stainless steel bead (qiagen, valencia, ca) was used with a tissuelyser lt (qiagen, valencia, ca) at hz for minutes. one ml of trizol was added to urine to to μl of urine. one ml of trizol was added to μl of serum from aj-z , aj-z , and aj-z . two-hundred microliters of serum-cell supernatants were added to one ml of trizol. samples were then incubated at room temperature for minutes. chloroform (thermo fisher scientific, waltham, ma) was added, samples were mixed, incubated for minutes at room temperature and centrifuged at , x g for minutes at ˚c. the aqueous phase was removed, μg of glycogen (thermo fisher scientific, waltham, ma) and % molecular grade isopropanol added (thermo fisher scientific, waltham, ma). samples were incubated at room temperature for minutes and then centrifuged at , x g for minutes at ˚c. supernatant was removed and % molecular grade ethanol (thermo fisher scientific, waltham, ma) was added to rna pellet. samples were vortexed and centrifuged at x g for minutes at ˚c. wash was removed and air-dried. rna was resuspended in rnase-free water and stored at - ˚c for future use. vero cells were grown to to % confluency in a -well tissue culture plate with % fbs-dmem. media was removed and ul of bat serum from dpi bats and dpi bats was inoculated onto cells. additionally, serum from each bat was diluted -fold in % fbs (millipore sigma) pbs supplemented with % calcium and magnesium, and inoculated onto cells. samples were incubated for one hour at ˚c. inoculum was removed and cells washed twice in sterile pbs. two-percent fbs-dmem was added to wells and plates were incubated at ˚c, % co . cells were assessed daily for cytopathology (cpe) through day but none was observed. two-hundred microliters of the supernatant was removed on day and used for rna extractions. an additional μl of supernatant was blind passaged onto vero cells at to % confluency. cells were incubated for one hour at ˚c, washed twice with sterile pbs and % fbs-dmem added. on day seven, supernatant was removed and trizol extractions performed for rna recovery. serum was treated as such in an attempt to amplify viral load and increase assay sensitivity serum may not be the most sensitive diagnostic sample [ ] [ ] [ ] [ ] . if any serum was remaining it was directly used for trizol rna extractions. serum samples remained from aj-z at dpi, and aj-z and aj-z at dpi. no serum remained from aj-z . roche real time ready rna virus master kit (roche, indianapolis, in) was used on rna extracted from serum-cell supernatants, serum, urine and tissue to assay for zikv rna according to manufacturers' instructions. primers used were zikv (ccgctgcccaa cacaag) and zikv c (ccactaacgttcttttgcagacat). probe was zikv -fam (agcctaccttgacaagcagtcagacactcaa) [ ] . two-hundred nanograms of sample rna was added to each reaction. reactions were performed in duplicate. standards were a non-infectious clone of full length zikv strain prvabc by which concentration was determined through optical density. molecular weight of the genome sequence was used to calculate copy number [ ] . a log dilution series of the standard was made and linear regression used to determine copy number equivalents of positive samples. amplification was performed according to manufacturers' protocol for roche real time ready rna virus master kit (roche diagnostics corporation, indianapolis, in) with pcr conditions as follows: min at ˚c, s at ˚c, and cycles of s at ˚c, s at ˚c and s at ˚c. tissues fixed in %-buffered formalin were cut in and submitted to colorado state university veterinary diagnostic laboratory (csu vdl, fort collins, co) for paraffin embedding, sectioning and staining with hematoxylin and eosin, as well as immunohistochemistry (ihc). tissues cut in on bats to assess for histology included: heart, lung, liver, kidney, testes, prostate, urinary bladder and brain. additionally, for aj-z and aj-z mandibular salivary gland was cut in. aj-z had esophagus and lymphoid tissue that included palatine salivary gland cut in. antibody for ihc was a polyclonal rabbit antibody that targets prem and e proteins of zikv and was provided by csu vdl's pathology department. the bond-iii automated instrument (leica biosystems, wetzlar, germany) was used for ihc staining. all slides were blindly read by a diplomat of the american college of veterinary pathologists. brain tissues was prepared for immunohistochemical and immunofluorescence staining as previously reported [ ] . tissue was dehydrated by using a graded ethanol series of % ethanol for h, % overnight, % for h and % for h. brain tissues were then post-fixed in dimethylbenzene for min and embedded in dimethylbenzene-paraffin at ˚c for h, after which samples were embedded in a metal frame. sagittal sections were collected at um thick. all dewaxing, antigen retrieval and immunofluorescence staining was automated using a leica bond rxm. in short, sections were dewaxed using ethanol and then boiled in antigen retrieval solution for minutes. the cooled sections were incubated in % h o for min at room temperature and then blocked with % donkey and goat serum (millipore sigma) for hour. rabbit anti-iba (wako chemicals usa, irvine, ca) and g- flavivirus e specific monoclonal antibodies (cdc, fort collins) were diluted in tbs to final concentrations of : and : , respectively. sections were incubated in primary antibodies concurrently at room temperature for one hour. following removal of unbound primary antibodies by washing, goat anti-rabbit secondary (alexafluor- ) and donkey anti-mouse secondary (alexafluor- ) was added and incubated for hour at room temperature. finally, dapi counterstain (vector laboratories, burlingame, ca) was applied and sections were washed with tbs prior to cover slipping for imaging. stained sections were imaged on a ziess lsm with airyscan laser-scanning confocal microscope (ziess, oberkochen, germany) using a × oil immersion objective. each field of view was imaged as a z-stack ( - planes, . -μm step size) transformed into a single maximum projection image using the ziess zen (blue) imaging software. zika virus. i. isolations and serological specificity zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria zika virus: history, emergence, biology, and prospects for control zika virus outbreak on yap island, federated states of micronesia rapid spread 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overview of the current status of zika virus pathogenesis and animal related research axl mediates zika virus entry in human glial cells and modulates innate immune responses microglia/macrophage-specific protein iba binds to fimbrin and enhances its actin-bundling activity entry sites of venezuelan and western equine encephalitis viruses in the mouse central nervous system following peripheral infection detection of zika virus in saliva biology of zika virus infection in human skin cells denge virus in mexican bats neotropical bats that co-habit with humans function as dead-end hosts for dengue virus detection of dengue virus neutralizing antibodies in bats from costa rica and ecuador sylvatic transmission of arboviruses among bornean orangutans zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently a sero-epidemiological survey for certain arboviruses (togaviridae) in pakistan investigating the potential role of north american animals as hosts for zika virus. vector borne zoonotic dis mammalian species: artibeus jamaicensis chimeric igg-binding receptors engineered from staphylococcal protein a and streptococcal protein g genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia rapid and specific detection of asian-and african-lineage zika viruses fluoxetine prevents lps-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress the authors would like to than brent davis, cdc, fort collins, co for supplying anti-zikv polyclonal and specific monoclonal antibodies. key: cord- - v qvhg authors: johansson, michael a.; reich, nicholas g.; meyers, lauren ancel; lipsitch, marc title: preprints: an underutilized mechanism to accelerate outbreak science date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: v qvhg in an essay, michael johansson and colleagues advocate the posting of research studies addressing infectious disease outbreaks as preprints. • despite the advantages of preprints and the endorsement of journals and funders in the context of outbreaks, less than % of ebola and zika journal articles were posted as preprints prior to publication in journals. • with broader adoption by scientists, journals, and funding agencies, preprints can complement peer-reviewed publication and ensure the early, open, and transparent dissemination of science relevant to the prevention and control of disease outbreaks. emerging public health threats, such as infectious disease outbreaks, require swift and evidence-based responses informed by science. however, the communication of scientific research is notoriously slow [ ] . when rapid dissemination of new information has the chance to prevent or control epidemics affecting hundreds or thousands of people, we must fast-track this process. preprints (manuscripts posted publicly prior to peer review) have been endorsed as a solution to this challenge, yet adoption remains very low and needs to be improved. on february , , more than of the world's largest and most prestigious public health journals and funding agencies issued a landmark statement on the importance of preprints and data sharing in public health emergencies such as the ebola and zika epidemics [ ] . journal signatories pledged to ( ) make related scientific content freely accessible, and ( ) allow data and preprint manuscripts to be shared prior to publication. here, we focus on preprints, which may include both new data and new analyses and offer an opportunity to speed and democratize further scientific analyses and the availability of evidence to inform outbreak responses. a a a a a the statement coincided with a proliferation of zika research in response to the epidemic in the americas (fig a) . between november and august , we identified a total of preprint manuscripts with zika in the title or abstract in recognized public preprint repositories, including general repositories: biorxiv ( , http://www.biorxiv.org/), arxiv ( , arxiv.org), f research ( , with other that was originally posted in biorxiv, f research.com), peerj preprints ( , peerj.com/preprints/), and the world health organization zika open repository ( , www.who.int/bulletin/online_first/zika_open/), which was established explicitly for the zika epidemic (s dataset). these likely represent the majority of zika preprints, though others may have been posted in ad hoc, lesser-known, or laboratory-or university-specific webpages or repositories. over a similar time period in the ebola outbreak (may to january ), there were ebola preprints ( fig b) . there were many more publications in peer-reviewed journals over these time periods: , and , publications indexed in pubmed (http://www.ncbi.nlm.nih.gov/pubmed, s dataset) with ebola or zika, respectively, in the title or abstract and of type "journal article" ( and additional publications were classified as letters, reviews, etc., respectively). this increase in publications ( %) cannot explain the increase in preprints alone ( %). over the same time period, there was a general increase in preprints for health sciences [ ] ; for example, biorxiv submissions with the word "outbreak" rose approximately -fold between ( ) and ( ). thus, the increase in preprints related to the zika epidemic likely reflects this general trend, some increase in research, and possibly the influence of the statement on data sharing. to assess changes in preprint posting during the epidemics, we examined the subset of all preprints that could be matched with an eventual publication. for ebola, we matched ( %) of the preprints to pubmed-indexed journal articles, and for zika, ( %). four additional zika preprints were matched to "review" ( ) or "comparative study" ( ) publications in pubmed. unmatched preprints may be preprints that were never submitted for peerreviewed publication (e.g., opinions or preliminary work that was abandoned), were never accepted for publication, or were still in peer review at the end of the selected time periods. preprints were increasingly used to disseminate new data during the zika outbreak. among the subset of preprints matched to journal articles described above, the proportion of preprints including original data increased substantially from % for ebola to % for zika (difference: %, % confidence interval [ci] % to %, -sample test of proportions). while a minority of preprints contained new data, the majority of preprints in both outbreaks included novel analyses, % for ebola and % for zika (estimated difference: %, % ci − % to %). the remainder were comprised of opinions, proposals for new lines of study, or research indirectly related to ebola or zika. the increase in data sharing between epidemics appears to represent a shift away from waiting for peer review and towards rapidly reported, open science. this shift should enable other researchers to build upon those findings and recognize the high value of data sharing (academic and otherwise), a common challenge in the midst of outbreaks. preprint posting also led to earlier access to those data and analyses. excluding manuscripts in f research, which uses the preprint posting date as the publication date, the median delay between preprint posting and publication was approximately days for both outbreaks, and less than % of the manuscripts were published within days of posting. notably, this delay, which may include submission to multiple journals, is quite similar to normal publication timelines [ ] . it is unclear to what extent journals are able to accelerate publication in outbreaks, but it is clear that every time there is an editorial or peer review decision, rejection, or revision there are delays, and that preprint posting precludes delays in broad access to the information. the successes of preprints in the ebola and zika epidemics belie a more complex story about preprint use during these outbreaks. while the number of preprints increased, data sharing was more common, and scientific findings were available earlier, adoption remained extremely low. publications with preprints represented a small minority of all pubmedindexed journal articles, at approximately . %. the proportion was slightly higher for zika compared to ebola ( . % versus . %), likely indicating a small increase between outbreaks (estimated difference: . %, % ci − . % to . %). among signatory publishers, who represented approximately % and % of the publications for ebola and zika, respectively, this proportion was approximately . % ( % ci . % to . %) higher than for nonsignatory publishers ( . % versus . %) (fig c and d ). this suggests that these publishers may have policies that are supportive of preprints irrespective of the statement [ ] . although preprint adoption in both outbreaks was very low, important advances were clear. first, the relatively higher preprint usage for signatory publishers both before and after the statement indicates that policy supportive of preprints can encourage their use. second, the number of preprints posted increased between the outbreaks, likely reflecting changing attitudes towards preprints in the life sciences and changing policies, including the data sharing statement [ ] [ ] [ ] . third, preprints generally contained new analyses and increasingly shared novel data. fourth, with preprints available months before peer-reviewed publications, preprint posting can accelerate the sharing of research. preprints also bring new challenges to outbreak responses [ ] . by definition, preprints are not peer reviewed prior to posting. while preprint posting is common practice in fields such as physics and statistics, it is a new concept to many scientists in public health and even more so to public health officials, the press, and the public, all of whom may be seeking the latest information during epidemics. until preprints are broadly recognized as pre-peer review manuscripts, they may be misinterpreted as peer-reviewed research. on the other hand, peer review faces its own challenges of subjectivity, bias, transparency, and speed [ , , ] . peer review is an integral component of scientific communication, but it does not intrinsically guarantee the quality of science. moreover, peer review is particularly challenging during major outbreaks when the most qualified reviewers are also immersed in urgent research. preprint posting may help mediate this process, providing an opportunity for broad and immediate community input that is not subject to the limits of traditional peer review [ ] [ ] [ ] . assuring ethical review, participant confidentiality, recognition of preprints as pre-peer review manuscripts, and finding mechanisms to enable transparent, open feedback will be essential to limiting possible negative impacts and maximizing the benefits of preprints for outbreak responses. immediate, open access to research prior to peer review raises the possibility of misinterpretation and the misuse of science in critical decision making when lives are at stake, but it also permits early and open criticism, discussion, and consideration of findings that may save lives. further adoption of preprints also requires changes in how funders, scientists, and publishers value scientific contributions. all stakeholders agree that the best science should be brought to bear against outbreaks, yet they are also keenly aware of the importance of peer review and the scientific accolades that come with publishing novel, impactful research in prestigious journals. in the context of outbreaks, the goal of impacting the epidemic by immediately sharing important scientific findings conflicts with career goals tied to the slower, traditional, peer review-centered publication process. the low adoption of preprints during the ebola and zika epidemics is a symptom of uncertainty about preprints and this conflict of incentives. the signatory funders and publishers clearly endorsed preprints, but the majority of scientists did not realize that they could or should post preprints or thought that posting a preprint would jeopardize publication opportunities. we advocate steps to improve the adoption of preprints and speed the dissemination of science in the context of outbreaks. first, scientists should promote preprints by posting them and choosing to submit manuscripts to journals that accept preprints; many journals have policies that explicitly allow preprint posting (tools for identifying these journals are provided by jisc: http://www.sherpa.ac.uk/romeo/search.php and wikipedia: https://en.wikipedia.org/ wiki/list_of_academic_journals_by_preprint_policy). second, all publishers should endorse preprint posting for research related to outbreaks. this would send a clear signal to all scientists that preprints are integral to scientific communication and help nonscientists identify preprints as a distinct form of communication compared to peer-reviewed publications [ , [ ] [ ] [ ] . publishers should actively encourage or require preprint posting at the time of submission, driving adoption directly as faculty of has for f research. third, preprint repositories and the scientific community should ensure that preprints contain appropriate content (e.g., maintaining ethics and privacy) and are readily identifiable as preprints, and that mechanisms exist to facilitate community input prior to or concurrent with peer review. these considerations can help reduce the risks of preprints and maximize their benefits. fourth, universities and funders should recognize preprints together with peer-reviewed publications and citations as an important part of an investigator's track record, especially for any scientist involved in outbreak responses. the scientific community should not ask why preprints are posted during outbreaks, we should ask why they are not posted and make early posting the standard rather than the exception. preprints offer numerous challenges and opportunities for science in general but represent a particularly important opportunity to accelerate the dissemination of science in the midst of infectious disease outbreaks, when early actions are critical and evidence is scarce [ , ] . despite this need and the statement on preprints and data sharing, less than % of ebola and zika journal articles were posted as preprints prior to publication in journals. this low adoption reflects an intrinsic and established, yet unnecessary, prioritization of the traditional publication process over the dissemination of science. further progress is essential to ensure that science can be rapidly and broadly disseminated in the context of outbreaks. it is incumbent upon scientists, publishers, and funders alike to recognize the value of preprints and embrace them as a critical component of outbreak science. supporting information s dataset. data on preprints included in the analysis. "preprint_date" is the initial preprint submission date from the respective repository. "pmid" is the pubmed identification number the preprint was matched to (if matched). "journal" and "pub_type" are from the pubmed entry. "publisher" indicates the publisher if the publisher signed the data sharing statement. "new_data" and "new_analysis" indicate whether the authors judged the manuscript to contain new data or analysis, respectively ( = true). (csv) s dataset. data on pubmed journal articles included in the analysis. "pub_date" is the journal publication date as indicated by pubmed or the entry creation date if a specific journal publication date was not supplied. "pmid" and "journal" are the pubmed identification number and journal indicated by pubmed for the article. "publisher" indicates the publisher if the publisher signed the data sharing statement. (csv) does it take too long to publish research? nature sharing data during zika and other global health emergencies the preprint dilemma time for a prepublication culture in clinical research? accelerating scientific publication in biology ten simple rules to consider regarding preprint submission peer review: a flawed process at the heart of science and journals estimating the reproducibility of psychological science improving the evidence base for decision making during a pandemic: the example of influenza a/h n middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility key: cord- - rl cyqj authors: de campos, thana cristina title: zika, public health, and the distraction of abortion date: - - journal: med health care philos doi: . /s - - - sha: doc_id: cord_uid: rl cyqj this paper suggests that the focus on abortion legalization in the aftermath of the zika outbreak is distracting for policy and lawmakers from what needs to be done to address the outbreak effectively. meeting basic health needs (i.e. preventive measures), together with research and development conducive to a vaccine or treatment for the zika virus should be priorities. on february th, the united nations (un) high commissioner for human rights mr zeid ra'ad al hussein urged abortion-banning latin american countries affected by the zika outbreak to legalize abortion, and allow pregnant women affected by the virus to choose whether to terminate their pregnancy (office of the united nations high commissioner for human rights ). in brazil, the country most affected by the zika outbreak, abortion is illegal, as in several other latin american countries affected by the outbreak, including colombia, el salvador, venezuela and peru. does the zika outbreak justify a change in abortion laws in latin american countries? although the ethics of abortion should certainly be debated, and both pro-abortion and anti-abortion sides should be free to voice their concerns, the plea to abortion in the context of the zika outbreak is problematic. the link between the zika outbreak and abortion could prove a major distraction from what would actually help the most vulnerable and affected: meeting their basic health needs (i.e. preventive measures), and fostering research and development (r&d) leading to a vaccine or treatment for zika. these should surely be the priorities when it comes to addressing such a poverty-related disease. as of february, , cases of microcephaly have been confirmed in brazil, of which only could be connected to the zika virus. of the pregnant women infected with the zika virus in colombia, none had given birth to an infant with microcephaly. although the causal relation between the mosquito-born zika virus and microcephaly has been recently established, (rasmussen et al. ; victora et al. ) prenatal tests and ultrasound may not necessarily detect of microcephaly until the third trimester (camosy ; romero ) . the fact that the zika virus is linked to grave birth defects like microcephaly in infants whose mothers contracted the virus during pregnancy is causing great despair, especially among pregnant women. pregnant women in brazil, colombia, el salvador, venezuela, and peru are being urged by many to terminate their pregnancies. the un high commissioner statement not only compounded existing fears, but prompted pro-abortion activists in latin america and beyond to advocate the legalization of abortion. in brazil, the country most affected by the zika outbreak, abortion is permitted only in cases of rape, anencephaly, or when continuing the pregnancy threatens the mother's life. although % of brazilians are in favor of the current law, and only % are in favor of a more relaxed law, (senra ; diniz ) pro-abortion activists in brazil, led by a group of law professors from university of brazilia, are about to petition the brazilian supreme court to legalize abortion (senra ; diniz ) . they claim that access to legal and safe abortion is an effective solution for dealing with an increased risk of birth defects (diniz ; gostin and phelan ; yamin ) . as such, they claim that the brazilian government have the duty to provide access to safe abortion services in public hospitals for all brazilian women-particularly for those in the poorest and most affected regions of the country, and who do not have the financial means to procure safe abortions in private clinics (diniz ; gostin and phelan ; yamin ) . other organizations abroad are echoing the same concerns. for example, the canadabased group women on web that sends abortifacient drugs like mifepristone and misoprostol to women wanting an abortion in countries where it is legally prohibited has voiced their 'worry that these women will turn to unsafe abortion methods, while we can help them with a safe, medical abortion' (miller ) . planned parenthood's international arm has developed a special zika virus fundraising campaign, (mora ) and amnesty international has warned about the 'devastating effect' of antiabortion laws (dawber and marters ) . the question whether abortion should be part of a constitutional right to health, and whether the public health care costs of abortion should be financed by public funds is contentious. health care resources are particularly scarce in the developing countries affected by the zika outbreak, requiring local governments and communities to define health care priorities carefully. the fact that the zika outbreak is a public health concern in need of an urgent remedy is not controversial. nor is the importance of finding, and implementing effective solutions to the zika virus outbreak. the controversy lies in whether abortion legalization should be conceived of as such an effective solution to the outbreak, particularly for pregnant women in the poorest and most affected regions, and if so, whether it constitutes a legitimate means to a worthwhile end. the latter question concerns what is due to human individuals in their earlier stages of development, and is at the center of the abortion debate. here i am interested in the former question, which is the one more specifically focused on issues relating to global health and poverty. zika is a 'neglected disease' (hotez and askoy ; parsons ) . as a poverty-related illness, it primarily affects the most marginalized and vulnerable populations in developing countries, living in remote rural areas and urban shantytowns, with poor access to basic health care services and goods (hunt ; paho ) . because neglected diseases affect mainly or exclusively poor populations with little or no purchasing power, there is insufficient medical knowledge about its causes, as well as little market incentive to foster research and development (r&d) for medical products addressing these medical conditions (hollis and pogge ) . typically, therefore, there is no adequate prophylactic and therapeutic medication for neglected diseases, mainly because the afflicted population cannot afford the treatment's price (hollis and pogge ) . being a neglected disease, zika has two main root problems directly linked to poverty: lack of basic health care (i.e. preventive measures such as basic sanitation), and lack of r&d conducive to a vaccine or treatment. when it comes to scarce health care resource allocation, the motto 'first things first' should be kept in mind, so that policy and lawmakers do not lose sight of their priorities when addressing neglected diseases. both basic health care (i.e. preventive measures such as basic sanitation) and r&d are priorities precisely because they tackle the root problems of the outbreak. the first and foremost need is the provision of basic health care (i.e. preventive measures) to affected populations, and most significantly, the poorest populations. the aedes aegypt mosquito carrying the zika virus breed in stagnant water where populations lack adequate plumbing and sanitation. therefore, sanitation, vector control measures, and appropriate personal protective measure are imperative to immediately reduce the risk of exposure, and prevent the spread of the zika virus (who ) . simple precautionary measures, such as cleaning and using nontoxic insecticides to eliminate areas that breed mosquitos, teaching people how to clean and reduce the breeding of mosquitos in their vicinities, emphasizing the importance of using protective covering such as adequate clothing and mosquitos nets, and distributing insects repellent to poor communities are examples of some measures that would immediately help in reducing the risks of infection. the other priority is r&d: only after researching and discovering how the virus is transmitted, and how it can be contained, can the population, and especially pregnant women, stop worrying about its potentially dire effects. true, r&d into effective drugs is costly, uncertain, and trials often fail. also, successful and timely r&d is even less probable with regard to neglected diseases, because they require greater research and coordination capacities, as well as generous funding, all of which is more likely in developed, rather than in developing countries. this in itself is an obstacle to be addressed. the assistance of the international community -particularly of developed countries is required and justified in this case, not least because the zika outbreak has the potential for spreading to developed countries. r&d for an effective zika virus vaccine or treatment may take several months, and it may well prove to be a complex process involving many different global stakeholders, who will have to coordinate their actions effectively. as with any other public health emergency of international concern (pheic) (who ), it will be challenging to mount a coordinated response involving different stakeholders, such as neighboring countries, regional organizations, the world health organization, and other international organizations, together with local authorities and local communities. yet, if the international community becomes as involved in the zika outbreak as it was in past global health threats, such as the sars, the h n , and the ebola outbreaks (sars ) , to which there were also no vaccines or treatments available when these outbreaks were declared pheic, it is plausible to believe that the spread of the zika virus may also be contained quickly. in short, there are good reasons for granting priority to both basic health care provision (including pre-natal, maternal, and infant preventive care) and r&d in the context of the zika outbreak: both are promising responses to the outbreak because they try to solve its root causes. abortion is not an effective solution to the zika outbreak, because it neither tackles the causes of the outbreak, nor does it prevent further spread of the infection. it does nothing to control the vector, or to improve sanitation and appropriate precautionary measures. the focus on abortion legalization by some as a response to the zika virus is misplaced. irrespective of the vexed moral controversy of abortion, importing that intractable debate into effective and morally uncontroversial responses to the zika outbreak risks distracting law and policymakers from strategies that would tackle the real causes of the outbreak. in short, the zika outbreak, therefore, is not the place to have this debate, because the moral controversies of abortion distract law and policymakers from what needs to be done to address the outbreak effectively. priorities in health care resources allocation need to be set carefully, given that resources are scarce. and not only material, but also human resources are finite: political will, and attention need to be focused on the main measures that can solve zika-related problems, rather than squandered in an intense moral debate. brazil is facing this important public health problem amidst a number of other difficult political, economic, and social challenges. to address the zika outbreak successfully, therefore, brazil-and particularly the frail rouseff/temer administration-needs to focus its energies on specific health questions, rather than on the intractable moral ones. is the call for zika virus abortions the new eugenics? zika virus: fear is pushing women into backstreet abortions the zika virus and brazilian women's right to choose the who must include access to birth control and abortion in its temporary recommendations for zika-associated public health emergency of international concern health impact fund-making new medicines accessible for all. incentives for global health neglected diseases-a human rights analysis will zika become the ntd of the year? with abortion banned in zika countries, women beg on web for abortion pills planned parenthood international using zika to fundraise for abortion advocacy upholding women's human rights essential to zika zika, public health, and the distraction of abortion ?newsid= &langid=e. accessed paho. . who. neglected, tropical and vector borne diseases we've neglected diseases like the zika virus for too long zika virus and birth defects-reviewing the evidence for causality surge of zika virus has brazilians re-examining strict abortion laws ebola outbreaks were different from the current zika outbreak: their modes of transmission as well as the ethical considerations they raised were all different. yet, what sars, h n , ebola and zika have in common is that they were all declared 'public health emergencies of international concern group prepares action in the supreme court for abortion in cases of microcephaly (in portugese) microcephaly in brazil: how to interpret reported numbers? ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations ihr procedures concerning public health emergencies of international concern (pheic) health, human rights and the zika virus acknowledgments i would like to thank john keown, christina lamb, david mulcair, alisha gabriel, joseph hattie, elizabeth cassidy, faye sonier, andrea mrozek, francisco javier urbina, steven hoffman, roojin habibi, and maxwell tran. conflict of interest i declare no conflicting interests. key: cord- -qtwcbn m authors: gao, yaning; tai, wanbo; wang, ning; li, xiang; jiang, shibo; debnath, asim k.; du, lanying; chen, shizhong title: identification of novel natural products as effective and broad-spectrum anti-zika virus inhibitors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qtwcbn m zika virus (zikv) infection during pregnancy leads to severe congenital zika syndrome, which includes microcephaly and other neurological malformations. no therapeutic agents have, so far, been approved for the treatment of zikv infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat zikv-caused diseases. after screening a natural product library, we have herein identified four natural products with anti-zikv activity in vero e cells, including gossypol, curcumin, digitonin, and conessine. except for curcumin, the other three natural products have not been reported before to have anti-zikv activity. among them, gossypol exhibited the strongest inhibitory activity against almost all zikv strains tested, including six recent epidemic human strains. the mechanistic study indicated that gossypol could neutralize zikv infection by targeting the envelope protein domain iii (ediii) of zikv. in contrast, the other natural products inhibited zikv infection by targeting the host cell or cell-associated entry and replication stages of zikv. a combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-zikv compound, exhibited significant combinatorial inhibitory effects against three zikv human strains tested. importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (denv) human strains in vitro. taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against zikv and other flaviviruses, such as denv. zika virus (zikv) is a mosquito-borne flavivirus in the same genus as other important human pathogens, including dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and tick-borne encephalitis virus (tbev) [ ] . zikv was originally isolated in a rhesus macaque in [ ] , but this virus has only recently claimed worldwide attention owing to its close association with congenital zika syndrome (czs), as represented by microcephaly, fetal demise, central nervous system abnormalities, and other neurological complications [ ] [ ] [ ] [ ] [ ] . no antiviral therapeutics for the treatment of zikv-associated human diseases, particularly congenital syndrome a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) ~ . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at °c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at °c for h. for gossypol, zikv (strain pan , ~ . × pfu; moi ~ . ) was incubated with serial dilutions of this natural product at °c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at °c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at °c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at °c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( l/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at °c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at • c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at • c for h. for gossypol, zikv (strain pan ,~ . × pfu; moi~ . ) was incubated with serial dilutions of this natural product at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at • c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at • c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at • c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( µl/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at • c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity using the calcusyn computer program [ , ] . the combinatorial cytotoxicity of gossypol with other natural products to vero e cells was detected using a similar approach as described above, except for mixing gossypol with one of the natural products (curcumin, digitonin, conessine, or bortezomib) throughly before adding them to the cells. this experiment was carried out as previously described, with some modifications [ ] [ ] [ ] [ ] [ ] [ ] [ ] . briefly, vero e cells ( /well) or zikv were incubated at different infection steps as described below, with or without the tested natural products at the specified concentrations of µm for gossypol, µm for curcumin, . µm for digitonin, or µm for conessine for h before zikv infection, h after infection, or the same time during infection. anti-zikv compounds, such as temoporfin [ ] , -hydroxycholesterol [ ] , bortezomib [ ] , and nitd [ ] , were included as controls for these steps. after the culture of the zikv-or compound-treated cells at • c for - days, plaques were visualized with crystal violet staining, as described above, and the percent inhibition of natural products was calculated. specifically, the following six stages of zikv infection were tested: (a) pretreatment of zikv (pan ,~ . × pfu; moi~ . ) with natural products at • c for h before incubation with cells; (b) pretreatment of cells with natural products at • c for h before incubation with zikv (pan ,~ pfu; moi~ . ); (c) cotreatment of cells with zikv (pan ,~ pfu; moĨ . ) and natural products at • c for h; (d) cotreatment of cells, zikv (pan ,~ pfu; moĨ . ), and natural products at • c for h; (e) preincubation of cells with zikv (pan ,~ pfu; moi~ . ) at • c for h and then incubation with natural products at • c for h; and (f) preincubation of zikv (pan ,~ pfu; moi~ . ) and cells at • c for h, followed by incubation with natural products at • c for h. the binding between natural products and zikv full-length e protein (aviva systems biology, san diego, ca, usa) or envelope protein domain iii (ediii) protein (e residues - fused with a c-terminal human fc) [ ] was carried out by elisa, as previously described [ , , , ] . briefly, elisa plates were precoated with the proteins described above at • c overnight and blocked with % fat-free milk at • c for h. serial dilutions of natural products or dmso (negative control) were then added to the plates and incubated at • c for h. the plates were washed with pbs containing tween- (pbst) and incubated at • c for h with zikv ediii-specific human monoclonal antibody (mab) zka -lala ( . µg/ml) for binding to zikv full-length e and ediii proteins. the plates were washed with pbst and incubated with horseradish peroxidase (hrp)-conjugated anti-human igg-fab ( : , abcam, cambridge, ms, usa) antibody at • c for h. the , , , -tetramethylbenzidine (tmb) substrate (sigma) was added to the plates, and the reaction was stopped by n h so . absorbance at nm (a value) was measured by elisa microplate reader (tecan). ec ( % effective concentration) was calculated based on the calcusyn computer program, as described above [ ] . to determine the ability of gossypol to inhibit binding between zikv ediii protein and ediii-specific human mabs (smzab , zka -lala, zv- , or z ) or edi/ii-specific human mab control (zka ) [ ] , elisa was carried out, as described above, except that serially diluted gossypol or dmso (negative control) was added in the presence of mabs ( . µg/ml), followed by sequential incubation with hrp-conjugated anti-human igg-fab antibody and tmb substrate and detection for a value. the percent inhibition of natural products was calculated, and ic (concentration causing % reduction in ediii-mab binding) was obtained using the calcusyn computer program [ ] , as described above. the interactions between natural products and zikv full-length e were analyzed at • c using the biacore t system (ge healthcare, port washington, ny, usa), as previously described [ , ] . briefly, zikv e was immobilized on a sensor chip (cm ) using the amine coupling kit (ge healthcare). natural products at various concentrations were injected as analytes, and pbs-p ( mm phosphate buffer containing . mm kcl, mm nacl, and . % surfactant p , ph . ) was used as the running buffer. the data were analyzed using biacore evaluation software (t version . ), and the curve was fitted with a : binding model. the potential combinatorial effect of gossypol with other natural products was carried out as previously described [ , ] . briefly, zikv (strain pan , flr, or prvabc , . × pfu; moĨ . ) was incubated with serially diluted gossypol at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was incubated with vero e cells at • c for h in the presence of dmem containing serial dilutions of each of the other three natural products identified, such as curcumin, digitonin, and conessine, or anti-zikv compound control (bortezomib). the unbound viruses and natural products were removed, the cells were cultured at • c for - days, and stained with . % crystal violet. the natural products without combinations were used as controls. the ic values of natural products were calculated using the calcusyn computer program [ ] , as described above. the natural products were then analyzed for combinatorial effects based on the combination index (ci) [ ] and ic values using the calcusyn computer program, as previously described [ , , ] . specifically, ci values < and > indicate synergy and antagonism, respectively. synergy was further identified as five different categories: ci values < . , . - . , . - . , . - . , and . - . indicate very strong synergism, strong synergism, synergism, moderate synergism, and slight synergism, respectively. fold enhancement of anti-zikv potency is expressed as the ratio of molar concentrations of natural products tested alone and in the mixture using the formula ((ic alone)/(ic in the mixture)- ). using a plaque-based assay, we initially screened natural products (at µm of concentration) from a natural product library for their inhibitory activity against infection of a recent zikv human strain (pan ). based on table , four "hit" natural products, including gossypol, curcumin, digitonin, and conessine ( figure a -d), were selected, since they demonstrated inhibitory activity against zikv infection with no obvious cytotoxicity in vero e cells when observed under a microscope. these natural products were subsequently ordered from sigma with ≥ % purity, and tested for their cytotoxicity using a cell-based cytotoxicity assay (cck kit), with cc values ranging from . to . µm (table , figure s ). among these natural products, curcumin has been previously reported to inhibit zikv infection [ ] , whereas the other three natural products have not been previously reported to have anti-zikv activity. gossypol, curcumin, digitonin, and conessine were retested to confirm their anti-zikv (strain pan ) activity, with ic values of . , . , . , and . µm, respectively ( table ). the ic values of these natural products against zikv pan strain were also calculated, equal to about . , . , . , and . µm, respectively, for gossypol, curcumin, digitonin, and conessine ( figure s ), which were slightly higher than the respective ic values, but much lower than the corresponding cc values. table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at µm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at µm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. viruses , , of table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. no. (%) natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at μm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at μm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values ( table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . m, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . m, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values (table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . µm, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . µm, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. comparison of zikv e protein sequences revealed that most of the amino acid sequences were highly conserved, but that some variations occurred among the zikv strains used for evaluation of the inhibitory activity of natural products, including the pan strain tested earlier ( figure s ). the above data demonstrate that the identified natural products, particularly gossypol, were able to block infections of divergent human, mosquito, and monkey zikv strains isolated from different time periods and countries, including six recent zikv human strains, confirming their broad-spectrum anti-zikv activity. to identify which steps of zikv infection in its life cycle were blocked by these natural products, we carried out a time-of-addition experiment by incubating natural products with zikv or cells at different time points during zikv and cell interaction, and then calculated the percent inhibition based on the number of plaques formed [ , , , ] . to test whether a natural product can neutralize zikv infection or inhibit viral entry by targeting the viral proteins, zikv was pretreated with the natural product at • c before incubation with the host cells ( figure a(a) ). to evaluate whether a natural product can bind to the cellular receptors or cofactors to block virus-receptor binding, cells were pretreated with the natural product at • c before incubation with zikv ( figure a(b) ). to determine whether a natural product can inhibit attachment of zikv to target cells, but cannot block the virus-cell membrane fusion, cells were cotreated with zikv at • c in the presence of the natural product ( figure a (c)). to assess whether a natural product can inhibit attachment of zikv to target cells and subsequent virus-cell membrane fusion, the cells were cotreated with zikv and the natural product at • c ( figure a(d) ). to investigate whether a natural product can inhibit zikv fusion with the cell membrane and then entry into the cell, cells were pretreated with zikv at • c first and then incubated with the natural product at • c ( figure a (e)). to study whether a natural product can inhibit zikv infection at postentry stages (i.e., viral replication, virion assembly, or release), cells were pretreated with zikv and then incubated with the natural product at • c ( figure a (f)). after completing the above approaches, we gained insight into the potential mechanisms of the natural products responsible for inhibiting zikv (pan ) infection. after pretreatment of zikv with gossypol at • c before incubation with the target cells, zikv completely lost its infectivity, whereas it maintained its infectivity after other treatments described above ( figure b ), suggesting that gossypol can effectively neutralize zikv infection by targeting the virus, rather than the cell or cell-associated entry or replication stages. the results from curcumin revealed that about - % of zikv infection was blocked when curcumin was incubated with zikv only at • c, or coincubated with zikv and cells at • c or • c, whereas there was low to no impact on zikv infection when curcumin was pretreated with cells or postincubated with zikv-treated cells at • c and • c, respectively ( figure c ). these results suggest that curcumin inhibits zikv infection at the early stages of viral entry, particularly the viral attachment stage. pretreatment of vero e cells with digitonin and then with zikv or cotreatment of cells with zikv and digitonin at • c significantly (≥ %) blocked zikv infection, whereas preincubation of cells with zikv and then with digitonin at • c, pretreatment of cells with zikv at • c and then with digitonin at • c, or cotreatment of cells with digitonin and zikv at • c inhibited about - % of zikv infection ( figure d ). in contrast, preincubation of digitonin and zikv had no effect on zikv infection ( figure d ). these results suggest that digitonin could not directly neutralize zikv infection, but inhibited zikv infection by binding to the viral receptors or inhibiting viral entry (i.e., attachment and membrane fusion or postentry steps). the data from conessine indicated that coincubation of cells with conessine and zikv at • c or postincubation of conessine with zikv-treated cells at • c or • c resulted in - % inhibition of zikv infection, whereas pretreatment of cells with conessine before zikv incubation blocked about % of zikv infection. nevertheless, preincubation of conessine and zikv at • c or cotreatment of cells with conessine and zikv at • c had very low to no effect on zikv infection ( figure e ). these data suggest that conessine does not block zikv attachment to the host cell, but inhibits zikv infection by targeting virus-cell fusion or postentry step. the above steps of inhibition of zikv infection were further proven inhibition of zikv infection were further proven by the respective anti-zikv compound controls ( figure f-i) . therefore, the above data confirm the potent inhibitory activity of the identified natural products, particularly gossypol, in blocking zikv infection at various stages of the viral life cycle. anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. (f) a potent anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. to identify the potential binding regions of the natural products in zikv proteins, we first carried out an elisa-based approach by coating the plates with zikv full-length e or ediii proteins. we then tested for binding affinity using a zikv ediii-specific mab, zka -lala, for e or ediii binding. results revealed that gossypol bound potently to the full-length e and ediii proteins, with ec values of . and . µm, respectively, whereas curcumin had much lower binding affinity to zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . µm to zikv e protein ( figure c ). zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . m to zikv e protein ( figure c ). since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . m, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the ability of gossypol to inhibit the binding between zikv ediii and ediii-specific neutralizing mabs. the concentrations of zikv ediii and mabs were . and . µg/ml, respectively. the percent inhibition in the ediii-mab binding was measured in the presence or absence of serially diluted gossypol using the formula (( -(ediii-mab-gossypol)/(ediii-mab) × ), which, in turn, formed the basis for calculating % inhibitory concentration (ic ) values. zikv edi/dii-specific mab (zka ) and dmso were used as controls. the data are expressed as mean ± s.e.m. (n = ) . the experiments were repeated twice, with similar results. since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . µm, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the data described here demonstrate that the identified natural product gossypol bound strongly to zikv e protein, potentially the conserved ediii, thus blocking ediii-mab binding at important neutralizing epitopes and inhibiting viral entry into the target cell. these data reasonably explain the potent broad-spectrum antiviral activity of gossypol against infections of multiple zikv strains. since gossypol demonstrated the highest antiviral activity individually against all zikv strains tested, we next investigated the potential combinatorial effects of the combination of gossypol with three other natural products identified, namely curcumin, digitonin, and conessine, as well as anti-zikv compound control (bortezomib). results demonstrated significant combinatorial inhibitory effects against three zikv strains (pan , flr, and prvabc ) tested when combining gossypol with any of these natural products, and the ci values ranged from . to . µm, . to . µm, and . to . µm for zikv pan , flr, and prvabc strains, respectively (tables - ). the combinations of gossypol with each of these natural products also resulted in the highest enhancement of anti-prvabc activity among the three zikv strains tested (table ). these data show that gossypol can be combined with other inhibitors described above to further increase overall inhibitory activity against current and future emergent zikv strains. the above results on the combinatorial effects against zikv infection could not exclude the possibility that the observed decrease in ic in the presence of another natural product might result from the enhanced cell death caused by the natural product in combination. therefore, we also assessed the change of cc of the natural products alone and in combination, and compared the enhancement of cc with that of ic against zikv strain prvabc . as shown in table , the cytotoxicity of gossypol in combination with curcumin, digitonin, or conessine was not enhanced. the cytotoxicity of gossypol in combination with bortezomib was slightly increased ( . -fold), but it was much lower than the enhancement in anti-prvabc activity of gossypol ( . -fold) in combination with bortezomib. in addition, the cytotoxicity of curcumin and bortezomib, respectively, in combination with gossypol was not enhanced. the cytotoxicity of digitonin or conessine in combination with gossypol was slightly increased by about . -and . -fold, respectively, while they were still lower than the enhancement in anti-prvabc activity of digitonin ( . -fold) or conessine ( . -fold) in combination with gossypol. moreover, the ci values of gossypol with any of the four natural products tested were greater than ( table ), indicating that there was no combinatorial effect on the cytotoxicity to the test cells. these results suggest that the observed decrease in the ic values of the natural products in combination was not due to the enhancement in cytotoxicity of these natural products. note: gossypol with one of the natural products, including curcumin, digitonin, conessine (three lead natural products) or bortezomib (anti-zikv compound control), were first mixed according to the molar ratio identified in the above combinational experiment against zikv strain prvabc , and then added to vero e cells to determine cytotoxicity. the combinatorial cytotoxicity of natural products to vero e cells is expressed as cc identification of broad-spectrum anti-flavivirus inhibitors is crucial to treat infections caused by zikv and other flaviviruses, such as denv. hence, using a plaque assay similar to zikv, we evaluated the antiviral activity of natural products against infections of four serotypes of denv human strains in llc-mk cells. even though all four natural products could inhibit denv- - infections, results showed that gossypol had the highest potency against denv- , denv- , and denv- infections, with ic values of . , . , and . µm, respectively (table ) . also, the anti-denv- activity of gossypol (ic value: . µm) was only slightly higher than that of curcumin (ic value: . µm) ( table ) . the cytotoxicity of these natural products on llc-mk cells was investigated by a cytotoxicity assay, with the cc values ranging from . to . µm ( table ) . the above data indicate the potent anti-denv activity of the natural products, particularly gossypol, against infections of four denv human strains with low to no cytotoxicity. the experiments were performed on llc-mk cells, and the cytotoxicity of natural products in this cell line is expressed as cc . the inhibitory activity of natural products against infections of denv- - is expressed as ic . the data are expressed as mean ± s.e.m. (n = ). the experiments were repeated twice, with similar results. as described earlier, gossypol targeted zikv e protein, potentially ediii. although a number of variations have been identified in the amino acid sequences of e proteins of zikv and denv strains tested in this study ( figure s ), gossypol could still inhibit all zikv and denv strains tested, suggesting that it potentially targeted the conserved sequences in zikv and denv ediii proteins. our data further explain the potent, broad-spectrum activity of gossypol against infections of at least two flaviviruses, including zikv and denv. development of safe and effective antiviral therapeutics is urgently needed to treat zikv infections and zikv-caused diseases, particularly zikv-associated microcephaly, fetal death, or neurological diseases [ , [ ] [ ] [ ] [ ] . here, we have identified four small-molecule-based natural products, namely gossypol, curcumin, digitonin, and conessine, with robust anti-zikv activities in vero e cells. among them, gossypol had the greatest potency to block infections of zikv for almost all strains isolated from to from nine countries and three hosts, including six recent human strains associated with congenital zika syndrome and other neurological malformations. time-of-addition-based mechanistic studies indicated that gossypol could neutralize zikv infection by targeting the virus rather than the cell or cell-associated zikv entry or replication steps. inhibition assays revealed that gossypol strongly bound to zikv e protein, particularly the conserved ediii, and blocked the binding between zikv ediii and ediii-specific neutralizing mabs, rationalizing its efficacious and broad-spectrum anti-zikv activity. it appears that the binding between gossypol and zikv e/ediii protein might be nonspecific, since gossypol is also active against other enveloped viruses, including herpes simplex virus type , parainfluenza virus type , influenza virus, and hiv- [ ] [ ] [ ] [ ] [ ] . because of the potential toxicity and side effects of gossypol to humans [ ] [ ] [ ] , it might not be a good idea to use this natural product as a drug to treat human diseases, including zikv-associated microcephaly and other flavivirus-caused diseases. nevertheless, the goal of this study is to identify the best active, natural product, then modify it to improve its antiviral activity and drug-like properties and reduce its toxicity. due to the symmetric nature of the gossypol molecule, there will be better opportunity to defragment its structure to smaller drug-like molecules with higher activity and lower toxicity. therefore, it is suggested not to use gossypol as the lead molecule, but rather the final drug. unlike gossypol, digitonin and conessine did not neutralize zikv directly, but instead inhibited zikv infection at various stages of the life cycle, including viral attachment, membrane fusion, or postentry steps. notably, digitonin is a glycoalkaloid saponin detergent. it is widely used as a cell membrane permeabilizing agent [ , ] and a detergent for selective solubilization of membrane proteins [ ] [ ] [ ] ; thus, the antiviral activity of this natural product is likely to be very nonspecific. by comparison, conessine appears to be a good candidate with a decent anti-zikv activity and very low cytotoxicity (higher selectivity). however, it is a steroidal alkaloid [ ] , which might not be ideal for further optimization. similar to gossypol, curcumin may directly neutralize zikv infection, but it seems to mainly inhibit the early stage (attachment) of virus infection, the mechanism of which is similar to that seen in a previous report using different approaches [ ] . our data have also demonstrated strong in vitro ability of these natural products-gossypol in particular-against four serotypes of denv human strains with low to no cytotoxicity, and the ic values against denv were similar to those against zikv. we have found that gossypol bound to zikv e protein, potentially ediii, suggesting that it may recognize highly conserved regions and sites in the e proteins of zikv and denv. previously, curcumin was shown to potentially inhibit denv- infection through direct effects on viral particle production or various cellular systems [ ] . we have not found studies in the literature reporting on the antiviral activity of curcumin on other serotypes of denv or the inhibitory effects of gossypol, digitonin, and conessine on zikv and other flaviviruses. thus, the present study provides a rationale for further understanding of anti-denv and anti-zikv mechanisms of these natural products and identifying their potential binding sites in the viral proteins. except for demonstrating the individual in vitro anti-zikv activity of natural products identified in this study, particularly gossypol, we have confirmed important combinatorial anti-zikv effects of gossypol in combination with other natural products. it is interesting to note that combining gossypol with curcumin, digitonin, or conessine resulted in increased combinatorial effect against prvabc strain compared to the flr strain of zikv, potentially because gossypol had more potent anti-flr activity than anti-prvabc activity when tested individually. also, such combinations enhanced the individual antiviral activity of curcumin, digitonin, and conessine against the test viruses. we have shown that the combination of gossypol and bortezomib, a licensed proteasome inhibitor previously reported inhibiting zikv infection [ , ] , led to significant combinatorial activity against the three epidemic zikv human strains tested. therefore, our study demonstrates the possibility of combining gossypol with other natural products or compounds to enhance antiviral activity. overall, this study has evaluated the antiviral activity of four natural products in vitro, among which three are newly identified natural products with strong anti-zikv ability. future observations will be needed to evaluate the protective efficacy of these natural products individually or in combination, or using their defragmented, small drug-like molecules (in the case of gossypol) against zikv-caused congenital infection and fetal demise in available animal models. modification of the structure of gossypol and identification of its derivatives with better antiviral activity, but without cytotoxicity, would be essential for developing a safe and effective anti-zikv agent for human use. also, future studies to evaluate the antiviral activity of modified gossypol or its derivatives against other flaviviruses, both in vitro and in vivo, will be helpful for identification of an effective and safe pan-flavivirus inhibitor. taken together, the broad-spectrum ability of the identified natural products, especially gossypol, against zikv and denv infections indicates the potential for further development of these small molecules or their derivatives as the lead compounds or effective anti-flavivirus inhibitors. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : association between inhibitory activity of natural products against zikv strain pan and their cytotoxicity. figure s : association between inhibitory activity of natural products against zikv strain prvabc and their cytotoxicity. figure s : multiple sequence alignment of amino acid (aa) sequences of e protein of zikv strains and denv- - human strains used in this study. author contributions: s.j., a.k.d., and l.d. designed the study. y.g., w.t., n.w., and x.l. performed the experiments and analyzed the data. y.g. and n.w. carried out sequence alignment analyses. y.g., s.j., a.k.d., l.d., and s.c. wrote and revised the manuscript. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus (i). isolations and serological specificity zika virus infection in pregnancy: maternal, fetal, and neonatal considerations implications of zika virus 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detergents specific release of membrane-bound annexin ii and cortical cytoskeletal elements by sequestration of membrane cholesterol anti-malarial property of steroidal alkaloid conessine isolated from the bark of holarrhena antidysenterica clinical use of proteasome inhibitors in the treatment of multiple myeloma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no competing interests. key: cord- -i jxn vj authors: morens, david m; fauci, anthony s title: pandemic zika: a formidable challenge to medicine and public health date: - - journal: j infect dis doi: . /infdis/jix sha: doc_id: cord_uid: i jxn vj nan it has been years since the noted german physician-scientist and public health advocate rudolf virchow made his prescient observation about new diseases. since then, the world has seen a dizzying succession of newly emerging and reemerging infectious diseases that have changed the landscape of medical practice and of biomedical research [ ] . the zika pandemic was first recognized in and is still unfolding in alarming and unprecedented ways. because of the pandemic's uniqueness and the insidious ability of zika virus to harm unborn children, the pandemic has captured the attention of infectious disease researchers and practitioners of clinical and public health medicine around the world, as well as the attention of allied colleagues working in entomology, vector control, informatics, teratology, immunology, and a host of other disciplines [ ] [ ] [ ] . zika virus has now spread to > countries or territories [ ] , including in the americas and the caribbean, causing millions of infections. an enormous body of information about zika has rapidly accumulated and the scientific community is now energetically organizing and translating new data into control and prevention approaches. in this issue of the journal, we review what we have learned so far and how we may best apply it to blunt zika's alarming progress. eighteen expert groups discuss multiple aspects of the zika pandemic, including its history, epidemiology, virology, vector characteristics, immunology, teratology, and vaccinology. our collective goal is to stimulate thought and discussion so we can more effectively work together in pursuit of preventing zika infections, developing treatments for those affected, and ending the pandemic. among the many important aspects of the zika pandemic covered in these reviews, its history, epidemiology, and vector biology are of fundamental interest [ ] [ ] [ ] [ ] [ ] . zika virus was first thought of as an almost completely inconsequential virus that had smoldered at a low level in remote african/asian sylvatic cycles for decades, if not centuries. zika virus then suddenly and unexpectedly surged, and the virus was spread around the world by a nonsylvatic-vector mosquito, the globally ubiquitous aedes aegypti. this is the same mosquito that also has transmitted yellow fever, dengue, and chikungunya viruses around the world. a. aegypti is intensely anthropophilic (preferring blood meals from human beings as opposed to other animal hosts). while human travel and population growth and crowding clearly have played an important role in zika's emergence, these factors alone seem insufficient to explain its sudden pandemicity, since the virus has apparently long persisted in populous and mobile urbanized asian/southeast asian countries with hyperabundant a. aegypti mosquitoes [ ] . it is conceivable that one or more viral mutation occurred to facilitate viral spread. this remains a critically important research question. preliminary data show no evidence for phenotypic changes related to human pathogenic or transmissibility properties of the virus [ ] and fortunately no evidence of viral evolution that would hinder diagnostic detection [ ] . however, surprising preliminary evidence is consistent with the new (asian lineage-derived) pandemic strain causing earlier and more-frequent viral infection of mosquito salivary glands [ , ] . most pandemic zika cases appear to have been acquired from a. aegypti, a tenacious mosquito vector intimately associated with human dwellings and extremely difficult to eliminate or even control effectively. some evidence also suggests that at least other aedes species have also vectored zika outbreaks. this is of great concern not only because of the global abundance of of these mosquitoes (aedes albopictus and aedes polynesiensis), but because of the possibility that zika virus may be able to adapt to become more efficiently transmitted by new vectors, as has been seen with the chikungunya alphavirus [ ] . this would have alarming implications for enhanced zika virus spread and more broadly for the possibility of emergence of other diseases via enhanced vector capacity. in addition to vector transmission, zika virus also is transmitted from mothers to fetuses and infants, via sexual transmission, via laboratory accident, and possibly through blood transfusion and organ transplantation [ ] . these realities greatly complicate public health control efforts. understanding the pathogenesis and natural history of zika virus infection has been facilitated by decades of research with flaviviruses [ ] . however, despite its genetic similarity to the other flaviviruses (including its close cousins, the dengue viruses), zika virus has some unique or at least uncommon phenotypic properties. these include the ability to cause congenital infections and marked teratogenicity; to be simultaneously viscerotropic and neurotropic, causing not only dengue-like febrile illnesses, but also neurologic conditions, such as guillain-barré syndrome and encephalitis [ ] ; to transmit sexually; and to persist for long periods in multiple sites of the body. furthermore, some studies have suggested that preexisting flavivirus immunity (eg, from prior dengue virus infection) might potentiate zika [ ] via antibody-dependent infection enhancement in some circumstances [ ] , while other research has countered this view [ ] . regardless, the serologic and immune responses to zika virus in the background of high population immunity to related flaviviruses is complex and raises difficult questions about diagnosis and pathogenesis [ , ] . whether this proves to be the case, the serologic and immune responses to zika virus in the background of high population immunity to related flaviviruses is complex and raises difficult questions about diagnosis and pathogenesis [ , ] . the emergence of zika virus has shown it to be among the most unique and least understood of flaviviruses, necessitating intensive experimental study to determine how it causes disease. as with most flaviviruses, small-animal models of zika virus infection and disease have been problematic, but considerable progress has nonetheless been made, including important new information bearing on teratogenicity and vaccine design strategy [ ] . moreover, exciting nonhuman primate work is yielding insights into the natural history not only of typical infection, but also of placentally acquired infection of fetuses [ ] . unquestionably, the most disturbing aspect of the zika pandemic is the secondary pandemic of zika-associated microcephaly and many other birth defects (congenital zika syndrome), as well as fetal losses that have affected a large but still unknown percentage of women infected with zika virus during pregnancy [ ] . a large body of experimental work, including epidemiological and clinical studies being conducted in south and central america, represents the first time that an epidemic of a human teratogenic/fetal infectious disease has been intensively investigated in real time. these efforts are critically important because of the unfolding tragedy of the loss of thousands of babies and the birth of thousands more who will be incapacitated, sometimes severely, throughout their lifetimes. to make matters worse, it is suspected that many infected babies born in apparent health will manifest delayed effects of intrauterine zika virus infection as they grow into infancy, toddler age, and early school age. the epidemic of congenital zika syndrome represents not only a profound medical tragedy, but a societal challenge, as well: thousands of parents and caregivers will need financial, medical, and social support for decades to come. in this regard, it is worth reflecting upon and studying the rubella epidemic of the mid s, when tens of thousands of babies were born with congenital rubella syndrome in the united states [ ] , the deadliest epidemic of infectious birth defects ever documented. study of this rubella epidemic and the ongoing occurrences of other congenital infections, such as cytomegalovirus infection, have made clear the importance of understanding zika through large-scale prospective epidemiologic studies, such as those now underway in the americas. effective antiviral therapy for zika virus infection is still an unrealized goal, but numerous compounds, including repurposed drugs that are already in use, as well as antiviral antibodies, are being examined in preclinical studies [ ] . because the most important clinical application of an anti-zika drug would be treatment of infected pregnant women and infected fetuses, unprecedented challenges are now being considered [ ] . another critically important research goal is the development of a preventive zika vaccine [ , ] . almost a century of work on flavivirus vaccines (going back to the s) [ ] has provided us with several viable vaccine approaches and platforms, including those using other flavivirus proteins, and could readily be switched to zika virus proteins. at least different zika vaccines are currently (in late ) in clinical trials, with ≥ more in preclinical development [ , ] . zika vaccine trials face significant challenges, including the fact that the zika pandemic is waning in many areas and that flavivirus outbreaks normally appear sporadically and unexpectedly. furthermore, it is unknown which of several possible vaccination strategies is optimal, eg, inducing sterilizing immunity in vulnerable individuals, protecting pregnant women by vaccine induction of population herd immunity, or vaccinating children to induce partial population immunity [ ] . in addition, as noted above, serious questions about vaccine-associated immunologically enhanced disease remain unanswered. finally, it is important that we not assume that pandemic zika is a one-time crisis that can be met, controlled, and then forgotten or relegated to historical review. over the past decades, we have seen the dawn of a new infectious disease era in which mostly quiescent arboviruses have begun to aggressively emerge and reemerge around the globe, including dengue virus, west nile virus, and chikungunya virus. all the tropical world and much of the temperate world is now at risk and is likely to remain at risk for the foreseeable future. how we deal with the zika pandemic is likely to become a roadmap for future challenges. this issue of the journal outlines some of the important thoroughfares on that roadmap and will hopefully contribute to the difficult journey ahead. notes supplement sponsorship. this work is part of a supplement sponsored by the national institute of allergy and infectious diseases (niaid), part of the national institutes of health (nih). potential conflicts of interest. all authors: no reported conflicts. ueber die neueren fortschritte in der pathologie, mit besonderer beziehung auf öffentliche gesundsheitspflege und aetiologie. (rede, gehalten in der zweiten allgemeinen sitzung der deutsches naturforscher-versammlung zu frankfurt a m am september ) in: virchow r, ed. gesammelte abhandlungen aus dem gebiete der öffentlichen medicin und der seuchen-lehre. erster band. xi. berlin: august hirschwald the perpetual challenge of infectious diseases zika virus in the americas-yet another arbovirus threat history and emergence of zika virus epidemiology of zika virus infection quantifying zika: advancing the epidemiology of zika with quantitative models modes of transmission of zika virus zika virus mosquito vectors: competence, biology and vector control zika virus evolution and spread in the americas diagnosis of zika virus infections: challenges and opportunities a zika virus from america is more efficiently transmitted than an asian virus by aedes aegypti mosquitoes from evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes meeting the challenge of epidemic chikungunya zika virus structure, maturation and receptors neurological implications of zika virus in the adult population enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity antibody-dependent enhancement of infection and the pathogenesis of viral disease zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus humoral immune responses against zikv infection and the importance of pre-existing flavivirus immunity small animal models of zika virus non-human primate models of zika virus infection, immunity and therapeutic development zika virus (zikv) infection in pregnancy: maternal, fetal and neonatal considerations pathogenesis of other congenital viral infections small molecules and antibodies for zika therapy zika virus vaccine development zika vaccines: role for controlled human infection clinical development strategies and considerations for zika vaccine licensure all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- - dljap authors: young, ginger; bohning, kelly j.; zahralban-steele, melissa; hather, greg; tadepalli, sambasivarao; mickey, kristen; godin, c. steven; sanisetty, srisowmya; sonnberg, stephanie; patel, hetal k.; dean, hansi j. title: complete protection in macaques conferred by purified inactivated zika vaccine: defining a correlate of protection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dljap a critical global health need exists for a zika vaccine capable of mitigating the effects of future zika epidemics. in this study we evaluated the antibody responses and efficacy of an aluminum hydroxide adjuvanted purified inactivated zika vaccine (pizv) against challenge with zika virus (zikv) strain prvabc . indian rhesus macaques received two doses of pizv at varying concentrations ranging from . µg − µg and were subsequently challenged with zikv six weeks or one year following the second immunization. pizv induced a dose-dependent immune response that was boosted by a second immunization. complete protection against zikv infection was achieved with the higher pizv doses of . µg, µg, and µg at weeks and with ug pizv at year following vaccination. partial protection was achieved with the lower pizv doses of . µg and . µg. based on these data, a neutralizing antibody response above . log( ) ec was determined as a correlate of protection in macaques. pizv elicited a dose-dependent neutralizing antibody response which is protective for at least year following vaccination. in and , large outbreaks of zika virus (zikv) occurred in the americas. these outbreaks were associated with clusters of congenital microencephaly and other severe neurological sequelae in infections in approximately of infants born to pregnant women with laboratory confirmed zika in the us and us territories . incidence of zikv infections subsequently declined in most of the americas throughout and . with the sporadic nature of zikv outbreaks and a very low incidence of symptomatic disease in both endemic and non-endemic areas, conducting phase clinical efficacy trials is not feasible. still, the risk of re-emergence and the severe consequences of infection in pregnant women demonstrate that the need for an effective zika vaccine remains. in such circumstances, alternative regulatory strategies such as animal rule approval or accelerated approval pathway may be relevant for licensure . non-human primate studies have contributed to the development of zikv vaccines by demonstrating protective efficacy and identifying biomarkers of protection against zikv. results to date have supported neutralizing antibodies as an immune marker that is reasonably likely to predict clinical benefit of several zikv vaccines , . indian rhesus macaques (macaca mulatta) are susceptible to zikv infection and have been used extensively as a model to study efficacy of zikv vaccines and pathogenesis of multiple zikv isolates [ ] [ ] [ ] [ ] [ ] . zikv infection can be performed by subcutaneous injection, which mimics infection via mosquito bite and causes consistent viremia [ ] [ ] [ ] [ ] [ ] . the kinetics of zikv infection are similar in rhesus macaques and humans where serum or plasma viremia typically peaks within the first six days of infection and resolves within - days , , . the purified inactivated zika vaccine (pizv) has previously been evaluated in mouse models and was immunogenic in ag and cd mice and protected ag mice against lethal zikv challenge . in those studies, baldwin et al. demonstrated that neutralizing antibodies correlate with protection in ag mice. pizv is currently being evaluated for safety and immunogenicity in phase trials (clinicaltrials.gov nct ). pizv elicits a dose dependent neutralizing antibody response. rhesus macaques were vaccinated with pizv on days and and challenged on day . serum neutralizing antibodies were tested using a zika reporter virus particle (rvp) assay. all macaques were seronegative on day and control macaques remained seronegative prior to zikv challenge (fig. ) . twenty-eight days following the first vaccine dose (study day ), little or no seroconversion was observed for the . and . µg pizv doses, while / macaques vaccinated with the . µg pizv dose seroconverted and / macaques receiving the and µg pizv dose seroconverted. immune responses were boosted in all groups after the second dose (all p-values ≤ . ). twenty-eight days after the second pizv dose (study day ), % of vaccinated macaques seroconverted. the magnitude of neutralizing antibody titers on day was similar after the second dose of . , , or µg, with no statistically significant difference detected among these groups. the titer on day was lower in the . (p = . ) and . µg groups www.nature.com/scientificreports www.nature.com/scientificreports/ (p = . ) compared to the . µg dose group. titers prior to zikv challenge on day were slightly decreased compared to day . following challenge, neutralizing antibody titers increased significantly in the placebo, . and . µg groups (all p-values ≤ . ), but not in the . , , and µg groups. neutralizing antibody levels after two pizv doses of . , , or µg were similar to post-challenge neutralizing antibody titers in the placebo group. in conclusion, we observed a dose-dependent neutralizing antibody response to one or two doses of pizv. after two pizv doses, the neutralizing antibody levels reached a plateau for vaccine doses above . µg, and the level of neutralizing antibody was comparable to infection with zikv challenge in the placebo group. the lack of anamnestic antibody responses after challenge in the . , , and µg pizv dose groups suggests that infection by zikv challenge virus may have been prevented by vaccination. in addition to evaluating neutralizing antibodies, we also quantified anti-zika-specific igg using a luminex-based assay. as with neutralizing antibodies, all pizv doses were immunogenic and no anti-zika igg was detected in the control group prior to zikv challenge. pizv elicited dose-dependent anti-zika igg responses (fig. ) in the vaccinated groups. anti-zika igg responses were significantly boosted after a second pizv dose for all vaccinated groups (all p-values ≤ . ). following zikv challenge, the anti-zika igg responses increased significantly in the placebo, . and . µg groups (all p-values ≤ . ), but not in the . , , and µg groups. similar to the neutralizing antibody response, the anti-zika igg titers following vaccination with the . , , or µg doses were similar to post-challenge anti-zika igg titers in the placebo group. pizv protects against zikv challenge. rhesus macaques were challenged subcutaneously on day with ffu prvabc . all macaques receiving the . , , or µg pizv dose were protected against zikv challenge, as no zika vrna could be quantified in any of the macaques in these groups. zika vrna was quantified in / macaques receiving the . µg dose, / macaques receiving the . µg dose, and in all macaques in the control group (fig. ) . the peak post-challenge zika vrna level decreased with increasing vaccine dose level. the peak zika vrna occurred on day ( days post-challenge), with a geometric mean level of . log copies/ ml in the control group (range . - . log copies/ml), . log copies/ml in the . µg dose group (range . - . log copies/ml) and . log copies/ml in the . µg dose group (range . - . log copies/ml). no clinical signs to the vaccine or challenge virus were seen throughout the studies. correlate of protection. pizv elicited a dose dependent neutralizing antibody immune response and an anti-zika igg response which correlated with a reduction in zikv vrna post-challenge (table ). an immune correlate analysis was subsequently performed to correlate zika vrna with neutralizing antibody titers (fig. ) and anti-zika igg (fig. ) , which demonstrated that an increase in both neutralization antibody and anti-zika igg titers correlates with a decrease in vrna concentration. correlation with protection was observed with both neutralizing antibodies ( . log ec ) and anti-zika igg ( . log u/ml). we chose the functional assay, neutralizing antibodies, to establish a correlate of protection in macaques as a neutralizing antibody titer of > . log ec , which conferred protection against zikv serum viremia in indian rhesus macaques. due to overlap among the distributions of neutralizing antibody titers between the protected and infected animals, titers in some protected animals were below the correlate of protection. to determine the kinetics of neutralizing antibodies over a year long period, a separate set of four male indian rhesus macaques were vaccinated with µg pizv on study days and and challenged with zikv prvabc on study day . zika neutralizing antibody levels were measured every days through day (except for days , , and ), as well as on day ( days post-zikv challenge). all macaques seroconverted following the first vaccine dose, with a significant boost in antibody titers following the second dose (p < . ; fig. a ). neutralizing antibody titers peaked on day , days following the second immunization, declined from day to day , and then remained stable from day www.nature.com/scientificreports www.nature.com/scientificreports/ to day . anti-zika igg levels also peaked on day and subsequently declined through study day (fig. b) . igg titers increased following zikv challenge. no zika vrna was detected in serum from any of the macaques following zikv challenge year following the second dose. in addition, no statistically significant increase in neutralizing antibodies was observed post-zikv challenge on day (log ec range of . - . on day compared to log ec range of . - . on day ). the combined results suggest that two doses of pizv prevented zikv infection year post-vaccination. in conclusion, two µg pizv vaccinations elicits persistent neutralizing antibodies and long-term protection in rhesus macaques. we demonstrated that pizv elicits a dose-dependent response of both zika neutralizing and anti-zika igg antibodies. pizv elicited neutralizing antibodies that persisted for at least year and protected against zikv challenge. vaccinating with a broad range of pizv dose levels enabled us to correlate both neutralizing and anti-zika igg antibody titers to protection against zikv infection. we determined a neutralizing antibody correlate of protection of . log ec , which we define as the maximum ec among the unprotected animals in the study. we chose the neutralizing antibody assay to establish a correlate of protection as the literature supports using functional neutralizing antibodies as a correlate of protection for zika and other flaviviruses. table . neutralizing antibody titers on day of zikv challenge, zika vrna levels post-challenge. summary of mean neutralizing antibody titers (log ec ), range of peak vrna post-zikv challenge (log copies/ml), and percent of macaques with quantifiable zika vrna post-zikv challenge. all reported and analyzed data is above the rt-qpcr lloq. < lloq = detected vrna was below the assay lower limit of quantitation. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ neutralizing antibodies directed against the envelope (e) protein have been identified as correlates of protection for vaccines japanese encephalitis virus (jev), yellow fever virus, and tickborne encephalitis viruses . other laboratories using different vaccine platforms (dna, rna, inactivated virus, protein subunit, adenovirus-vectored, vlp) have reported induction of zikv-specific neutralizing antibodies that conferred protection against live zikv challenge in animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these data support zikv e-specific neutralizing antibodies as a mechanism of protection against zikv infection. in flavivirus vaccine development, neutralizing antibody titers that can confer protection against viremia have be reported in animal models using several different assay methods: microneutralization (mnt), rvp, and plaque reduction neutralization (prnt). studies of other candidate zikv vaccines have demonstrated that a neutralizing antibody titer as low as , using a mnt assay, or a titer of , using an rvp assay can confer protection against viremia in animal models , , . these results are qualitatively similar to those of licensed vaccines against flaviviruses such as jev, where a neutralizing antibody titer of > (as measured by prnt) is considered to determine the correlate of protection, the neutralizing antibody titers (log ec ) of infected (positive for vrna at any given timepoint) and protected (negative for vrna) macaques were plotted. the correlate of protection was defined as the maximum neutralizing antibody titer across all unprotected macaques in this study. based on this definition, the correlate of protection was established as > . log ec . cop = correlate of protection. www.nature.com/scientificreports www.nature.com/scientificreports/ to correlate with protection [ ] [ ] [ ] . further supporting this correlate of protection, it has been reported that passive transfer of zika neutralizing monoclonal antibodies or antisera from vaccinated humans and animals to mice is sufficient to protect against zikv challenge , , , . adoptive transfer of serum from immunized mice fully protected against viremia, while splenocytes from the same donors provided marginal protection demonstrating that the mechanism of protection against zikv infection is antibody mediated. while zikv exists as three genotypes (west african, east african and asian), they all behave as a single serotype , . our previous studies demonstrated that pizv is capable of eliciting antibodies that neutralize both african and asian zikv isolates in vitro . others have shown that the magnitude or duration of viremia in unvaccinated macaques challenged with zikv isolates from brazil or puerto rico is similar , and that vaccinated macaques are protected against challenge with heterologous zikv strains , , . protection of animal models against heterologous challenge and cross-neutralization capabilities of antisera from multiple vaccine platforms , , , suggest that data from a single challenge strain may be sufficient to show cross protection against zikv strains from other lineages. several groups have developed rhesus macaque challenge models using zika strain prvabc , , , , which is a well-documented and characterized isolate from human serum and is representative of viruses that were circulating in the americas during the - outbreak . we therefore selected prvabc as the challenge strain for our studies. in this study, we extended our observation of pizv efficacy in mice to demonstrate efficacy in prevention of zikv vrna and to evaluate anamnestic antibody responses after zikv challenge in non-human primates. we did not assess presence of vrna in tissues. our working hypothesis is that prevention of serum zikv vrna may be a surrogate for prevention of the most serious sequelae of zikv infection in humans -fetal infection. by employing a pizv dose titration study design and a zikv rvp assay, we have established a minimum protective vaccine dose of . µg in rhesus macaques and established a neutralizing antibody correlate of protection against zikv challenge of . log ec . finally, we demonstrated that zikv neutralizing antibodies persist and are capable of preventing zikv infection for at least year post-vaccination. in the event that human phase efficacy studies are not feasible, identifying a correlate of protection in an appropriate non-human primate challenge model may be important to support licensure of a zika vaccine , , . altogether, these data support neutralizing antibodies as an immune marker that is associated with efficacy in a relevant animal model and that may predict a reasonable likelihood of clinical benefit in humans. vaccine. pizv is an aluminum hydroxide adjuvanted whole purified inactivated virus vaccine based on zikv strain prvabc which was originally isolated from serum from a human infected in puerto rico (genbank accession number ku ). pizv has been previously described and characterized by baldwin et al. . the same lot of pizv used in this study was also used in clinical trial zik (clinicaltrials.gov nct ). rhesus macaque challenge studies. thirty-five indian rhesus macaques were separated into groups. three male and three female macaques per group were vaccinated intramuscularly with either . , . . . , or µg pizv on study days and . to achieve statistical significance comparing vaccine doses, six animals/ group were selected to obtain > % power if the true infection rate among controls is ≥ % and the true infection rate among vaccinated animals is ≤ % based on a one-sided fisher's exact test with % type error rate . two male and three female macaques were vaccinated with placebo control (pbs) on study days and . one female from the control group was euthanized on the day of zikv challenge due to reasons unrelated to the study, resulting in macaques in the control group. macaques were challenged subcutaneously with ffu/ . ml zikv prvabc on study day . serum samples were collected and tested for antibody titers on study days , , , and , and , and for zikv rna on study days - , and study day . www.nature.com/scientificreports www.nature.com/scientificreports/ a second study was conducted to assess long-term pizv immunogenicity and efficacy. flavivirus seronegative male rhesus macaques (n = ) were vaccinated with the µg pizv dose on days and and challenged subcutaneously with ffu/ . ml zikv prvabc on day . serum samples were collected and tested for antibody titers monthly up to year post-vaccination and days following zikv challenge (study day ). to assess replication of zika vrna following zikv challenge, serum samples were collected on study days - , and . challenge virus. zika virus puerto rico strain prvabc was used for the challenge ( . ml containing a nominal dose of ffu). the seed stock was passaged three times on vero cells at cdc, fort collins, colorado. virus stocks were prepared by passaging two times on c / mosquito cells. stocks were from a master working virus bank and tested for sterility, mycoplasma and endotoxin prior to use. screening. to select flavivirus naïve macaques, baseline igg serostatus was determined using a multiplex luminex kit (ampersand biosciences flavivirus serological panel). briefly, macaque serum was diluted : , in sample diluent and added to multiplexed magnetic beads coupled with zika, dengue, yellow fever, japanese encephalitis, west nile, usutu, saint louis encephalitis, and chikungunya virus antigens. serum and beads were incubated on a plate shaker for hour at room temperature and then washed three times with assay buffer. detection antibody, anti-igg pe, was added to each sample and incubated on a plate shaker for minutes at room temperature. following another three wash cycles, beads were resuspended in assay buffer and read on the bio-plex magpix to measure median fluorescent intensity (mfi) for each bead set. macaques were considered flavivirus naïve and selected for the study if the mfi was below pre-defined assay cutoff criteria for seronegativity for all antigens. neutralizing antibodies. a zika rvp assay (sonnberg et al., manuscript in preparation) was used to determine neutralizing antibody titers in serum following the administration of pizv (study days , , , and ), and days post-zikv challenge (day ). briefly, macaque serum was heat inactivated at °c for minutes and then serially diluted -fold in assay media for an -point dilution series. diluted serum and rvp were plated in duplicate in a -well assay plate and incubated for hour at °c. vero cells were added to each well and incubated at °c for hours. renilla-glo substrate (promega, wi, usa) was then added to the plate and incubated for minutes at room temperature. finally, plates were analyzed by a luminometer. the effective concentration at % (ec ), was determined by a non-linear regression curve fit with the lower asymptote constrained to in graphpad prism. the lloq for the nhp assay is . log ec , below which the serum matrix interfered with the measurement. the upper limit of quantitation (uloq) of the standard assay is . log ec . any samples returning titers >uloq were retested with a higher initial pre-dilution. anti-zika igg binding antibodies. anti-zika igg antibody levels were measured for study days , , , and (prior to zikv challenge) using a luminex based anti-zika igg assay. heat-inactivated serum samples were diluted : in assay buffer and then serially diluted -fold for an -point dilution curve. magnetic beads covalently coupled with pizv antigen were added to each sample dilution in a -well plate and each sample dilution was tested in duplicate. plates were incubated on a plate shaker for minutes at room temperature and then washed two times with assay buffer. diluted anti-ig-pe detection antibody was then added to each sample and incubated on a plate shaker for hour at room temperature. after two wash cycles, beads were resuspended in assay buffer and read on the luminex flexmap d to measure the mfi. the igg antibody concentration was quantified using a reference standard serum with an assigned igg concentration in units/ml (u/ml). the lloq for the assay is . log u/ml. total rna from serum samples was extracted using the qiagen biorobot universal instrument and qiaamp virus biorobot mdx kit (qiagen; valencia, ca). extracted and purified rna was evaluated in two separate rt-pcr assays. primarily, zika vrna was detected and quantified using rt-qpcr with primers and probe specific to known zikv genotypes as described in lanciotti et al. and a standard curve generated from synthetic reference zika vrna (atcc). a qualitative extraction control rt-pcr was also performed. the extraction control rt-pcr utilized a primer/probe set, specific to macaca mulatta c galt c l mrna, confirmed adequate nucleic acid extraction from serum samples independent of zika vrna detection. the geometric mean, range of peak zika vrna detection, and the respective percentage of protected macaques (as defined by absence of vrna) was calculated using quantities greater than the assay lower limit of quantitation of . log copies/ml. samples with vrna concentration lower than the assay limit of detection of . log copies/ml were considered negative. correlate of protection. the mean neutralizing antibody titers (log ec ) determined by zika rvp assay was computed for each dose group at day (day of zikv challenge). zika vrna copies/ml determined by rt-qpcr assay were computed for each dose and timepoint post-challenge (study days - and ). peak vrna for each macaque was defined as the highest observed vrna concentration across all timepoints tested. macaques were considered protected if vrna was not detected or was below the assay lloq for all timepoints tested. the correlate of protection was defined as the maximum neutralizing antibody titer across all unprotected macaques in this study. this definition was conservative in that some protected macaques could have neutralizing antibody titer levels below the correlate of protection due to overlap between the distributions of protected and unprotected macaques. since the correlate of protection was not a statistical estimate, no confidence intervals were reported. statistical analysis. statistical tests were performed using the r software version . . .; log neutralization titers were compared for selected pairs of days for each dose group using a paired two-sided t-test. at day , tukey's test was used to compare all dose groups with each other. a two-sided spearman's test was applied to the unprotected macaques to check for an association between day neutralizing antibodies titers and peak vrna. comparisons between selected pairs of days were also performed with the log igg antibody levels in the main study and with the log neutralization titers from the long-term immunogenicity study using paired two-sided t-tests. a p-value threshold of . was used to determine statistical significance. the data that support the findings of this study are available from the corresponding author upon reasonable request and with permission of takeda vaccines, inc. vital signs: zika-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital zika virus infection -u.s. territories and freely associated states who region of the americas/pan american health organization. plisa health information platform for the americas: cases of zika virus disease clinical development strategies and considerations for zika vaccine licensure correlates of protection induced by vaccination demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and licensure of zika vaccine candidates protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys rapid development of a dna 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western hemisphere a tale of two viruses: does heterologous flavivirus immunity enhance zika disease? zika viral dynamics and shedding in rhesus and cynomolgus macaques what is the predictive value of animal models for vaccine efficacy in humans? the importance of bridging studies and species-independent correlates of protection genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia correspondence and requests for materials should be addressed to g.y.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - s vh authors: delvecchio, rodrigo; higa, luiza m.; pezzuto, paula; valadão, ana luiza; garcez, patrícia p.; monteiro, fábio l.; loiola, erick c.; dias, andré a.; silva, fábio j. m.; aliota, matthew t.; caine, elizabeth a.; osorio, jorge e.; bellio, maria; o’connor, david h.; rehen, stevens; de aguiar, renato santana; savarino, andrea; campanati, loraine; tanuri, amilcar title: chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: s vh zika virus (zikv) infection in utero might lead to microcephaly and other congenital defects. since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. here we show that chloroquine exhibits antiviral activity against zikv in vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. we demonstrate that chloroquine reduces the number of zikv-infected cells in vitro, and inhibits virus production and cell death promoted by zikv infection without cytotoxic effects. in addition, chloroquine treatment partially reveres morphological changes induced by zikv infection in mouse neurospheres. zika virus (zikv) is an arthropod-borne virus, transmitted by aedes mosquitoes, that belongs to the flavivirus genus, which also includes other pathogens such as west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and dengue virus (denv). zika virus was first isolated from a sentinel monkey in the zika forest in uganda in [ ] . since then, zikv has been isolated from humans and mosquitoes throughout africa and southeast asian countries. phylogenetic analysis of the nonstructural protein encoding region has disclosed three zikv lineages: east african, vero cells (atcc, manassas, va, usa) are derived from the kidney of african green monkey and were grown in dmem high glucose (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with % fetal bovine serum (fbs) (gibco). human brain microvascular endothelial cells (hbmec) were a kind gift from dr. julio scharfstein (federal university of rio de janeiro, rio de janeiro, brazil), and hbmec isolation was performed as previously described [ ] . these cells were cultured in dmem high glucose supplemented with % fbs. the c / cell line is derived from aedes albopictus. c / cells (atcc, manassas, va, usa) were grown in leibovitz l- medium (gibco) supplemented with . g/l tryptose phosphate broth (sigma aldrich, boston, ma, usa), mm glutamine (gibco), . % sodium bicarbonate (gibco), x non-essential amino acids (gibco) and % fbs. neural stem cells (nscs) were derived from human induced pluripotent stem cells (ipscs). ipscs were provided by the biobank of ipscs of the brazilian ministry of health (conep b- # . / - ). according to the supplier, fibroblasts were reprogrammed using the protocol developed by paulsen et al. [ ] , and transduced with the cytotune-ips sendai kit (thermo fisher scientific, waltham, ma, usa). ipscs presented a normal karyotype and the expression of pluripotency markers. these cells were cultured with e culture media (gibco) on a matrigel (bd biosciences, san jose, ca, usa) coated surface. ipsc colonies were manually passaged every - days until they reached %- % confluence and were maintained at • c in humidified air with % co . to produce nscs, human ipscs were exposed to the serum-free neural induction medium (gibco), containing neurobasal medium (gibco) and the pluripotent stem cell neural induction supplement (gibco), according to the manufacturer's protocol [ ] . briefly, the medium was changed every other day until day , when initial nscs are split and grown on neural expansion medium (advanced dmem/f and neurobasal medium ( : ) with neural induction supplement; gibco). nscs were used after four passages in neural expansion media. mouse central nervous system (cns) cells were harvested from swiss mouse embryos at embryo day (e ) and grown for h as free floating neurospheres in neurobasal culture media (gibco) supplemented with x b (gibco). chloroquine diphosphate was kindly supplied by farmanguinhos (fiocruz, rio de janeiro, brazil). the lyophilized powder was diluted in double distilled water to mm. the chloroquine solution was filtered through a . µm membrane and stored at − • c. zikv strain mr (uganda/africa, accession no.: nc . , kindly provided by dr. davis ferreira, federal university of rio de janeiro, rio de janeiro, brazil) was isolated from a rhesus monkey and injected intracerebrally on swiss mice for several passages [ ] and zikv br (recife/brazil, zikv pe/ , accession no: kx . , kindly provided by dr. marli tenório cordeiro, centro de pesquisas aggeu magalhães, recife, brazil) was isolated from a patient presenting classical symptoms of zikv infection [ ] . these viruses were propagated in vero and c / cells, respectively. briefly, the cells were infected with zikv at a multiplicity of infection (moi) of . and incubated at • c. after h, the inoculum was removed and replaced with dmem high-glucose (vero) and leibovitz l- (c / ) growth media supplemented with % fbs. after to days, the conditioned medium was harvested, centrifuged at × g, and sterile-filtered to remove cells and cellular debris. virus stocks were stored at − • c. virus titers were determined by plaque assay performed on vero cells. virus stocks or samples were serially diluted and adsorbed to confluent monolayers. after h, the inoculum was removed and cells were overlaid with semisolid medium constituted of alpha-mem (gibco) containing % carboxymethyl cellulose (sigma aldrich) and % fbs (gibco). cells were further incubated for days when cells were fixed in % formaldehyde. cells were stained with % crystal violet in % ethanol for plaque visualization. titers were expressed as plaque forming units (pfu) per milliliter. vero cells, hbmec, and nsc were infected with zikv mr or zikv br strain at an moi of . after h, the inoculum was removed and medium containing chloroquine ( . to µm) was added to the cells. after to days, cells were fixed with % paraformaldehyde (sigma aldrich) in phosphate buffered saline (pbs) for min at room temperature and washed with pbs. cells were permeabilized with . % triton x- (sigma aldrich) in pbs, washed with pbs, and blocked with pbs with % fbs. cells were incubated with g , a pan-flavivirus antibody raised against the zikv envelope e protein produced in g - - hybridoma (atcc), diluted : in pbs with % fbs. cells were labeled with donkey anti-mouse alexa fluor antibody (thermo scientific, waltham, ma, usa) diluted : in pbs with % fbs, and were analyzed by flow cytometry in a bd accuri c (becton, dickinson and company, franklin lakes, nj, usa) for zikv infection. vero, hbmec, and nsc were seeded on black -well plates with clear bottoms and infected with zikv mr or zikv br strain at an moi of for h. neurospheres were seeded on coverslips and infected with zikv mr with . × pfu after infection, the viral inoculum was removed and cells were incubated with medium containing chloroquine ( . to µm) for to days, depending on cell type. cells and neurospheres were fixed with % paraformaldehyde in pbs for min at room temperature. the fixative was removed and the samples were washed three times with pbs. blocking of unspecific binding of the antibody and permeabilization were performed with pbs supplemented with % bovine serum albumin (bsa, sigma aldrich) and . % triton x- for min at room temperature. incubation with anti-map antibody was performed following the manufacturer's instructions (abcam, cambridge, cambridgeshire, uk, # ; : ) and the g antibody was diluted : in pbs with % bsa and incubated with cells for h. after washing three times with pbs, cells were incubated with secondary antibodies coupled to alexa fluorochromes (thermo fisher scientific) for min and washed five times. coverslips with neurospheres were mounted with prolong gold mounting medium (thermo fisher scientific). samples were imaged using either leica sp or leica spe (leica biosystems, wetzlar, hesse, de) confocal microscopes and a nikon te (tokyo, japan) inverted microscope coupled to a leica dfc fx camera (leica biosystems, wetzlar, germany). vero, hbmec, and nsc were exposed to zikv mr at an moi of . after h, inoculum was removed and chloroquine-containing medium ( . to µm) was added to the cells. five days post-infection, µl of celltiter blue reagent (promega, madison, wi, usa) was added in each well, incubated for - h, and fluorescence was measured ( / nm), except for nscp cells when celltiter blue was added at three days post-infection. mean fluorescence intensity (mfi) and standard deviation were displayed. in order to calculate the half maximum effective concentration (ec ) that protects cells from death caused by zikv infection, mfi values from zikv mr control were subtracted from every condition and then these values were normalized over the mock control. these data were plotted and hill parameter sigmoidal regression was performed on sigma plot v. . (systat software inc., san jose, ca, usa). vero cells, hbmec, and nsc were incubated with medium containing chloroquine ( . to µm) for days (nsc) or days (vero and hbmec). celltiter blue reagent (promega) was added in each well, incubated for - h, and fluorescence was measured ( / nm). in order to calculate the % cytotoxicity concentration (cc ), mfi values were normalized over the untreated control. these data were plotted and hill parameter sigmoidal regression was performed on sigma plot v. . (systat software inc.). vero cells were inoculated with zikv mr at an moi of for h at • c. cells were washed three times with cold pbs to remove unbound virus and treated with µm chloroquine that was added at different time points: , . , , , and h post-infection. conditioned media were collected at h post-infection to analyze the production of virus particles through viral rna content or the amount of infectious virus particles. viral rna was extracted and quantitative reverse transcription polymerase chain reaction (rt-qpcr) was performed. the titer of infectious virus particles was determined by plaque assay. vero cells were infected with zikv mr or zikv br strain at an moi of for h at • c. virus input was washed three times with cold pbs and cells were treated with chloroquine ( . to µm) for h, and then the supernatant was collected and the rna was extracted and analyzed by relative quantification by rt-qpcr. the supernatant was also evaluated by plaque assay to quantitate the infectious virus particles. viral rna was extracted from µl supernatant of infected cells using qiamp minielute virus spin (qiagen, hilden, düsseldorf ,de), following the manufacturer's recommendations. zikv detection was performed using one step taqman rt-qpcr (thermo fisher scientific) on a real-time pcr system (applied biosystems) with primers and the probe described elsewhere [ ] . threshold cycle (ct) was determined and ∆ct (ct chloroquine treated − ct untreated) was calculated. the fold reduction of virus particles' release, including defective viral particles, were calculated by ∆ct . mean and standard deviation (sd) were calculated for each assay. one way analysis of variance (anova) was conducted using the non-parametric test (kruskal-wallis) followed by dunn's multiple comparisons test. a p-value of < . was considered significant. all analyses were performed on graphpad prism v. (graphpad software, san diego, ca, usa). the sample size is provided in the respective figure legends. we characterized the antiviral properties of chloroquine in vero cells, a model widely used to study viral infections. vero cells were infected with zikv mr at an moi of (i.e., pfu/cell) and were then treated for days with chloroquine in concentrations ranging from . to µm. viral infectivity was assessed using the g antibody, which detects flavivirus envelope e protein. we observed that chloroquine treatment decreased the number of zikv-infected cells in a dose-dependent manner. flow cytometry analysis showed a reduction of % and % in zikv-infected cells when cultures were treated with µm and µm chloroquine, respectively, compared to untreated infected cells ( figure a ). immunofluorescence staining corroborated these results ( figure b ) and additionally, chloroquine decreased the production of infectious ( figure c ) and total ( figure d ) virus particles, including defective viral particles, by zikv-infected cells. to confirm that viral inhibition is independent of chloroquine cytotoxicity, the viability of uninfected cells treated with chloroquine ( . to µm) for days was analyzed. chloroquine did not impact cell viability at concentrations of µm or lower ( figure e ). we further analyzed whether chloroquine treatment could protect vero cells from zikv infection as assessed by cell viability. chloroquine, ranging from . to µm, increased cell viability from % up to % ( figure f ). microcephaly cases and neurological disorders have only been associated with infection with strains of zikv from the asian lineage, detected in french polynesia and in the americas [ , , ] . to determine the inhibition spectrum of chloroquine against zikv infection, vero cells were infected with the brazilian isolate of the asian lineage (zikv br). chloroquine decreases the percentage of cells infected with the brazilian isolate from % to % and % at . and µm, respectively ( brazilian strain at an moi of and exposed to chloroquine for h. the supernatant was collected and viral rna was relatively quantified over the untreated infected control (b) or infectivity was analyzed by immunofluorescence with g antibody (c). the dashed line represents fold reduction on virus production of . data are represented as mean ± sd from two independent experiments. statistical significance was assessed by kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < . ). inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [ , ] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr . the supernatant was collected h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at h postinfection had a greater impact on the production of zikv particles, decreasing viral rna -fold over the controls ( figure a ). the addition of chloroquine from min to h post-infection was able to reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [ , ] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr . the supernatant was collected h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at h post-infection had a greater impact on the production of zikv particles, decreasing viral rna -fold over the controls ( figure a ). the addition of chloroquine from min to h post-infection was able to reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. considering that zikv infects hbmecs [ ] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced % and % of the number of zikv mr -infected hbmecs at and µm, respectively ( figure a,d) . these concentrations are non-cytotoxic ( figure b ), and protected approximately % of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure c ). considering that zikv infects hbmecs [ ] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced % and % of the number of zikv mr -infected hbmecs at and μm, respectively ( figure a,d) . these concentrations are non-cytotoxic ( figure b) , and protected approximately % of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure c ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [ ] . to evaluate if chloroquine could protect these cells from zikv mr infection, nscs were exposed to up to μm chloroquine for days. chloroquine treatment decreased the number of nscs infected with zikv mr by %, and, through cell viability assessment protected % of nscs from zikv infection, without cytotoxicity effects ( figure a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure e ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [ ] . to evaluate if chloroquine could protect these cells from zikv mr infection, nscs were exposed to up to µm chloroquine for days. chloroquine treatment decreased the number of nscs infected with zikv mr by %, and, through cell viability assessment protected % of nscs from zikv infection, without cytotoxicity effects ( figure a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure e ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [ ] . as we previously observed, neurospheres infected with zikv strain mr showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein (map ) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure a-c) . furthermore, zikv infection decreased when neurospheres were treated with . μm chloroquine, as evaluated by g staining (figure d-f) . data are represented as mean ± sd from two to three independent experiments. statistical analysis was performed with kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < . ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [ ] . as we previously observed, neurospheres infected with zikv strain mr showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein (map ) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure a-c) . furthermore, zikv infection decreased when neurospheres were treated with . µm chloroquine, as evaluated by g staining (figure d-f) . chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in , from january to april , zikv transmission has been reported in countries and territories [ ] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the figure . chloroquine inhibits zikv infection in mouse neurospheres. mouse neurospheres were infected with zikv mr ( . × pfu and were treated with chloroquine for days. neurospheres were analyzed by phase contrast microscopy (a-c), and triple stained for envelope viral protein (green), microtubule-associated protein (map- , red), a neuron-specific protein, and dapi (blue) (d-f). chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in , from january to april , zikv transmission has been reported in countries and territories [ ] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the chance of vertical transmission in viremic pregnant women as was shown for hiv-infected pregnant women treated with antiretroviral therapy [ ] . here we demonstrated that chloroquine decreases the number of zikv-infected cells and protected cells from zikv infection as measured by cell viability at non-cytotoxic concentrations (figures , and ) . the ec or concentration of chloroquine that protected % of the cells from zikv infection assessed by cell viability, was . - . µm depending on the cell model and the cc ranged from . to . µm ( table ) . the values of ec obtained for zikv mr are lower than those obtained for denv inhibition (~ µm) and hiv inhibition ( µm) [ , ] . furthermore, we observed similar zikv inhibitory effects of chloroquine when tested on different zikv lineage infections (figures and ) , supporting the idea that chloroquine could help to manage recent infections caused by asian zikv lineage. although chloroquine has shown antiviral activity against a large spectrum of viruses in vitro, few clinical studies have been performed to evaluate chloroquine effects on patients with viral infections. two clinical trial studies of chloroquine have been conducted to assess chloroquine treatment in patients infected with denv [ , ] . one of the trials evaluated the benefits of chloroquine treatment for days in patients infected with denv and showed no reduction in the duration or intensity of denv viremia or nonstructural protein (ns ) antigenemia clearance [ ] . however, a trend towards a reduction in the number of dengue hemorrhagic fever cases was noticed in the chloroquine-treated group [ ] . a more recent clinical trial of chloroquine administration to denv-infected patients, also for days, showed that % of the patients in the chloroquine-treated group reported feeling less pain and showed improvement in the performance of daily chores during treatment [ ] . moreover, the symptoms returned after medication withdrawal. however, chloroquine treatment did not reduce the duration and intensity of the fever or duration of the disease [ ] . the antiviral effect of chloroquine may be insufficient to produce a decrease in viral load or improvement of the disease progression when chloroquine/hydroxychloroquine is used in monotherapy. however, chloroquine may produce a significant antiflaviviral effect when used in combination therapies, as recently shown in a clinical trial of hydroxychloroquine plus ribavirin and interferon alpha in individuals infected with hepatitis c virus (hcv) [ ] . in regard to the potential antiviral therapeutic combinations for zika, a freshly published screening of drugs already approved for other clinical indications has resulted in the identification of more than candidate drugs [ ] . of note, one of these is mefloquine, a compound related to chloroquine. in terms of safety for pregnant women, however, mefloquine is included in the b category, i.e., a drug for which the animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women. be that as it may, the aforementioned study corroborates our results using chloroquine, and provides new anti-zikv drugs that could be tested in combination with chloroquine. differing from mefloquine administration, the use of chloroquine during pregnancy was thoroughly evaluated and when prophylactic doses of chloroquine were administered for malaria ( mg/week), no increment in birth defects was observed [ ] . higher concentrations of chloroquine ( mg to mg/day) were administered to pregnant women with severe diseases, such as lupus or rheumatoid arthritis. in these studies, few cases of abortion and fetal toxicity were observed. however, fetal toxicity or death could not be discarded as direct consequences of the disease itself. in addition, all term deliveries resulted in healthy newborns [ , ] . chloroquine has been successfully tested in animal models. twice daily administration of chloroquine ( mg/kg) has been shown to increase the balb/c mice survival rate to %- % after infection with ebola virus (ebov) [ ] . in a c bl/ mice model for coronavirus infection, chloroquine ( mg/kg) protected % of -day-old suckling mice infected with human coronavirus oc (hcov-oc ) when administered to pregnant mice day prepartum [ ] . a survival rate of % was observed in balb/c mice infected with avian influenza h n virus treated with chloroquine at mg/kg/day [ ] . these results suggest that chloroquine has the potential to inhibit zikv in in vivo mouse studies. chloroquine is widely distributed to body tissues as well as its analogue hydroxychloroquine. the concentration of hydroxychloroquine in the brain is - times higher than in the plasma [ ] . the concentration of chloroquine in the plasma reached µm when a daily intake of mg was prescribed to arthritis patients [ ] . chloroquine is able to cross the placental barrier and is supposed to reach similar concentrations in both maternal and fetal plasma [ ] . concentrations of chloroquine, similar to the ec values calculated here (table ) , are achieved in the plasma in current chloroquine administration protocols and might reach the brain. different mechanisms for the chloroquine inhibition of viral infection have been described [ ] [ ] [ ] . we observed a strong reduction in the release of zikv particles when the drug was added at h post-infection (figure ) , suggesting a higher impact on early stages of infection, possibly during fusion of the envelope protein to the endosome membrane. chloroquine inhibits acidification of the endosome, consequently inhibiting the low ph-induced conformational changes required for the fusion of the envelope protein of flaviviruses with the endosomal membrane [ ] . chloroquine was also effective in decreasing virus release, although less pronouncedly and not statistically significant, when added after the early stages of virus infection (from . to h post-infection), suggesting that later stages of the zikv replication cycle might also be affected (figure ) . zikv was detected in the cerebrospinal fluid of zikv-infected adult patients that manifested meningoencephalitis, indicating that zikv invades the central nervous system through yet unknown mechanisms. transcytosis through the endothelial cells of the blood brain barrier is a known mechanism of viral access to the central nervous system [ , ] . here we demonstrated, by different methodologies, that chloroquine protects hbmec, an immortalized cell line widely used as in vitro model of the blood-brain barrier [ ] , from zikv infection (figure ) . recent studies showed that neural stem cells are highly permissive for zikv infection and one of the mechanisms proposed for the cause of microcephaly would be the depletion of the stem cell pool induced by zikv [ , , ] . our data showed that chloroquine inhibits the infection of human neural stem cells ( figure ). using the mouse neurospheres model to study neural stem cell differentiation into neurons, 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bases affect late stages of mayaro virus replication cycle in vertebrate cells effects of chloroquine on viral infections: an old drug against today's diseases? rodenhuis-zybert, i.; wilschut, j. flavivirus cell entry and membrane fusion human immunodeficiency virus- uses the mannose- -phosphate receptor to cross the blood-brain barrier mechanism of west nile virus neuroinvasion: a critical appraisal brain-region-specific organoids using mini-bioreactors for modeling zikv exposure chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids acknowledgments: this work was supported by conselho nacional de desenvolvimento e pesquisa (cnpq), key: cord- -roxa ix authors: i. sardi, silvia; h. carvalho, rejane; c. pacheco, luis g.; p. d. almeida, joão p.; m. d. a. belitardo, emilia m.; s. pinheiro, carina; s. campos, gúbio; r. g. r. aguiar, eric title: high-quality resolution of the outbreak-related zika virus genome and discovery of new viruses using ion torrent-based metatranscriptomics date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: roxa ix arboviruses, including the zika virus, have recently emerged as one of the most important threats to human health. the use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. herein, we used rna-based metatranscriptomics associated with ion torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related zika virus (zikv) genome ( , nt), with extended ′-utr and ′-utr regions, using a newly-implemented bioinformatics approach. besides allowing for the assembly of one of the largest complete zikv genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting c / cell lines, namely: alphamesonivirus strain salvador ( , nt) and aedes albopictus totivirus-like ( nt); the latter likely represents a new viral species. altogether, our results demonstrate that our bioinformatics approach associated with ion torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of rna deep sequencing for virus identification. arthropode-borne viral infections (arboviroses), transmitted to mammals by hematophagous arthropod vectors, have recently emerged as one of the most important threats to human health. due to their worldwide occurrence and fast adaptation to environmental changes, mosquitoes from the aedes genus play an important role in the transmission of viral etiological agents of emerging human infections, including dengue virus (denv), zika virus (zikv), chikungunya virus (chykv) and of yellow fever virus (yfv) [ , ] . many epidemic events have been reported, which are associated with the lack of efficient vaccines, the rapid evolution of viral genomes, and inefficient control strategies. one of the last episodes was the zika outbreak in brazil in the period - , which impacted more than , individuals by the end of [ ] . the impact of this outbreak was amplified due to the zika virus infection's relationship to many neurological disorders, such as guillain-barre and microcephaly syndromes, which led the world health organization to declare the brazilian zika outbreak as one of international concern by . different from african lineage, the asian lineage of zika virus, supposedly the origin of the strains circulating in brazil, exhibits a different profile with lower pathogenicity and prolonged infections [ ] . these differences have been attributed to mutations in the viral genome, mainly in untranslated regions [ , ] . the long-term infection, which could facilitate mosquito transmission, associated with the high susceptibility of the population likely explains the rapid spread of the virus that reached almost all brazilian states in only a few months, different, for example, to the dengue virus whose spread took decades [ ] . in addition to arboviruses, aedes mosquitoes are also known to carry a vast range of viruses, most of them insect-specific [ ] [ ] [ ] . although they do not cause direct harm to human health, they have been described as impacting mosquito susceptibility to other viral infections and vector competence [ ] [ ] [ ] . therefore, the knowledge of the viruses circulating in mosquitoes (virome) is important in understanding mosquito-arbovirus interactions as well as in developing new strategies of control, and in the prevention of epidemic events. in this context, metagenomics associated with deep sequencing dramatically increased the resolution of viral genomes and the identification of new viruses [ , ] . however, while dna sequencing restricts the identification of viruses that use dna intermediates in the replication cycle, rna sequencing allows for the identification of all classes of viruses, since almost all of them generate rna intermediates during their replication. indeed, metatranscriptomics has been successfully applied to the identification of rna and dna viruses in a vast range of organisms, such as plants, insects, fungi, and mammals [ , [ ] [ ] [ ] . despite being a powerful strategy, there are technical limitations that hamper confident identification of whole viral genomes, such as length of sequenced reads, repetitive genomes, differential expression of virus segments, and natural variation in the virus population that make the confident assembly of contiguous sequences a challenging task [ , ] . here, we applied rna-based metatranscriptomics associated with ion torrent deep sequencing and a newly developed bioinformatics approach to the high-quality reconstitution of viral genomes. using this strategy, we successfully reconstituted an outbreak-related zika virus genome with extended -utr and -utr regions and the genomes of two arthropod-infecting viruses co-infecting c / cell lines. altogether, our data demonstrate that our bioinformatics approach associated with ion torrent sequencing allows for high-quality reconstruction of known and unknown viral genomes. c / cells (aedes albopictus) were maintained in leibovitz's l medium (gibco ® , ca, usa), supplied with % fetal bovine serum (fbs) (gibco ® ), % tryptophan broth (sigma-aldrich ® , boston, ma, usa) at • c. the cells were inoculated with a zika virus strain (partial fragment of e protein deposited at genbank kr from zika virus isolate br/ufba/labviro/ex -see figure b ) derived from a serum sample collected from a patient in the brazilian outbreak of with moi , and after observation of the cytophatic effect ( days), the supernatant of the infected cells was centrifugated ( . g at • c for min) and then submitted to ultracentrifugation ( . g at • c for . h). the pellet was resuspended in phosphate saline solution (pbs) and used to perform rna extraction through the qiaamp viral rna mini kit (qiagen, hilden, germany). quantification of the total extracted rna was performed by fluorimetry using qubit equipment (life technologies, carlsbad, ca, usa). the rna library was constructed from the extracted rna using ion total rna-seq kit v viruses , , of according to the manufacturer's protocols. sequencing was performed using an ion ™ chip kit (life technologies) in the ion onetouch™ system and the ion s instrument. sequencing data were deposited at the sra database under accession number: srr . a. albopictus genome was downloaded from vectorbase (www.vectorbase.org). all complete bacterial genomes used to remove possible contamination in sequenced reads were downloaded from the refseq database [ ] . sequence and metadata of the zika virus were downloaded from virus pathogen resource (vipr) [ ] , and only complete genomes were analyzed. for phylogenetic analysis, top blast hits for each new viral contig were retrieved from the ncbi database ordered by score. pre-processing: reads derived from rna deep sequencing were submitted to quality checking and filtering using fastqc [ ] and fastx toolkit [ ] ; reads with an average phred quality below were discarded. to enrich the viral sequences, filtered reads were aligned against the a. albopictus genome, and bacterial genomes present on the refseq database using bowtie [ ] . only unaligned reads were used for the subsequent analysis. assembly: assemblies were performed using spades assembler [ ] with the following variations: ( ) standard: filtered reads were used as input and standard parameters kept; ( ) meta: filtered reads were analyzed as derived from the metagenomics sample, using the parameter "-meta"; and ( ) meta (trusted) + regular: viral contigs assembled in "meta" strategy were used as trustable contigs, the parameter "-trusted-contigs," together with filtered reads. all strategies were run with the parameter "-iontorrent." in strategies and , the parameter "-cov-cutoff" was set to "auto." in strategy , "-cov-cutoff" was set to "off." all contigs with significant hit with viral sequences were submitted to manual curation. manual curation of assembled genomes: assembled viral genomes were curated based on structure (presence of orfs, conserved domains, secondary structure) and rna coverage (evaluation of continuous coverage along segment). the viral genome sequences produced in this work were deposited on genbank under accession numbers mn -mn . characterization of viral contigs: assembled contigs were submitted to sequence similarity searches using blast software [ ] against non-redundant ncbi databases of nucleotide (nt) and aminoacids (nr), requiring minimum e-values of e − and e − , respectively. contigs that showed significant similarity to viral sequences were further analyzed. conserved domains were analyzed using the hmmer tool [ ] with the pfam database. orf prediction: the prediction of the open read frame of the assembled sequences was performed using the ncbi orf finder (https://www.ncbi.nlm.nih.gov/orffinder/) with "standard" genetic code defining minimum orf length to nt. rna coverage analysis: reads were re-aligned to assembled viral genomes with bowtie ; sam files were processed and quantified using samtools [ ] and bedtools [ ] , and for visual inspection of reads coverage we used igv [ ] . secondary structure analysis: analyses of the secondary structure of and utr of the zika virus genomes were performed using the rnafold webserver from the vienna platform [ ] with standard parameters. phylogenetic and genotypic analysis: phylogenetic analysis was performed using mega software [ ] . maximum likelihood phylogenetic trees were constructed based on the whole viral genome with the kimura -parameter method, or rdrp protein using the poisson substitution model for alphamesonivirus and totivirus-like virus, respectively. the trees were drawn with branch sizes measured in the number of substitutions per site. the percentage of trees in which the associated taxa clustered together in the bootstrap test ( replicates) is shown next to the branches. investigation of the zika virus genotype was performed using well defined phylogenetic clusters with information of geographic region integrated in the zika virus typing tool [ ] . rna coverage analysis: preprocessed reads were compared to each reference sequence using bowtie , allowing a maximum of two mismatches. bowtie 's sam output was used to compute per base coverage with the bedtools package. per base coverage was plotted using r language [ ] with the ggplot package [ ] . it is important to highlight that this viral genome extended in bp ( bp in ′-utr and bp in ′-utr including coding regions) is the closest viral sequence derived from the outbreak, the zika virus strain bahia , that shows ~ % of coverage with ~ % of identity (figure b) [ ] . since confident assembly of utr regions is challenging due to the lower number of reads in comparison to non-utr regions, we further manually cured these regions by investigating aligned reads using a genome browser. as expected, we noted that utr regions presented a considerably lower number of reads in comparison to non-utr regions, , compared to , , , respectively. however, although the number of reads was restricted, we observed concordantly alignments in both utr regions, and , for ′ and ′ extremities, respectively ( figure s ) . furthermore, analysis of the utr region sequences suggests that both of them form secondary structures, which has been shown previously to be essential to the virus driving viral fitness and pathogenesis ( figure ) [ ] . this demonstrates that our strategy based on ion torrent metatranscriptomics associated with de novo assembly is able to reconstitute whole viral genomes. indeed, performing a global overview of complete zika virus genomes, it is noticeable that our newly total rna from c / culture cells was extracted using qiamp ® rna minikit and submitted to one-step rt-pcr using the following primers (f: acgtgccagctgtgtctatg and r: acaccagccatcaaggacg) and the access rt-pcr system (promega, ma usa). cycling conditions were defined as follows: • c for min, • c for min, and cycles of • c for s, • c for s, and • c for min, the final extension step was at • c for min. the primers amplified a fragment of bp when visualized in % agarose gel, stained with ethidium bromide and detected by an ultraviolet-transilluminator system. rt-pcr product was cleaned up using the qiaquick pcr purification kit, according to the manufacturer's instructions (qiagen, germany). sequencing was performed by the genetic analyzer using the big dye terminator method, and it was carried out in ng of purified rt-pcr product and . pmol of the same primers used for the rt-pcr. rna deep sequencing produced , , reads. after preprocessing, , , were kept for posterior analysis. since the goal was to reconstitute the zika virus genome that only has a small fragment of the protein e available, we discarded all sequences derived from mosquito genome ( , , ) . the remaining reads ( , , ) were used to perform assembly using different strategies described in figure a , in the top panel. we first tried to use spades with the default parameters (highlighted in gold in figure a ) referred here as 'regular.' although we identified many contigs derived from the zika virus, we were not able to reconstitute the viral genome as a single segment. since our data are derived from deep sequencing of all rnas present in the cell, we performed the assembly using the "-meta," a variant of the assembler optimized for metagenomic data. using this strategy, we considerably improved the assembly of the zika virus genome, reducing the number of contigs from to , and increasing size average from . to nt (highlighted in dark blue in figure a ). although we assembled longer contigs, the genome was still fragmented. interestingly, we observed that fragmentation in the zika genome correlated with the positions that had high variability in the density of coverage (figure a-bottom panel) . this is consistent with the literature showing that differences in coverage of overlapping sequences can lead to the split of supposedly contiguous sequences into different contigs [ ] . this is a problem mainly when assembling viral genomes from rnas since viral genes can show different transcription profiles [ , ] . to overcome this limitation, we decided to use contigs derived from the "meta" strategy as trusted contigs trying to extend these contiguous sequences. this new strategy, which is hereafter referred to as "meta (trusted) + regular," resulted in the complete reconstitution of the zika virus genome containing , nt (figure a ,b-top panel). an overview of the final approach is shown in figure s and described in detail in the material and methods section. it is important to highlight that this viral genome extended in bp ( bp in -utr and bp in -utr including coding regions) is the closest viral sequence derived from the outbreak, the zika virus strain bahia , that shows~ % of coverage with~ % of identity (figure b) [ ] . since confident assembly of utr regions is challenging due to the lower number of reads in comparison to non-utr regions, we further manually cured these regions by investigating aligned reads using a genome browser. as expected, we noted that utr regions presented a considerably lower number of reads in comparison to non-utr regions, , compared to , , , respectively. however, although the number of reads was restricted, we observed concordantly alignments in both utr regions, and , for and extremities, respectively ( figure s ). furthermore, analysis of the utr region sequences suggests that both of them form secondary structures, which has been shown previously to be essential to the virus driving viral fitness and pathogenesis ( figure ) [ ] . this demonstrates that our strategy based on ion torrent metatranscriptomics associated with de novo assembly is able to reconstitute whole viral genomes. indeed, performing a global overview of complete zika virus genomes, it is noticeable that our newly assembled genome figures are among the top in length out of more than viral sequences available, which seems to be independent of collection year or country (figure c) . indeed, recent work has shown that strategies based on amplicon are still preferentially used to reconstruct zikv genomes, which create a bias to the coding region since amplicons are mostly designed to anchor at genic regions with lower mutation rates [ ] . viruses , , x for peer review of assembled genome figures are among the top in length out of more than viral sequences available, which seems to be independent of collection year or country (figure c) . indeed, recent work has shown that strategies based on amplicon are still preferentially used to reconstruct zikv genomes, which create a bias to the coding region since amplicons are mostly designed to anchor at genic regions with lower mutation rates [ ] . similarly, for the previous zika virus strains derived from the outbreak in , the viral strain assembled in this work clustered together with other strains with asian genotype (figure d ). comparative analysis among all complete sequences deposited at the vipbrc database revealed that differences among viral genomes are mainly in untranslated regions, which is consistent with the literature [ , , ] . curiously, untranslated regions have been linked to differences in virulence of different arboviruses [ , , ] . in addition, these regions are also suggested to be involved in other viral characteristics, such as host preference and tissue tropism [ , , ] . this result highlights the need for achieving complete viral genomes to better our understanding of virus-host interactions. similarly, for the previous zika virus strains derived from the outbreak in , the viral strain assembled in this work clustered together with other strains with asian genotype (figure d ). comparative analysis among all complete sequences deposited at the vipbrc database revealed that differences among viral genomes are mainly in untranslated regions, which is consistent with the literature [ , , ] . curiously, untranslated regions have been linked to differences in virulence of different arboviruses [ , , ] . in addition, these regions are also suggested to be involved in other viral characteristics, such as host preference and tissue tropism [ , , ] . this result highlights the need for achieving complete viral genomes to better our understanding of virus-host interactions. notably, besides the identification of contigs showing sequence similarity to the zika virus, with the new assembly strategy, we were also able to reconstruct two large contigs showing similarities to viral sequences. one contig of , nt showed a sequence similarity at the nucleotide level ( % identity with e-value = ) with viruses from alphamesonivirus genus while one contig with nt presented a similarity at the protein level (~ % identity with e-value = e- ) with viruses from the totiviridae family, which likely represents a new viral species. phylogenetic analysis based on the nucleotide sequence revealed that the alphamesonivirus identified in c / cell lines is a new strain of a previously identified alphamesonivirus from the mesoniviridae family, a group of single-stranded rna viruses that have been described in mosquitoes (figure a ) [ ] . this is the first report of this viral family in a. albopictus mosquitoes. it was named alphamesonivirus strain salvador. phylogenetic analysis based on rdrp protein of contig related to the totiviridae family suggested its relationship to a newly proposed family, totivirus-like, which is different from totiviruses that have fungi as natural hosts, and have been described in a vast range of organisms (figure b ) [ ] [ ] [ ] [ ] [ ] . this new virus was named aedes albopictus totivirus-like. for both of the two new viruses identified, we observed an orf structure consistent with their closely related viruses (figure c,d) . in addition, we observed a considerable number of reads aligned with the totivirus and alphamensonivirus assembled genomes, , and , respectively, representing . % and . % of the library. furthermore, rna density along viral genomes showed continuous coverage, suggesting that they were correctly assembled (figure c,d) . to confirm the detection of the newly identified totivirus-like sequence, experimental validation was performed by rt-pcr, and we were able to detect the viral genome in the same c / population we used to perform deep sequencing. we also retrieved a fragment of its genome through sanger sequencing in order to compare with the contiguous sequence obtained through metatranscriptomics ( figure s a,b) . we observed a high similarity between the fragment retrieved through sanger sequencing and the assembled using deep sequencing ( % identity), suggesting the high accuracy of our metatranscriptomics strategy to reconstitute viral genomes. assembled in this work clustered together with other strains with asian genotype (figure d ). comparative analysis among all complete sequences deposited at the vipbrc database revealed that differences among viral genomes are mainly in untranslated regions, which is consistent with the literature [ , , ] . curiously, untranslated regions have been linked to differences in virulence of different arboviruses [ , , ] . in addition, these regions are also suggested to be involved in other viral characteristics, such as host preference and tissue tropism [ , , ] . this result highlights the need for achieving complete viral genomes to better our understanding of virus-host interactions. altogether, our results indicate that our strategy based on rna deep sequencing using ion torrent technology allows the confident reconstitution of known and unknown viruses. using this strategy, we were able to reconstruct the complete genome of the outbreak-related zika virus genome, including and utr regions, that would not be possible using standard approaches. furthermore, we also assembled the full genomes of two unknown viruses co-infecting the stock, alphamesonivirus and the newly described totivirus-like, highlighting the potential of our strategy for monitoring viral contaminations. therefore, our work highlights the importance of the development of new unbiased metagenomic approaches for the identification of viral sequences. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : bioinformatics approach to reconstruct viral genomes based on ion torrent data; figure s : overview of reads aligned to utr regions of the zika virus genome assembled using igv genome browser; figure the global distribution of the arbovirus vectors aedes aegypti and ae. albopictus. elife global risk mapping for major diseases 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members of the genus flavivirus secondary structure of the -untranslated region of yellow fever virus: implications for virulence, attenuation and vaccine development virus pathogenesis and tissue tropism mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range a novel totivirus-like virus isolated from bat guano co-infection by two distinct totivirus-like double-stranded rna elements in chalara elegans (thielaviopsis basicola) virome of > thousand culex mosquitoes from throughout california southern tomato virus: the link between the families totiviridae and partitiviridae expanding our understanding of the seaweed holobiont: rna viruses of the red alga delisea pulchra this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank all the members of virology and allergy and acarology laboratories. the authors declare no conflict of interest. key: cord- -hzes mwt authors: mcguinness, sarah l.; wu, henry m. title: pretravel considerations for non-vaccine-preventable travel infections date: - - journal: travel medicine doi: . /b - - - - . - sha: doc_id: cord_uid: hzes mwt pretravel advice should be tailored to the individual following a thorough review of his or her itinerary, planned activities, and host characteristics. in addition to vaccinations and malaria chemoprophylaxis, a pretravel consultation should include advice on regionally endemic or emerging non–vaccine-preventable infections that can cause severe illness or chronic morbidity. these include mosquito-borne infections such as dengue, chikungunya, and zika, and regionally endemic severe respiratory infections such as middle east respiratory syndrome (mers) and some strains of avian influenza. zika virus is notable given its capacity for sexual transmission and association with congenital birth defects. preventive advice for other potentially relevant infections associated with specific exposures or activities (e.g., schistosomiasis and leptospirosis from freshwater exposure) should be provided where relevant. understanding the epidemiology and prevention of these infections is crucial to providing a comprehensive pretravel consultation. to provide optimal advice, travel health providers should be able to educate the traveler on preventive measures against key travel-related infections, including those for which no vaccine is available. tailoring this advice for an individual requires a thorough review of the travelers' itinerary and planned activities, consideration of the travelers' host characteristics, and a working knowledge of the epidemiology of relevant diseases. travelers play an important role in the global epidemiology of infectious diseases; therefore ensuring that travelers are aware of specific preventive measures not only protects the health of the individual but has the potential to protect the health of their communities. in this chapter, pretravel considerations for major non-vaccine-preventable infectious diseases are covered, including specific advice for dengue, chikungunya, zika, middle east respiratory syndrome coronavirus (mers-cov), and avian influenza. dengue virus (denv), chikungunya virus (chikv), and zika virus (zikv) are globally important mosquito-borne viruses spread via aedes aegypti and a. albopictus. the public health impact of these viruses has increased dramatically over the last years, with epidemics of increasing size, geographic reach, and severity recorded. with factors such as population growth, urbanization, globalization, travel, and climate change facilitating increased transmission, travel medicine practitioners in temperate countries are increasingly likely to see returned travelers with these infections. furthermore, since the ranges of aedes vectors extend into temperate areas, infected returned travelers can precipitate outbreaks of these viruses in nonendemic regions. aedes mosquitoes are typically daytime biters and have a preference for the morning and late afternoon hours (crepuscular periods). a. aegypti, the primary mosquito vector for dengue, chikungunya, and zika, is found in tropical, subtropical, and some temperate climates and has adapted to cohabit with humans in both urban and rural environments. a. aegypti typically lays eggs in manmade or artificial containers in or around the home and can bite indoors. a. albopictus (the asian tiger mosquito) can live in a broader temperature range and at cooler temperatures than a. aegypti and thus has a wider geographic distribution, extending into temperate regions. a. albopictus feeds on animals as well as humans, prefers natural habitats, usually bites outdoors, and is generally considered a less efficient vector of human disease than a. aegypti. aedes mosquitoes can be found in temperate areas, including southern europe (a. albopictus), northern queensland in australia (a. aegypti), and southeastern regions of the united states (both species) , ( fig. . ). denv is a flavivirus that is the most common and arguably most important arbovirus globally. originating in africa, denv is now endemic in more than countries across africa, southeast asia, the americas, the western pacific, and the eastern mediterranean regions. , estimates suggest million infections occur worldwide annually, with % of cases occurring in asia. denv has four distinct serotypes (denv - ), with most endemic countries reporting circulation of all four serotypes. primary infection provides lifelong serotype-specific protection but only short-lived cross-protection to other serotypes. , broadly neutralizing antibodies are produced following a second dengue infection, and symptomatic disease is rarely seen with subsequent infections. pretravel advice should be tailored to the individual following a thorough review of his or her itinerary, planned activities, and host characteristics. in addition to vaccinations and malaria chemoprophylaxis, a pretravel consultation should include advice on regionally endemic or emerging non-vaccine-preventable infections that can cause severe illness or chronic morbidity. these include mosquito-borne infections such as dengue, chikungunya, and zika, and regionally endemic severe respiratory infections such as middle east respiratory syndrome (mers) and some strains of avian influenza. zika virus is notable given its capacity for sexual transmission and association with congenital birth defects. preventive advice for other potentially relevant infections associated with specific exposures or activities (e.g., schistosomiasis and leptospirosis from freshwater exposure) should be provided where relevant. understanding the epidemiology and prevention of these infections is crucial to providing a comprehensive pretravel consultation. between nonhuman primates, small mammals, and aedes mosquitoes. however, in outbreaks chikv can spread without the need for animal reservoirs. among populations with no prior immunity, chikv outbreaks can be explosive, and attack rates as high as % have been documented. introduction of chikv into asia occurred during or before the s and led to outbreaks in india and southeast asia. reemergence of chikv from africa in resulted in major outbreaks involving millions of people across the islands of the indian ocean in (including the comoros islands, la reunion, and mauritius) and india in - . furthermore, introduction of chikv to temperate areas in this period resulted in autochthonous transmission in italy and france. , the first report of local transmission of chikv in the americas occurred in in saint martin, with subsequent spread to > countries and territories across north, central, and south america. the unprecedented magnitude of chikv outbreaks in recent years is probably attributable to several factors including increased urbanization, global travel, and a series of adaptive mutations in the virus which have resulted in enhanced transmission by a. albopictus. the incubation period of chikungunya is typically - days (range - days). most chikungunya infections are symptomatic, with more than % of people with serologic evidence of infection reporting a history of symptoms. chikungunya infection is characterized by sudden onset of fever and severe, potentially disabling arthralgia. notably, the name chikungunya is derived from a makonde word describing the bent posture that can be seen with severe arthralgia. the arthralgia/ arthritis is usually symmetric and affects multiple joints, with fingers, wrists, ankles, elbows, toes, and knees the most often affected. additional symptoms include headache, myalgia, conjunctivitis, and rash. the case fatality rate of chikungunya is < %, but disease can be associated with significant acute and long-term morbidity secondary to debilitating polyarthralgia/arthritis. although self-limiting in most individuals, some of those affected develop chronic joint pain that may last for months to years, with older people (> - years) more predisposed. , unlike dengue, in which nonsteroidal antiinflammatory drugs (nsaids) are contraindicated, antiinflammatory drugs are indicated for symptomatic management of chikungunya infections. zikv is a flavivirus that was first isolated in from a rhesus monkey in the zika forest of uganda, with the first human cases detected in uganda and tanzania in . , a following its discovery, the virus remained in relative obscurity for over years, with only cases reported until , when an explosive outbreak infected approximately three-quarters of the population of yap, federated states of micronesia. subsequent outbreaks occurred across the pacific islands from to ; spread of the virus to brazil in march preceded subsequent transmission throughout latin america, the caribbean, mexico, and florida and texas in the united states. , as of february , countries, territories, or subnational areas have reported evidence of vectorborne zikv transmission. unlike denv and chikv, there is now substantial evidence that direct person-to-person transmission of zikv is possible-both horizontally through sexual transmission, and vertically from the mother to the fetus during pregnancy. transmission through blood transfusion has been reported, as well as a single case report of transmission probably resulting from close contact with bodily fluids from an infected patient. the incubation period is thought to be similar to other mosquito-borne flaviviruses with an estimated range from - days. male-to-female, male-to-male, and femaleto-male transmission to unprotected sexual contacts of returning following a short incubation period, with symptoms typically beginning - days (range - days) after exposure, dengue can present with a wide spectrum of illnesses, from asymptomatic infection to severe and fatal disease. most infections are asymptomatic or subclinical; symptomatic infections occur in approximately one-third of cases. patients who recover after a self-limited febrile illness, typically characterized by fever, headache, retroorbital pain, arthralgia, and myalgia, are classified as having dengue. the small proportion who progress to capillary (plasma) leakage with or without bleeding, circulatory collapse, or severe end organ impairment are designated as having severe dengue. epidemiologic risk factors for severe dengue include young age, secondary infection with a different serotype, and infection with a more virulent strain of virus. , severe dengue occurs in approximately %- % of dengue cases, with case fatality rates ranging from < %- %; the greatest burden of severe dengue occurs in children and infants in endemic countries. dengue is a common diagnosis in travelers, accounting for > % of presentations to geosentinel surveillance clinics. most infections are acquired in asia, followed by the americas, with only a small proportion acquired in africa. the incidence of dengue infection in travelers ranges from . - infections per person months, and varies according to travel destination, duration, and season of travel. regionspecific peaks of travel-related dengue infections have been demonstrated for southeast asia (june and september), south central asia (october), and south america (march). viraemic travelers can introduce dengue into new areas, with autochthonous transmission documented in the continental united states, europe, and australia. , some travelers with dengue may require hospitalization or even evacuation. studies of dengue in travelers have reported a dengue hemorrhagic fever prevalence of . %- %, though this is likely an overestimate as patients experiencing more severe symptoms are more likely to seek medical attention. epidemiologic studies in endemic settings have shown that the risk of severe disease is significantly higher during a second denv infection than during a primary infection. however, a lack of consensus exists regarding risk factors for severe disease in travelers. results of one study in travelers suggest that severe dengue may occur at similar rates among cases with primary and secondary infections. given that most dengue infections are asymptomatic, and that severe dengue in travelers is rare, travelers with a history of dengue infection need not avoid known dengue areas but rather should be advised to use the personal protection strategies outlined in box . to prevent subsequent infection. chikv is a mosquito-borne alphavirus first isolated in tanzania in . in africa, chikv exists in an enzootic sylvatic transmission cycle • wear an insect repellent containing an active ingredient such as deet or picaridin, particularly during daylight hours when the mosquitoes are most active. • wear long-sleeved shirts and long pants to help protect yourself from bites. light-colored clothes are best. • treat clothes and shoes with an insecticide such as permethrin or purchase pretreated clothing. • use mosquito coils, plug-in mosquito repellent devices, or insecticide surface sprays inside your accommodation; or stay in screened or air-conditioned accommodation. testing may also be considered in asymptomatic potentially exposed pregnant women after considering risk of infection, patient preferences, and clinical judgment. nonpregnant individuals and couples traveling to zika-affected areas should also be counseled on measures to prevent sexual transmission and congenital infection. due to the risk of prolonged viral shedding in semen, public health authorities advise men with risk of zika exposure to wait months from the last possible exposure to zika (or after onset of symptoms following symptomatic infection) before attempting procreation. most authorities advise women to wait weeks from the last possible exposure to zika (or after onset of symptoms following symptomatic infection) before attempting to conceive ; one exception is the who, which advises women to wait months before attempting conception. because many pregnancies are unplanned, all sexually active travelers and female partners of travelers should also practice measures to prevent congenital zika infection. readers are encouraged to review the most updated recommendations for prevention of sexual transmission of zikv and congenital zika infection from public health authorities including who and the us centers for disease control and prevention (cdc) ( the severe acute respiratory syndrome (sars) outbreak highlighted the potential for travelers to introduce novel respiratory infections into their home countries. more recently, concern has focused most on the emergence of middle eastern respiratory syndrome (mers) and certain strains of avian influenza. travelers has been reported with the former felt to be most prominent. sexual transmission from patients with both symptomatic and asymptomatic disease has been described. currently it appears that zika can remain in semen longer than in other body fluids (including cervical mucus, vaginal fluids, urine, and blood). in semen, zikv rna has been detected as long as days after the onset of symptoms, and infectious virus has been cultured up to days after symptom onset. most zikv infections are asymptomatic, with serosurvey studies indicating that only % of those infected report clinical illness. in symptomatic cases, illness is generally mild and self-limiting with symptoms including fever, rash, pruritus, arthralgia, myalgia, conjunctivitis, and headache. zikv has been associated with neurologic complications including guillain-barré syndrome (gbs) and adverse fetal outcomes including congenital microcephaly. an association with gbs was first reported in - during the french polynesian outbreak. more than countries have now reported an increased incidence of gbs and/or laboratory confirmation of a zikv infection among gbs cases. in february , following reports from brazil of microcephaly in babies whose mothers had been exposed to zika during pregnancy, the world health organization (who) declared that zika constituted a public health emergency of international concern (pheic). microcephaly is one of several neurologic and musculoskeletal birth defects described in congenital infection; this constellation of findings is now known as congenital zika syndrome. although a live attenuated tetravalent dengue vaccine (cyd-tdv; dengvaxia) has been registered in several countries, and several other dengue vaccine candidates are in clinical development, at this time no dengue vaccine is licensed for travelers. likewise no vaccines are licensed for chikungunya or zika; therefore prevention of these viruses largely relies on personal protection strategies that limit contact between humans and aedes mosquitoes (see box . and chapter ; insect protection) as well as avoidance of travel during peak transmission or outbreak periods. travelers returning to nonendemic areas with aedes mosquitos (see fig. . ) should also be advised to avoid mosquito bites on their return to prevent local transmission. symptomatic travellers should seek medical evaluation immediately. infection with chikv is thought to result in lifelong protective immunity. duration of zikv immunity following infection is currently unknown. in contrast, due to the multiple serotypes, individuals can be infected with dengue up to four times, and travelers with a history of infection should be educated about the potential risks of subsequent infections. due to the high prevalence of asymptomatic infections and the risk of sexual and vertical transmission, zika-specific preventative advice is important for those traveling to zika affected areas. pregnant women who do not reside in zika transmission risk areas should be advised not to travel to areas with risk; if travel cannot be avoided, advice to prevent mosquito bites and sexual transmission should be given. measures to prevent sexual transmission include abstaining from sexual activity or use of condoms during sexual activity (including vaginal, anal, and oral sex, and sharing of sex toys) during the entire pregnancy. pregnant women possibly exposed to zikv due to travel or sexual contact should discuss the potential exposure with their although no vaccines are available at this time for prevention of mers and avian influenza (h n and h n ) in travelers, providers should routinely review the most recent epidemiology of severe respiratory infections reported by authorities such as the who and cdc (see table . ) and promote general hygiene and other preventative measures to travelers to these areas (box . ). while seasonal influenza vaccination does not protect against avian influenza or mers, vaccine-related prevention of seasonal influenza may reduce the chance of coinfections and overall risk of respiratory infections. travelers should also be advised to inform their health care providers of their travel history whenever seeking medical care for respiratory (and other illnesses) acquired during or soon after travel (see chapter ). travel medicine providers should also be familiar with risk areas and specific preventive advice for other non-vaccine-preventable infectious diseases with regional distributions. some of the more important of these infections are presented in table . , along with specific preventive advice, such as insect bite avoidance (e.g., for prevention of african trypanosomiasis) or freshwater contact avoidance (e.g., for prevention of schistosomiasis or leptospirosis). mers is a respiratory infection caused by mers coronavirus (mers-cov). first described in , mers is an endemic infection in the arabian peninsula with epidemic potential in health care and travel settings. following an incubation period of - days, initial symptoms of mers are similar to many common viral respiratory infections and include fever, rhinorrhea, sore throat, and muscle aches. rapid progression to acute respiratory distress syndrome may follow, but mild and asymptomatic infections have also been described. , nausea, vomiting, or diarrhea, and acute kidney injury can also occur. risk factors for severe mers include age > years and comorbid conditions such as hypertension, diabetes, heart disease, end stage renal disease, chronic lung disease, cancer, or those receiving immunosuppressive therapy. among confirmed cases reported to who up to july , % have been fatal. a seroepidemiologic studies indicate that mers-cov circulates in dromedary camels in the middle east and africa, and direct contact with camels has been described in % of primary cases without known exposure to mers cases or health care settings. the route of transmission from dromedaries to humans is unclear, but contact with infectious bodily secretions and fluids are suspect, and consumption of raw dromedary products has also raised some concern. person-to-person transmission is primarily described in health care settings, although transmission among household close contacts has been described. all cases of mers reported outside of the arabian peninsula have occurred in returned travelers, or as a result of secondary transmission from a patient with recent travel to the arabian peninsula, as was the case for the health care-associated outbreak in south korea that resulted in cases and deaths. , , in this outbreak, delayed mers diagnoses and inadequate infection control precautions led to multiple generations of infections affecting other patients, visitors, and health care workers. , the risk of traveler-initiated health care-associated outbreaks with mers mirrors the global experience of sars. like mers, the viral agent of sars is a coronavirus that emerged in southern china in and subsequently caused over infections and deaths in more than countries. , the reservoir of sars coronavirus (sars-cov) is unknown, but some cases appear to have resulted from contact with animals used for human consumption such as civet cats. although no cases of sars have been reported since , the potential for reemergence is possible. while seasonal influenza is a common infection among travelers, other influenza types including certain strains of avian influenza can also pose a risk to travelers. although avian influenza typically affects birds, human cases and outbreaks occur sporadically. avian influenza strains associated with severe respiratory infections with high mortality rates in humans include the highly pathogenic avian influenza a h n , and more recently, novel avian influenza a h n . although the majority of human infections caused by avian influenza are linked to direct contact with infected birds (primarily poultry), unsustained person-to-person transmission has been reported for h n and h n . , furthermore cocirculation of different influenza a viruses in humans and animals raises the concern of reassortment leading to new strains that spread more readily from person to person. h n , first described in southern china in , has since been documented in over countries in asia and africa. long-term sequelae of chikungunya virus disease: a systematic review zika: the origin and spread of a mosquito-borne virus zika virus: history of a newly emerging arbovirus zika virus outbreak on yap island, federated states of micronesia zika virus epidemiology, prevention, and potential future treatments of sexually transmitted zika virus infection zika virus classification tables fatal zika virus infection with secondary nonsexual transmission estimated incubation period for zika virus disease persistence of zika virus in body fluids-preliminary report congenital zika virus infection beyond neonatal microcephaly update: interim guidance for health care providers caring for pregnant women with possible zika virus exposure-united states (including us territories) world health organization. prevention of sexual transmission of zika virus-interim guidance update isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome asymptomatic mers-cov infection in humans possibly linked to infected dromedaries references the efficacy of repellents against aedes, anopheles, culex and ixodes spp.-a literature review the dengue vector aedes aegypti: what comes next the global distribution of the arbovirus vectors aedes aegypti and ae. albopictus the global distribution and burden of dengue global spread of dengue virus types: mapping the year history dengue vaccine: who position paper a travel medicine view of dengue and dengue hemorrhagic fever geographic expansion of dengue: the impact of international travel dengue fever and international travel geosentinel surveillance network. geosentinel surveillance of illness in returned travelers geosentinel surveillance network. seasonality, annual trends, and characteristics of dengue among ill returned travelers chikungunya: a re-emerging virus epidemiology of chikungunya in the americas chikungunya virus infections infection with chikungunya virus in italy: an outbreak in a temperate region arrival of chikungunya virus in the new world: prospects for spread and impact on public health increase in human infections with avian influenza a(h n ) virus during the fifth epidemic-china avian influenza a (h n ) infection with respiratory failure and meningoencephalitis in a canadian traveller avian influenza a(h n ) virus infection in travelers returning from china to canada human african trypanosomiasis leishmaniasis in travelers: a literature review histoplasmosis infections worldwide: thinking outside of the ohio river valley leptospirosis: an emerging disease in travelers rickettsial infections in the tropics and in the traveler myiasis in travelers schistosomiasis in travelers and migrants multicenter geosentinel analysis of rickettsial diseases in international travelers mers-cov global summary and assessment of risk risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia transmission of mers-coronavirus in household contacts the clinical and virological features of the first imported case causing mers-cov outbreak in south korea preliminary epidemiological assessment of mers-cov outbreak in south korea ii, transmission characteristics of mers and sars in the healthcare setting: a comparative study who guidelines for the global surveillance of severe acute respiratory syndrome (sars) lessons from the severe acute respiratory syndrome outbreak in hong kong global epidemiology of avian influenza a h n virus infection in humans, - : a systematic review of individual case data epidemiology of avian influenza a h n virus in human beings across five epidemics in mainland china key: cord- -r mskri authors: magnani, diogo m.; silveira, cassia g. t.; rosen, brandon c.; ricciardi, michael j.; pedreño-lopez, núria; gutman, martin j.; bailey, varian k.; maxwell, helen s.; domingues, aline; gonzalez-nieto, lucas; avelino-silva, vivian i.; trindade, mateus; nogueira, juliana; oliveira, consuelo s.; maestri, alvino; felix, alvina clara; levi, josé eduardo; nogueira, mauricio l.; martins, mauricio a.; martinez-navio, josé m.; fuchs, sebastian p.; whitehead, stephen s.; burton, dennis r.; desrosiers, ronald c.; kallas, esper g.; watkins, david i. title: a human inferred germline antibody binds to an immunodominant epitope and neutralizes zika virus date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: r mskri the isolation of neutralizing monoclonal antibodies (nmabs) against the zika virus (zikv) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. here we describe the characterization of human mabs from the plasmablasts of an acutely infected patient. one of the mabs had the unusual feature of binding to and neutralizing zikv despite not appearing to have been diversified by affinity maturation. this mab neutralized zikv (neut( ) ~ μg/ml) but did not react with any of the four dengue virus serotypes. except for the expected junctional diversity created by the joining of the v-(d)-j genes, there was no deviation from immunoglobulin germline genes. this is a rare example of a human mab with neutralizing activity in the absence of detectable somatic hypermutation. importantly, binding of this mab to zikv was specifically inhibited by human plasma from zikv-exposed individuals, suggesting that it may be of value in a diagnostic setting. zika virus (zikv) belongs to the genus flavivirus of the flaviviridae family and is related to dengue virus (denv), yellow fever virus (yfv), japanese encephalitis virus (jev), and west nile virus (wnv) [ ] . the globally distributed mosquito species of the aedes genus, vectors for many flavivirus, can also transmit zikv [ , ] . however, zikv remained a relatively minor and obscure cause of human disease for most of the second half of the th century and was featured in a limited number of scientific reports. in fact, it was not until that autochthonous human infection was described outside africa and continental asia-in the federated states of micronesia [ ] [ ] [ ] . since then, reports from brazil have chronicled a rapidly spreading epidemic that co-exists with denv and chikungunya virus (chikv). the epidemic has spread north with mosquito-borne transmission being reported in many nations of the americas as far north as mexico and southern florida [ ] [ ] [ ] . more ominously, zikv has been implicated as the causative agent in fetal developmental problems [ , ] . there are reports of zikv-associated birth defects, primarily brain abnormalities and microcephaly in infants born to mothers infected with zikv [ ] . virus has been recovered from amniotic fluid, placental, and brain tissues [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . zikv infection has been classified as an ongoing threat by the world health organization. in the united states, the centers for disease control and prevention has issued guidance for the management of the infection in the general population, pregnant women, and infants [ ] [ ] [ ] . due to recent reports of sexually transmitted zikv infection, the cdc has also developed guidelines for prevention of this mode of transmission [ ] [ ] [ ] [ ] [ ] [ ] . more recently, zikv transmission has also been described in miami, florida [ ] , suggesting that autochthonous spread could occur in any region of the u.s. inhabited by aedes spp. treatment of a variety of human ailments using mabs is revolutionizing our ability to ameliorate human suffering. for infectious disease, the ebola epidemic highlighted the potential utility of a cocktail of three neutralizing (n)mabs that block infection by the ebola virus [ ] . most convincingly, the administration of a single nmab up to five days post infectious virus exposure prevents the development of disease in ebola-infected macaques [ ] . because mabs can be engineered to prevent antibody-dependent enhancement by incorporating the l a and l a (lala) mutations which reduce fcγr binding [ ] , they are a promising intervention in flaviviral therapies. our long-term goal is to use a cocktail of lala-mutated nmabs to prevent zikv infection in at-risk individuals, primarily pregnant women. therapeutic nmabs must be potent in order to be clinically viable, and most nmab isolation strategies are based on the identification of high-titer, antigen-selected repertoires. somatic hypermutation (shm) in germinal center (gc) b cells provides the basis for selection of b cells producing abs with increased affinity-a hallmark of the adaptive humoral response. this feature is conserved among mammals, highlighting the importance of ab affinity enhancement for evolutionary fitness [ ] . thus, it is unsurprising that the vast majority of human abs in the memory immunoglobulin (ig)g pool have undergone affinity maturation and have, on average, - nucleotide substitutions from precursor genes [ ] . the contribution of shm to ab-mediated viral neutralization is particularly clear for the chronicallyinduced broadly neutralizing antibodies to hiv [ ] [ ] [ ] [ ] . reversion of these anti-hiv nmabs to precursor germline antibodies results in a drastic reduction or complete loss of viral neutralization [ ] [ ] [ ] [ ] . although mutated mabs are found after secondary denv infection, the role of these mutations in acute virus-neutralization and clearance is less clear [ ] [ ] [ ] [ ] . still, the prevalent thought is that antiviral ab response involves the engagement of poor-or non-neutralizing germline clones generated by v(d)j rearrangement, followed by shm-mediated refinement in germinal centers to enhance neutralization potency. here we describe the isolation of plasmablast-derived human mabs, sorted days post onset of symptoms from a zikv-patient in são paulo, brazil. the patient reported a previous history of dengue infection and yellow fever vaccination (table ) . a few of the isolated abs neutralized zikv, most of them at relatively high concentrations. interestingly, one of these mabs (p f ) exhibited no nucleotide mutations when compared to its corresponding germline sequences, but still recognized a zikv immunodominant epitope and neutralized the virus. these results suggest unforeseen roles for gc-independent responses against zikv and possibly other viruses. blood samples were collected from volunteer , a -year-old woman who reported a pruriginous skin rash that started six days prior to the beginning of acute neurological deficits suggestive of gbs. zikv infection was confirmed by a positive real-time reverse-transcriptase pcr assay for zikv rna in urine samples collected at days and after the onset of the first rash symptoms. blood and cerebrospinal fluid were negative for zikv rna. previous history of a single dengue infection and yellow fever immunization were also reported. peripheral blood mononuclear cells (pbmcs) were obtained from blood samples collected days post onset of symptoms. blood samples from patient were obtained after signing a written consent form approved by the university of são paulo's institutional review board (cappesq / ). anonymized plasma samples from volunteers in brazil and us were obtained from naïve and convalescent subjects with rt-pcr-confirmed zikv or denv infection (s table) . four volunteers donated samples post yellow fever vaccination. we conducted reverse transcription followed by a nested pcr to amplify the variable region of the immunoglobulin (ig) chains using described protocols with minor modifications [ ] . briefly, cdna was synthesized in a μl reaction using the original sort plates. each reaction contained μl of ng random hexamer (idt), μl of mm dntp (life technologies), μl of superscript iii reverse transcriptase (life technologies), μl molecular biology grade water, and μl of single sorted cell sample in lysis buffer (described above). the reverse transcription reaction was performed at ˚c for min, ˚c for min, ˚c for min, c for min. after the reaction was completed, cdna was stored at - ˚c. heavy and light chains were amplified in three different nested pcr reactions, using a mix of ' v-specific primers with matching ' primers to the constant regions of igg, igl, and igk. pcr reactions were conducted using hotstartaq plus dna polymerase (qiagen). the second set of pcr reactions was carried out with primers redesigned to incorporate restriction sites compatible with subcloning into rhesus igg expression vectors, instead of the original human vectors [ ] . we sequenced the amplified and cloned products using primers complementary to the ig constant regions. sequences were analyzed using igblast and imgt/v-quest to identify v (d) j gene rearrangements, as well as shm levels [ , ] . we expressed mabs in expi f (thermofisher) human cell lines. the plasmids encoding heavy and light chains were co-transfected using the expifectamine transfection kit (a , thermofisher). after - days, we harvested the secreted mab in the supernatant. ig concentration in the supernatant was determined by an anti-rhesus igg elisa, before we proceeded with the functional assays. for the experiments with purified mabs, we used protein a plus (pierce)-containing columns to remove the impurities. the concentration of purified protein was determined by measurement of absorbance at nm (nanodrop, thermo scientific). virus capture assay and recombinant e protein elisa p f binding was determined by both virus capture assay (vca) and recombinant (r)e elisas. the vca plates were coated overnight with the mouse-anti-flavivirus monoclonal antibody g (clone d - g - - , emd millipore) followed by incubation with viral stocks (zikv or denv). the re elisa plates were coated with zikv e protein (mybiosource, mbs ) diluted to μg / ml in pbs. after the coating step, the plates were washed with pbs and mab samples diluted to μg / ml were added to designated wells and incubated for h at ˚c. subsequently, the plates were washed and detection was carried out using a goat anti-human igg hrp secondary ab (southern biotech), which was added to all wells at a dilution of : , . following a h incubation at c, the plates were washed and developed with tmb substrate at room temperature for - min. the plates were developed with tmb substrate at room temperature for - min. the reaction was stopped with tmb solution and absorbance was read at nm. the neutralizing potency of the mabs was measured using a flow cytometry-based assay [ , ] . in brief, recombinant mabs (transfection supernatant or purified) were diluted and preincubated with zikv (paraiba) or the reference denv serotypes in a final volume of μl for h at ˚c. the virus and mab mixture ( μl) was added onto wells of a -well plate of % confluent vero cell monolayers in duplicate. a new seed of vero cells (ccl- tm) was obtained from the american type culture collection (atcc) repository for this study. the inoculum was incubated in a ˚c incubator at % co for one hour with agitation of the plates every min. after one hour, the virus and mab-containing supernatants were aspirated and the wells were washed with media. fresh media was then added and the plates were incubated for a total of hours. cells were trypsinized with . % trypsin (life technologies), fixed (bd cytofix), and permeabilized (bd cytoperm). viral infection was detected with the g antibody (millipore) recognizing zikv or denv, followed by staining with an anti-mouse igg a apc fluorophore-conjugated secondary reagent (biolegend). the concentration to achieve half-maximal neutralization (neut ) was calculated using a nonlinear regression analysis with prnts were conducted as previously described [ ] . briefly, purified p f was serially diluted in optimem supplemented with % human serum albumin (vwr), % fetal bovine serum, and gentamicin. zikv paraiba was diluted to a final concentration of~ - pfu / ml in the same diluent added to equal volumes of the diluted ab. the virus/mab mixture was incubated at ˚c for min. cell culture medium was removed from % confluent monolayer cultures of vero cells on -well plates and μl of the virus/ab mixture was transferred onto duplicate cell monolayers. cell monolayers were incubated for min at ˚c and overlaid with % methylcellulose in optimem supplemented with % fbs mm glutamine + μg / ml gentamicin. samples were incubated at ˚c for four days after which plaques were visualized by immunoperoxidase staining, and a % plaque-reduction neutralization titer was calculated. inhibition of p f mab binding was determined by elisa. to begin, the elisa plate was coated with mouse anti-flavivirus monoclonal antibody g (emd millipore) diluted : , in carbonate binding buffer and incubated overnight at ˚c. the next day, the plate was washed five times with pbs-tween and wells were blocked with % skim milk in pbs for h at ˚c. after the block step, the plate was washed and virus samples were added to designated wells for h incubation at room temperature. subsequently, the plate was washed with pbs only and corresponding blocking plasma samples were added for h at ˚c. following the plasma block, the plate was washed and p f was added to corresponding wells for h at c. p f was detected using a rhesus igg-specific antibody (mouse anti-monkey igg-hrp clone sb a; southern biotech). thereafter, the plate was washed and wells were developed with tmb substrate at room temperature for - min before the reaction was stopped with tmb stop solution. absorbance was determined at nm. we isolated plasmablasts from patient who presented with suspected guillain-barré syndrome (gbs) ( table ) (first day of symptoms = d ). the patient had a previous history of dengue infection and yellow fever vaccination ( table ). the previously healthy -year-old woman presented to the emergency room (d ) reporting a progressive paresthesia mainly in the extremities of her hands, along with acute, intermittent pain in her left forearm during the previous four days. at physical examination, the patient presented with a grade iv asymmetric muscular weakness and hypoesthesia in her left limbs, with abolished deep tendon reflexes in the lower limbs. a mild weakness of her left facial muscles was also noted. the patient reported no respiratory disorders and no hoarseness, and no signs of dysautonomia were detected at the clinical evaluation. fever, conjunctivitis, and myalgia or joint pain were absent during the illness. afterwards, the patient was hospitalized with a clinical diagnosis of gbs, for which an intravenous human ig (ivig) treatment was initiated at a dosage of . g / kg / day for days. cerebrospinal fluid analysis and an electroneuromyogram were performed on fourth (d ) and fifth (d ) days after neurological symptom onset, respectively; the results were within normal limits. the electroneuromyogram was repeated on the th day of neurological symptoms, but no significant abnormalities were noted despite the persisting weakness in the patient's left leg and arm. during the treatment with ivig, the patient presented with transient worsening of her hemiparesis, but progressively recovered over the course of weeks after discharge from the hospital. at days post-neurological symptom onset (d ), a physical exam revealed significant improvement of muscular strength and abolished deep tendon reflexes in the lower limbs. the remittent skin rash cleared completely days after its initial emergence. blood, cerebrospinal fluid and urine samples were collected on the th day of neurological symptoms (d ) for detection of zikv by rt-pcr. the urine sample was zikv-positive by pcr, while blood and cerebrospinal fluid were negative. a saliva sample collected on d was negative for zikv. we isolated plasmablasts from peripheral blood mononuclear cells (pbmcs) collected on day (table ) . from wells containing single-sorted cells, we amplified, cloned, and sequenced heavy and light ab chains using ' primers complementary to the v gene segments and a ' primer annealing to the constant igg region [ ] . this resulted in paired heavy and light chains (s table) . eight of the mabs bound to zikv (fig ) . seven of these mabs exhibited cross-reactivity to one or more of the denv serotypes, and a single mab-p f -bound exclusively to zikv. interestingly, two mabs bound to denv but not zikv. we tested the neutralization potency of the zikv-specific p f mab in a flow-based neutralization assay and a plaque reduction neutralization test (prnt) and found that it neutralized zikv at approximately μg / ml (prnt ) (fig ) . analysis of the isolated antibody variable (v) domain sequences revealed five mabs with average gene mutation levels ( - nucleotide modifications), two mabs with over nucleotide substitutions, and mabs with unusually low levels of shm for isotype-switched mabs (lower than changes) (s table) . the most highly mutated mabs (p b and p c ) were not zikv-specific by binding (s table) . in fact, the eight zikv-binding mabs had the lowest shm levels, including four mabs lacking clearly recognizable mutations when compared with putative heavy and light chain germline precursors (s table, fig ) . except for junctional diversity, the zikv-neutralizer p f mab heavy chain did not exhibit signs of antigenselected ig diversification. p f had an identical sequence to the ig heavy chain variable (ighv) genes segment ighv - à up to the amino acid c , prior to the cdr-h (international immunogenetics information system [imgt]) [ ] . however, position g -the site of the junction between ighv and the igh diversity (ighd) genes-differed from the germline reference. interestingly, this region is part of a segment (n ) with non-germline nucleotides corresponding to six amino acids identified between the ighv and ighd genes (fig c) . this segment is likely the result of n nucleotide additions generated during b cell ig gene rearrangement, prior to antigen selection. because of the lack of mutations elsewhere in the sequences, it is likely that the r g substitution was also generated during this developmental step. the downstream sequence corresponding to the junction between ighd - à and the igh joining (ighj) ighj à genes also revealed similar nucleotide insertions. likewise, the kappa (k) chain junction between the igkv - à and igkj à genes also contained one insertion. although we cannot rule out the possibility of shm-mediated nucleotide changes in the n insertion regions, no mutation was identified in the remainder of the regions of the heavy and light chains. thus, the p f mab is likely very close or identical to the original v-(d)-j gene rearrangement in the naïve b cell before antigen contact. to investigate whether p f recognizes an immunodominant zikv epitope, we used a serological blocking assay. in brief, this assay detects the presence of competing abs that can inhibit the p f mab binding to its epitope. because p f did not bind to recombinant e protein (fig ) we used whole virus in our binding assays. we captured zikv on the plate using the g mab (pan-flavivirus), and incubated zikv with plasma from patients with diverse histories of denv and zikv exposure (s table) . we added unlabeled p f (engineered with rhesus igg constant regions) and detected binding of the mab using a hrplabeled mouse anti-rhesus mab (fig ) . nine of ten plasma samples from individuals that had been infected with zikv blocked the binding of p f in a blinded test (fig , s table) . similar blocking activity was observed regardless of whether individuals had been previously infected with denv or had been vaccinated for yellow fever. in contrast, little or no blocking activity was observed by denv+ plasma in the absence of prior zikv exposure (fig ) . furthermore, this recognition was specific in that it was not observed in of denv-only infected individuals. thus, the p f serological blocking assay accurately predicted previous zikv exposure, as confirmed by rt-pcr, in all but one of the patient plasma samples tested. although this patient, donor , had a positive urine rt-pcr result for zikv, plasma from did not block p f binding to zikv (s table) . interestingly, the plasma did not exhibit detectable zikv-neutralizing activity, suggesting that this patient did not mount a measurable antibody response against zikv. in conclusion, only the plasma that inhibited zikv infection of vero cells contained p f -blocking antibodies. here we show that a igg mab with no detectable shm was generated against zikv early in infection. remarkably, despite being germline-encoded, this mab is zikv-specific and does not bind to any of the four denv serotypes. furthermore, this mab not only neutralizes zikv, but it also binds to an immunodominant epitope on the virus. remarkably, despite being germline-encoded, p f binds specifically to zikv and does not cross-react with any of the four denv serotypes. our results also suggest that p f recognizes a unique epitope on zikv. it is unclear how this ab developed such specificity without shm. finally, these findings suggest that affinity maturation is not necessary for the generation of isotype switched virus-neutralizing abs. low levels of shm in abs possessing neutralizing activity have been previously reported in mice and humans [ ] [ ] [ ] , supporting the idea that germline-encoded mabs can indeed neutralize. abs with low levels of shm have also been reported during the acute phase of human denv infection, but it was not clear that these abs contributed to the antiviral neutralization activity [ ] . in studies in mice, vsv-specific mabs lacking shm have been isolated previously [ ] . interestingly, secondary, but not primary, mouse abs against vsv had mutations [ ] . furthermore, the reversion of these mutated abs to non-mutated precursors reduced, but did not abrogate, vsv binding and neutralizing activity. the binding differences between the mutated and germline abs were much less pronounced than might be expected [ ] . additionally, mice that cannot conduct shm due to aid knockout still mounted neutralizing ab responses against friend virus, a strain of murine leukemia virus [ ] . it has been suggested that these abs lacking extensive shm undergo a gc-independent developmental pathway [ ] , although the mechanistic basis for this phenomenon remains to be elucidated. rapid, gc-independent responses might be particularly relevant in the control of acute cytopathic viruses [ , , ] . the gc-independent abs would arise quickly after infection and then curtail viral replication, preventing virus-mediated damage [ ] . even more provocatively, hangartner et al. have argued that cytopathic viruses specifically evolved to retain binding to these germline sequences to decrease host lethality and increase fitness. on the other hand, chronic viruses may have evolved to avoid germline-binding and development of neutralizing responses to persist [ ] . so far, these hypotheses remain unsubstantiated by the lack of evidence for strictly germline neutralizing ab responses in humans. while our experiments were not specifically designed to detect gc-independent responses, it seems likely that the isotype-switched p f originated directly from a germline precursor. we isolated p f from a zikv-infected individual that developed neurological complications compatible with gbs and was treated with ivig. underlying factors that influence the potential association of gbs and zikv infection might involve an autoimmune process, which could influence the development of immune responses [ ] . additionally, ivig may have had a role in the selection of the ab responses mounted by peripheral b cell repertoires [ ] . this is unlikely, however, since the patient initiated ivig treatment on the same day that the plasmablasts were isolated. it is possible, then, that gbs or ivig-treatment influenced the development of p f . these potential associations are difficult to determine and were outside the scope of this study. it is clear, however, that these responses were not exclusive to volunteer , as p f binding can be blocked by the serum of most zikv-infected individuals (fig ) . recently [ ] . these mabs were isolated from memory cells sorted with soluble and monomeric zikv e proteins and, in contrast to p f , bind to the recombinant protein [ ] . in contrast, we isolated zikv-specific mabs from circulating plasmablasts at d . the peak recall of memory b-cell derived plasmablasts is thought to occur within the first week post-secondary infection [ , ] . thus, it is probable that most of the isolated mabs did not have a memory-b cell origin, and it remains possible that some of the plasmablasts were sorted from the basal population that circulate in low frequencies in the blood. in conclusion, the isolation of mabs using different b cell methods suggest that anti-zikv mabs with germline characteristics are not limited to specific b cell subtypes [ , ] . notably, the anti-zikv mabs isolated to date are less mutated than the mabs isolated after related denv infections [ ] [ ] [ ] [ ] . together, these findings suggest possible differences in the development of ab responses against zikv. unfortunately, despite our efforts, we were unable to map p f 's binding site. we first employed an in vitro escape assay [ ] , which did not result in a single mutated consensus sequence. also, p f did not bind to the prm/e proteins expressed in cells, precluding our ability to map this interaction using an ala-mutated envelope panel [ , ] . characterizing this interaction will, thus, require a significant effort that is beyond the scope of the current manuscript. because the p f mab retains the ability to bind virions, our conclusion is that it binds to a conformational epitope. based on the cohort of human plasma samples tested in this study, it appears that most zikv-infected individuals mount ab responses against the epitope recognized by p f . this epitope is recognized by abs in individuals previously infected by zikv, thereby preventing the binding of p f . by contrast, abs in the plasma from individuals previously infected by any of the denv serotypes, do not prevent binding of p f . p f may, therefore have potential as a diagnostic. several diagnostic options for testing for zikv exposure exist, including rt-pcr, igm elisa, and prnt methods [ , ] . while it is relatively straightforward to detect zikv nucleic acid during the acute phase in blood, urine, saliva, and semen, it has proven more difficult to design rapid and effective diagnostics for zikv exposure in the chronic phase. for samples collected after the first week of symptoms, the initial test is an anti-zikv, anti-denv, anti-chikv virus igm elisa [ ] . however, in patients who have received a flaviviral vaccine (denv, yfv, or jev) and/or have been infected with any flaviviruses in the past, these assays may be difficult to interpret due to the cross-reactivity of the abs [ ] [ ] [ ] [ ] [ ] [ ] . thus, a positive igm test needs to be confirmed with a laborious prnt assay. igm antibodies persist for - weeks in serum, and sera from individuals previously infected for more than weeks would also have to be confirmed with a virus neutralization-based method [ ] . our plasma inhibition assay may, perhaps, provide an alternative to these other techniques. in this study, we isolated plasmablast-derived abs from a zikv-infected individual with unusual characteristics. the human igg p f has no or limited shm yet binds to an immunodominant zikv epitope that is not present on any of the four denv serotypes. furthermore, this mab can neutralize the virus with a neut of approximately μg / ml. our results suggest that shm-independent pathways may generate neutralizing abs in the responses against zikv. molecular evolution of zika virus during its emergence in the (th) century a simple technique for infection of mosquitoes with viruses; transmission of zika virus twelve 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of autoimmune diseases rapid and massive virus-specific plasmablast responses during acute dengue virus infection in humans origin and function of circulating plasmablasts during acute viral infections structure and function analysis of therapeutic monoclonal antibodies against dengue virus type a high-throughput shotgun mutagenesis approach to mapping b-cell antibody epitopes neutralizing human antibodies prevent zika virus replication and fetal disease in mice interim guidance for interpretation of zika virus antibody test results fields virology. th ed. philadelphia: wolters kluwer health/lippincott williams & wilkins structural basis of potent zika-dengue virus antibody cross-neutralization cross-reactivity and function of antibodies elicited by zika virus infection high specificity of a novel zika virus elisa in european patients after exposure to different flaviviruses diagnostics for zika virus on the horizon we thank the patients for their volunteer donations. we also thank the laboratory and clinical personnel responsible for collecting the plasma samples used in this study. conceptualization: dmm diw egk. key: cord- - xk authors: mutso, margit; st john, james a.; ling, zheng lung; burt, felicity j.; poo, yee suan; liu, xiang; Žusinaite, eva; grau, georges e.; hueston, linda; merits, andres; king, nicholas j.c.; ekberg, jenny a.k.; mahalingam, suresh title: basic insights into zika virus infection of neuroglial and brain endothelial cells date: - - journal: j gen virol doi: . /jgv. . sha: doc_id: cord_uid: xk zika virus (zikv) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and guillain–barré syndrome. the pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. cellular responses to infection are also poorly understood. this study characterizes the in vitro infection of laboratory-adapted zikv african mr and two asian strains of ( ) brain endothelial cells (hcmec/d cell line) and ( ) olfactory ensheathing cells (oecs) (the neuroglia populating cranial nerve i and the olfactory bulb; both human and mouse oec lines) in comparison to kidney epithelial cells (vero cells, in which zikv infection is well characterized). readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. although not as high as in vero cells, viral titres exceeded ( ) plaque-forming units (p.f.u.) ml(− ) in the endothelial/neuroglial cell types, except hoecs. despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. immunolabelling of infected cells revealed localization of the zikv envelope and ns proteins in the cytoplasm; ns staining overlapped with that of dsrna replication intermediate and the endoplasmic reticulum (er). infected oecs and endothelial cells produced high levels of pro-inflammatory chemokines. nevertheless, zikv was also able to establish persistent infection in hoec and hcmec/d cells. taken together, these results provide basic insights into zikv infection of endothelial and neuroglial cells and will form the basis for further study of zikv disease mechanisms. zika virus (zikv) is a recently emerged mosquito-borne virus belonging to the genus flavivirus, family flaviviridae. the virus was initially isolated from a rhesus monkey in the zika forest near entebbe, uganda, during yellow fever investigations in and was subsequently isolated from wild-caught aedes spp. mosquitoes collected in uganda in [ ] . the recent emergence of this virus as a cause of larger outbreaks of disease was first reported in when an outbreak of zikv was identified on yap island, federated states of micronesia, in the southwestern pacific ocean [ ] . three-quarters of the population of yap island were estimated to be infected during the outbreak, with the majority of the patients presenting with mild disease [ ] . in october the virus was identified as the cause of an outbreak of dengue-like illness in french polynesia, located in the south pacific [ , ] . thousands of suspected cases of zikv infection were reported during the outbreak, with most patients presenting with mild disease, fever, arthralgia, maculopapular rash and conjunctivitis. during these outbreaks, an increase of neurological complications in the form of guillain-barré syndrome (gbs) in zikvinfected patients and microcephaly associated with zikv infection during pregnancy were noted [ , ] . the pathogenesis of disease caused by zikv, including the mechanisms of neuroinvasion and host cell responses to infection, are currently not clearly delineated. the pathway by which zikv gains access to the central nervous system (cns) is unknown. the mechanism of neuroinvasion may involve multiple routes, as is seen with other neurotropic flaviviruses, such as west nile virus, for which hypotheses of both haematogenous and transneural entry have been proposed [ , ] . olfactory ensheathing cells (oecs), the glial cells of the primary olfactory nervous system, are found in the olfactory nerve and bulb, and have crucial roles in the regeneration of olfactory axons, which occurs throughout life. transneuronal transmission of neurotropic virus such as rabies virus [ ] has been shown to involve the olfactory system, but it remains unknown whether other neurotropic viruses such as zikv can enter the cns via this path. further, whilst it is known that oecs are highly phagocytic cells that can phagocytose microorganisms and be pathogen hosts [ ] [ ] [ ] [ ] , their roles in virus dissemination or as immunoregulatory cells in vivo are not clearly defined. another potential neuro-invasion model for microorganisms that has been proposed is crossing the bloodbrain barrier (bbb). the bbb prevents virus circulating in blood from entering the brain. the human cerebral microvascular endothelial cell line (hcmec/d ) is a stable, easily grown bbb cellular model used in a wide range of research areas, including passage of infectious micro-organisms across the bbb [ ] [ ] [ ] [ ] [ ] . there are reports showing that hcmec/d cells are susceptible to zikv infection, leading to the speculation that zikv has the ability to cross the bbb [ , ] . in the present study, the permissiveness of human and mouse neuroglial cells, including oecs and hcmec/ d s, to zikv strains belonging to asian genotypes and the highly adapted mr was investigated. we show that brain endothelial cells and neuroglial cells are permissive for zikv infection, may potentially serve as a persistent reservoir of infection, and could contribute to cns inflammation through the production of pro-inflammatory chemokines. immortalized mouse olfactory ensheathing cells (moecs) [ ] and human olfactory ensheathing cells (hoecs) [ ] were maintained in dulbecco's modified eagle's medium (dmem) with % foetal calf serum (fcs). vero cells (african green monkey epithelial cells; atcc, ccl- ) were maintained in dmem with % fcs. human brain endothelial cells (hcmec/d ) [ ] were maintained in endothelial cell growth basal medium (ebm- medium) with % fcs, . µm hydrocortisone, μg ml − ascorbic acid, % chemically defined lipid concentrate, mm hepes and ng ml − basic fibroblast growth factor. all cells were cultured at °c in a humidified atmosphere of % co and were passaged at -day intervals after attaining - % confluency. three zikv strains were used. (i) mr (uganda), an african genotype, is a rhesus monkey origin virus isolate from the centers for disease control and prevention (cdc), which is strongly adapted to mice. while mr does not represent the natural african lineage due to it being highly adapted, it nevertheless has been used in many other studies, thus allowing a good comparison with those studies. (ii) beh , an asian genotype, derived from an infectious clone based on the sequence of the brazilian strain [ ] . (iii) prvabc , an asian genotype, is a human isolate from a puerto rico patient [ ] and was kindly provided by dr jill carr, flinders university, australia. these viruses were propagated and plaque-titred in vero cells. viral titres were determined by plaque assay on vero cells. briefly, vero cells were seeded in -well plates. tenfold dilutions of each virus stock were prepared in culture medium and µl of each dilution was added to the cells. cells were incubated with virus dilution for h at °c and then the inoculum was removed and the cells were overlaid with ml of dmem supplemented with % fcs containing . % carboxymethyl cellulose (sigma life science). cells were incubated at °c for days prior to staining with crystal violet fixing solution ( . % crystal violet in % ethanol and . % formaldehyde). plaques were counted and the virus titre expressed as plaque-forming units (p.f.u.) ml − . all multiplicity of infection (m.o.i.) values used in subsequent experiments were calculated based on virus titres in vero cells. multistep growth curves were performed using moec, hoec, hcmec/d and vero cell lines. briefly, cells were seeded in -well plates and infected with zikv at an m.o.i. of . in serum-free media. cells were incubated for h, the inoculum was removed and the cells were washed twice with phosphate-buffered saline (pbs). the cells were overlaid with . ml of complete media and incubated at °c. cell culture media were collected at , . , , , , , and days post-infection (p.i.). at each time point, all media were removed and replaced with fresh medium. virus titres were determined by plaque assay. cells were cultured on glass coverslips, infected at an m.o.i. of in serum-free medium for h, washed once with pbs and covered with complete growth medium. mock-infected cells were used as controls. at indicated time points, the cells were washed with pbs, fixed with % paraformaldehyde and permeabilized with . % triton x- for min at room temperature. fixed cells were washed with pbs and blocked with % fcs in pbs. cells were immunolabelled using the following primary antibodies: rabbit anti-ns (obtained in-house) at : dilution [ ] , mouse anti-flavivirus group antigen antibody, clone d - g - - (millipore, usa), monoclonal anti-dsrna antibody (english and scientific consulting; szirak, hungary) or golgi marker mouse anti- k (abcam, usa) at : dilution for h or pdi-fitc (thermo fisher, usa). following the removal of primary antibodies, cells were washed and treated with the respective anti-rabbit/mouse alexa- / / -conjugated goat antibodies (life technologies); ′, ′-diamidino- -phenylindole (dapi) (life technologies) was used to counterstain nuclei. slides were mounted using slowfade gold reagent. immunofluorescence images were obtained and analysed using a nikon a r+ confocal microscope. images were analysed using imaris software. to determine the infectivity of zikv in the selected cell lines, confluent monolayers of each cell type were infected at an m.o.i. of . samples were harvested at h post-infection (p.i.) and stained with live/dead cell viability dye (life technologies). cells were fixed in % paraformaldehyde in pbs at room temperature for min, washed twice with facs buffer (pbs with % fbs) and permeabilized using . % tween in facs buffer for min at °c. cells were labelled with rabbit anti-ns primary antibody, diluted : in . % tween in facs buffer (diluent) for min on ice and washed twice in diluent. the primary antibody was detected using anti-rabbit alexa antibody in . % tween in facs buffer. samples were examined using the bd lsr fortessa cell analyser and the resulting data were analysed with flowjo software. the live and ns -positive population of each cell line was determined and used as an indication of the efficiency of viral infection. the viability of infected cells was determined using a cell proliferation assay (mtt, sigma-aldrich, usa). briefly, -well plates were seeded with × cells per well. the cells were infected with mr or beh strain at an m.o.i. of . after h the media was replaced with µl of complete medium. at , , or days p.i. µl of mtt reagent ( mg ml − in pbs) was added into each well; cells were incubated for to h until a purple precipitate become visible. the medium was removed, the precipitates were dissolved by adding µl of dmso and absorbance was measured at nm. mouse and human oecs and hcmec/d cells were infected as described above. infected cultures were maintained for months, with : dilution of the culture when reaching - % confluency. the cell lines obtained using this procedure are referred to as moec_mr , hoec_ mr , hcmec/d _mr , moec_beh , hoec_ beh , hcmec/d _beh , moec_prvabc , hoec_prvabc and hcmec/d _prvabc . to analyse the presence of zikv rna in moec_mr , hoec_ mr , hcmec/d _mr , moec_beh , hoec_ beh , hcmec/d _beh , moec_prvabc , hoec_prvabc and hcmec/d _prvabc cells, the cells were seeded in six-well plates at a density of approximately cells/well. after h incubation, cells were washed twice with pbs and replaced with ml of fresh media. after another h incubation cells were collected and total rna was extracted for determination of virus genome copy number. to analyse the growth of zikv-infected cells, hoec, hoec_mr , hoec_prvabc , hcmec/d , hcmec/ d _mr and hcmec/d _prvabc cells were seeded in six-well plates at a density of cells/well. day , and post-seeding total cell numbers in the well were counted using the standard haemocytometer cell counting method according to protocol. briefly, cells were trypsinized and collected into ml medium to obtain a cell suspension. fifty microlitres of cell suspension was mixed with µl of . % trypan blue solution and was applied into the haemocytometer chamber. the total cell number was counted using the following formula: cells/ml=(total cells counted/number of boxes counted)×dilution factor× ×total sample volume. total rna were extracted from persistently infected cells using the rneasy kit (qiagen, germany) according to the manufacturer's protocol. the obtained rna was reversetranscribed using random nanomer primers and moloney murine leukaemia virus reverse transcriptase (sigma-aldrich). serial dilutions of pcci-sp zikv plasmid [ ] were used to generate a gene copy standard curve. viral genome copy number was determined by rt-qpcr using ssoadvanced universal probes supermix (bio-rad) in a . µl reaction and primers targeting the ns region (mr , ′ aaat acac atac caaa acaa agtggt and ′ tcca ctcc ctct ctgg tgttg) or envelope region (prvabc and beh , ′ agatgtcggccctg-gagttc and ′ ttgccacaccgtccttgagg). all reactions were performed in -well plates using a bio-rad cfx touch real-time pcr detection system. the samples were amplified using the following conditions: °c for min, and cycles of °c for s, °c for s and °c for s. a dissociation curve was acquired using cfx manager software and used to determine the specificity of the amplified products. the copy numbers of the amplified products were interpolated from the standard curve using graphpad prism software. media collected from infected hoec and hcmec/d cells were assessed for ccl , ccl , ccl , ccl and cxcl using enzyme-linked immunosorbent assay (elisa) (r and d systems) as specified by the manufacturer. all statistical analyses were performed with graphpad prism software. statistical differences were analysed using two-way analysis of variance (anova) with bonferroni's post hoc and one-way anova with a tukey's post hoc test. to characterize infection by different zikv strains (mr , prvabc and beh ), brain endothelial cells (hcmec/d ) and neuroglial olfactory ensheathing cells (hoec and moec) were infected at an m.o.i. of . . vero cells, for which zikv infection is well characterized, were used as a positive control. although not reaching titres as high as those in vero cells ( fig. a ; peaked ~ p.f.u. ml − at days p.i.), all zikv strains replicated in brain endothelial cells and neuroglial cells, with the highest titres being observed in hcmec/d ( fig. b ; peaked ~ p.f.u. ml − at days p.i.) followed by moec ( fig. c ; peaked around p.f.u. ml − at days p.i.). in hoec cells, titres clearly above the detection limit were only observed for the mr strain ( fig. d ; peaked ~ p.f.u. ml − at days p.i.). these observations show that both african and asian zikv strains are able to infect and replicate in neuroglial and brain endothelial cells. with the exception of moec cells, mr appeared to be the fastest replicating zikv strain, with titres that were consistently higher at days p.i. compared to prvabc and beh (fig. a,b d) . consistent with this observation, mr also induced more prominent cytopathic effect (cpe) in vero cells than beh : the viability of mr -infected cells was reduced to ~ % by days p.i., while, in contrast, the viability of beh -infected cells remained at > % even at days p.i. (fig. a, b) . in contrast to vero cells, both mr and beh strains had no significant effect on the viability of neuroglial or brain endothelial cells (fig. a, b) . combined, these findings suggest that neuroglial and brain endothelial cells may harbour an intrinsic mechanism of suppressing or controlling infection, viral replication and virus-induced cytotoxicity. we next determined zikv infection efficiency in different neuroglial cell lines by detecting the percentage of cells that were positive for zikv non-structural protein (ns ). the viral growth curve revealed that in vero cells the release of new viral particles only occurred after h p.i. (fig. s , available in the online version of this article) and the highest levels of ns were observed at - h p.i. (fig. ) . therefore, infected hoec and hcmec/d cells were harvested at h p.i.; at this time point only primarily infected cells had detectable expression of ns . flow cytometry revealed that the infection efficiency of all zikv strains in the neuroglial cells (hoec) and endothelial cells (hcmec/d ) was much lower than could be counted from the m.o.i. calculated based on titres obtained using vero cells. unlike in the growth curve experiment (fig. d) , all three strains performed similarly in hoec cells and infected approximately % of the total cell numbers (fig. ) . interestingly, in hcmec/ d cells both asian strains showed significantly higher infectivity (~ % infected cells) compared to the mr strain (~ . % of infected cells). these results clearly show a much lower susceptibility to virus infection in neuroglial and brain endothelial cells compared to non-neural vero cells; this finding is consistent with the observed lower growth efficiency (fig. ) . furthermore, there is no clear correlation between efficiency of infection and efficiency of replication (virion production) in neuroglial and brain endothelial cells. thus, mr has the lowest infection efficiency in hcmec/d cells (fig. ) and yet it produced more virions than any of the asian strains (fig. b) . these data, again, may also suggest that intrinsic mechanism(s) responsible for reduced viral entry or/and replication and/ or virion formation/release are present in neuroglial and endothelial cells. to study the intracellular localization of zikv ns and envelope proteins, zikv-infected vero cells were examined by immunofluorescence assay (ifa). viral proteins were visualized at , , , , and h p.i. (fig. ) . at h p.i., the ns and envelope proteins were distributed evenly in the cell cytoplasm. the highest levels of envelope and ns protein were detected at h p.i. and h p.i. at these time points, localization of both ns and envelope protein appeared to be in cytoplasmic membranes that may represent endoplasmic reticulum (er) and/or golgi apparatus. ns , albeit at reduced levels, was also detected at both and h p.i (fig. ) . at h, there were signs of cell death (fig. ) , an observation consistent with the results from the viability assay (fig. ) . next, hcmec/d cells infected with different zikv isolates were examined by ifa at h p.i. using antibodies against golgi apparatus ( k protein), er membranes (pdi), ns and dsrna that served as a marker for viral rna replication. consistent with previous observations ns was localized in the cytoplasm (fig. a, b) . the widespread punctate staining pattern of dsrna overlapped in some areas with ns staining and showed clear co-localization with er membrane (fig. b) , a site of likely localization of zikv replication complexes. overlap of ns was also observed with golgi marker, but not to the same extent as for er marker (fig. a) . the ifa results obtained for zikv infected vero, hoec and moec cells (fig. s ) were almost identical to those obtained for hcmec/d cells (fig. ) . to examine the virus persistence in neuroglia and human brain endothelial cell cultures, the viral rna copy numbers in hcmec/d , hoec and moec cells infected with mr , prvabc and beh for months were quantified using rt-qpcr (fig. ) . the viral genome copy numbers were high in mr -and prvabc -infected hcmec/d and hoec cells. for beh the viral copy number was only high in hcmec/d cells. beh was also detected in moec cells, albeit at low levels. release of infectious virions was only detected for hcmec/d infected with any of the zikv strains (data not shown). these data indicated that all three cell lines can become persistently infected. furthermore, at least hcmec/d cell line produced zikv virions after months of infection. analysis of the percentage of ns -positive cells using staining with anti-ns antibody and facs analysis revealed that ns levels were only above the detection limit in some of the cells; the percentage of ns positive cells showed large variation (from - %) depending on cell line and clone. a correlation between virus titre and the percentage of ns -positive cells in the culture was also observed. these data indicate that cells do not harbour zikv in a uniform manner in persistently infected cultures and contain a variable percentage of cells in which the presence of zikv cannot be revealed. mtt assays did not reveal any cytotoxic effect of zikv acute infection in brain endothelial and neuroglial cells (fig. ) . interestingly, all these cells still produced infectious virions, in some cases even at days p.i. (fig. b-d) , and, at least in the case of hcmec/d cells, continued to do so months later. to understand the effect of zikv persistence in these cells, we compared the proliferation of hcmec/d and hoec cells that had been preinfected for months with zikv-mr or zikv-prvabc with the growth of the same type of uninfected cells over a day period. it was found that the proliferation of hcmec/d cell lines infected with either zikv strain was clearly lower than that of uninfected cells, and the differences become prominent at day and further increased by day after the cells had been seeded (fig. a) . similarly, the proliferation of hoec_mr cells was slow. in addition, these cells exhibited poor attachment, with the result being a loss of ~ % of cells observed by day . in contrast, persistent infection of hoec cells by prvabc had no detectable effect on cell proliferation (fig. b) . pro-inflammatory chemokines were determined in hcmec/ d and hoec cell cultures infected with zikv mr and prvabc at an m.o.i. of . at h p.i. cell culture media were collected, clarified by centrifugation and used for the detection of ccl , ccl , ccl , ccl and cxcl using elisa. all chemokines were significantly upregulated in the samples from infected cultures compared to mock-infected cultures (fig. ) . in particular, all these chemokines were produced at slightly higher levels in mr -infected cells compared to cells infected with prvabc ; however, these differences did not reach statistical significance. the recent outbreak of zikv associated with neurological complications and microcephaly in newborns has stimulated a significant amount of research to investigate cell tropism, to develop suitable in vitro and in vivo models and to understand the mechanisms of zikv-induced pathology. both asian and african strains of zikv have been shown to replicate in a variety of human cell types, including epidermal, neural and placental cells, although there are differences in replication efficiencies, the type of cellular immune response induced and the induction of apoptosis and autophagy [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the african strain mr is very heavily adapted for mouse neurons and multiple studies confirm a higher infectivity and replication rate for mr using in vitro models compared to strains belonging to asian genotype. a direct in vitro comparison of the infectivity of neural stem cells and cellular responses induced by strains belonging to african and asian genotypes suggested that the african strain had a higher infection rate and induced stronger anti-viral responses compared with the strain belonging to the asian genotype [ ] . although neurological complications have been more evident with the recent outbreak of zikv in south america, caused by the asian genotype, it is unclear if there are strain differences in neuropathology and it is possible that the disease associated with strains of african genotype are less frequently confirmed and characterized. in the present study, the permissiveness of human and mouse oec and hcmec/ d cells to african mr and asian zikv, the kinetics of viral growth, the localization of virus proteins and dsrna in the neural cells, and zikv persistence as well as cytokine responses were investigated. as expected, vero cells supported high levels of viral replication. however, zikv also replicated very efficiently in hcmec/d cells, which is in accordance with other studies [ , ] . it is suggested that this efficient zikv replication in endothelial cells is due to the specific receptor axl present in these cells [ ] and that a potential mechanism for zikv invasion of the cns via the bbb involves affecting brain capillary permeability [ ] . zikv infection and replication has previously been shown for neuroglia within the cns radial glia [ ] , myelinating oligodendrocytes [ ] and astrocytes [ ] , but never for oecs, the neuroglia of the primary olfactory nervous system. oecs surround and support the axons of olfactory sensory neurons as they extend from the olfactory mucosa into the olfactory bulb in the brain, forming the olfactory nerve. oecs are also present in the outer layer of the olfactory bulb termed the nerve fibre layer, where they are thought to mediate the organization of primary olfactory axons to their correct bulbar targets [ ] [ ] [ ] [ ] . the olfactory nerve is a route by which certain bacteria and viruses can reach the brain [ , [ ] [ ] [ ] . thus, oecs provide a potential route for access of pathogens and consequently have also been shown to be the main innate immune cells in the olfactory nerve [ ] . in this study, we clearly showed the permissiveness of this cell type to support zikv infection. compared to vero or hcmec/d , viral titres were lower in hoec and moec, suggesting reduced replication efficiency in these cells. despite the detection of reasonably high viral titres in the growth medium of neuroglial cells, the facs analysis of infected cells suggested that only a small percentage of the cells contained detectable amounts of zikv ns . nevertheless, ifa using antibody against dsrna, a marker for viral genome replication, confirmed the permissiveness of the cell lines for zikv rna replication. the localization of ns in the cytoplasm is classical for flaviviruses. colocalization of dsrna and ns with er membrane marker indicates that zikv replication complexes are, as expected, localized at er membranes. identifying other proteins interacting with ns and the role of these complexes in the coordination of replication steps and pathways could provide a target for impeding or interrupting viral replication. the infection of vero cells resulted in readily visible cpe from to days p.i.; this coincided with a decline in cell viability. interestingly, no cpe was detected in neuroglial cells. some studies have given insights into the possibility of persistent zikv infection. zikv was shown to persist in primary human foetal neural progenitors for at least month with no activation of cytokine responses despite some cell death occurring [ ] . recently, a study on sertoli cells showed that even after weeks, cells were producing virions, suggesting that sertoli cells are a major reservoir of virus [ ] . in vivo studies in rhesus monkeys provide evidence of zikv persistence in the cns and lymph nodes [ ] . mladinich et al. [ ] reported the presence of zikv in primary human brain microvascular endothelial cells and hcmec/d cultures at days p.i. without cpe development and suggested that these cells could be susceptible to persistent infection. here, we confirmed that zikv indeed develops persistent infection in hcmec/d cells; even after months of infection zikv rna is present in these cells at high copy numbers and production of infectious virions was observed. in contrast, no particle production was observed for persistently infected moec or hoec cells. there are two possible explanations for the absence of virus production in these cell lines. it is possible that these cells harbour defective but replicating viral genomes and lost the ability to produce plaques. it may also be that it was not possible to detect virus produced in these cells using standard assays (plaque assay) as the yield of infectious virus was below the detection level of the plaque assay. these results suggest that brain endothelial cells may act as a reservoir for zikv and a potential route for virus to reach to the brain. additionally, persistent infection could be detected after months in hoecs, albeit to a lesser extent and in a zikv strain/genotype-specific manner. the fact that the african strain, mr , is heavily adapted for neurons compared to recent strains of the asian genotype fig. . zikv can establish persistent infection in moec, hoec and hcmec/d cell cultures. moec, hoec and hcmec/d cell cultures were infected with three different zikv strains for months. after this, cells were collected, total rna was isolated and viral genome copy numbers were determined using rt-qpcr. the data represent the mean±sem from two independent experiments. nd, not detected. the dotted line indicates the detection limit of the assay ( viral rna copies µg − total rna). might explain the observed advantage of mr in these types of studies. mladnich et al. reported persistent zikv infection of primary human brain microvascular cells and suggested the involvement of the isg /ifn pathway in persistence, similar to that reported for hepatitis c virus [ ] . how zikv is able to sustain stable infection without cytotoxicity remains to be resolved. acutely infected cells produced high levels of proinflammatory chemokines, including ccl , ccl , ccl , ccl and cxcl , which could contribute to cns inflammation during zikv infection in vivo by promoting the recruitment of various leukocytes into the cns. cxcl (il- ) and ccl (mip- α) could potentially recruit neutrophils, whilst ccl (mip- β) may recruit monocytes and nk cells. in particular, ccl (mcp- ), induced by flavivirus infection in various cells [ ] [ ] [ ] , could drive the recruitment of bone marrow-derived ccr hi inflammatory monocytes into the cns in neuronal infection, as it does in other flavivirus models, to promote immunopathology [ , ] . in the case of foetal neuronal infection, however, the extent to which this would recruit foetal monocytes or even maternal inflammatory monocytes is unclear. later in infection sustained ccl production would likely drive maximal recruitment of the adaptive virus-specific t cell effectors that could clear virus. all these chemokines are chemoattractant for innate immune cells, consistent with their induction by flaviviruses early in infection, but each chemokine also has multiple functions in addition to chemoattraction [ , ] . given that many chemokines engage with multiple receptors, and all of the receptors for pro-inflammatory chemokines engage with multiple chemokines, it is presumably the combination of different receptors on individual cell types, activated by the differential concentration of each chemokine produced, that finally determines what cells migrate to the infected tissues and how each cell responds. overall, the growth kinetics analysis, replication efficiency studies, localization studies and cytokine expression studies suggest that neuroglial and brain endothelial cells are susceptible to zikv infection. specifically, the fact that both olfactory ensheathing neuroglial cells and brain microvascular cells could support zikv replication may relate to the ability of the virus to enter the cns via the olfactory nerve or via the bbb. the persistent infection detected without demonstrable cpe suggests that neuroglial cells and brain endothelial cells could provide a useful model for further investigation of intrinsic mechanism(s) responsible for reduced viral entry, rna replication and/or virion formation. use of these models can further promote our understanding of zikv-induced neuropathology. the authors declare that there are no conflicts of interest. zika virus. i. isolations and serological specificity zika virus outbreak on yap island, federated states of micronesia zika virus outside africa rapid spread of emerging zika virus in the pacific area zika virus infection complicated by guillain-barre syndrome--case report guillain-barré syndrome ( cases) 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article at microbiologyresearch.org. key: cord- - nnyq p authors: imran, mudassar; usman, muhammad; malik, tufail; ansari, ali r. title: mathematical analysis of the role of hospitalization/isolation in controlling the spread of zika fever date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: nnyq p the zika virus is transmitted to humans primarily through aedes mosquitoes and through sexual contact. it is documented that the virus can be transmitted to newborn babies from their mothers. we consider a deterministic model for the transmission dynamics of the zika virus infectious disease that spreads in, both humans and vectors, through horizontal and vertical transmission. the total populations of both humans and mosquitoes are assumed to be constant. our models consist of a system of eight differential equations describing the human and vector populations during the different stages of the disease. we have included the hospitalization/isolation class in our model to see the effect of the controlling strategy. we determine the expression for the basic reproductive number r( ) in terms of horizontal as well as vertical disease transmission rates. an in-depth stability analysis of the model is performed, and it is consequently shown, that the model has a globally asymptotically stable disease-free equilibrium when the basic reproduction number r( ) < . it is also shown that when r( ) > , there exists a unique endemic equilibrium. we showed that the endemic equilibrium point is globally asymptotically stable when it exists. we were able to prove this result in a reduced model. furthermore, we conducted an uncertainty and sensitivity analysis to recognize the impact of crucial model parameters on r( ). the uncertainty analysis yields an estimated value of the basic reproductive number r( ) = . . assuming infection prevalence in the population under constant control, optimal control theory is used to devise an optimal hospitalization/isolation control strategy for the model. the impact of isolation on the number of infected individuals and the accumulated cost is assessed and compared with the constant control case. the zika virus is transmitted to humans primarily through aedes mosquitoes and through sexual contact. it is documented that the virus can be transmitted to newborn babies from their mothers. we consider a deterministic model for the transmission dynamics of the zika virus infectious disease that spreads in, both humans and vectors, through horizontal and vertical transmission. the total populations of both humans and mosquitoes are assumed to be constant. our models consist of a system of eight differential equations describing the human and vector populations during the different stages of the disease. we have included the hospitalization/isolation class in our model to see the effect of the controlling strategy. we determine the expression for the basic reproductive number r in terms of horizontal as well as vertical disease transmission rates. an in-depth stability analysis of the model is performed, and it is consequently shown, that the model has a globally asymptotically stable disease-free equilibrium when the basic reproduction number r < . it is also shown that when r > , there exists a unique endemic equilibrium. we showed that the endemic equilibrium point is globally asymptotically stable when it exists. we were able to prove this result in a reduced model. furthermore, we conducted an uncertainty and sensitivity analysis to recognize the impact of crucial model parameters on r . the uncertainty analysis yields an estimated value of the basic reproductive number r = . . assuming infection prevalence in the population under constant control, optimal control theory is used to devise an optimal hospitalization/isolation control strategy for the model. the impact of isolation on the number of infected individuals and the accumulated cost is assessed and compared with the constant control case. the zika virus spreads among humans primarily through an infected mosquito bite, which has been increasing at an alarming incidence rate worldwide over the past few years (dick et al., ) . it belongs to the family of flaviviruses which includes more than fifty viruses, such as dengue, yellow fever, and the west nile virus. this virus was first identified in the zika forests of uganda and east africa during the investigations on the ecology of the yellow fever (anderson et al., ) . the first isolation was made in april of from the serum of pyrexia rhesus monkey caged. the second isolation was made in in the same forest (dick et al., ) . just a year later, in , the virus was recovered from mosquito aedes africanus from the zika forest. the first human case of zika fever was reported in uganda in . the first outbreak of zika fever was reported in on the pacific island of yap, this outbreak caused symptomatic cases. another epidemic outbreak occurred in french polynesia between and . during this time, it was estimated that about , people were reported to have zika like symptoms (anderson et al., ) . epidemic, reporting the most cases of people infected with the zika virus worldwide. in , the state of rio de janeiro alone reported over , probable zika virus infections, however, this number dropped to only cases in . sexual transmission of the zika virus has also been reported from both males and females to their partners (cdc, ; ecdc, ; hastings and fikrig, ; summers and acosta, ) . zika virus can be sexually transmitted from a person who is infected by the virus, even while they are not symptomatic. furthermore, it has been suggested that zika virus can be transmitted from a pregnant woman to her fetus during pregnancy. zika virus can be transferred horizontally as well as vertically. during the zika epidemic, brazilian health officials reported an increase in the number of cases of microcephaly disease, a condition in which a baby's head is smaller than normal in zika affected areas. the main symptoms of zika fever include a fever, the maculopapular rash often spreading from the face to the body, joint and muscle pain, vomiting, or bilateral non-purulent conjunctivitis (ecdc). the first well-documented case of zika fever was reported in and started with a mild headache with later development of a maculopapular rash, fever, and back pain (hayes, ) . the symptoms of zika fever are thus quite similar to those of dengue fever and there is a strong possibility of misdiagnosis in regions where dengue virus is common. the incubation period for the zika virus is between and days (krow-lucal et al., ) . disease-related symptoms are developed within one week of infection for % of the infected individuals and within weeks among % (krow-lucal et al., ) of the infected individuals. the vast majority of infections are not contagious from person to person; however, it may be passed person to person during sex. the virus infection is usually diagnosed by a blood test. the disease symptoms are usually mild and short lasting ( days), and infection may go unrecognized or be misdiagnosed as dengue fever (ecdc). unfortunately, there is no vaccine, antiviral drug, or other modality available to prevent or treat the zika virus infection. zika fever is a preventable but not a curable disease. thus, the only means of controlling the zika virus is to control the mosquitoes that spread the disease and protection during sex. in the past several years, a number of deterministic models for the transmission dynamics of the dengue virus have been studied and analyzed (esteva and vargas, ferguson et al., ; garba et al., garba et al., , kautner et al., ; wearing and rohani, ) . after the zika outbreak, models for zika transmission have been developed (agustoa et al., ; maxiana et al., ; wiratsudakul et al., ) and analyzed. in these models, the authors included the effect of sexual transmission of the disease. in this work, we formulate and study a deterministic model for zika virus transmission including vertical and horizontal transmission of the disease. although esteva (esteva and vargas, ) discussed vertical disease transmission it was among vectors in a dengue transmission. our deterministic model for zika virus transmission includes horizontal and vertical transmission in both humans and vectors. as stated previously, it has been suggested that zika virus can spread to newborns from their mothers, and we therefore feel that an accurate model must include the vertical transmission. our work is an extension of our previous model (imran et al., ) by including a population group that is using controlling measures. in the previous work, we considered death due to the infection and the total human and vector populations were functions of time. the previous model did not possess global stability for both disease-free and endemic equilibriums. we showed a backward bifurcation phenomenon. the current model has constant population size. since death cases reported from zika fever were negligible, we take disease-induced mortality to be zero. there is no backward bifurcation and the steady states results are global. since the only way to control the disease is to isolate patients who have been infected with the zika virus, we included a new population compartment consisting of hospitalized individuals. we have calculated the basic reproductive number associated with our model that guarantees the elimination of the disease. finally, using optimal control techniques, we also propose and analyze the control strategies for decrease infected individuals while minimizing the costs and resources simultaneously. the rest of this paper is organized as follows, the proposed model is presented in section . basic properties and a detailed steady-state analysis of the model are presented in section . in section , we perform a sensitivity and uncertainty analysis of the model parameters and reproductive number associated with our model. section uses ideas from optimal control theory to propose various controlling strategies to overcome zika are proposed. finally, section presents our conclusions and contains a brief discussion of our results. we consider two types of populations in this model one for the humans and for the mosquitoes. the total human and mosquitoes populations at time t, denoted by n h and n v , are constant. the human population is divided into five mutually exclusive groups, susceptible humans s h (t), exposed humans e h (t), infected humans i h (t), isolated or hospitalized individuals h h (t) and recovered humans r h (t). the total vector population is divided into three mutually exclusive classes comprising of susceptible vectors s t the model assumes that the susceptible human population s h (t) has a recruitment rate μ h n h , where n h is total human population and μ h is the natural birth rate of humans. we assume that the birth rate of human population is same as the natural death rate. susceptible individuals get infected with zika fever virus (due to contact with infected vectors) at a rate λ h and thus enter the exposed class e h . in order to consider vertical transmission in our model, we make the assumption (see, e.g., li et al., ) that a fraction of newborn individuals from parents in the e h (t) and i h (t) classes will be infected, and thus remain in e h class before becoming infectious. we have assumed that the hospitalized individuals do not contribute to vertical transmission. population in each class is removed at the natural death rate μ h . we assumed a lifelong immunity for humans who recovered from zika virus. the exposed individuals who got an infection, move to infectious class at a rate ξ. the infected population recovers from the zika fever at a rate θ i and some infected individuals are transferred to hospitalized class at a rate τ. the hospitalized population recovered at a rate of θ h . the susceptible vector population s t ( ) v has a recruitment rate μ n v v , and μ v is a natural death rate of vector population. a fraction of offsprings in the e t ( ) v and i t ( ) v classes will be infected, and thus remain in e h class before becoming infectious. because of this vertical transmission, a fraction of susceptible individuals will enter the exposed class. susceptible vectors are infected with zika virus (due to effective contact with infected humans) at a rate of λ v and thus move to the exposed vector class e v . the susceptible, exposed and infected vectors have natural death rate μ . v in addition, exposed vectors develop symptoms and move to the infected vector class i v at a rate of σ v . it is assumed that infected vectors do not recover, and die at the natural death rate of μ v . as mentioned earlier there is no vaccine available for zika fever. the only way to control this disease is to reduce the contact rate either by killing the mosquitoes or using the protective measure like mosquito repellents, nets etc. the effective contacts will be further reduced by isolating the infected humans. isolation of individuals with disease symptoms constitutes what is probably the first infection control measure since the beginning of recorded human history (hethcote, ) . over the decades, these control measures have been applied, with varying degrees of success, to combat the spread of some emerging and re-emerging diseases such as leprosy, plague, cholera, typhus, yellow fever, smallpox, diphtheria, tuberculosis, measles, ebola, pandemic influenza and, more recently, severe acute respiratory syndrome (gumel et al., ; imran et al., ; lipsitch et al., ; lloyd-smith et al., ) . chavez et al. analyzed a saiqr model in detail, to investigate the effect of isolation on influenza (vivas-barber et al., ) . they used the isolation (quarantine) i-q model, where the infected population is isolated. we have included epidemiological factors like permanent or partial immunity after recovery as well as intervention control measures through the inclusion of a hospitalized (or isolated) class, h h . in this case both the total host population and the vector population are constant. the forces of infection λ h and λ v are given as (garba et al., ) : here we assumed that an individual in h class still can transmit the disease but with lower rate. the value of modification parameter ≤ η < . fig. presents schematic diagram of the model ( ). a description of the variables and parameters of the model ( ) is given in tables and respectively. the model ( ) will be studied on the closed set: is positively invariant and attracting with respect to the model ( ). it can be seen that the solutions are always positive. the right sides of ( ) are smooth, so that initial value problems have unique solutions that exist on maximal intervals. since paths cannot leave , solutions exist for all positive time. thus the model is mathematically and epidemiologically well posed. in this section, we will perform a detailed steady state and stability analysis of the zika fever model presented in section . the model ( ) has a disease free equilibrium (dfe) given by in order to investigate the local stability of the dfe ( ) , the next generation operator method (van den driessche and watmough, ) will be used. following the notation of van den driessche and watmough ( ), the matrix f (for the new infection terms) and the matrix v (of the transition terms) are given, respectively, by progression rate of humans from exposed to infected class τ hospitalization rate of infected individuals σ v progression rate of vectors from exposed to infected class c hv effective contact rate η modification parameter for relative infectiousness of hospitalized humans the basic reproduction number r for our model is given by ( ) when p = q = , vertical transmission is not present in the model; the above r reduces to the basic reproduction number r of a seir model for a vector disease (garba et al., ) . to get a better understanding of the basic reproduction number r in ( ), we rewrite it using taylor expansion about p and q: where ) and . note that r c is the basic reproductive number for horizontal transmission (khan et al., ) . the square root means that the two generations required for an infected vector or host to reproduce itself (van den driessche and watmough, ) . r p + r q is the sum of the number of infected individuals during the mean latent period and the number of infected individuals during the mean infectious period. similarly, r r + r s is the sum of the number of infected vector offsprings during the mean latent period and the number of infected vector offsprings during the mean infectious period. the expression r = r c r p + r c r q + r c r r + r c r s ) represents the total contribution to the infective class made by the exposed and infective individuals of first generation (li et al., ) . the local stability of the disease free equilibrium follows directly from van den driessche and watmough ( ) . we have following result about local and global stability of disease free state: lemma . . the dfe ( ) of the model ( . ), is locally-asymptotically stable if r < , and unstable if r > . ( ) is globally-asymptotically stable in whenever r < . be a solution of ( ) with x = x( ). a comparison theorem will be used for the proof. the equations for the infected components of ( ) can be written as (where the prime denotes the derivative with respect to time), lemma . established the local asymptotic stability of the dfe when r < , or equivalently, ρ(fv − ) < , which is equivalent to all eigenvalues of f − v having negative real parts when r < (van den driessche and watmough, ) . also, f − v has all off-diagonal entries non negative. then this means that the omega limit set of x , ω(x ), is contained in the disease-free space. but, on the other hand, it is straightforward to check that every solution with the initial condition in the disease-free space converges to the epidemiological implication of the above result is that the disease can be eliminated from the population if the basic reproduction number r can be brought down to a value less than unity (that is, the condition r < is sufficient and necessary for disease elimination) irrespective of the size of the initial populations in each class. the stability of the dfe is demonstrated in fig. a . if r > the dfe is unstable in this case and the solutions are attracted to an (apparently unique and stable) endemic equilibrium, as depicted in fig. b . in this section, the existence and stability of endemic equilibrium (ee) of the model ( ) will be discussed. we define endemic equilibrium to be those fixed points of the system ( ) in which at least one of the infected compartments of the model are non-zero. theorem . . the endemic state, , of the model ( ) exists whenever r > . denote an arbitrary endemic equilibrium of the model ( ). also, let solving the equations of the model ( ) for steady-state by setting right hand asides of the model ( ) . where and all k′s are defined above. substituting ( ) in ( ) and simplifying gives, also, substitute ( ) into ( ), we have the following equation in λ h . clearly the model ( ) has no endemic state if r < and one unique endemic state when r > . □ fig. shows the variation in r with the relative fraction of newborn exposed individuals (p) and newborn infected individuals (q). we notice that no less than % of newborn exposed individuals (p) and no more than % of the fraction of newborn infected individuals to bring r less than : fig. shows the effect of hospitalization rate τ of infected individuals on the basic reproductive number r . from this figure, we can see that effective isolation will help to reduce the basic reproductive number. about % of infected individuals should be effectively isolated to bring the basic reproductive number to less than . this plot shows that the effective isolation is helpful in controlling the epidemic of zika virus. ( ) showing the contour plot of r as a function of fraction of newborn exposed individuals (p) and newborn infected individuals (q). theorem . . if r > then the disease is strongly uniformly ρ-persistent: be a solution of model ( ). in this case there exists an endemic steady state. let (that is, x is the disease-free subspace). note that x, as well as + x ℝ \ , are positively invariant. also, all solutions originating in x converge to n as t → ∞. n is asymptotically stable in x. hence n is isolated in x. corollary . in salceanu ( ) )), together with proposition . and lemma . in salceanu ( ) , imply that {n } is also uniformly weakly repelling. then, from theorem . in smith and thieme ( ) we have that the semiflow generated by ( . ) is uniformly weakly ρ-persistent. from the positive invariance of , we have that ( . ) is point dissipative. then, according to theorem . in smith and thieme ( ) , there exists a compact attractor of points for ( ). this, together with uniformly weakly ρpersistent imply ( ) (see smith and thieme, , theorem . ) . in this case there exists an endemic steady state (smith and thieme, ) . □ the local stability of the endemic steady state of the model ( ) is given in the lemma below. theorem . . if r > , then the endemic state of the model ( ) is locally asymptotically stable in for model ( ). for the proof of this above local stability theorem see imran et al. ( ) . for the global stability of the endemic steady state , we consider the ( ) with no hospitalization and a small incubation period so that we can assume that susceptible individuals after infection move to infected class. in this case, it is easily seen that both for the host population and for the vector population the corresponding total population sizes are asymptotically constant. we assumed that in our model the total population is constant. previous results (thieme, ) imply that the dynamics of systems ( ) is qualitatively equivalent to the dynamics of system given by: theorem . . if r > , then the endemic state of ( ) is globally asymptotically stable in . o proof. we will use geometric approach to global-stability method given in li et al. ( ) . let x = (s, i h , i v ) and f(x) denote the vector field of ( ). the jacobian matrix and its second additive compound matrix j [ ] is (see li et al., ) : where now from the system ( ), we can write second and third equation, the variation in the values of the parameters of our model ( ) is a source of uncertainty and sensitivity. in this section, we carry out a parameter base global uncertainty and sensitivity analyses on r . there are a lot of reasons for the sensitivity of the parameters, for example, inadequate data, lack of information about the vertical transmission. we use the latin-hypercube sampling based method to quantify the uncertainty and the sensitivity of r as a function of the model parameters. we use the latin-hypercube sampling based method to quantify the uncertainty and the sensitivity of r as a function of model parameters, namely μ μ θ θ ξ σ p q r s c τ η , , , , , , , , , , , , . the partial rank correlation coefficient (prcc) measures the impact of the parameters on the output variable using the rank transformation of the data to reduce the effects of nonlinearity. the uncertainty analysis (figs. and ) yields an estimated value of r = . with % ci ( . , . ) for the zika fever. the sensitivity analysis suggests that r is highly sensitive to the parameters c θ θ μ τ , , , , . the accuracy and precision in the values of these parameters is vital for the accurate predictions of the model. the estimated parameters are presented in table . one of the goals of this study is to come up with a time-dependent hospitalization/isolation strategy that would minimize the infected population while keeping the costs to a minimum at the same time. m. imran et al. virus research ( ) - optimal control is a very useful mathematical technique that can help us to address these questions. here our goal is to put down infection from the population by increasing the recovered class and to minimize the required resources to control zika fever infection using isolation or hospitalization. the optimal control algorithm we use is based on pontrygain's maximum principle which appends the original model to an adjoint system of differential equations with terminal conditions. the optimal objective system, which characterizes the optimal controls, consists of differential equations of the original model (state system) along with the adjoint differential equations (the adjoint system). the number of equations in the adjoint system is same as that of in the state system. the detailed mechanism of forming the necessary conditions for the adjoint and optimal controls are discussed in fleming and rishel ( ) and pontryagin and boltyanskii ( ) . an important decision while formulating an optimal control problem is deciding how and where to introduce the control in the system of differential equations. the form of the optimal control primarily depends on the system being analyzed and the objective function to be optimized. in this paper, we will propose various strategies to eradicate zika fever using optimal control techniques. the first step is to find an optimal hospitalization schedule that minimizes the number of infectious individuals and the overall cost of hospitalization during a fixed time. we define the control set as u= {τ(t) : ≤ τ(t) ≤ ζ, ≤ t ≤ t, < ζ ≤ , τ(t) is lebesgue measur-able}. here ζ is a positive number and is defined as the maximum value attained during optimal control procedure. our aim is to minimize the associated cost function which is given as: here a i and a n are positive constants used to balance in the size of i(t) and n t ( ) v . further, we used a nonlinear cost function in order to accommodate the impact of variety of factors associated with hospitalization, documented widely in literature, see for instance kirschner et al. ( ) . w is weight associated with quadratic cost due to hospitalization. moreover a linear function has been chosen for cost incurred by infected individuals and the mosquitoes population. our objective is to find an optimal control for hospitalization rate τ * (t) such that . the lagrangian of the optimization problem is given the associated "hamiltonian" is given by where k i represents the right hand side of the ith equation in our original model. w depends on the relative importance of the control measures in mitigating the spread of the disease as well as the cost incurred (such as material resources and human effort) during the implementation of control measure per unit time. pontrayagin's maximum principal converts model ( ) and objective function ( ) into minimizing the hamiltonian ( ) with respect to τ. now we prove the following theorem to elicit the effect of optimal control of hospitalization. theorem . . there exists a unique optimal control τ * (t) which minimizes j over u. also, there exists an adjoint system of ϕ i 's such that the optimal treatment control is characterized as for some positive number ζ. the adjoint system is given as the above adjoint system also satisfies the transversality condition, proof. we can easily verify that the integrand of j is convex with respect to τ(t). also, the solutions of our model are bounded above. in addition, it is verifiable that the model has the lipschitz property with respect to the state variables. using the properties mentioned above along with corollary . of fleming and rishel ( ) , the existence of an optimal control is established. now using the pontryagin's maximum principle, we obtain . therefore on the set {t : < τ * (t) < ζ} we obtain now, we discuss the numerical solutions of the optimality system and the corresponding optimal control obtained using ζ = . . the optimal strategy is obtained by solving the optimal system consisting of both the state system as well as the adjoint system. since there are initial conditions present for the state equations, we start solving them with a guess for τ using the fourth-order forward runge-kutta method. the adjoint equations are then solved using the fourth-order backward runge-kutta method because of the presence of final conditions. then, the controls are updated by using a convex combination of the previous control and the value from the characterization given above. this process is repeated until we obtain a desired accuracy of convergence (fig. ) . fig. represents the optimal isolation(hospitalization) strategy to be employed to minimize the cost and the infected population. considering the practical constraints, an upper bound of . was chosen for the optimal hospitalization control. the figure shows that initially, the optimal level remains at the upper bound of . after which it declines steadily to . this implies that in the early phase of the endemic breakout, keeping the control at the upper bound would help in decreasing the number of infected individuals. fig. captures the dynamics of the infected population (i h ) by virtue of a comparison between the infected host population under optimal control and constant control. it can be seen that the decrease in the number of infected individuals is greater with optimal control as compared to that with constant control. furthermore, in contrast with a constant control, the infected population remains lower when an optimal control is applied. fig. shows a comparison between the costs associated with optimal and different constant control strategies. it is clear that the cost associated with different control strategies is higher as compared to that of optimal control. it is important to note that high constant isolation rate (τ = . ) incurs almost the same cost as the of optimal control. however, practically it is highly unlikely to implement these high constant controls primarily due to the lack of required resources and facilities. fig. captures the effect of the change in effective contact rate over the optimal control strategy. it is clear from the simulation that an increase in the contact rate may or may not lead to higher rates of hospitalization. in this paper, we have presented a zika fever epidemic model comprising of eight compartments consists of vector and human population. the dynamics of zika fever epidemic model have been considered. furthermore, using optimal control theory, we proposed control strategies to eliminate the infection from the population. a vertical and horizontal transmission model for zika fever is constructed in the form of a system of ordinary differential equations. this model features the study of zika fever by considering vertical transmission in both humans as well as vectors. the basic reproductive number r is formulated by using a next-generation matrix. this reproductive number is simplified in order to better understand the effect of vertical transmission parameters. it is shown that the disease-free steady state is globally asymptotically stable when the basic reproductive number (r ) is less than . the model has a unique endemic equilibrium when the reproduction number r exceeds unity. this equilibrium is shown to be globally asymptotically stable when the reproduction number r exceeds unity under the reduced model. it is locally asymptotically stable when we consider a full model. we performed a parameter based global uncertainty and sensitivity analysis on r . the uncertainty analysis yields an estimated value of the basic reproductive number r = . with % confidence interval ( . , . ). this estimated value of r is close to the calculated value of basic reproductive using real data (villela et al., ). our previous model had an estimated basic reproductive number r = . with % confidence interval ( . , . ) given in imran et al. ( ) . our sensitivity analysis on the zika model parameters showed that the most influential parameters are the effective contact rates, the recovery rate of the infected individuals and the birth rate of mosquitoes. we proposed an optimal controlling strategy to eliminate zika fever from the population. we observed that optimal control strategy is most effective in terms of eliminating infection as it minimizes our cost and resources at the same time. moreover, the control measures themselves may take time to implement, once the outbreak has been realized. despite these points, our analysis can help public health authorities to determine quasi-optimal strategies they might want to adopt, especially as our work highlights the relative effectiveness of different control strategies. mathematical model of zika virus with vertical transmission zika virus background zika virus (i). isolations and serological specificity estimated incubation period for zika virus disease analysis of a dengue disease transmission model a model for dengue disease with variable human population influence of vertical and mechanical transmission on the dynamics of dengue disease coexistence of different serotypes of dengue virus microcephaly in brazil potentially linked to the zika virus epidemic the effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens deterministic and stochastic optimal control backward bifurcations in dengue transmission dynamics effect of cross-immunity on the transmission dynamics of two strains of dengue modelling strategies for controlling sars outbreaks zika virus and sexual transmission: a new route of transmission for mosquito-borne flaviviruses zika virus outside africa the mathematics of infectious diseases a comparison of a deterministic and stochastic model for hepatitis c with an isolation stage transmission dynamics of zika fever: a seir based model dengue virus infection: epidemiology, pathogenesis, clinical presentation, diagnosis, and prevention estimating the basic reproduction number for single-strain dengue fever epidemics optimal control of the chemotherapy of hiv global dynamics of an seir epidemic model with vertical transmission transmission dynamics and control of severe acute respiratory syndrome curtailing transmission of severe acute respiratory syndrome within a community and its hospital zika virus dynamics. when does sexual transmission matter the mathematical theory of optimal processes pan american health organization, zika cumulative cases robust uniform persistence in discrete and continuous dynamical systems using lyapunov exponents dynamical systems and population persistence zika virus in an american recreational traveler convergence results and a poincar bendixson trichotomy for asymptotically autonomous differential equations zika in rio de janeiro: assessment of basic reproduction number and comparison with dengue outbreaks dynamics of an saiqr influenza model ecological and immunological determinants of dengue epidemics dynamics of zika virus outbreaks: an overview of mathematical modeling approaches the authors declare that there is no conflict of interests regarding the publication of this article. we study the following feasible region of the new system ( ) key: cord- -wdh jiry authors: ishtiaq, farah title: a call to introduce structured zika surveillance in india date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: wdh jiry india has the climatic conditions conducive to year-round transmission of zika virus, and a structured disease surveillance program should be implemented to prevent an outbreak. such a program should (i) start screening before an outbreak arises; (ii) collect baseline data to assess future disease risk and monitor potential birth defects; and (iii) provide new insights into the ecology of the disease and inform public health policy following the one health concept. antileishmanial immunity to be able to explore the therapeutic potential. another potential target would be the transcription factor hif- a, the master regulator to the response to hypoxia. hif- a alters myeloid cell functions during l. donovani infection, leading to inhibition of t cell responses. it is also required by leishmania to survive inside macrophages. in the case of hif- a, the therapeutic window is very small, since this transcription factor is involved in many vital functions that are unrelated to the immune response. similarly to b cells, we first need to dissect specific pathways of activation and downstream effects of hif- a to better exploit the therapeutic potential of this target. what is the current status of visceral leishmaniasis? which pressing steps should be taken to reduce it? leishmaniasis is on the who list of neglected tropical diseases, despite the fact that the disease is spread over countries and, in the case of visceral leishmaniasis (vl), is still taking a human toll. over a billion people living in endemic areas are at risk of infection worldwide. there are about one million estimated cases of cutaneous leishmaniasis each year, and of visceral leishmaniasis. some % of the vl cases occur in india, bangladesh, sudan, south sudan, brazil, and ethiopia. leishmaniasis mainly affects poor people in tropical and subtropical regions of the world, and is also associated with factors that are independent of genetics, such as malnutrition, poor housing, and population migration. hence, an effective vaccine would be an ideal solution for controlling disease and limiting transmission, especially for species that can be transmitted from humans to humans, such as l. donovani. unfortunately, to date there is no vaccine available on the market. another important point is the constant emergence of drug resistance and consequently the urgent need to develop new drugs. better epidemiological data are also required to thoroughly understand various reservoirs of infection, and gather information on vectors and leishmania strains and species affecting a determinate region in correlation with disease outcome and severity. finally, we certainly need more information on disease transmission and the role of asymptomatic carriers in parasite transmission. the who aims to reduce the overall burden of these diseases by . in your opinion, are we on track to achieve those goals? why? a reduction in the overall burden could definitely be feasible for some countries. unfortunately, as i mentioned before, leishmaniasis is a disease that is associated with poverty, malnutrition, poor housing, and population displacement. the increasing incidence of conflicts and political and economic instability in certain regions of the world has actually worsened the disease burden in those areas in the past decade. a vaccine would be a cost-effective and a long-term solution to limit the overall spread of the disease in endemic countries. unfortunately, i think that we are still far away from having one. leishmanization (subcutaneous immunization with live parasites) has actually conferred a good degree of protection against cutaneous leishmaniasis. however, this method has never been officially approved because of the possibility that large scars may remain after immunization in certain individuals. moreover, the efficacy of leishmanization against visceral strains has not yet been proven in humans. for the moment, control of infection relies on the systemic treatment of patients and on reservoir and vector control. this method will definitely reduce disease burden in the short term, but only until the emergence of drug-resistant strains. a long-term solution is warranted. farah ishtiaq , * india has the climatic conditions conducive to year-round transmission of zika virus, and a structured disease surveillance program should be implemented to prevent an outbreak. such a program should (i) start screening before an outbreak arises; (ii) collect baseline data to assess future disease risk and monitor potential birth defects; and (iii) provide new insights into the ecology of the disease and inform public health policy following the one health concept. the recent outbreak of zika in south america brought zika under the spotlight and led the world health organization (who) to declare it an international public health emergency in , with an immediate need to prepare for, and respond to, any future epidemic. the who has since declared the end of the public health emergency i , but zika, a mosquito-borne flavivirus, remains a cause of global concern, particularly in resource-limited countries in africa and the asia-pacific region. in these countries, the presence of competent mosquito vectors and suitable climatic conditions could support local transmission of the virus. in india, four cases of zika have now been reported in the media. in november and february , three cases of zika were confirmed from a densely populated area (bapunagar) in the city of ahmedabad in western gujarat state, india ii . whilst the three patients (two pregnant women and an elderly man) showed no complications, and had not travelled outside the country, the government confirmed these cases only several months later iii . more recently, in july , a fourth case, reported from tamil nadu state, was confirmed in a man iv . these are not the first cases of zika virus reported from india; a study conducted in detected the neutralizing antibodies of zika virus in one-sixth of blood sera samples, suggesting exposure to the virus [ ] . as i discuss below, india has the ideal ecological conditions to support transmission and to host a zika outbreak; however, no measures were taken to combat future outbreaks and there is as yet no structured disease surveillance program. zika is thought to be primarily transmitted by aedes mosquitoes v at elevations < m above sea level [ ] , but the scientific literature is dichotomous on whether culex mosquitoes can transmit the virus. in india, aedes aegypti is the only mosquito species that is screened for zika, albeit at a low frequency (> aedes mosquito samples screened until september vi . recently, a controversial report originating from china [ ] , and a second report from brazil [ ] , suggested that culex quinquefasciatus could also potentially transmit the virus. cu. quinquefasciatus is widely distributed and prevalent in india and should be screened for zika virus to increase the likelihood of predicting any potential outbreak. furthermore, vector control is key to prevention and control of mosquito-borne infections and, in india, wolbachia-based vector control strategies for aedes mosquitoes are currently being proposed and prioritized for zika [ ] . a recent study [ ] further emphasises the zika transmission potential of aedes mosquitoes, but also highlights sexual transmission via travellers returning from zika virus-affected areas. the role of human movement has clearly been central to disease transmission following a string of recent outbreaks, including ebola, h n influenza, and severe acute respiratory syndrome (sars). the same study [ ] reported that global human mobility places large populations at risk of mosquito-borne zika virus infection. in india, an estimated . billion people ( air travellers arriving per year from outside the country) are susceptible to zika virus exposure at the time of peak seasonal risk, which coincides with dengue outbreak in the rainy season (august). even though these predictions are based on very conservative estimates [ ] , such modelling efforts may offer useful insights to time-sensitive public health decisionmaking in india. in africa and south east asia, many other host species may support zika virus infection; forest-dwelling birds [ ] , horses, goats, cattle, ducks, and bats [ ] have been reported based on serology, but were not verified by viral isolation. the question of whether birds transfer the virus over long distances remains unanswered. in india, infectious disease ecology with respect to wildlife is completely neglected and, consequently, the potential for the zika virus as spillover infection from other animals to humans has never been considered. furthermore, assessing future zoonotic disease risk requires baseline datainformation about where infectious diseases are distributed geographically, taxonomically (with respect to animal reservoirs), and in relation to human populations. this is illustrated in figure ; the key ecological drivers of zika virus and seasonality in india, where warm temperatures prolong the mosquito seasonhigh mosquito density, densely populated areas, and a continuous flow of airline travellersare likely to facilitate virus transmission by increasing humanmosquito-human contact. the indian zika cases have been reported outside the monsoon season and august, which is modelled as month of peak exposure to zika virus [ ] . in addition, the presence of wild animals, such as rhesus macaques and other animals within human habitation, blurs the boundaries between reservoir hosts and spillover infections, further complicating the prediction of zoonotic disease events in india. of the four cases of zika reported in the media, none has been published in a scientific journal. a confirmed case of zika virus disease requires laboratory confirmation of infection by either the presence of zika virus rna or of antigen in serum or other samples (e.g., saliva, tissues, urine, whole blood); or alternatively the detection of igm antibodies against zika virus. the four cases of zika reported in the media in india seem to have been caused by the asian strain of the virus, the same strain that caused the zika outbreak in brazil vii . however, without a confirmed record, it is difficult for the international scientific community to understand the extent of the disease or even to acknowledge the indian government's reluctance to report these cases. furthermore, flavivirus genetic data are key to understanding the parasite's movement and can be used to infer whether a given strain is the result of locally acquired transmission or importation based on relatedness to locally isolated strains (e.g., [ , ] ). without these data we cannot learn (i) if there has been an outbreak of zika virus at the reported sites in india, and (ii) why the extent of outbreak was not as amplified as in south america or french polynesia. the ecological modelling study [ ] predicts august as the time of peak seasonal risk for transmission of zika in india; however, three of the four reported zika cases in india appeared between november and february in areas with relatively poor access to health services. this discrepancy could be due to an extended mosquito transmission season or delayed appearance of symptoms. malone et al. [ ] reported that up to % of cases of adult human infection with zika virus remain asymptomatic, and it is reasonable to assume that the total number of cases in india is likely to be underreported as they can easily be confused with chikungunya and dengue [ ] . the sporadic and small number of zika cases from india probably suggests either apparent immunity to the virus or a lower number of susceptible hosts to maintain transmission between humans and mosquitoes. a similar trend is now being observed in the americas where transmission persists at low but steady levels with no symptoms [ ] . however, the virus may persist in acute or chronic stages in humans or as zoonotic infection in reservoir hosts, which can be a source for future outbreaks. the concept of disease ecology, or the idea that host-pathogen interactions can be studied within the context of their environment, which is central to understanding the epidemiology and to preventing future outbreaks, has remained neglected in india. in fact, india is an ideal place to explore the coevolutionary dynamics of this host-parasite system because of several factors: (i) the high volume of human movements [ ] , (ii) the apparent immunity to zika from circulating strains of the virus [ ] , and (iii) the possibility of transmission in less immunocompetent hosts, such as pregnant women and the elderly viii , and (iv) adults with a prior history of malaria or dengue infections, which may help facilitate transmission and pathogenesis of zika, potentially resulting in a positive feedback loop [ ] . if there is a positive feedback loop, it may result in a malaria or dengue outbreak and/or the spread of strains of zika adapted to hosts with prior infections. whilst there is no influence of pre-existing immunity to dengue in the kinetics of zika infection in macaques [ ] , this work does not rule out a possible role of preimmunity in the immunologic enhancement in zikaassociated neurological and congenital abnormalities. these factors place india in a privileged place to better understand zika evolution in the asian context and whether reported microcephaly cases are zika-linked or not. but to achieve this, there is an urgent need to collect data on microcephaly and other birth defects ix . finally, to reduce the risk of a zika outbreak in the future, india needs a structured disease surveillance program that will go beyond sample screening postoutbreak; the program should include widespread serosurveillance for underlying population immunity, predictive sampling to collect baseline data to assess future disease risk, and should follow the 'one health' approach with the collaboration of public health officials, clinicians, social scientists, and local government. such work, together with enhanced health care infrastructure aimed at improved diagnostics and maternal care, could provide novel insights into the ecology of the disease and protect women of childbearing age from the potential devastating fetal complications. from zika virus-affected areas provide ample opportunity for virus transmission in densely populated areas. in india, three out of four cases occurred outside of the period of peak seasonal risk (august) predicted in [ ] , and none of the patients had travelled outside the country. (b) human habitation is interspersed with wildlife and livestock, blurring the boundaries between reservoir and incidental hosts for zika virus. (c) susceptible or immunologically challenged hosts act as amplifiers in low to moderate transmission. ii www.who.int/csr/don/ -may- -zika-ind/en/ iii www.business-standard.com/article/ economy-policy/why-was-public-kept-indark-for- -months-after-first-zika-case-in-india- _ .html iv www.thehindu.com/sci-tech/health/there-seemsto-be-low-level-transmission-of-zika-virus-in-india/ article .ece v www.who.int/mediacentre/factsheets/zika/en/ vi www.icmr.nic.in/zika/zika% update% -% th% september% .pdf vii https://scroll.in/pulse/ /india-is-expandingzika-surveillance-but-there-was-an-element-of-luckin-finding-the-tamil-nadu-case viii www.who.int/csr/don/ -may- -zika-ind/en/ ix www.thehindu.com/todays-paper/tp-national/ govt-finally-goes-ahead-to-test-microcephalyzika-link/article .ece proliferation of malaria parasites in a host requires mechanisms to spread between red blood cells (rbcs). we discuss here the implications for biology and antimalarial drug development of companion studies that establish the requirement of two plasmodium spp. proteases of the plasmepsin family in parasite egress from, and invasion into, rbcs. protozoan parasites of the plasmodium spp. cause malaria through cyclic waves of replication within the rbcs of infected individuals, followed by infection of new host cells resulting in large numbers of parasites within circulation. the mechanisms that guide malaria parasites through egress from infected host cells, and invasion into uninfected rbcs, are a subject of intense interest, not least because this critical step in proliferation has been little explored for the development of new drugs for the treatment of malaria and combatting the emergence of drug resistance. plasmodium spp. employ intracellular proteases to break down the surrounding vacuolar and rbc membranes to initiate egress into the bloodstream [ ] . two recent papers identify essential roles in egress and invasion for two previously uncharacterized members of the plasmepsin (pm) family of aspartic proteases, pmix and pmx [ , ] . the studies further demonstrate the potential for pmix and pmx as drug targets to block malaria infections in vivo. plasmodium spp. express multiple plasmepsin protease genes during the blood stage that regulate distinct functions through the cell cycle, including hemoglobin digestion and the export of parasite proteins for remodeling of the host cell (e. g. [ , ] ). pmix and pmx, two homologs of a subclass of the plasmepsin family [ ] , are specifically expressed during the schizont stage of development late in the asexual cell cycle, and had not been previously characterized. in parallel, nasamu et al. [ ] and pino et al. [ ] studied the biological functions and potential drug susceptibility of these proteases in plasmodium parasites. the researchers employed a combination of reverse genetics and chemical biology to discover novel, essential functions for these plasmepsin genes in egress and rbc invasion. the two studies use either conditional knockdown [ ] or conditional knockout [ ] in plasmodium falciparum to show that pmix is specifically required for rbc invasion. pmix is enriched at rhoptries [ , ] , organelles released into the rbc during host cell attachment to initiate formation of a parasitophorous vacuole (pv). pmix is required for efficient proteolytic processing of rhoptry factors in vivo [ , ] , with biochemical evidence in vitro for direct activity toward rhoptry-specific substrates [ ] . based on morphology, proteolytic processing by pmix appears to mediate biogenesis of functional rhoptry organelles [ ] . nasamu et al. employed conditional knockdown in p. falciparum to identify an essential function for pmx in rbc egress, with evidence for function also in invasion [ ] . pmx is targeted to exonemes [ ] , among the earliest parasite organelles discharged during egress, and is required in vivo for proteolytic maturation of the serine protease neutralizing antibodies against certain viruses in the sera of residents of india elevation as a proxy for mosquitoborne zika virus transmission in the americas culex pipiens quinquefasciatus: a potential vector to transmit zika virus zika virus replication in the mosquito culex quinquefasciatus in brazil potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study arbovirus survey in wild birds in uganda a survey for arboviral antibodies in sera of humans and animals in lombok, republic of indonesia genomic epidemiology reveals multiple introductions of zika virus into the united states zika virus: medical countermeasure development challenges zika virus infection and associated neurologic disorders in brazil the missing pieces: lack of zika data from africa complicates search for answers enhancement of zika virus pathogenesis by pre-existing antiflavivirus immunity impact of prior flavivirus immunity on zika virus infection in rhesus macaques spotlight targeting plasmodium proteases to block malaria parasite escape and entry aditya s. paul and manoj t. duraisingh resources i www.who.int/mediacentre/news/statements/ / zika-fifth-ec/en/ key: cord- - k f tw authors: parker, elaine l.; silverstein, rachel b.; verma, sonam; mysorekar, indira u. title: viral-immune cell interactions at the maternal-fetal interface in human pregnancy date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: k f tw the human decidua and placenta form a distinct environment distinguished for its promotion of immunotolerance to infiltrating semiallogeneic trophoblast cells to enable successful pregnancy. the maternal-fetal interface also successfully precludes transmission of most pathogens. this barrier function occurs in conjunction with a diverse influx of decidual immune cells including natural killer cells, macrophages and t cells. however, several viruses, among other microorganisms, manage to escape destruction by the host adaptive and innate immune system, leading to congenital infection and adverse pregnancy outcomes. in this review, we describe mechanisms of pathogenicity of two such viral pathogens, human cytomegalovirus (hcmv) and zika virus (zikv) at the maternal-fetal interface. host decidual immune cell responses to these specific pathogens will be considered, along with their interactions with other cell types and the ways in which these immune cells may both facilitate and limit infection at different stages of pregnancy. neither hcmv nor zikv naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. here, we will highlight new approaches using placenta-on-a-chip and organoids models that are providing functional and physiologically relevant ways to study viral-host interaction at the maternal-fetal interface. pregnancy is a unique immunological phenomenon in which the semiallogenic fetus is able to grow in the maternal uterine environment. in order for a successful pregnancy to occur, healthy placentation is necessary to create an environment that is protective for the developing fetus and promotes growth. how immune balance is maintained by maternal and fetal cells to promote the survival of the genetically distinct fetus, while preventing infection by a large number of pathogens, is yet to be fully elucidated ( ) . this little understood enigma has been the subject of interest and research for decades ( ) . fertilization leads to the creation of single celled embryo which undergoes several successive divisions to form a blastocyst. the blastocyst is made up of two types of cells: the outer trophoblast or trophoectoderm (te) layer forming the placenta and chorion, and the inner layer or inner cell mass (icm) forming the embryo proper and amnion ( ) . the decidua underlying the embryo is called the decidua basalis, which composes the maternal side of the placenta. the maternal-fetal interface is made up of the maternal decidua and fetally-derived placenta. during implantation, the blastocyst attaches to the decidualized endometrium and the outer layer of the blastocyst differentiates into different lineages. the te gives rise to cytotrophoblast cells (ctbs) which follow villous and extravillous pathways to form the placenta. in the villous pathway, the mononuclear ctbs fuse, creating multinucleated syncytiotrophoblasts (stbs) that establish floating villi (fv). the fv are surrounded by maternal blood, with stbs aiding the provision of nutrients by enabling gas exchange and exchange of secreted pregnancy-related hormones (human chorionic gonadotropin, hcg, human placental lactogen, hpl) at the maternal-fetal interface. furthermore, ctbs act as anchoring villi for the attachment of the embryo to the uterus. the ctbs present in the cell column of the anchoring villi follow the extravillous pathway and differentiate into interstitial (ictbs) and endovascular extravillous trophoblast cells (ectbs). the ictbs further invade up to the inner third of the myometrium and ectbs remodel the spiral arteries in low resistance high blood flow to provide nutrients to the developing embryo ( ) ( ) ( ) ( ) . the invasion of trophoblast cells at the maternal-fetal interface occurs in the presence of a large population of maternal immune cells ( ) . this includes % decidual natural killer (dnk) cells, %- % macrophages, %- % t cells and . % dendritic cells ( ) ( ) ( ) . the abundance of decidual cytotoxic t cells and macrophages can vary through the course of pregnancy ( ) . the abundance of nk cells in the decidua during the first trimester, and through the pregnancy (albeit at lower abundance), implicates them as an essential element in both the promotion of an immunotolerant environment and the control of pathogenic infection during pregnancy ( figure ) . thus, the paradoxical maternal-fetal interface is admired for both its immunotolerance to semiallogeneic trophoblastic invasion (leading to a successful pregnancy) while remaining remarkably resilient to pathogenic infections. nevertheless, several pathogen, termed torch pathogens (described below), successfully cross the placental barrier and cause devastating infection in the developing fetus ( ) . in this review, we will look at the interactions between decidual immune cells and specific viral torch pathogens and review known mechanisms which may enable viral pathogenesis within the placental environment. torch is an acronym defining some of the most common infections associated with vertical transmission. initially described in , this group contained just pathogens; toxoplasmosis, rubella, cytomegalovirus (cmv) and herpes simplex type and ( ) . since then this group has been broadened to comprise a host of other infections including listeria monocytogenes, syphilis, varicella zoster virus, human immunodeficiency virus (hiv), enteroviruses and parvovirus b ( ) . most recently, following the zika virus (zikv) epidemic in south america resulting in observed congenital anomalies, this group has been further expanded to include zikv, with some suggesting renaming this group "torchz" ( ) . the mechanism by which these "torchz" pathogens are able to circumvent typical clearance by groups of immune cells (e.g. nk cells, macrophages and others) has been studied by many groups over the last few decades in order to elucidate not only routes of pathogenicity but also roles of immune cells within this immune-privileged environment ( ) . it remains to be proven whether the new emerging viral threat by sars-cov which causes covid- including in pregnant women, will be included in this group of vertically transmitted pathogens ( ) . in this review, we will focus on maternal and fetal macrophages, t cells, and nk cells and their relationship with each virus. we will focus on the viruses human cytomegalovirus (hcmv) and zikv, which are known causes of adverse pregnancy outcomes and delve into how they interact with various decidual immune cells to promote their survival and replication. we will examine the timings of pregnancy that appear to be most permissive to pathogenic infection by these viruses and we will look at the role of various immune cells in this context ( figure ). in the early first term decidua, %- % of resident leukocytes are t cells with approximately %- % of these t cells being cd + (t helper cells) and %- % being cd + (cytotoxic) t cells ( ) ( ) ( ) . further studies have estimated the decidual cd + population to be comprised of about % activated memory cd dim t cells and % cd + cd bright foxp + treg cells. unlike the peripheral circulation, the decidua has a higher ratio of cd + t cells to cd + t cells and an overall higher number of cd + t cells ( ) . in addition, approximately % of the decidual cd + population are effector-memory t cells with reduced perforin and granzyme b in comparison to their peripheral counterparts ( ) . one study published in described a small percentage of cd + t cells found in uncomplicated term decidua to be viral specific. though these populations of viral specific cd + t cells were . % and . % in the decidual basalis and decidual parietalis respectively, they demonstrated that this was higher than that seen in peripheral blood and postulated a role for their presence in the decidua as one of immunoprotection for the fetus. this study could not conclude upon the origin of these t cells, and whether they were recruited from the periphery or activated in the decidua. in addition, more work remains to be done to establish whether these virus specific cd + t cells exist early in pregnancy ( ) . another study has described the presence of a small population of cd + hla-g+ t cells which are thought to acquire hla-g through trogocytosis from decidual dendritic cells. it is thought that these t cells promote immunotolerance at the maternal-fetal interface, and they have been shown to be downregulated in pathologies such as preeclampsia (pe) ( ) . therefore, it appears t cells play specific roles in immunity and tolerance. to this end we will look at the role that various populations of t cells may play in either enabling or preventing infection by torch pathogens at the maternal-fetal interface. macrophages constitute - % of all leukocytes in the first trimester decidua and play an important role in tissue remodeling, angiogenesis, host defense and immunotolerance ( ) . macrophages are considered a key link between adaptive and innate immunity, communicating to other immune cells and modulating their activity ( , ) . these cells are therefore vital throughout pregnancy, adapting their phenotype to address the changing requirements of the evolving decidua ( ) . tissue resident decidual macrophages are thought to be recruited from monocytes in the peripheral circulation ( ) . distinct subtypes of macrophages have been shown to be present in first-trimester decidual tissue exhibiting immunomodulatory, proinflammatory, and tissue remodeling phenotypes and play key roles in protective immunity as well as fetal tolerance ( ) . decidual macrophages are known for their highly immunosuppressive phenotype at the maternal-fetal interface, expressing cd , dc-sign and tim- among other receptor markers ( , ) . in addition to these maternally derived macrophages exist fetal-derived macrophages called hofbauer cells (hcs), which sit in the stroma of the chorionic villi ( ) . these hcs are resident in close proximity to fetal vessels and trophoblast cells from the first trimester until birth. hcs could serve as a portal of entry for pathogens from the infected mother ( ) . initially during implantation, they appear to have an inflammatory m phenotype which has both microbicidal activity and promotes a cell-mediated th cytokine response. later, they shift to a mixture of both m and m phenotypes following trophoblastic invasion and remodeling ( , ) . several studies have implicated hcs in host viral interactions. here, we look at the reciprocal interactions between hcs, maternal macrophages, and hcmv and zikv. the nk cell population in the peripheral circulation is predominately made up of cd dim cd + cells, which are believed to have a more cytotoxic phenotype ( ) . approximately % of the peripheral circulation is constituted by cd bright nk cells, which have a more immunotolerant phenotype ( ) . in the decidua, these nk cell proportions are reversed; - % of the total lymphocytes are cd bright cd - ( ) . research has demonstrated a number of dnk subsets within the cd +cd -population. it is believed that this distinct immunotolerant population is fundamental to the maintenance of a successful pregnancy, with research postulating both an ability to enable the semiallogenic fetus to thrive while at the same time responding to pathogenic infections. these nk cells reside in the decidua basalis close to invading evts and express specific receptors (e.g. kir receptors, cd /nkg a, ilt ) to activate or inhibit evt function ( ) . this large population of dnk cells are known to be sustained during the first and second trimester, with their numbers declining toward term ( , ) . despite the unique immunotolerant phenotype demonstrated by dnk cells, it is evident that this cell population displays a high level of plasticity, gaining cytotoxic function in the presence of specific pathogens ( ) . one way by which this happens is through activation of dnk cell cytotoxcity via killer cell ig-like receptor ds (kir ds ). reduced expression of this receptor has been associated with adverse pregnancy outcomes such as miscarriages and fetal growth restriction and individuals with increased kir ds expression have shown better outcomes post-viral infections ( ) . we will explore further the role that nk cells play in specific viral infections in pregnancy torch pathogens hcmv human cytomegalovirus (hcmv) was first described in by margaret smith, who replicated a virus from two newborn babies who had died from cytomegalic inclusion disease (cid) ( ) . what we now know as hcmv first came to the attention of ribbert et al. in , where intranuclear inclusions within large cells were noted in renal and parotid gland cells of stillborn fetuses. these inclusions, often described as 'owl's eye inclusions', were noted to be surrounded by a clear halo ( ) . hcmv was identified in the s when smith, weller and rowe isolated and cultured hcmv from salivary glands, adenoid tissue and liver biopsies respectively ( , ) . mechanisms of vertical transmission of hcmv can either be transplacental during gestation or transvaginal during parturition; additionally, there is some evidence for breastmilk transmission ( ) . hcmv infection is most likely to occur in the third trimester, demonstrating a % risk of mother to child transmission in the first trimester compared to a % risk in the third trimester ( ) ( ) ( ) . congenital hcmv has been estimated to affect - in every , live births, with % of hcmv positive infants suffering neurological consequences from birth ( ) . hcmv infection during pregnancy therefore poses a substantial risk to the developing fetus, leading to congenital disease including cerebral abnormalities such as periventricular calcifications, microcephaly, visual impairment, sensorineural hearing loss, neurodevelopmental delay and hepatomegaly ( ) . congenital hcmv affects , - , pregnancies annually in the united states and accounts for % of all incidents of pediatric sensorineural hearing loss ( ) ( ) ( ) . it is estimated that the burden of morbidity associated with congenital hcmv infection is greater than that of other common congenital pediatric conditions such as down's syndrome or fetal alcohol syndrome ( ) ( ) ( ) . hcmv is also associated with intrauterine growth restriction and miscarriage. there is a great need to understand maternal immunity pathways involved in hcmv infection to develop effective vaccines ( ) . hcmv is associated with asymptomatic infection of most of the world's population and subclinical illness in pregnant mothers. in the us, an estimated % of unexposed pregnant women experience primary infection during pregnancy, resulting in congenital infection in % of cases from this population ( , ( ) ( ) ( ) ( ) ( ) . however, vertical transmission of hcmv is not only seen in mothers with primary infection but also igg seropositive mothers, who exhibit a % rate of congenital hcmv infection. mechanisms of infection have been studied through analysis of placental tissue from all three trimesters of human gestation. in placental tissues from those suffering from hcmv, necrosis and oedema has been noted associated with severity of congenital disease symptoms. it has also been noted that hcmv infection is often associated with bacterial coinfection with a potentially pathogenic synergism ( ) . hcmv resides in the chorionic villi, specifically infecting ctbs, stbs and hcs. it is believed that the ability to travel between stbs in the decidua is key to hcmv pathogenesis ( ) . many studies have explored the role of the adaptive and innate immune system in hcmv infection. below we review established interactions between hcmv and immune cells (figure ). hcmv's ability to infect different populations of macrophages has been demonstrated by several studies. hcmv has been shown to be sequestered by hcs, with placentas from confirmed cases of hcmv infection demonstrating significant hyperplasia of this cell population ( ) ( ) ( ) . a study investigating vaccine development showed that when neutralizing antibodies are produced against hcmv, rates of hcs infection are decreased ( ) . a different study utilizing placental explants showed hcmv-igg immune complexes to undergo fc receptor mediated transcytosis as a mechanism to traverse the syncytium to ctbs. hcmv is then taken up by hcs in the placental villi ( ) . furthermore, another study by loenen et al., supports the idea that hcmv genes are able to increase fcr expression on infected cells ( ) . another study suggested that hcmv replication in stbs is upregulated in the presence of macrophages ( ) by analyzing hcmv replication in stbs alone or when infected stbs were cultured with uninfected placental macrophages. this study also demonstrated elevated levels of hcmv viral titres in co-cultured supernatants when compared to those from stbs cultured alone. this demonstrates that not only do macrophages have the capacity to be infected by hcmv, but also that they may amplify hcmv infection of surrounding cells in the decidua. some studies have depicted a role for latently infected maternal decidual macrophages in congenital hcmv infection, describing how microbial infections or insults in the placenta may reactivate these macrophages and in turn reactivate hcmv infection ( ) ( ) ( ) . the maternal-fetal interface is unique in respect to allogenic interactions with cd + t cells. evts are known to invade the decidua, evading destruction despite the intrinsic ability of cd + t cells to recognize foreign antigen via mhc class i molecules. as discussed previously, one mechanism by which evts are believed to evade cd + t cell recognition is through a lack of expression of hla-a and hla-b, which are key to cd + cytotoxic activity. during pregnancy, many viruses have been shown to upregulate maternal cd + t cell activity, leading to migration of highly differentiated effector memory t cells to the decidua. despite many descriptions regarding the role of t cells in hcmv infection in the fetus and the mother, there are few studies identifying their tissue specific role at the maternal-fetal interface ( ) . hcmv is thought to limit cd + t cell activity through restriction of mhc class ii expression on apcs, which in turn may prevent activation of cd + and cd + t cells ( ) . this is thought to be mediated through the hcmv protein gpus , which may degrade mhc class ii glycoproteins or disrupt downstream ciita/jak/stat signaling pathways ( ) . crespo et al., in demonstrated that hcmv did not induce a significant difference in hla-g expression on either jeg- cells or primary evts. hla-g expression has been associated with immunotolerance, and therefore its persistence despite infection may act to protect infected trophoblast cells from cytotoxic destruction ( ) . studies looking at the role of t cells in viral infection at the maternal-fetal interface demonstrated lower t-cell numbers and response in mothers who vertically transmitted hcmv to their offspring when compared to infected mothers who did not transmit hcmv, potentially suggesting an active role for t cells in vertical hcmv transmission ( ) . more specifically, a reduction in the number of cd +cd ra +ifn-g+ treg cells and cd +cd ra+ifn-g+ t cells in mothers who transmitted hcmv to their fetus was noted when contrasted with mothers who were hcmv positive but did not transmit the infection. there was also a measurable blunted t cell response in hcmv infected mothers who vertically transmitted infection, compared to infected mothers who did not transmit the virus ( , ) . in infected mothers, hcmv virus specific t cells have been shown to be elevated in the final trimester when compared to uninfected mothers ( ) . congenital hcmv infection risk is highest for the fetus in the third trimester, with a % transmission risk compared to a % risk in first trimester ( ) . this is despite the abundance of immune cells, specifically nk cells, in early pregnancy. dnk cells exposed to hcmv infected decidual fibroblasts are known to alter their phenotype to express higher levels of activating receptors (such as nkg d and cd /nkg c or nkg e). uniquely, utilizing in vitro studies, it was noted that decidual nk cells had targeted cytotoxic activity against hcmv infected autologous decidual fibroblasts and heterologous uninfected fibroblast cells, but appeared to spare trophoblast cells ( , ) . this demonstrates a clear cytotoxic effector response by decidual nk cells to hcmv, switching from their typically immunotolerant phenotype with high levels of inhibitory receptor expression (cd /nkg a, lir- , kirs), to a cytotoxic phenotype ( , ) . this group also studied the interaction between dnk and hcmv-infected cells using hcmv positive and hcmv negative decidual villous explants. this investigation revealed through fluorescent staining of dnk cells that colocalisation of dnk cells to cells throughout the hcmv positive explant occurred, including synaptic connections which was not seen in hcmv negative explants. this was thought to suggest that the dnk cells were unable to connect with uninfected trophoblasts. this also demonstrates that dnk cells are able to localize and target hcmv infected cells while sparing fetal derived semiallogenic trophoblast cells ( ) . dnk cells are unique in their function, both contributing to immunotolerance at the maternal-fetal interface, thereby enabling invasive trophoblastic activity, as well as controlling pathogenic infection ( ) . this is thought to be mediated by secretion of specific cytokines ( , ( ) ( ) ( ) ( ) . the relatively limited vertical transmission of hcmv during the first trimester of pregnancy, when the population of nk cells is abundant, has led many to speculate about the role nk cells may play in hcmv control ( ) . tilburgs and colleagues have recently demonstrated distinct cytotoxic responses in dnk cells to hcmv in first trimester versus at term wherein term pregnancy dnks harbor reduced efficacy in responding to hcmv-infected cells ( ) . siewera et al., suggested that dnk cells undergo a phenotypic transformation to acquire cytotoxic function in the presence of hcmv-infected cells ( ) . this study proved, through antibody mediated abrogation of the fas ligand (fasl) and tumor necrosis factor-related apoptosis-induced ligand (trail) on dnk cells, that death of hcmv infected cells is not initiated by dnk cells through these death receptor-ligand pathways. however, this study demonstrated that dnk cells form immunological synapses with hcmv infected fibroblasts, enabling the delivery of perforin/granzyme for cellular destruction. furthermore, the ability of dnk cells to degranulate in the presence of hcmv infected fibroblasts was demonstrated to be through high levels of cd a expression, a key cell surface molecule in the mechanism of lytic granule release. dnk cells have also been found to secrete higher quantities of granulysin when compared to peripheral blood nk cells. upon incubation with infected fibroblast cells, it was noted that cd bright nk cells decreased from . % to %, while there was an elevation in cd expression by nk cells, denoting a transformation to a cytotoxic phenotype. hcmv infected cells have been noted to upregulate expression of natural cytotoxicity receptor (ncrs) nkp by almost -fold on dnk cells as well as increasing expression of nkg c. ncrs are associated with activation of the cytotoxic profile of nk cells. accompanying this was a reduction in nkg a, kir dl , kir dl , and ilt receptor expression, receptors aligned with nk effector inhibitory function ( ) . activating dnk cell receptors such as kir ds , kir ds , kir ds and kir ds have been correlated with antiviral activity ( ) . a study by crespo et al., demonstrated an increased population of kir ds + nk cells in the decidua, suggesting an increased activating dnk cell capability in response to hla-c , and thereby increased cytotoxic potential. these cells also displayed higher levels of cytolytic molecules when compared to peripheral nk cells. this study demonstrated that kir ds + dnk cells showed increased cytotoxicity to hcmv infected decidual stromal cells (dscs) positive for hla-c when compared to kir ds -dnk cells. this was not the case for infected jeg- and primary evt cells, which did not appear to initiate degranulation or cytokine secretion from dnk cells. despite this, a reduction in the number of infected evts in the presence of co-cultured dnk cells was noted, suggesting that dnk may be clearing virus infected evts by other means ( ) . hcmv has been seen to reduce expression of mhc class i, thereby potentially evading cd + t cell destruction ( ) ( ) ( ) . one study reports an initial reduction in hla-c expression on evts in hcmv infection. the possible reason for this is not clear, however this study suggests it could prevent inhibition of nk cells through the hla-c/kir dl route, with an additional suggestion of potentially other unknown ligands being upregulated for activation of kir ds , leading to cytotoxic action against infected cells ( ) . another study showed that the potential effect of dnk cell activation on t-cell activation could be mediated via an upregulation in hla-dr expression upon exposure to hcmv infected fibroblasts ( ) . therefore, dnk cells may play a role in congenital hcmv infection by potentially protecting the first trimester fetus from infection via activation of t cell function. collectively, these studies indicate varied interactions between dnk cells and hcmv, with many routes by which hcmv may evade clearance as well as a number of ways through which dnk cells may be activated in the presence of hcmv infected cells. additionally, dnk cells are seen specifically to modulate activity in the context of t cell activation. zika virus (zikv) was first isolated in in zika forest, uganda, from infected rhesus monkey serum during epidemiological yellow fever research ( , ) . however, the first case of human infection was not reported until , when three patients presented with jaundice and were later confirmed to have rising levels of zika antibodies ( ) . initially, zikv was associated with innocuous prodromal illness on the african and indian subcontinents transmitted by the aedes aegypti mosquito, leading to an asymptomatic or self-limiting course of infection ( ) . in , a mild disseminated infection was identified to be zikv in over % of the population of the island of yap ( ) . concerns regarding human zikv infections were not aroused until when incidences of neurological deficits associated with zikv infection were first described, with almost , recorded infections noted in french polynesia ( , ) . shortly following this in , a zikv epidemic began in south america where not only were neurological deficits such as guillain-barreś yndrome seen, but also spontaneous abortion and congenital malformations such as microcephaly in infants from infected mothers ( ) . by the end of the epidemic in brazil, there were more than , notifications of zikv cases ( ) . estimates for infants born with congenital zika syndrome (czs) after the - epidemic ranged from to in every , births ( ) . the threat of a zikv epidemic lingers, with who reporting countries affected by aedes aegypti mosquitoes, therefore carrying the potential for zikv infection and transmission ( ) . zikv demonstrated continuing global epidemic capacity in india in ( ) . zikv belongs to the flavivirus family alongside west nile virus, dengue virus, and yellow fever virus. zikv is an enveloped and icosahedral virus with a nonsegmented, . kb single stranded positive sense rna genomes ( ) . this virus is composed of several proteins, categorized as three structural (capsid, pre-membrane and envelope) and seven nonstructural proteins. the seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) are essential for viral replication and assembly, as well as being responsible for the pathogenicity of the virus by binding to transcription and restriction factors ( ) . the biggest risk of congenital zikv infection is for mothers infected during their first trimester ( ) . zikv infection demonstrates wide tissue tropism, with zikv successfully infecting the central nervous system, blood, retinal, genital and reproductive tissues including placenta ( ) ( ) ( ) ( ) ( ) . zikv was thought to be exclusively arthropod transmitted until cases of human-human transmission emerged in nonendemic regions, illustrating a role for sexual transmission ( ) ( ) ( ) . the presence of zikv rna has also been found in breast milk of zikv infected mothers ( ) ( ) ( ) . however, there are reports which suggest that vertical transmission of zikv by breastmilk does not occur in most cases, which suggests the possibility that breastmilk does not have a high enough viral load to infect the newborn ( , ) . despite some knowledge regarding zikv pathogenesis, its mechanism of infection in placental immune cell types remains limited ( ) ( ) ( ) ( ) . histopathology of zikv infected placentae has shown zikv infection in first trimester villous stromal tissue cells, which includes immune cells in the chorionic villi ( , , ) . uniquely, zikv was also found to infect ctbs, endothelial cells, fibroblasts and hc in chorionic villi, as well as amniotic epithelial cells and trophoblast progenitor cells ( , ( ) ( ) ( ) ( ) ( ) ( ) (figure ). similar to hcmv, zikv has also been shown to infect hcs and ctbs ( , , ) . during the first trimester of pregnancy, zikv infects hcs, entering the fetal blood stream in order to reside in the placenta. zikv uses hcs as a "trojan horse". this strategy is utilized by several viruses in order to cross the blood brain barrier, where the virus infects leukocytes, leading to them being carried across barriers and thereby enabling the propagation and spread of infection ( ) ( ) ( ) . hcs have been associated with zikv spread to the fetus through the "trojan horse" route ( ) . the presence of zikv-specific antigen was demonstrated in hcs in confirmed maternal infection. multiple studies suggest hcs are a crucial step in vertical transmission of zikv to fetal cells, demonstrating that hcs are preferentially infected when compared to ctbs ( , , ) . infection of hcs with zikv is thought to propagate infection through hyperplasia and proliferation of these cells, leading to persistence of this hc population into later trimesters ( , , ) . a study performed on first trimester fetal and maternal tissue showed that zikv can replicate in different cell types, such as decidual fibroblasts and macrophages. it can also infect trophoblasts and hcs as well as umbilical cord mesenchymal stem cells, suggesting that the route of zikv infection may move from the decidua basalis to the anchoring villi ( ) . a study performed using blood from + asian zikv infected pregnant women shows that cd + monocytes are the primary target of zikv infection. these monocytes are resistant to change in m phenotype and downregulate type ifn signaling, which induces the expression of different host genes involved in pregnancy complications ( ) . in the decidua basalis, zikv infects evts, macrophages and stromal cells. zikv also targets proliferative ctbs in the anchoring villi, however is unlikely to infect stbs due to ifnl-mediated antiviral defense mechanisms ( ) . zikv achieves replication within macrophages through fcr, tlr and dc-sign receptors ( , ) . in vitro studies have demonstrated zikv infection to be augmented in hcs by igg from prior flavivirus exposure through antibody dependent enhancement (ade) ( ) . there remain many gaps in knowledge regarding the role for macrophages targeted and infected by zikv. a study performed using decidual and chorionic villous tissue from early and mid-gestation human pregnancy shows that zikv appears to elevate type i and iii ifn expression, which does not occur in hcmv infection ( ) . studies looking at the interaction between zikv and t cells in humans are scarce although zikv infection has been demonstrated to activate both cd and cd t cells ( ) with specific increases in vd tcr+ cells which have been implicated in recurrent miscarriages but not associated with zikv-induced fetal complications. there have not been notable studies looking specifically at t cell zikv communication at the human maternal-fetal interface ( ) . a recent study examined peripheral t cell responses of confirmed cases of zikv infection that had been stimulated with pooled zikv peptides from all viral components ( ) . this study demonstrated responses from both cd + and cd + t cells to both structural and nonstructural zikv components. however, this study particularly showed that cd + t cells exhibited a strong response to nonstructural proteins ns , ns and ns , and cd + t cells a strong response to cap and env proteins. this response was demonstrated by marked ifn-g production from both cell subtypes indicating cell activation ( ) . another case looking at a zikv infected individual from the united states demonstrated interactions between the zikv ns and env proteins with cd + and cd + t cells, respectively. ( ) . furthermore, in a different study, cd + t cells of two a b d c fibroblasts occurs through fcgr and results in increased expression of some interferons, such as ifna, and decreased expression of others, such as ifn-g and ifnb. infection is associated with increased secretion of proinflammatory cytokines such as ip- , il- , and mcp- . ns viral proteins are thought to downregulate interferon-stimulated genes (isgs) and reduce interferon signaling via stat degradation, while the viral proteins ns and ns b inhibit ifn signaling by downregulating tbk . (c) cd + t cells exhibit a strong response to nonstructural ns , ns , and ns zikv proteins, while cd + t cells respond to cap and env zikv proteins. in both cases, response to zikv proteins was characterized by increased ifn-g production. (d) systemic dnk cells exhibit increased activation, including increased ifng production and cd a expression when incubated with zikv infected monocytes. frontiers in immunology | www.frontiersin.org october | volume | article zikv infected individuals showed activity in response to nonstructural proteins (ns , ns and ns ). consistently, cd + t cells were seen to raise activity against the structural protein env ( , ) . these studies demonstrate consistency in the response of cd + t cells and cd + t cells to zikv proteins, revealing cd + t cells to specifically respond to particular nonstructural proteins and cd + t cells to react to structural proteins, particularly cap and env ( , , ) . several studies have looked at the ability of denv-specific cd + and cd + t cells to be stimulated by the presence of zikv peptides in humans ( , ) . these studies showed viral epitopes for specific peptides located in similar regions and structurally conserved across flaviviruses; however, they displayed differences in their sequences ( ) . nonetheless, these studies indicated cross-reactivity between the viruses regarding their cd + and cd + t cell activity. one study demonstrated that cd + and cd + t cells from denv positive donors reacted to zikv viral peptides, resulting in an upregulation of ifng secreting cells. this group also showed that stimulation with zikv peptides for those in acute phase of zikv infection resulted in recruitment of elevated levels of cd + ifn-g+ t cells ( ) . a recent transcriptomics study investigated transcriptional signatures in cd /cd t cells, b, and nk cells and plasmacytoid dendritic cells in patients (nonpregnant) infected with zikv ( ) . interestingly, they did not note significance transcriptional changes in nk or cd t cells in a zikv infected background but noted significance alterations in pdcs. whether pregnancy plus zikv infection would affect the immune cell transcriptome in humans remains to be determined. studies specifically analyzing interactions between dnk cells and zikv in humans once again are lacking. however, studies have looked at zikv and its communication with peripheral nk cells. one such study postulated crosstalk between monocytes and nk cells in zikv infected patients. the activation of nk cells was associated with the presence of monocytes, which induced expression of ifn-g and cd a, key markers of nk cell function. depletion of monocytes in the peripheral blood reduced the levels of these markers and thus the activation of nk cells ( ) . there are few studies showing the interaction between zikv with nk cells. glasner et al., showed that zikv infection led to activation of mhc class i, which was somehow not sensed by dnk cells and their activating receptors, allowing the virus to escape nk cell-mediated killing. mhc class i expression is triggered through the ifn-b pathway via activation of rigi-irf ( ) . however, the mechanism by which nk cells may promote an immunosuppressive environment in the face of zikv infection is not clear. some studies have indicated that interactions between other aspects of the innate immune system and nk cells may be at play in zikv pathogenesis. there are several studies suggesting that pathogenesis of zikv is not mediated through decidual immune cells alone but rather conducted, at least in part, through the activation of interferonstimulated genes (isgs), which in turn leads to activation of innate host cell immunity ( ) . isgs act to specifically target viral replication. multiple studies have indicated zikv stimulation of interferons (ifn) to vary depending on the type of ifn. while type i and iii ifns have been shown to be inhibited by zikv, specifically the ns component of the pathogen, type ii ifns have been shown to be upregulated by the virus ( , ) . one study demonstrated that when type iii ifns were upregulated, specifically ifn-l , trophoblast cells were infected with zikv at a lower rate. further, ns , ns a and ns b have been demonstrated to inhibit ifn type i response. this leads to suppression of the tank binding kinase (tbk )/irf and jak-stat pathway, which in turn results in reduced activation of innate immune responses ( ) . interferon induced transmembrane protein (ifitm ) and ifitm specifically are isgs which act as restriction factors to inhibit zikv replication. the mechanism by which the inhibition and activation of innate immunity impacts the recruitment of innate immune cells to the site of infection is not clear. little is known about the role of nk cells in human zikv infection. one study has noted interactions between tlr , cd and ifitm , postulating that the restriction of zikv is associated with inhibitory activity of ifitm , potentially through activation of nk cells ( , ) . another group looking at isgs showed that viperin played a role in zikv pathogenesis, with data revealing that when viperin levels were high, zikv mrna levels were low and vice versa ( ) . ns is seen to target directly the akt-mtor pathway, leading to reduced signaling from this pathway and subsequent activation of autophagy in host cells ( ) . zikv has been shown to co-opt the autophagy pathway for post-rna replication capacity and survival ( , ) . importantly, the ns b-ns protease activity of zikv can be blocked by an inhibitor of autophagy, hydroxychloroquine (hcq) ( ) . hcq is an fda approved drug considered safe to use during pregnancy and could serve as an effective treatment for preventing zikv congenital syndrome ( ) . the relationship between zikv infected cells and attenuated ifn production has been extensively reported, leading to questions regarding the mechanism underlying this association. it has been proposed that zikv may infect cells through ade of infection. many cells express the fcg receptor, and it is thought that viral particles may complex with antibodies and thereby enter into cells via fcg receptors ( ) . host cells (such as trophoblasts and fibroblasts) infected with zikv demonstrate innate immune system activation with a rise in specific ifns (e.g. ifn-a), but falling levels of others such as ifn-l and ifn-b ( ) . the elevated levels of proinflammatory cytokines and chemokines, namely il- , mcp- and ip- which are linked to recruitment of immune cells such as monocytes and t cells ( ) . zikv has been shown in multiple studies to downregulate type i ifn signaling and to be active in suppression of antiviral signaling. zikv nonstructural proteins ns and ns b inhibit ifn signaling by downregulating levels of tbk . however, ns b downregulates the jak-stat pathway and inhibits apoptosis of zikv, and hence inhibits innate antiviral responses ( ) . one study specifically has implicated the role of the nonstructural zikv protein ns in promoting zikv propagation by targeting stat for degradation, thereby reducing isg levels ( ) . this is thought to promote viral replication through a dampened host innate immune cell response. there remains much to be elucidated in terms of zikv infection in human pregnancy. new studies are identifying metabolic reprogramming pathways underpinning innate immune responses to zikv which opens additional avenues of investigation ( ) . we refer readers to recent reviews highlighting zikv-immune interactions in adverse pregnancy outcomes ( ) and ade ( ) . the limited availability of placental tissues during early pregnancy has always been a challenge for the reproductive biologists, hampering the study of placental physiology and cell to cell interactions. in vitro cell line models can often be biologically distinct and therefore unable to demonstrate enough similarity to replicate the conditions of human pregnancy. in addition, the use of cell line models can fail to reproduce the complexity of the number of cell types and cell interactions present within the decidua. therefore, functional in vitro d models being are developed, for example placenta-on-a chip and organoid cultures, which can mimic in vivo conditions and would be useful to understand the mechanisms of viral host interactions. the 'placenta-on-a-chip' is a microfluidics model utilizing human trophoblast cells (bewo) and fetal derived cells (huvecs and hpvecs) ( , ) . these cell lines are cultured and separated by a semipermeable membrane within flow conditions with the purpose of understanding placental mechanisms and barrier function ( ) . recent reports have described the faithfulness of placenta-on-a-chip model to in vivo placental conditions ( ) . for example, glucose transport using a placenta-on-a-chip model was demonstrated by lee et al., and blundell et al., highlighting significant similarity to in vivo glucose transport in the human placenta ( , ) . placenta-on-a-chip models have also been used to investigate the transport of heparin and anti-hyperglycemic agents such as glyburide using bewo and human placental villous endothelial cells ( ) . recently, the transport of the xenobiotic compound caffeine across the placenta has been studied using this model system, providing new insights into the extent of caffeine transfer from mother to fetus ( ) . bacterial infections have also been studied using this model. zhu et al., showed that in the presence of escherichia coli (e. coli), trophoblast cells (bewo) activated the circulating macrophages on the "maternal" side of the chip to secrete several inflammatory cytokines that mimicked in vivo conditions during pregnancy ( ) . the impact of common environmental exposures such as titanium dioxide nanoparticles (tio -nps) has also been studied using this d placental model showing a series of different placental responses (barrier permeability, oxidative stress, cell apoptosis, and maternal immune cells behavior ( ) . they showed placental barrier permeability and maternal immune cells to be influenced by even low concentration of nps ( ) . therefore, this simple in vitro model can prove useful in understanding the environmental exposure of nps during pregnancy and can help in a range of biological studies ( ) . recent studies report generation of an organ-on-a-chip model, wherein decidualized human endometrial stromal cells and macrophage cell lines are co-cultured in a microfluidic device and shown to inhibit secretion of tnf-a in response to lps stimulation ( ) . these devices have also been used to determine the impact of cytokine secretion by dnk cells on the migration of primary trophoblast cells. these studies illustrate the functionality of microfluidic organ on chip devices to elucidate importance of maternal immune cells in the placenta ( ) . thus, the use of fetal membrane on organ-ona-chip provides a suitable model to explore the impact of pathogenic infections during pregnancy ( , ) . the use of in vitro trophoblast organoids as a d culture model also provides a new tool to understand the mechanism of implantation at the maternal-fetal interface. recent studies have shown the characterization of these organoids derived from sttrimester ctbs ( to weeks) and suggest their resemblance to primary trophoblast cells ( ) ( ) ( ) . due to similarity with the placental architecture, these organoids could be used to study physiological, metabolic and hormonal changes that occur during pregnancy. the viruses we highlighted in this review, hcmv and zikv, do not naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. in particular, there remains a paucity of understanding zikv-immune cell interactions during pregnancy. thus, the employment of placenta-on-a-chip or organ-on-a-chip, and organoid models will be pivotal in providing functional and physiologically relevant ways to study the interaction of immune cells at the maternal-fetal interface with viral pathogens that affect pregnancy. both hcmv and zikv can be sequestered into fetal macrophages. hcmv implicates hcs in the potential infection of other decidual cells, leading to the promotion of hcmv transcytosis in trophoblasts. zikv preferentially infects hcs, persisting in this cell population and potentially mediating infection of other fetal-derived cells. more poignant is the suggestion that decidual macrophages may mediate reactivation of hcmv by acting as a latent reservoir for infection. these studies collectively indicate a central role for macrophages in the pathogenesis of torch viruses. dnk cells have been seen to alter their phenotype to express higher levels of various activating receptors when in the presence of infected decidual fibroblast cells. they also are known for their plasticity in the face of specific pathogens, acquiring more cytotoxic function. kir dl /hla-c has been identified as a mechanism by which dnk cells are activated and display cytoxicity toward hcmv infected cells. it has also been suggested that dnk cell activation may trigger activation of t-cells through upregulating hla-dr expression on infected fibroblast cells. zikv viral components demonstrate capacity to elicit strong responses from peripheral cd + and cd + t cells, with ns , ns , and ns being associated with cd + stimulation whereas cap and env proteins being associated with cd +. we also see in this review the importance of interferon stimulating genes in the restriction of zikv replication. thus, the implications and outcomes of viral interactions with immune cells at the maternal-fetal interface are varied. we see the importance of the host immune response and recognize the importance of studying mechanisms of pathogenesis in detail to enable targeted therapeutic interventions including vaccines to mitigate the adverse outcomes of viral infections during pregnancy ( figure ) . finally, we posit that better understanding of the immunological underpinnings of infections at the maternal fetal interface can support the inclusion of pregnant women in trials testing vaccines and therapeutics to compact existing and emerging viral infections. ep, sv, and im wrote 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interactions self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta trophoblast organoids as a model for maternal-fetal interactions during human placentation derivation of trophoblast stem cells from naive human pluripotent stem cells conflict of interest: im serves on the scientific advisory board of luca biologics.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © parker, silverstein, verma and mysorekar. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -yrt fq m authors: pinchoff, jessie; serino, arianna; merritt, alice payne; hunter, gabrielle; silva, martha; parikh, priya; hewett, paul c. title: evidence-based process for prioritizing positive behaviors for promotion: zika prevention in latin america and the caribbean and applicability to future health emergency responses date: - - journal: glob health sci pract doi: . /ghsp-d- - sha: doc_id: cord_uid: yrt fq m since the zika outbreak in latin america and the caribbean, a plethora of behavior change messages have been promoted to reduce zika transmission. one year after the united states agency for international development (usaid) initiated its zika response, more than variants of preventive behaviors were being promoted. this situation challenged social and behavior change (sbc) programming efforts that require a coordinated response and agreed upon set of focus behaviors to be effective. to support usaid implementing partners in harmonizing prevention efforts to reduce zika infection, we developed an evidence-based process to identify behaviors with the highest potential to reduce zika infection and transmission. we compiled a full list of behaviors and selected the most promising for a full evidence review. the review included systematic keyword searches on google scholar, extraction of all relevant published articles on aedes-borne diseases between and , review of seminal papers, and review of gray literature. we examined articles to determine each behavior's potential effectiveness in preventing zika transmission or reducing the aedes aegypti population. we also developed assessment criteria to delineate the ease with which the target population could adopt each behavior, including: ( ) required frequency; ( ) feasibility of the behavior; and ( ) accessibility and cost of the necessary materials in the setting. these behaviors were refined through a consensus-building process with usaid's zika implementing partners, considering contextual factors. the resulting evidence-based preventive behaviors have high potential to strengthen sbc programming's impact in usaid's zika response: ( ) apply mosquito repellent, ( ) use condoms during pregnancy, ( ) remove standing water, ( ) cover water storage containers, ( ) clean/remove mosquito eggs from water containers, ( ) seek antenatal care, and ( ) seek family planning counseling. this case study documents a flexible process that can be adapted to inform the prioritization of behaviors when there is limited evidence available, as during many emergency responses. z ika virus is a communicable disease primarily transmitted by the aedes aegypti mosquito, a vector that also transmits other arboviruses including dengue, chikungunya, west nile virus, and yellow fever. the first outbreak of zika detected in the americas occurred in , with a spike in suspected congenital malformations and other neurological complications such as guillain-barré syndrome. by august , , there were approximately , confirmed zika cases, and about , cases of associated congenital zika syndrome. zika is now considered endemic throughout latin america and the caribbean (lac), parts of africa, and asia. between % and % of zika infections are asymptomatic according to the u.s. centers for disease control and prevention (cdc). infection during pregnancy is linked to congenital zika syndrome in newborns, which is characterized by severe microcephaly (small head size), decreased brain tissue mass, and subcortical calcification. other health abnormalities, including developmental delays, associated with the zika virus have been reported. research on the impact of the virus on mothers and children is ongoing. the outbreak in lac demanded a concerted regional response, given the wide distribution of the mosquito vector, the lack of population-level immunity, the absence of a vaccine or rapid diagnostic test, uneven access to water due to low quality water and sanitation infrastructure, water shortages, lack of information about the disease, and inadequate health systems to respond to the health impacts. on february , , the world health organization declared zika a public health emergency of international concern. the united states agency for international development (usaid) and other u.s. government entities and international partners began working together through existing country systems to reduce the risk of new zika infections, particularly in pregnant women, and to provide care for those affected through interventions in vector control, social and behavior change (sbc), and health service delivery. the focus for this process was sbc for individuals, households, and communities in zika-affected regions, only. usaid's sbc programming was comprised mainly of mass and social media, community engagement, and interpersonal communication, with the goal to "raise awareness, reduce misinformation, and address the barriers that prevent individuals, families, and communities from practicing lifesaving behaviors to improve health outcomes." the sbc literature suggests that behavior change is more likely to occur when clear and concise messaging is repeated frequently through multiple channels. [ ] [ ] when too many preventive behaviors are promoted or messages lack precision, adopting prevention behaviors can be inhibited or done in a way that is either ineffective or counterproductive. messaging can be particularly challenging during emergency responses when data may be unavailable to inform programming and time constraints inhibit collective planning, leading to the promotion of messages before a concerted and harmonized response can be organized. in a non-systematic, rapid desk review of sbc messages approximately year after the usaid zika response began, we identified more than variants of prevention behaviors that were being promoted. the prevention messages for these behaviors were not consistently presented, lacked cohesion in their packaging, and offered little specificity regarding how the behavior should be implemented to effectively reduce zika infection and transmission. too many behaviors with insufficiently specific instructions in the messages could have resulted in confusion, information overload, and incorrect performance of the behaviors among individuals and communities. the behaviors promoted were also not always based on available evidence around their effectiveness in relation to zika transmission. facing an outbreak of a disease new to the americas, public health institutions and organizations found themselves conducting research while simultaneously launching interventions and programs. at the time programs were rolling out, there were limited data to guide sbc programming and messaging for the most effective preventive actions for individuals and communities. these circumstances often led to a lack of cohesion in promoted behaviors and sbc messages. to more effectively coordinate the zika response among implementing partners and increase the rate of behavior adoption among target populations, the breakthrough action þ research projects, in collaboration with usaid, developed an evidence-based process to identify priority behaviors with the highest potential for preventing zika acquisition and transmission. stakeholders across disciplines and involved in various levels of programming were engaged throughout to ensure buy-in, harmonize priority behaviors and their sbc messages, and ensure a more effective zika response. existing research could be leveraged because the transmission dynamics for zika were similar to other arboviruses and sexually transmitted infections; preventive behaviors targeting the vector (the aedes aegypti mosquito) and practices to reduce sexual transmission had already been identified in the literature and could be assessed for zika. understanding the transmission dynamics was critical to identifying behaviors to consider for prevention. the exercise, referred to as the "behavior prioritization process," focused on a range of individual-and household-level behaviors to reduce the risk of zika acquisition and transmission. messages were developed by partners based on the set of behaviors identified and prioritized in this process. establishing an evidence base and a refined set of preventive behaviors tailored to the specific context can greatly improve the success of sbc programming by reinforcing promotion of consistent behaviors and using evidence to add specificity to the desired actions. the process combined available evidence and a consensus-building approach to allow for adaptation based on local an evidencebased process identified priority behaviors with the highest potential for preventing zika. context. this article summarizes our experience in prioritizing the behaviors with highest potential for zika prevention, identifies specific target audiences for each behavior, and documents the design and implementation of the behavior prioritization process developed to achieve these aims in a flexible way. we also consider the applications of this process for strengthening future public health emergency responses. a list of more than zika preventive behaviors (or their variants) that usaid implementing partners across usaid-supported countries were promoting was compiled by informally reviewing numerous project materials and documents. all of these preventive behaviors were related to the transmission dynamics of zika virus-transmission by a vector (aedes aegypti) that also transmits arboviruses such as dengue and sexual transmission. a team of experts in sbc programming and vector control was enlisted to categorize and refine the behaviors. all of the zika prevention messages were first grouped together by behavior to create a condensed version of about behaviors. through an iterative review process including experts and discussions with partners, the list was distilled to key behaviors ( table ) . behaviors were excluded if they had limited effectiveness preventing aedes aegypti-borne diseases (such as zika) or reducing aedes aegypti mosquito populations (after a quick literature scan and input from experts) or due to other criteria. applying mosquito repellent (deet, picaridin, ir , or lemon eucalyptus oil, only), using each product as directed, for duration of pregnancy, to reduce risk of zika transmission through mosquito bites. application of mosquito repellent is highly efficacious in preventing mosquito bites, and thus the potential of vector transmission of zika to an individual. this behavior is within the control of pregnant women and their male partners. users should be thoroughly counseled on proper product application. women intending to become pregnant should also consider using repellent. using condoms to prevent sexual transmission of zika in pregnancy. condom use to prevent sexual transmission of zika is highly efficacious, but sexual transmission may be a small portion of overall transmission. this behavior should be prioritized for pregnant women and their partners because pregnant women are at risk for negative pregnancy outcomes. regularly removing unintentional standing water both inside and outside the house and in communal areas. this is a potentially efficacious behavior to reduce mosquito populations, and thus reduce the potential for individual-and population-level risk of zika transmission. promotion of the behavior must be accompanied by specific, focused instructions that target the highest density breeding sites and be conducted weekly in homes and communal areas to be effective. efficacy is highest in areas where there is strong community engagement, including active mosquito searches in homes and communities and awareness of the mosquito life cycle. covering water storage containers at all times with a tight-fitting cover that does not warp or touch the water. covering long-term water storage containers has moderate potential efficacy in reducing breeding sites if a tight-fitting, long-lasting lid is available. covering short-term water storage containers has less potential efficacy, as frequent lid use can result in wear and tear and render the lids ineffective or counterproductive. scrubbing walls of water storage containers weekly to remove mosquito eggs. scrubbing walls of water storage containers weekly is efficacious in removing mosquito eggs and can thus reduce the potential for individual-and population-level risk of zika transmission. however, the specific cleaning steps that eliminate mosquito eggs must be explicitly described. seeking antenatal care to monitor pregnancy and discuss zika risk and prevention. seeking antenatal care enables providers to counsel pregnant women on zika prevention, which can increase the chances of pregnant women taking protective measures and reducing the risk of vertical transmission of zika from mother to child. seeking counseling from a trained provider on modern family planning methods if not planning on getting pregnant. family planning use (for those not intending on getting pregnant) is directly linked to reducing the risk of vertical transmission of zika. family planning counseling should be done by a trained health care provider. in determining the most promising behaviors to review further, behaviors were excluded from the list if: . the behavior was largely outside the control of the individual or household (e.g., indoor residual spraying or applying larvicide, which require trained technicians). . there was limited evidence of the behavior's efficacy (e.g., bed net use as the aedes mosquito mainly bites in the daytime). . the behavior had only been implemented in a geographically limited pilot stage intervention (e.g., larvivorous fish in water storage containers). . the behavior was not supported by usaid (i.e., usaid was not procuring or distributing required materials to carry out the behavior) because of the lack of effectiveness of the behavior (e.g., bed nets are not considered effective for zika because of the vector behavior) or because it was not feasible there is also limited evidence that wearing regular clothing that has not been treated with insecticide is effective in preventing mosquito bites. application of larvicide while considered highly efficacious, larvicides should be applied by vector control technicians, rather than household members, so control over implementation of this behavior does not lie at the household level. larvivorous fish application of larvivorous fish to water storage containers is still in the pilot phase; limited data available on efficacy. additionally, usaid is not procuring larvivorous fish, and the behavior is outside the locus of household control since it is currently being done by vector control specialists who visit the home. indoor residual spraying this behavior is implemented by vector control technicians and therefore does not lie within the control of the household. there is limited literature on the efficacy of this intervention as it is traditionally only used for anopheline mosquitoes; some pilots are in progress to test for effectiveness for aedes mosquitoes. use of insecticide-treated curtains/screens there is some evidence that insecticide-treated curtains or screens are effective in preventing aedes abundance indoors; however, usaid is not procuring these. use of coils to repel mosquitoes efficacy appears limited upon initial review, with some studies even suggesting they increase dengue risk. planting basil plants while some research suggests that essential oils extracted from plants may have a repellent effect, no studies were identified that assess the repellent effect of basil plants. within the scope of the program (e.g., installing screens on windows). table lists the excluded behaviors and reasons for not including them. at this stage, behaviors were selected for a full evidence review that fell into the broad categories of ( ) personal protection and ( ) household and community vector control. a third category, prevention-enabling behaviors, was created for additional behaviors that did not undergo a full evidence review here but were still recommended. prevention-enabling behaviors create the potential for exposure to zika prevention counseling by trained and trusted health care providers and include antenatal care and family planning counseling. investigation of enabling behaviors was not pursued in the next phase of the process, as extensive existing literature supported the efficacy of antenatal care and family planning counseling on uptake of sexual and reproductive health services (generally), and their promotion was already ongoing through reproductive health programs in the region. although it is not necessarily clear how the zikaspecific context may have affected uptake of these behaviors, there was no research available at the time to review. to conduct a systematic evidence review for the personal protection and vector control preventive behaviors, google scholar results were compiled for articles on aedes-borne diseases published between and . any seminal papers published before and zika-related gray literature and unpublished data (from sources including unicef, the cdc, and usaid implementing partners) were also reviewed. because of the recency of the zika outbreak, a limited number of relevant papers had reached publication, so relevant articles on any aedesborne diseases (dengue, chikungunya, yellow fever, west nile) were also compiled. literature on malaria was excluded because it is transmitted by anopheles mosquitoes, not aedes, calling for different interventions. a prisma diagram ( figure) shows the selection criteria and screening process results for articles included in the review. each article was summarized in an annotated bibliography. as noted in the top panel of table , each behavior's efficacy and, if available, effectiveness in preventing zika transmission were investigated through the literature review process. we considered a behavior efficacious if it had one of the following impacts: a reduction in mosquito bites, a reduction in the mosquito population (as measured by number of eggs, pupae, or adult mosquitoes), or a reduction in the sexual transmission of zika. we considered a behavior effective if programs promoting the behavior had an impact on the outcome at the population level and/or measured a public health impact. the effectiveness or public health impact was not always measured or reported in the published literature; in those cases, other sources were explored for reasonably extrapolating this information. if gray literature was available, this was explored. otherwise, logical assumptions were tentatively made; for example, if a study found that mosquito abundance was reduced, we extrapolated that zika transmission may also be reduced. the evidence regarding efficacy and effectiveness from the literature for each behavior was assessed against each criterion as being "high," "medium," or "low." the evidence was also qualitatively weighted by the rigor of the studies reviewed and how recently the study was conducted. our literature review also considered the locus of control (who was primarily responsible for implementing the behavior); how the outcome was measured (e.g., number of mosquito bites, population density); whether programs targeted specific sub-populations (e.g., pregnant women, male partners); and whether interventions targeted multiple behaviors (e.g., larvicide application, removing stagnant water). these additional factors were considered to guide interpretation of the study findings based on context and better understand the generalizability of results (e.g., if a study assessed multiple behaviors being promoted at once and the impact of a single behavior could not be isolated). if the article had insufficient detail, we also contacted research authors to clarify the specific steps required in the behaviors assessed in their studies. in addition to efficacy and program effectiveness criteria, a third criterion was developed to assess whether the behavior was easy to do and amenable to change to consider the contextual realities of how behaviors were being promoted and adopted. as noted in the bottom panel of table , this criterion was defined by the ( ) frequency of performance required to be effective; ( ) feasibility of the behavior (e.g., single versus multiple steps, required negotiation or engagement of others); and ( ) availability and accessibility of required materials (e.g., a brush to scrub a water-storage container). the locus of control was also considered here and whether an individual could carry out the behavior independently. as was done in the evidence review, each behavior's ease of use/amenability to change was rated as "high," "medium," or "low" (excepting "feasibility," which was ranked as "easy," "medium," or "complex"). to evaluate each behavior against this programmatic criterion, a consensus-building approach was implemented at meetings of the usaid partners zika sbc technical working group and through consultations with technical experts. the sbc technical working group aims to support collective sbc efforts by creating a forum to coordinate, share, and discuss challenges, solutions, and best practices for zika prevention and promote evidence-based sbc practices. members include usaid, unicef, and implementing partners working directly with households and communities in the region. implementing partners are ngos receiving usaid funding for the implementation of the zika response. to summarize, the prioritization process took place over months between october and april . more than variants of behaviors and messages were identified and narrowed down to through extensive discussion with the technical working group and input from technical experts to reach an initial consensus. to narrow down to these behaviors, we used a combination of expert opinion, initial scan of available evidence, and factors such as whether usaid was supporting the behavior by procuring the materials necessary. two of the final behaviors were considered enabling behaviors and recommended but not included in the evidence review, given the substantial existing evidence on these behaviors. five behaviors were explored in a full literature review to ascertain their relative effectiveness. during the literature review phase, partners provided additional gray literature where applicable. the full findings were presented at a subsequent sbc technical working group meeting to ensure a consensus on the findings. the initial purpose of presenting the findings was to further narrow down the prioritized behaviors, but after partners expressed significant pushback, the full list of behaviors were agreed upon. any feedback from implementing partners regarding how behaviors were carried out, contextual challenges, or unpublished effectiveness findings was integrated into the final recommendations, and the results were disseminated to all usaid implementing partners. the findings and conclusions for zika sbc programming drawn from this process are presented below for each of the preventive behaviors reviewed, as well as the enabling behaviors. applying mosquito repellent: application of mosquito repellent is highly efficacious in preventing mosquito bites, and thus the potential of vector transmission of zika to an individual. this behavior is within the control of pregnant women and male partners of pregnant women. it is recommended that users be thoroughly counseled on proper product application. women intending to become pregnant should also consider using repellent. applying mosquito repellent (deet, picaridin, ir , or lemon eucalyptus oil) and using each product as directed is a highly effective method in preventing mosquito bites and reducing risk of zika transmission through mosquito bites. deet is considered the gold standard repellent, showing greater than % efficacy in preventing mosquito bites for - hours. usaid and cdc approved additional repellents (picaridin, ir , lemon eucalyptus oil) based on evidence suggesting they have an efficacy comparable to deet. these repellents are the only ones for which evidence of effectiveness has been recorded. the use of these repellents was also considered safe for use during pregnancy. [ ] [ ] [ ] we did not find program or intervention studies that assessed the impact of mosquito repellent on arbovirus disease transmission, but deduced a strong likelihood of effectiveness because it prevents mosquito bites. concerning the third criterion-"easy to do and amenable to change"-repellent must be applied frequently (multiple times per day) to be effective, particularly if the person is sweating, swimming, or changing clothes. , the use of repellent was considered to have medium feasibility because individuals decide whether to use repellent and therefore have control; however, those who have low literacy or limited access to trained antenatal care or pharmacies may have a disadvantage in following the written package instructions. mosquito repellents are sold on the market in most countries in lac and generally available in these settings. contextual information regarding repellents was reported from partners working in the field with local knowledge. usaid also has procured repellents for distribution at antenatal care clinics in a few select countries. the price may be a barrier, particularly for low-income households. using condoms to prevent sexual transmission: condom use to prevent sexual transmission of zika is highly efficacious, but sexual transmission may be a small portion of overall transmission. this behavior should be prioritized for pregnant women and their partners because pregnant women are at risk for negative pregnancy outcomes. there is evidence that zika is transmitted sexually. [ ] [ ] [ ] according to our evidence review, condoms are highly effective in preventing sexually transmitted infections. condom use is the only behavior that can prevent sexual transmission of zika to sexually active women who may become pregnant or already are pregnant. although statistical modeling suggests that sexual transmission of zika is only %- % of total transmission in the general population, - the attributable risk of exposure among sexually active women may be twice as high. this increased risk of exposure, combined with the severity of outcomes in pregnancy, led to the identification of pregnant women and their partners as a target population for messaging about condom use. we found that condom-use behavior had a mixed amenability to change for reasons: first, condoms must be worn consistently and correctly to be effective (on the basis of findings from the sexually transmitted infection literature), including throughout pregnancy , ; second, the behavior is complex, requiring negotiation between partners [ ] [ ] [ ] [ ] ; and third, since condom use is not considered a normative behavior during pregnancy, it may be challenging to promote and adopt. condoms are widely available in pharmacies and health centers in lac, but access may be limited for women of low income. regularly removing unintentional standing water both inside and outside the house: this is a potentially efficacious behavior to reduce mosquito populations and reduce the potential for individual-and population-level risk of zika transmission. promotion of the behavior must be accompanied by specific, focused instructions that target the highest density breeding sites and be conducted weekly in homes and communal areas to be effective. efficacy is highest in areas with strong community engagement, including active mosquito searches in homes and communities and awareness of the mosquito life cycle. according to the evidence, performing this behavior is highly efficacious in reducing the adult aedes mosquito population. one study found a greater than % reduction in the adult mosquito population following a very strict intervention, involving community campaigns and visits from trained volunteers, to remove stagnant water. however, individuals and households may find it challenging to perform this behavior correctly and consistently. to have an impact on the aedes mosquito population, and thus zika transmission, it requires an ongoing, collective effort, [ ] [ ] including households as well as common areas, such as schools, clinics, cemeteries, and others. to maximize the potential impact, efforts need to focus on the highest-density mosquito-breeding sites as identified by entomological data collection. [ ] [ ] [ ] general clean-up campaigns in which communities receive information to clean their yards or communal areas without specificity on targets for removal often are only effective if they target the most productive breeding sites. in addition, these kinds of interventions are challenging to measure since clean-up campaigns are often conducted in conjunction with other interventions, and studies do not isolate the effect of any one of them. despite these concerns, when instructions are clear and focused, regular removal of standing water is relatively easy to do and amenable to change; special materials are unnecessary in most cases. however, the feasibility is somewhat complex; the targeting of breeding sites should depend on how productive they are (to identify the highest-density breeding sites), and those sites are often either difficult to access (e.g., in storm drains) or located in communal areas requiring collective effort and engagement to attempt (e.g., in schools or construction sites). in addition, this behavior requires weekly action, based on the life cycle of aedes mosquitoes. covering water storage containers at all times with a tight-fitting cover that does not warp or touch the water: covering longterm water storage containers has moderate potential efficacy in reducing breeding sites if a tight-fitting, long-lasting lid is available. covering short-term water storage containers has less potential efficacy, as frequent lid use can result in wear and tear and render the lids ineffective or counterproductive. the focus of this behavior is on long-term storage items such as barrels or other large household water-storage containers used less than once per week. a small number of studies suggest that the correct use of lids is associated with a significant reduction in pupal infestation if the containers are used infrequently. however, correct use and adequate lids are critical; if the lid is broken or touches the water in the container, the lid itself can spawn a breeding site for aedes mosquitoes. the beneficial effect of correctly using a lid is mixed or even reversed if the water-storage container is used very often, constantly opened and closed, or often left open. this behavior, when done correctly, may reduce transmission at the population level, but in most published evidence, it is combined with community mobilization and cleaning of containers, making it challenging to isolate its effect. the behavior itself is relatively easy to implement. for long-term storage containers (from which water is accessed infrequently), the frequency of removing lids is low and relatively feasible, assuming the lids are used correctly. however, as research is ongoing to determine what type of lids are the most effective, access to proper lids was rated low. for short-term water storage, the frequent opening and closing of lids and additional requirement of monitoring the quality of the cover reduces ease of implementation, and therefore, its effectiveness. long-term water storage containers coupled with correct use of tight-fitting, long-lasting lids, may enable this behavior to have moderate potential efficacy in reducing aedes breeding sites. eliminating mosquito eggs from waterstorage container walls weekly: thorough cleaning of water-storage containers can remove mosquito eggs, significantly reducing the population and, thus, zika transmission, but easy access to effective materials cannot be assumed. aedes aegypti mosquitoes lay their eggs in water storage containers, such as washbasins and metal drums, located inside or outside the house, increasing the risk of transmission of diseases such as zika to households. as a result, cleaning containers is often recommended, but historically a lack of specific instruction has led to mixed results. for example, the world health organization recommends scrubbing containers with a brush, but does not mention whether a cleaning solution (such as bleach or detergent) should be applied, and cites studies that do not isolate the effect of cleaning from other behaviors (such as using lids). research from the early s reported manual cleaning of containers was ineffective in removing mosquito eggs, but it is unclear exactly how the containers were being cleaned and if eggs were targeted incorrectly. [ ] [ ] since that time, several new methods have been developed. in our review, we judged methods to be effective based on available efficacy evidence and consultations with entomologists with field experiences in the region (listed here in decreasing order of effectiveness). . the untadita method, tested in a randomized controlled trial, was found to be more effective than scrubbing alone. in this method, a specific bleach and non-ammonia detergent mixture is applied to container walls that are then scrubbed with a brush and rinsed out after minutes. although this method has been promoted, there have been concerns in the field regarding potential toxicity of mixing non-recommended types of detergent with chlorine and the need to fully empty the container, which is challenging in water-scarce areas. in one study, % of surveyed households stored water and cited interruption of water services, poor water pressure, or costsaving concerns as reasons for not wanting to empty their water-storage containers. . the second method, developed in the negociación de prácticas mejoradas trial, requires applying bleach to water-storage container walls without being emptied if they are partially filled. no field-based results on effectiveness or the intervention at scale were available, but small and experimental tests suggested positive ovicidal results. . if the first cleaning methods are not possible to carry out due to lack of bleach, the third technique of cleaning the walls of the container with detergent alone (using a brush, if available) should be implemented. this technique requires fully emptying the container. . lastly, scrubbing the container walls with a brush (only) is recommended if neither detergent nor bleach is available. seeking antenatal care. seeking antenatal care is known to contribute to healthy pregnancies. in this setting, seeking antenatal care enables counseling on zika prevention by trained health care providers, allowing for early diagnosis and treatment, as well as access to information about effective protective measures to reduce the risk of transmission of zika from mother to child. using contraception voluntarily. seeking family planning (for those not intending to get pregnant) is also a critical behavior, linked to prevention of sexually transmitted infections and prevention of sexual transmission of the zika virus. both of these behaviors-seeking family planning and antenatal care-are routinely promoted for adoption of healthy behaviors among pregnant women and women of reproductive age. the results of the literature review and the consultative process discussed in this article helped to identify behaviors with the greatest promise for preventing the acquisition and transmission of zika. a critical part of this process was the consensus-building process with partners, ensuring input from those working on the ground. the sbc technical working group engaged partners throughout the process. partners requested focusing on behaviors that families and communities could do themselves ("locus of control" at the household). points of contention often centered around behaviors that were being promoted already and were perceived by partners to be effective but had mixed evidence. for example, covering water storage containers was found to effective in the literature only for long-term water storage containers, but ultimately short-term containers were included in the guidance based on conversations with partners. this was mainly because partners perceived this behavior (covering water storage containers with a lid) to be effective based on this behavior being implemented already and being received positively. however, for other behaviors, for example use of bed nets, we clarified that although these are effective for malaria, they are not effective for zika, and this was ultimately agreed upon and the guidance accepted. through the process, partners were reminded that choices had to be made to prioritize key behaviors; although all of them were potentially effective, we were looking for relative effectiveness to prioritize the most effective ones and focus on them. where there was pushback, we asked for field data to inform the decision to modify the final guidance. these results were summarized in the zika prevention behavior matrix, a document widely disseminated through the zika communication network (zcn), a platform for sharing zikarelated resources and media products in the lac region. a technical specifications content guide (a companion to the matrix) was also developed to detail the evidence-based technical requirements and steps to follow for each of the behaviors (described here in table , and in more detail in the technical specifications content guide) to reduce transmission. the guide was made available on the zcn website to guide implementing partners in developing sbc content. both documents, available in english and spanish, guide prevention messages and prioritize calls to action to harmonize partner efforts and clarify specific messages to families, communities, and health care providers targeted by sbc programs for prioritized behaviors. the documents will be available to the public via an interactive digital platform that will guide users through the evidence, messaging, and technical specifications in a user-friendly way. because aedes aegypti vector control and mosquito bite prevention behaviors are included in this resource, partners working on dengue and chikungunya can use it. the documents will be continually updated as new developments emerge, reflecting input from implementing partners, to ensure that the materials address realities on the ground. for example, based on input from the field, guidance on correct disposal of repellents was recently added. additional resources have been developed by breakthrough action to support the promotion of the prioritized behaviors with the needed specificity described in the technical specifications content guide. sbc program teams in the field can use these tools to adapt their efforts to the latest findings and recommendations. a job aid has been developed to guide outreach workers and volunteers during household visits to better target audience segmentation for behaviors to maximize the uptake of the recommendations in the zika prevention behavior matrix. to facilitate effective use of the job aid and messaging around the prevention behaviors, a training-of-trainers curriculum on interpersonal communication skills for outreach workers has also been developed. this curriculum has been used to train health promoters, volunteers, and field technicians in lac countries and adapted for context-specific variation with usaid implementing partners and/or ministry of health personnel. both the job aid (in english and spanish) and curriculum are available on the zcn. country-specific adaptations to the job aid, such as including language for dengue and chikungunya prevention, are also available on the zcn. the conditions brought on by climate change, international travel, urbanization, deforestation and other global and regional trends may result in new emerging diseases, as well as the spread of existing diseases to previously unexposed populations. ministries of health, international organizations, and ngos must respond rapidly to outbreaks by coordinating an effective public health response. under these circumstances, institutions rarely have sufficient data to guide sbc messaging for the most effective preventive actions for individuals and communities and may be forced to launch interventions or programs at the same time data are being gathered, assessed, and synthesized. this situation often leads to a lack of cohesion in the promoted behaviors and sbc messages. the behavior prioritization process documented in this article was developed to help usaid implementing partners identify focal behaviors for prevention to harmonize sbc programming efforts for greater impact. the process combined available evidence and a consensusbuilding approach to allow for adaptation based on local context. the consensus-building approach was critical for selecting behaviors and necessitated coordination across all stakeholders involved in the response across disciplines (e.g., public health, entomology, medical, and other technical area experts) and across response partners (e.g., those involved in mass media, service delivery, and community engagement). types of behaviors were selected based on the transmission dynamics of the disease; zika is transmitted by vector (mosquito) and sexually, highlighting preventive behaviors to consider. behavior change is complex, and each behavior is comprised of many different behaviors and decisions that have to be addressed to successfully change. each behavior we identified was rated against criteria, related to supporting evidence and related to feasibility and amenability of the behavior to change. the first criteria of the process assessed the state of the evidence available for selecting key behaviors to promote. the availability and quality of evidence depends on how long the disease has been around and whether it is occurring in a new region or sub-population. for example, in the severe acute respiratory syndrome outbreak, there was no evidence as the world was contending against a newly encountered disease. during the west africa ebola outbreak, researchers were attempting to understand the behaviors to target to interrupt transmission, while programmers were developing prevention and treatment programs and messages in real-time; research and evidence generation came later. in the case of zika, the disease was previously known in east asia and the pacific but was entirely new to lac. however, other aedes aegypti mosquito viruses were prevalent in the region, specifically, dengue, which had been circulating in the region for more than years, and chikungunya had emerged in . thus, information regarding best practices to prevent transmission could be gleaned from previous mosquito control and behavior change programming from lac and southeast asia and could be adapted to zika prevention efforts. two unique characteristics of zika presented themselves when applying the evidence. first, unlike dengue or chikungunya, zika is mostly asymptomatic or presents mild symptoms; lac is the first setting where it has been widely associated with adverse pregnancy outcomes (and guillain-barré syndrome). second, it was found that zika can be transmitted sexually, but other arboviruses, such as dengue and chikungunya, cannot. although we were able to adapt an extensive evidence base for vector control of the aedes aegypti population, there were little to no published data available regarding sexual transmission of zika or its impact on pregnancy outcomes. sexual transmission and the link with congenital malformations meant we needed to consider specific behaviors and messaging directed to pregnant women and women of reproductive age. to address this gap, evidence was gleaned and adapted the behavior prioritization process was developed to identify focal behaviors for prevention to harmonize sbc programming efforts for greater impact. from the literature on sexually transmitted infections and from research findings as they emerged from the field (before publication). the third criteria of the process comprised a set of components related to behavior amenability to change in the lac setting. this step was an important contextual step because the effectiveness of a behavior is largely dependent on whether people are able and willing to perform it and was additionally complicated because zika was new to the region. because the behavior prioritization process was started after a year of zika response, local partners had field experience to advise on which priority behaviors were feasible to implement and which materials were available and affordable. a consensus-building approach with usaid and its implementing partners was developed to categorize the relevant behaviors by criteria. although the evidence review highlighted behaviors with potential to prevent diseases with the same modes of transmission or reduction of the same vector population as zika, it was critical for each focal behavior to be assessed through the lens of this third criteria to ensure partner buy-in-that recommendations were realistic and reflected the context on the ground. the result of the process was a set of priority behaviors based on a combination of research evidence and context from partners with local knowledge. the behavior prioritization process was developed to streamline the zika response but began mid-implementation, by which time many partners were already deploying various behavior change recommendations. any suggested changes to sbc programming that stemmed from the findings of this necessitated midcourse corrections. all behaviors were prioritized by the usaid zika response across countries, and included interventions such as regional mass media campaigns, household and school-based sbc activities, household visits by vector control technicians, community fairs, and health care provider counseling. several key products were developed and made available to implementing partners to facilitate the incorporation of the findings of the process, including a list of priority behaviors and detailed guidance on how to correctly perform each behavior for maximum effectiveness (through a behavior matrix [supplement ], technical specifications content guide [supplement ], and a job aid). these products were useful for sbc programs to reconfigure and refine their messages as they continued to implement their sbc interventions. many partners reported that the guidance provided a basis to focus their limited remaining resources and to attain the needed specificity for each behavior to be effectively implemented. the iterative, collaborative process of defining behaviors across all stakeholders was critical to ensuring a more harmonized and feasible response. this process strives to incorporate evidence where it is available and was refined as the work developed, allowing it to be responsive to new evidence and contextualized based on input and expertise from those on the ground. this process can identify and select behaviors with the most potential to reduce transmission, is designed to be adapted to local contexts, and is flexible and based on consensus building with local and international stakeholders. our experience developing this process provides a potential model for future public health emergencies, as it highlights a way forward in prioritizing behaviors in evidence in situations where direct evidence is limited or absent, time is constrained, and there are many key stakeholders. the result of the process was a set of priority behaviors based on a combination of research evidence and context from partners with local knowledge. prevención del zika en américa latina y el caribe priorización de comportamientos clave basado en la evidencia y su aplicabilidad a futuras respuestas de emergencia de salud pública en el propósito de maximizar el impacto de los esfuerzos de prevención del zika, se estableció un proceso de priorización para los programas de cambio social y de comportamiento en base a una combinación de evidencia sólida y proveniente de investigaciones y experiencia programática. los comportamientos priorizados fueron: aplicación de repelente de mosquitos, uso de condones, eliminación de agua acumulada alrededor de la casa y comunidad, cubrir los recipientes de almacenamiento de agua, fregar las paredes de los recipientes de almacenamiento de agua, asistencia a las consultas de atención prenatal, y la busqueda de consejería sobre planificación familiar si no planea un embarazo. desde el brote del zika de en américa latina y el caribe, se venian promoviendo una gran cantidad de mensajes de cambio de comportamiento para reducir la transmisión del zika. un año después de que la agencia de los estados unidos para el desarrollo internacional (usaid) iniciara su respuesta al zika, se encontró que se venían promoviendo más de variantes de conductas preventivas. esta situación planteó un desafió a los esfuerzos de los programas de cambio social y de comportamiento (csc) que requieren de una respuesta coordinada y de común acuerdo sobre el conjunto de comportamientos a promover para lograr máyor efectividad. para apoyar a los socios implementadores de usaid en la armonización de los esfuerzos de prevención para reducir la infección por zika, se desarrolló un proceso basado en evidencia que identificara aquellos comportamientos con mayor potencial en reducir la infección y transmisión del zika. se recopiló una lista completa de comportamientos y se seleccionaron los más prometedores para luego llevar adelante una revisión de la evidencia presentada. la revisión incluyó búsquedas sistemáticas de palabras clave en google scholar, identificación de todos los artículos publicados entre y que fueran relevantes a las enfermedades transmitidas por el mosquito aedes, y revisión de documentos pioneros y de literatura gris. se examinaron una serie de artículos para determinar la efectividad potencial de cada comportamiento en prevenir la transmisión del zika o en reducir la población de aedes aegypti. igualmente se desarrollaron criterios de evaluación para medir la facilidad con la que la población objetivo podría adoptar cada comportamiento, incluyendo: ( ) frecuencia requerida; ( ) viabilidad del comportamiento; y ( ) accesibilidad y costo de los materiales necesarios en el contexto inmediato. estos comportamientos se refinaron con los socios implementadores de usaid de la respuesta al zika a través de un proceso de creación de consensos considerando factores contextuales. el resultado fué la selección de comportamientos preventivos que según la evidencia presentaban el mayor potencial para lograr el impacto de los programas de csc en la respuesta al zika de usaid: ( ) aplique repelente de mosquitos, ( ) use condones durante el embarazo, ( ) elimine agua acumulada alrededor de la casa y comunidad, ( ) cubra los recipientes de almacenamiento de agua, ( ) elimine los huevos de mosquito de los recipientes de agua, ( ) busque atención prenatal, y ( ) busque consejería en planificación familiar si no planea un embarazo. el presente estudio de caso documenta un proceso flexible que puede ser adaptado para llevar adelante ejercicios de priorización de comportamientos cuando se cuenta con una evidencia limitada, como bien sucede en muchas de las respuestas de emergencia. zika strategic response plan. geneva: who after the epidemic: zika virus projections for latin america and the caribbean prevalence of asymptomatic zika virus infection: a systematic review congenital zika syndrome & other birth defects. centers for disease control and prevention website the zika virus epidemic in brazil: from discovery to future implications united states agency for international development website united states agency for international development website health communication: lessons from family planning and reproductive health health communication capacity collaborative. sbcc for emergency preparedness i-kit website the efficacy of repellents against aedes, anopheles, culex and ixodes spp.: a literature review prevent mosquito bites. centers for disease control website mosquito repellents, effectiveness in preventing diseases and safety during pregnancy insect repellants during pregnancy in the era of the zika virus zika virus infection-the next wave after dengue? sexually acquired zika virus: a systematic review sexual transmission of zika virus: current status, challenges and research priorities sexual transmission and prevention zika virus and pregnancy: an overview higher incidence of zika in adult women than adult men in rio de janeiro suggests a significant contribution of sexual transmission from men to women zika risk and pregnancy in clinical practice prevalence and incidence of zika virus infection among household contacts of zika patients: puerto rico risk communication and community engagement for zika virus prevention and control. new york: unicef measures taken to prevent zika virus infection during pregnancy-puerto rico valongueiro alves s. women's reproductive intentions and behaviors during the zika epidemic in brazil pregnancy: parallel stories and new challenges s. pregnant women's knowledge and attitudes about behavioral strategies and vaccines to prevent zika acquisition. vaccine a simple periodic-forced model for dengue fitted to incidence data in singapore assessing the effects of interventions for aedes aegypti control: systematic review and meta-analysis of cluster randomised controlled trials understanding water storage practices of urban residents of an endemic dengue area in colombia: perceptions, rationale and socio-demographic characteristics ecological, biological and social dimensions of dengue vector breeding in five urban settings of latin america: a multi-country study low entomological impact of new water supply infrastructure in southern vietnam, with reference to dengue vectors is dengue vector control deficient in effectiveness or evidence?: systematic review and meta-analysis effectiveness of dengue control practices in household water containers in northeast thailand aedes aegypti breeding ecology in guerrero: cross-sectional study of mosquito breeding sites from the baseline for the camino verde trial in mexico world health organization (who) effect of a community-based aedes aegypti control programme on mosquito larval production sites in el progreso dengue: a worldwide problem, a common strategy. mexico city: mexican ministry of health and rockefeller foundation trial of a communitybased intervention to decrease infestation of aedes aegypti mosquitoes in cement washbasins in el progreso negociación de prÁcticas mejoradas -nepram (negotiation of improved practices): the development of a national behaviour change strategy for community-based prevention of dengue fever in the dominican republic efecto ovicida del cloro casero puro (hopoclorito de sodio al acknowledgments: the authors would like to acknowledge the hard work of the numerous organizations working to prevent zika transmission in communities in latin america and the caribbean. competing interests: none declared. key: cord- -tu xrt x authors: li, cui; gao, fei; yu, lei; wang, ruoke; jiang, yisheng; shi, xuanling; yin, chibiao; tang, xiaoping; zhang, fuchun; xu, zhiheng; zhang, linqi title: a single injection of human neutralizing antibody protects against zika virus infection and microcephaly in developing mouse embryos date: - - journal: cell rep doi: . /j.celrep. . . sha: doc_id: cord_uid: tu xrt x zika virus (zikv) is a mosquito-transmitted flavivirus that is generally benign in humans. however, an emergent strain of zikv has become widespread, causing severe pre- and post-natal neurological defects. there is now an urgent need for prophylactic and therapeutic agents. to address this, we investigated six human monoclonal antibodies with zikv epitope specificity and neutralizing activity in mouse models of zikv infection and microcephaly. a single intraperitoneal injection of these antibodies conveyed distinct levels of adult and in utero protection from zikv infection, which closely mirrored their respective in vitro neutralizing activities. one antibody, zk b , showed the most potent neutralization activity, completely protected uninfected mice, and markedly reduced tissue pathology in infected mice. thus, zk b is a promising candidate for the development of antibody-based interventions and informs the rational design of zikv vaccine. zika virus (zikv) is a mosquitotransmitted flavivirus that can cause severe neurological defects in humans. li et al. have identified a human monoclonal antibody capable of protection against zikv infection and related diseases when tested in mouse models. this antibody serves as a promising candidate for clinical development against zikv. the recent, widespread neurological deficits caused by an emergent strain of zika virus (zikv) have caught the world off guard wikan and smith, ) . zikv was first identified in the forests of uganda, and infection was generally benign in humans (dick et al., ) . however, this new strain of zikv is far more virulent and causes a range of clinical anomalies rasmussen et al., ; wikan and smith, ) . most notable are microcephaly and other congenital defects in infants born to mothers infected with zikv during pregnancy (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . although the exact mech-anism of neuropathogenesis remains uncertain, clinical abnormalities have been linked to the aberrant development and loss of neural progenitor cells (npcs) (cugola et al., ; gabriel et al., ; garcez et al., ; li et al., a li et al., , b tang et al., ) . the contemporary strain of zikv has enhanced replication capacity and a specialized tropism for npcs (cugola et al., ; dang et al., ; garcez et al., ; li et al., b; tang et al., ) , although other types of cells are susceptible (tabata et al., ; weisblum et al., ) . the infection inhibits npc proliferation and differentiation and can trigger apoptosis or autophagy. critically, the highest rates of birth defects occur in pregnant mothers who are infected during their first and second trimesters. this is presumably because, during the early stages of gestation, npcs have a greater susceptibility to zikv infection, and there is more viral transfer across the placental barrier (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . to fully protect the developing fetuses, an intervention must occur before this period or, ideally, prior to infection (marston et al., ) . neutralizing antibodies are the essential mediator of immunity against viral infection (burton and hangartner, ; corti and lanzavecchia, ) . for zikv and other flaviviruses, human neutralizing monoclonal antibodies target the surface envelope glycoprotein (e) that facilitates infection (dejnirattisai et al., ; fernandez et al., ; fibriansah et al., ; heinz and stiasny, ; magnani et al., ; pierson and diamond, ; pierson and graham, ; robbiani et al., ; rogers et al., ; sapparapu et al., ; stettler et al., ; wang et al., wang et al., , b zhao et al., ) . we previously reported on a panel of monoclonal antibodies (mabs) derived from the longitudinal samples of a zikv-convalescent individual and characterized their neutralizing activities, epitope specificities, and development timeline over the course of infection . we also reported on mouse models of zikv infection and microcephaly, with enhanced specificity for neurological infection figure . experimental design to evaluate the prophylactic potential of six human mabs against zikv infection in developing fetuses and ag mice (a) timeline for mab injection, zikv inoculation, infant delivery, and the monitoring of a complementary set of clinical, virological, and neuropathological outcomes from embryonic day . to p . the six mabs tested are shown alongside their ic values and epitope specificities. zikv-inoculated fetuses and neonates are indicated by zikv + in red; those left unexposed are indicated by zikv À in blue. the cartoon mice on p include zikv + (small and large indicated in red) and zikv À (large indicated in blue); the size representations reflect potential body weight outcomes. each mab was tested, and the outcomes were monitored in three littermate neonates of three pregnant mice on p and p . (b) different levels of protection conferred by the six mabs, shown with the number (n) of neonates monitored for survival in each mab treatment group. (c) the body weight of neonates among the different mab groups. zikv + neonates indicated in red are presented as a percentage of the blue zikv À littermates. the zk b -treated group had no discernable differences between the zikv + and zikv À littermates and is, therefore, shown as a single red/blue checkered bar. the number of neonates for each analysis is indicated above the respective mab, with zikv + in red and zikv À in blue. (legend continued on next page) using a contemporary zikv asian strain (gz ). in the model of microcephaly, the virus was inoculated directly into the lateral ventricles of the fetal mouse brain (li et al., a) . zikv replicated in the fetal brain, with preferential infection of npcs. infection resulted in cell-cycle arrest, differentiation defects, and a large number of cell deaths, as well as clinical presentations of microcephaly (li et al., a) . here, we use the mouse models of zikv infection and microcephaly to analyze the in vivo protective activities of six human mabs and compare the findings with our reported in vitro neutralization activity, as measured by plaque reduction neutralization test (prnt). our results offer compelling evidence that the in vivo protection is directly associated with in vitro neutralization. one antibody, zk b , provides complete protection in pre-exposure treatment and partial protection in post-exposure therapy, with markedly reduced tissue pathology. we believe that zk b is a promising candidate for the development of antibody-based interventions and informs vaccine design specific to zikv infection. in vivo protection correlates with in vitro neutralizing activity of mabs we evaluated and compared the protective potential of six representative human mabs following the protocol highlighted in figure a . all these (zk - , zk - , zk f , zk c , zk c , and zk b ) were isolated by our team. two additional zikv domain iii (diii)-specific mabs (zv and z ) isolated by others were included for comparative analysis (robbiani et al., ; zhao et al., ) . a human antibody, middle east respiratory syndrome- (mers- ), targeting the middle east respiratory syndrome coronavirus (mers-cov), was used as a negative control. our mabs were initially isolated from the peripheral b cells of a convalescent chinese man who acquired zikv while traveling to venezuela during the outbreak in . specimens were derived from sequential blood samples collected on day (zk - ), day (zk - ), day (zk f ), or day (zk c , zk c , and zk b ) after symptom onset. they targeted the zikv e with varying degrees of binding and neutralizing activities and epitope specificities . for example, zk b and zk c were strictly zikv specific and demonstrated the most potent neutralizing activities, as measured by prnt. of these, zk b recognized diii, while zk c was specific for domain i/domain ii (di/dii). in comparison, zk - , zk - , zk f , and zk c were much less potent against zikv but cross-neutralized other members of the flavivirus family, such as dengue virus serotypes denv and denv . on the zikv envelope, they targeted either di/dii (zk - , zk - , and zk f ) or di/dii/diii (zk c ). in terms of genetic analyses, these antibodies demonstrated diverse heavy-chain variable regions. two, zk - and zk f , fell into the immunoglobulinheavy variable (ighv) - family, while zk - and zk c belonged to the ighv - family. finally, zk b and zk c belonged to the ighv - and ighv - families, respectively. to test prophylactic activity against zikv infection, mg/kg of each mab was administered intraperitoneally to a group of three pregnant mice on embryonic day . ( figure a ). the following day (e . ), the developing littermate fetuses in each pregnant mouse were either left untouched (zikv À ; figure a , blue) or inoculated with plaque-forming units (pfus) of zikv asian strain gz directly into the lateral ventricles of the brain (zikv + ; figure a , red). the neonates were closely monitored after delivery and analyzed for a complementary set of clinical, virological, and neuropathological outcomes on post-natal day (p) and for survival up to p . as shown in figure b , the levels of protection conferred by the different mabs varied considerably but clearly correlated with their neutralizing activities, as measured by half-maximal inhibitory concentrations (ics ) in the prnt . the most potently neutralizing mab, zk b , conferred complete protection. survival rates of infected fetuses were as high as % on p . the next potent, zk c , however, conferred only partial protection with median survival of days. the two antibodies (zv and z ) isolated by others, together with the rest of the mabs isolated by us, offered negligible protection and were virtually similar to the isotype control mers- . we also assessed impact on developing body weights and found weights measured on p closely correlated with the neutralizing activity. the zikv + neonates treated with the most potent mabs, zk b and zk c , had weight gain similar to that of their zikv À littermates. the zikv + neonates treated with zv , z , and the remaining mabs failed to do so. the control zikv + mers- group had the greatest loss in body weight. next, we studied whether the protection pattern observed in the pregnant mice also occurred in non-pregnant mice. we administered mg of each mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- to a group of four -to -week-old ag mice via the intraperitoneal (i.p.) route. the following day, animals were challenged with pfus of zikv asian strain gz via the i.p. route. survival rates, body weight, and viral rna copies in the blood were monitored for up to days ( figure d ). consistent with the outcomes for pregnant mice, the level of protection in nonpregnant mice correlated with antibody neutralizing activities as measured by half-maximal inhibitory concentrations (ics ) in the prnt ( figure e ). for example, the most potent and protective mab in pregnant mice, zk b , provided complete protection in non-pregnant mice, with survival rates of % days after the challenge. the next potent and protective, zk c , conferred only partial protection, with median survival of days. the rest of the tested mabs, including the diii-specific ones (zv and z ) isolated by others (robbiani et al., ; zhao et al., ) , offered even lower levels of protection. zk - was virtually identical to the isotype control mers- . (d-g) shown here: (d) timeline for mab injection, zikv inoculation, and monitoring for (e) survival, (f) body weight, and (g) blood zikv rna up to days in ag mice. the body weight and zikv rna in the whole blood derived from a single measurement showed distinct results among the study groups. the number of animals used in each group was four. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. similarly, changes in body weight measured over the -day follow-up also correlated with the neutralizing activities. the zikv + mice treated with zk b maintained relatively stable body weight throughout the study, although there was a noticeable decline after the first blood sample collection on day (figure f ). in contrast, zikv + mice treated with zk c lost substantial body weight beginning on day post-challenge. animals treated with the remaining mabs had severe and rapid losses in body weight that coincided with a clinical deterioration (figure e) . lastly, for zikv + mice treated with zk b , blood levels of viral rna were indistinguishable from those measured in uninfected animals on both day ( . ± . versus . ± . ) and day ( . ± . versus . ± . ) after challenge ( figure g ). however, rna levels measured in zk c -treated mice were, on average, log greater on day ( . ± . versus . ± . ) and more than logs greater on day ( . ± . versus . ± . ), compared to zk b . the remaining mabs failed to suppress viral replication. the measured zikv rna load on day ranged from . to . logs higher than that of zk b , depending on the mab used ( figure g ). taken together, these results demonstrated that the protective patterns of each mab were similar in both pregnant and non-pregnant mice and closely mirrored their respective in vitro neutralizing activities. the protective effects of each mab on body weight and overall survival mirrored their respective virological and neuropathological outcomes in treated mice. for instance, rna loads measured in zikv + neonates treated with zk b were suppressed in the blood ( . ± . log copies per milliliter) and the brain ( . ± . log copies per gram) to levels indistinguishable from those in zikv À littermates. in fact, zk b was able to reduce the zikv rna by . logs in the blood and . logs in the brain, relative to that in the zikv + mers- group (figures a and b ). in comparison, zk c was equally effective at suppressing replication in blood ( . ± . log copies per milliliter) but only moderately protective in the brain ( . ± . log copies per gram). the mabs zv and z only moderately suppressed zikv replication in the blood ( . ± . and . ± . log copies per milliliter, respectively) and the brain ( . ± . and . ± . log copies per gram, respectively). none of the remaining mabs notably suppressed replication in either blood or brain tissue. all of the zikv + neonates treated with these mabs had high levels of zikv rna, which were similar to levels in the zikv + mers- control group and significantly higher than those in their zikv À littermates (figures a and b ). importantly, low levels of zikv rna were associated with the healthier development of the neonatal brains. of the six representative mabs selected for in-depth neuropathological study, zikv + mice treated with either zk b or zk c maintained normal brain growth and structure. there were no notable differences in cerebral size, cortical thickness, or lateral ventricle area between these zikv + neonates and their zikv À littermates ( figures d- h ). however, zikv + mice treated with zv and z received only moderate protection, while zk f and mers- failed to provide any protection. low protection was measured by reduced cerebral size, thinner cortices, and enlarged lateral ventricles in zikv + neonates compared to their zikv À littermates ( figures d- h ). it is interesting to note that the e-specific human immunoglobulin g (igg) levels in the brain were higher in mice treated with zk b and zk c compared to the other mab groups. in particular, the levels of zk b ( . ± . mg/kg, which is translated into . ± . mg/ml) in zikv + neonates were well above the ic neutralization values measured in the prnt, whereas igg levels in the other mab groups measured less than or similar to those in the negative control pbs group ( figures a and c) . these results indicate that the tested mabs were able to transport across both the maternal-fetal placental barrier and the primitive blood-brain barrier of developing fetuses (saunders et al., ) . we also conducted immunohistochemical analyses of brain sections collected from p neonates in each group. as shown in figure a , in zk b and zk c groups, no zikv-infected cells (green) or apoptotic cells (cas + , red) were detected in the cortices of either zikv + neonates or their zikv À littermates. the mature neurons (neun; figure a , gray) and nuclei (dapi; figure a , blue) appeared healthy, with a tightly packed structure throughout the tissue sections. in distinct contrast, a large number of zikv-positive cells were identified throughout the cortices of the zikv + neonates in zv , z , zk f , and mers- groups. this high prevalence of infection was associated with significant cell death ( figure a , red) and degradation of the cortical structure ( figure a , gray and blue). we also quantitatively assessed the protective activity of zk b and zk c using marker cells from multiple tissue sections. as shown in figures b and c , treatment with zk b and zk c in zikv + neonates reduced zikv infection and cell death to levels indistinguishable from those of their zikv À littermates. furthermore, in the zk b and zk c groups, counts of mature neurons were equivalent between zikv + neonates and their zikv À littermates ( figure d ). however, for the zikv + neonates treated with either zv , z , zk f , or mers- , zikv-positive cell and apoptotic cell counts were as high as , cells and , cells, on average, per square-millimeter tissue section, respectively ( figures b and c) . counts of mature neurons were also significantly reduced in these groups ( figure d ). these results support the enhanced therapeutic activity of zk b and zk c at the cellular and tissue levels relative to zv , zv , zk f , and mers- and also offer an explanation for their impressive in vivo protection of developing fetuses. as zk b demonstrated the greatest prophylactic efficacy against zikv infection, we went on to evaluate its treatment efficacy after zikv infection of the developing fetuses. specifically, after the littermate fetuses were either left untouched (zikv À ) or inoculated with pfus of zikv gz in the lateral ventricle (zikv + ) on e . , we administered mg/kg zk b i.p. to the pregnant mice. this was done either on the same day (e . , day ), day later (e . , day ), or days later (e . , day ). the littermate neonates were closely monitored after delivery and analyzed for the same outcomes measured in the prevention experiments ( figure a ). as shown in figure a , delaying treatment from day to either day or day reduced the survival percentages. treatment on day offered the most effective treatment, with a median survival of about days. this is signif-icantly higher than that of mice treated on either day ( days) or day ( days). the survival advantage conferred by treatment on day corresponded with a clear reduction of viral load in the brain ( figure d ) and less damage to the cortical thickness and lateral ventricles (figures f and g) . however, the effect was less obvious when measured by body weight or each red dot represents a zikv + ; each blue dot represents a zikv À neonate. the red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c -treated groups. the numbers of neonates for each mab analyses are indicated above each group, with zikv + in red and zikv À in blue. in (g) and (h), each dot represents the mean value of at least two slices from one neonate. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. blood serum viral loads on p (figures b and c ). considering that, in our mouse model, the brain is the initial and active site of viral replication, we expected that antibody treatment effects would be more sensitive and immediate in this organ and would be apparent before effects on other organs and body weight as a whole. this hypothesis was supported by immunohistochemical analyses of brain sections derived from p neonates. as shown in figure a , zikv-positive cells (green) were only sporadically detected in the day- treatment group. however, they were detected throughout the cortices if treatment was delayed to day or day . a greater level of infection was associated with increased cell death ( figure a , red) and cortical abnormalities ( figure a , gray and blue). in particular, the tightly packed and well-organized structure of matured neurons in the cortices became loosely connected and disorganized. furthermore, a quantitative assessment of marker cells derived from multiple each red dot represents a zikv + neonate; each blue dot represents a zikv À littermate. red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c treated groups. each dot represents the mean value of at least two slices from one neonate analyzed. the numbers of neonates for each analysis are indicated above each group, with zikv + in red and zikv À in blue. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . . tissue sections supported the evidence that the time of treatment administration impacted treatment efficacy. for instance, in the zikv + neonate group, treatment on day significantly suppressed the number of zikv-positive cells to ± and apoptotic cells to ± per square millimeter of tissue section. treatment on day , however, allowed widespread cell infection and cell death counts as high as , ± , and ± per square-millimeter tissue section, respectively ( figures b and c ). however, although tissue samples from all three temporal treatment groups showed structural abnormalities in the cortices, there were no obvious reductions in the total number of mature neurons ( figure d ). treatment with the isotype mers- control was worst among all groups, with no signs of figure . evidence of therapeutic potential of zk b against zikv infection in developing fetuses (a) decreased survival when treatment administration is delayed from day (e . ) to day (e . ) or to day (e . ). the number (n) of neonates monitored for survival is indicated. (b-d) after treatment with zk b on day , day , or day , we analyzed zikv + neonates (indicated in red) and zikv À littermates (indicated in blue) at p for (b) body weight, (c) zikv rna copies in the blood, and (d) zivk rna copies in the brain. the number of neonates analyzed for each treatment time point is indicated above each group. (e-g) shown here: (e) the representative coronal sections of the neonatal brains at p was visualized with nissl staining and analyzed for (f) cortex thickness and (g) ventricular area. in (f) and (g), each dot represents the value of one slice. the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and , respectively. the number of neonates analyzed for each treatment time point is indicated above each group. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. protection (figures and ) . these results highlight the critical role of zk b in attenuating disease progression by inhibiting cell infection and death at the cellular and tissue levels. furthermore, the time of administration relative to infection impacts efficacy. treatment has the greatest protective effect when initiated immediately after infection. we report here the systematic analyses of the prophylactic and therapeutic potential of a panel of human mabs against zikv infection in a mouse model of microcephaly and non-pregnant ag mice. we showed that different mabs demonstrated distinct protective activity against zikv infection and that the major determinant of efficacy is their intrinsic neutralizing activities. a single i.p. injection of pregnant and non-pregnant mice with the most potent mab, zk b , provided developing fetuses and adult animals with a complete protection against zikv infection. treatment with zk b markedly reduced fetal infection and tissue pathology and significantly delayed mortality. thus, zk b is a promising candidate for the development of antibody-based intervention and informs rational design of zikv vaccine. two unique aspects of our study are worth highlighting here. one is based in the tested mabs that are all derived from the same zikv convalescent individual at different time points in the course of natural infection. each has distinct neutralizing activity, epitope specificity, and lineage ancestry . this diversity not only allows us to pinpoint the major determinants of protection in the mouse model but also provides evidence for when protective potential arises during the course of a natural infection in humans. our results clearly show that neutralizing activity, rather than other features, is the critical biomarker for protection against zikv infection, although the role of fc-mediated antiviral functions still need further investigation (dejnirattisai et al., ; pierson and graham, ; sapparapu et al., ; stettler et al., ). among the mabs tested here, zk b is the most potent, and its epitope is located in the lateral ridge region within the diii of e . mabs that target diii with potent neutralizing activity have also been isolated by other groups, derived from either infected humans or mice, and have been shown to be effective in various models of zikv pathogenesis (fernandez et al., ; magnani et al., ; robbiani et al., ; stettler et al., ; wang et al., b; zhao et al., ) . some weakly neutralizing mabs have also been reported against the same domain (robbiani et al., ; sapparapu et al., ; stettler et al., ; zhao et al., ) . it would be hard to determine whether this convergence on diii is a purely random event or whether diii epitopes are somehow more exposed and, therefore, more accessible by the mabs. it would be interesting to compare the exact epitopes of the highly potent mabs to see whether the vulnerable spots are widely spread or confined to restricted regions on the diii. this information would undoubtedly contribute to our understanding of the immune recognition mechanisms and assist in the rational design of vaccines against zikv infection. similar to other reported diii-specific mabs, zk b was isolated late after the onset of symptoms ( days), when the production of diii-specific antibodies had become more prevalent the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and in (b) and (d), and , , , , and in (c), respectively. the total number of neonates analyzed is indicated above each group. all data are presented as mean ± sem. **p < . ; ***p < . ; ns, not significant. . however, why the diii-specific response seems to be delayed relative to di/ii-specific responses, and whether a vaccine could induce the preferred diii-specific response ahead of those targeting di/ii, requires more in-depth study. nevertheless, the potent prophylactic and therapeutic activities demonstrated by zk b and other diii-specific mabs will serve as the key criteria for future antibody-based intervention against zikv infection. the other unique aspect of our study was the mouse model of microcephaly, in which the virus was directly inoculated into the lateral ventricles of the fetal brain. despite the surgical sophistication required with this technique, the model has been standardized with remarkable reproducibility and captures the phenotypic features that are key to zikv infection in humans (li et al., a wang et al., a; yuan et al., ) . direct inoculation eliminates the need for immune-deficient pregnant mice and allows for control over the developmental stage at which infection occurs. as a result, both pathogenesis and intervention strategies can be evaluated with more precision. critically, it is likely the most stringent model for brain infection, as it tests the immediate protective activity of mabs at the injection site, as well as their capacity in preventing subsequent dissemination throughout the body. in this regard, the levels of protection conveyed by zk b are, indeed, remarkable. furthermore, direct inoculation on e . also allows for the study of immediate and long-term impacts of zikv infection in the developing fetuses. in this study, neonates were followed up to p , but this could have been extended to investigate the long-term sequelae of zikv infection, be they physiological, cognitive, or behavioral. this feature is complementary to the existing model, in which zikv infection was initiated earlier in embryonic development and was frequently associated with fetal demise before or shortly after delivery (fernandez et al., ; morrison and diamond, ; sapparapu et al., ) . in the future, it would be interesting to test the panel of mabs identified here in the early infection model in order to evaluate their protective potential. in conclusion, our study provides compelling evidence that the protective potential of tested mabs against zikv infection in pregnant and non-pregnant mice was directly associated with their neutralizing activities measured in the prnt. the most potent neutralizing antibody, zk b , demonstrated impressive prophylactic and therapeutic activities and, therefore, could serve as a promising candidate for antibody-based interventions and inform rational vaccine design against zikv infection. we previously reported on the isolation and characterization of a panel of human mabs from longitudinal samples of a zikv convalescent individual and showed their distinctions in neutralizing activities, epitope specificities, and time of development during nature infection . we also reported on one of the most stringent mouse models of microcephaly, in which a contemporary zikv asian strain is inoculated directly into the lateral ventricles of the fetal brains (li et al., a) . the present study combines these methodologies in order to evaluate the protective potential of six representative human mabs against zikv infection and microcephaly. using a single i.p. injection of mabs in pregnant mice prior to zikv infection, we showed that these antibodies convey distinct levels of protection to the developing fetuses and newborns and that these levels closely mirrored their respective in vitro neutralizing activities. the most potent neutralizing antibody tested, zk b , provided a complete protection from zikv infection in pre-exposure treatment and partial protection in post-exposure therapy, as measured by markedly reduced tissue pathology. the tested mabs (zk b , zk c , zk f , zk c , zk - , and zk - ) targeted the zikv e and were initially isolated from a zikv convalescent chinese traveler visiting venezuela during the viral outbreak in . the details of their neutralizing activity, epitope specificity, lineage ancestry, and the time of isolation during natural infection were previously reported . two zikv diii-specific mabs, zv and z , were isolated by others (robbiani et al., ; zhao et al., ) and used here for comparative analyses. all mabs were in the human igg backbone, manufactured in f cells (atcc) by transient transfection, and purified by affinity chromatography using protein a agarose (thermo scientific). the mab concentration was determined using the bca protein assay kit (thermo scientific). we previously isolated the human mab mers- , which targets against the mers-cov and was used as a negative control (jiang et al., ) . approximately mg/kg of each tested mab or isotype control mers- was administered i.p. to a group of three pregnant mice at e . . all icr pregnant mice were purchased from beijing vital river laboratory animal technology. the experimental protocol and procedure were approved by the institutional animal care and use committee at tsinghua university ( -zlq ). the pregnant mice were kept in separate cages and maintained on a -hr/ -hr light/dark cycle throughout the experiment. for each tested mab, a group of three pregnant mice were included, and their littermate neonates were monitored for a complementary set of clinical, virological, and neuropathological outcomes on p and for survival up to p ( figure a) . specifically, the first (i) and second (ii) groups of littermate neonates were sacrificed on p . brain and blood samples were immediately collected, processed, and frozen at À c until use. group i was used to measure for viral loads in the blood serum and the brain, as well as human igg in the brain. group ii was used for blood serum viral loads, brain size, and section analysis. the third (iii) group littermate neonates were monitored for body weight at p and for survival up to p . non-pregnant c bl/ mice deficient in interferon (ifn)a, -b, and -g receptors (ag mice) were purchased from institute pasteur of shanghai, chinese academy of sciences. the mice were bred and maintained in a pathogenfree animal facility at tsinghua university. specifically, mg of each tested mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- was administered to a group of four -to -weekold ag mice via the i.p. route. the following day, the animals were challenged with pfus of zikv (gz strain) via i.p. injection and monitored for survival, body weight, and viral rna copies in the blood up to days. on day and day after challenge, blood was collected from each animal for viral load measurement. microinjection of zikv into the brain ml zikv asian strain gz (genbank: ku and virus stock concentration . pfu/ml) was injected into the right side of the lateral ventricle of the embryonic mouse brains at e . , as described previously (li et al., a) . approximately half of the littermate neonates were injected with zikv (zikv + ), while the rest remained untouched (zikv À ). gz was derived from the same chinese traveler from whom the tested mabs were isolated . phylogenetic analysis of the complete coding sequences indicated that gz was tightly clustered with those circulating in the americas and belonged to the asian lineage, including those identified from french polynesia in . the whole blood ( ml) and right brain of the neonates were collected on p , immediately transferred to an eppendorf tube containing lysis buffer (qiagen), and kept in À c until use. each brain sample was weighted and homogenized using a stainless steel blender (next advance). total rna from the homogenized brain and the whole blood was extracted using an rneasy mini kit ( , qiagen) and reverse-transcribed into cdna using an iscript cdna synthesis kit ( - , bio-rad). as previously described, viral rna copies were quantified through taqman qpcr amplification of zikv envelope genes and expressed as log viral rna copies per gram for the brain or per millimeter for the blood samples calculated against the standard curve. the sequences for the primers and probes used for the analysis are shown as follows: zikv-fccgctgcccaacacaag, zikv-r ccactaa cgttcttttgcagacat, zikv-probe agcctaccttgacaagcartcagac actcaa ( fam, tamra). the neonatal right brains were collected on p and immediately frozen at À c until use. the frozen brain samples were weighted and homogenized with a stainless steel blender (next advance) before being mixed with ml pbs and allowed to stand at À c for hr. the tested mabs, suspended in the pbs, were collected by centrifugation at , g for min and quantified by serial dilution and application to elisa plates pre-coated with recombinant full-length e glycoprotein of zikv (gz , ku ), as previously described . bound mabs were detected using goat antihuman igg (fc specific) conjugated with horseradish peroxidase (promega, madison, wi, usa) and , , , -tetramethylbenzidine (tmb) substrate (cwbio, beijing, china). the mabs levels in the neonatal brains were calculated against a curve that was standardized using the corresponding mab suspended in pbs and expressed as milligrams per kilogram or micrograms per milliliter. brains were fixed in % pfa, dehydrated in % sucrose, and frozen in tissuefreezing medium before being sliced into -mm-thick tissue sections. for nissl staining, brain sections were stained with . % toluidine blue for min; dehydrated, in turn, by %, %, and % ethanol ( s, twice for each); and then hyalinized by xylene for more than min. for immunohistochemical staining, sections were blocked for hr at room temperature (rt) and incubated with the primary antibody for one night at c. after three rounds of extensive washing with phosphate-buffered saline with . % tween- (pbst), the secondary antibody was added and incubated at rt for hr. the section was then washed once more with pbst. the tissue sections were then imaged on an lsm (carl zeiss) confocal microscope and analyzed with imaris and imagej, as described previously (li et al., a) . the antibodies used for immunohistochemical analysis were caspase- (abcam, ab , : , ) and neun (abcam, ab , : , ). zikv serum ( : , ) was derived from the same convalescent patient. nuclei were stained with dapi (invitrogen). for experiments involving pregnant and non-pregnant mice, - mice were included in each assessment group to ensure representation and consistency of the data. all data were analyzed using prism software (graphpad). statistical evaluation was performed by student's unpaired t test. data were presented as mean ± sem. *p < . ; **p < . ; and ***p < . . broadly neutralizing antibodies to hiv and their role in vaccine design broadly neutralizing antiviral antibodies the brazilian zika virus strain causes birth defects in experimental models zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus zika virus. i. isolations and serological specificity antibody-mediated neutralization of flaviviruses: a reductionist view a dynamic landscape for antibody binding modulates antibody-mediated neutralization of west nile virus human antibodies to the dengue virus e-dimer epitope have therapeutic activity against zika virus infection a highly potent human antibody neutralizes dengue virus serotype by binding across three surface proteins recent zika virus isolates induce premature differentiation of neural progenitors in human brain organoids zika virus impairs growth in human neurospheres and brain 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and cross-reactive memory b cell responses in dengue-experienced donors neutralizing human antibodies prevent zika virus replication and fetal disease in mice the rights and wrongs of blood-brain barrier permeability studies: a walk through years of history specificity, crossreactivity, and function of antibodies elicited by zika virus infection zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus infects human cortical neural progenitors and attenuates their growth molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus transfer of convalescent serum to pregnant mice prevents zika virus infection and microcephaly in offspring a human bi-specific antibody against zika virus with high therapeutic potential zika virus infects early-and midgestation human maternal decidual tissues, inducing distinct innate tissue responses in the maternal-fetal interface zika virus: history of a newly emerging arbovirus delineating antibody recognition against zika virus during natural infection a single mutation in the prm protein of zika virus contributes to fetal microcephaly excretion of infectious zika virus in urine structural basis of zika virus-specific antibody protection we are grateful to the zikv convalescent patient for donating his blood samples and drs. jiang wang, wenxin hong, and lingzhai zhao for providing the patient with treatment and care. we thank drs. cheng-feng qin and gong cheng for providing the zika virus isolate gz and for assisting with evaluations of antibody protective activity in ag mice. we also thank ms. angela fan for editing the manuscript. all authors contributed to the present study, including intellectual input, results analysis, and actual writing and editing of the manuscript. specifically, c.l., f.g., r.w., y.j., l.y., and x.s. performed the experiments. c.y., x.t., f.z., z.x., and l.z. designed the study, and c.l., f.g., z.x., and l.z. analyzed the data and wrote the paper. patents have been filed for the isolated antibodies, and they are all available for collaboration in research and development through material transfer agreement. key: cord- -qdbcdn j authors: bassi, maria rosaria; sempere, raquel navarro; meyn, prashansa; polacek, charlotta; arias, armando title: extinction of zika virus and usutu virus by lethal mutagenesis reveals different patterns of sensitivity to three mutagenic drugs date: - - journal: antimicrob agents chemother doi: . /aac. - sha: doc_id: cord_uid: qdbcdn j flaviviruses constitute an increasing source of public health concern, with growing numbers of pathogens causing disease and geographic spread to temperate climates. despite a large body of evidence supporting mutagenesis as a conceivable antiviral strategy, there are currently no data on the sensitivity to increased mutagenesis for zika virus (zikv) and usutu virus (usuv), two emerging flaviviral threats. in this study, we demonstrate that both viruses are sensitive to three ribonucleosides, favipiravir, ribavirin, and -fluorouracil, that have shown mutagenic activity against other rna viruses while remaining unaffected by a mutagenic deoxyribonucleoside. serial cell culture passages of zikv in the presence of these compounds resulted in the rapid extinction of infectivity, suggesting elevated sensitivity to mutagenesis. usuv extinction was achieved when a -fold dilution was applied between every passage, but not in experiments involving undiluted virus, indicating an overall lower susceptibility than zikv. although the two viruses are inhibited by the same three drugs, zikv is relatively more susceptive to serial passage in the presence of purine analogues (favipiravir and ribavirin), while usuv replication is suppressed more efficiently by -fluorouracil. these differences in sensitivity typically correlate with the increases in the mutation frequencies observed in each nucleoside treatment. these results are relevant to the development of efficient therapies based on lethal mutagenesis and support the rational selection of different mutagenic nucleosides for each pathogen. we will discuss the implications of these results to the fidelity of flavivirus replication and the design of antiviral therapies based on lethal mutagenesis. h uman disease caused by flaviviruses represents a growing source of global health concern, with elevated numbers of deaths and cases of severe disease ( , ) . the incidence of flavivirus-related disease has increased during recent years. this is possibly related to multiple environmental and socioeconomic factors, such as long-distance spread of pathogenic flaviviruses (e.g., introduction to a different continent) and broader dissemination in temperate climate regions ( , , ) . despite having had limited relevance to public health prior to (only cases reported), recent large epidemics of zika virus (zikv) in asia and the americas have had a major socioeconomic impact. it is estimated that there have been over million cases of infection, leading to several thousand people suffering from severe disease ( , ) . in addition to severe neurological conditions, such as guillain-barré syndrome and congenital microcephaly, a wide range of disorders linked to the establishment of persistent infection in different tissues have been documented ( - ). without attracting the same level of attention as zikv, other emerging flaviviruses are affecting an increasing number of people. in particular, viruses infecting birds, such as west nile virus (wnv) and usutu virus (usuv), have been related to recent cases of neurologic disease in temperate countries ( ) ( ) ( ) ( ) . increased incidence of flaviviral disease seems to be connected to climate change and its impact on the migratory dynamics of birds and the geographic spread of mosquito vectors ( ) ( ) ( ) ( ) . usuv has caused recent epidemics in birds across europe, with elevated mortality in some species, such as blackbirds, owls, and other wild and captive animals ( , ) . recent sporadic cases of human disease geographically connected to these outbreaks are raising concerns of usuv becoming a potential threat to global health ( , , , , ( ) ( ) ( ) ( ) ( ) . lethal mutagenesis has long been proposed as a broad-spectrum strategy to control viral infections, with recent data supporting its feasibility and efficacy in vivo ( , ) . the rationale for antiviral therapies based on mutagenesis stems from theoretical studies by eigen and colleagues ( , ) . these investigations led to the proposal that the elevated error frequencies during rna virus replication are in the proximity of a maximum tolerated value for viability, namely, the error threshold ( ) ( ) ( ) . mutation rates beyond this value would be incompatible with maintaining meaningful genetic information, and thus virus propagation, leading to the extinction of the population in a process known as error catastrophe ( ) ( ) ( ) . the theory was empirically proven using mutagenic agents that induce increased mutation frequencies in viruses ( ) ( ) ( ) ( ) . a vast repertoire of molecules that exert broad-spectrum antiviral activities linked to viral mutagenesis have been identified, including nucleoside and nonnucleoside compounds ( , , ( ) ( ) ( ) ( ) ( ) ( ) . mutagenic nucleosides can be incorporated into newly synthesized viral rna genomes after their intracellular conversion into phosphorylated nucleoside analogues ( , ( ) ( ) ( ) ( ) ( ) ( ) . some of these compounds are currently used at the clinical level; e.g., ribavirin has been extensively used for the treatment of hepatitis c virus (hcv) infection, and favipiravir (also known as t- and commercialized as avigan), has been trialed against influenza virus and ebola virus disease ( , ( ) ( ) ( ) . several recent studies have indicated an association between mutagenesis and antiviral activity in vivo. we have demonstrated that the ribonucleoside favipiravir can cure persistent murine norovirus infection in the mouse intestine. this antiviral activity is accompanied by increased mutation frequency and decreased specific infectivity, both signatures of error catastrophe, in samples isolated preceding complete viral clearance ( ) . additional evidence of antiviral mutagenesis in vivo has been obtained from the analysis of hcvinfected patients treated with ribavirin ( ) . larger mutation frequencies accompanied by decreased specific infectivity were also observed in hantaan virus recovered from infected mice treated with ribavirin ( ) . several other studies have provided additional indirect proof of antiviral mutagenesis in vivo, further stimulating the development of therapies based on this strategy ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . several nucleoside analogues have demonstrated antiviral activities against a broad number of flaviviruses, which include zikv and wnv ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in particular, ribavirin and favipiravir efficiently inhibit zikv infection in different cell culture systems, including human neuronal progenitor cells ( ) . a correlation between the antiviral activity elicited by these molecules and larger mutation frequencies has been observed for some flaviviruses ( , , , ) . however, the possible mutagenic activity of these molecules on zikv and usuv has not been yet investigated. it also remains unclear whether two different although related pathogens can have different responses to the treatment with the same mutagenic compounds or whether they show distinct sensitivity to them. this information could be relevant in the design of broad-spectrum antiviral therapies against the flaviviruses based on lethal mutagenesis. here, we examine the antiviral activities displayed by three nucleoside analogues, all licensed for human use, in cell culture infection with zikv and usuv. we observe that ribavirin, favipiravir, and -fluorouracil are all inhibitors of both zikv and usuv, and that consecutive passage of virus in the presence of these drugs can lead to the complete extinction of infectivity. notably, the efficacies of these drugs vary depending on the virus. the molecules exhibiting better antiviral efficacies are typically associated with higher mutagenicity of the corresponding virus. however, the relative increases in mutation frequency observed for each drug treatment differ in usuv and zikv. we observed the highest mutation frequencies in zikv when treated with ribavirin and favipiravir, and in usuv when treated with -fluorouracil. the relevance of these results to a better understanding of flavivirus replication, genetic diversity, and the development of prospective antiviral therapies will be discussed. zika virus replication is suppressed by different ribonucleoside analogues. to investigate whether zikv replication could be affected by increased mutagenesis, we tested four different compounds known to be mutagenic for diverse viruses, -fluorouracil, ribavirin, favipiravir, and decitabine ( , , , ( ) ( ) ( ) . decitabine is an inhibitor of human immunodeficiency virus ( , ) . its antiviral activity has been associated with lethal mutagenesis, and it is possibly related to the incorporation of the phosphorylated deoxyribonucleoside derivative into viral cdna during reverse transcription ( , ) . we have included this analogue as a negative control for antiviral mutagenesis in our rna viral targets, as it is not predicted to be a substrate of rna polymerases. we first investigated the toxicities of these compounds on vero cells (fig. ) . only prolonged treatments with -fluorouracil ( h) led to cell death rates above %. the remaining drugs only showed modest or no effect on cellular viability even after prolonged exposures to the highest concentration tested ( m). treatment of infected cells with all the ribonucleosides, i.e., favipiravir, ribavirin, and -fluorouracil, led to significant inhibition of zikv replication (fig. ) . these molecules antimicrobial agents and chemotherapy exhibited similar antiviral activities when using an epidemic strain isolated in the americas (asian lineage; fig. a ) and an african isolate (fig. b ). as predicted, decitabine showed no effect on zikv replication, further supporting the idea that the antiviral activity elicited by the ribonucleosides is directly related to their incorporation by the viral rna polymerase (fig. c ). further analysis of zikv replication kinetics in the presence of different concentrations of each drug suggests that these molecules exhibit a similar inhibitory capacity. however, favipiravir seems to elicit a stronger inhibitory activity than ribavirin and -fluorouracil when higher concentrations are used ( fig. d to f). favipiravir, ribavirin, and -fluorouracil cause effective zikv extinction after serial passage in vero cells. we further analyzed the antiviral efficacies of these drugs against zikv during prolonged treatment by testing their capacity to abolish infectivity during consecutive viral passages in cell culture. sequential infections of zikv in the presence of these drugs resulted in a total loss of infectivity when a final concentration of m was used (except for decitabine; fig. ). the complete extinction of zikv infectivity was replicated in three independent lineages of passages in each drug ( fig. d and g). favipiravir eliminated zikv in a faster manner (undetectable viral levels reported after passages for all the three independent lineages) than ribavirin and -fluorouracil (all three lineages were extinct after passages). the extinction of zikv populations in these samples was confirmed by performing an additional blind passage in cell culture in the absence of mutagens (data not shown). we did not observe any detectable infectivity or viral rna in the samples recovered, confirming that these three nucleosides completely eliminate zikv infectivity. serial passage of zikv in cells treated with ribavirin or favipiravir at lower concentrations ( to m) also resulted in a gradual decrease in infectivity, with the two drugs presenting similar efficacies ( fig. a to c, e, and f). unlike favipiravir and ribavirin, zikv exhibited lower susceptibility to passages in the presence of -fluorouracil, suggesting that this molecule is a weaker inhibitor or mutagen for this virus. as anticipated, decitabine had no effect on infectivity, as the viral titers measured during serial transfers were similar to those found in passages of virus in the absence of drug ( fig. d and g). a typical signature of lethal mutagenesis is that viral populations isolated in passages preceding extinction show reduced specific infectivity ( , ) . to investigate whether zikv populations treated with these compounds manifested any alteration in their specific infectivity values, we determined the proportion of infectious particles relative to the total number of viral rna molecules in the sample. we confirmed that viral populations rescued after treatment with all three drugs exhibited lower specific infectivity than the untreated viruses, and most significantly those treated with favipiravir ( fig. ) . usuv shows a different sensitivity pattern to nucleoside analogues from that with zikv. to elucidate whether these drugs are also broadly effective against other flaviviruses, we analyzed their antiviral activity on vero cells infected with usuv. all three nucleosides (favipiravir, ribavirin, and -fluorouracil) that inhibited zikv replication also manifested antiviral activity on usuv (fig. ) . likewise, treatment with decitabine exhibited no effect on usuv replication ( fig. b and c). in contrast to what we had observed with zikv, -fluorouracil was the most effective compound against usuv (fig. ). single-cycle infection kinetics experiments with drugs at m revealed that -fluorouracil antiviral activity becomes more prominent at later replication time points (fig. a ). this is also observed when the same drugs are tested against usuv during multiple cycles of infection (infections at low multiplicity of infection [moi]). the . three independent lineages of passages were performed for each drug and concentration tested. serial passages were carried out with l of the cell culture supernatant recovered from the previous infection passage (corresponding to / of the total volume collected). the different graphs show the virus titers determined by tcid assay (a to d) and the genome copy equivalents obtained by quantitative pcr (qpcr) assays (e to g) that were found along serial passages of zikv. a black diamond represents zikv titers found in the supernatants of cultures treated with decitabine (dec); dark-gray squares illustrate -fluorouracil (fu)-treated series; light gray triangles, ribavirin (rbv); and white inverted triangles, favipiravir (fav). every value represents the average of virus titrations or viral genome copy equivalents (grna eq) from at least three biological replicas obtained from independent series of passages (Ϯ sem). in panel d, individual values obtained from each lineage are represented to better illustrate independent events of virus extinction. relative activity of -fluorouracil compared to those of ribavirin and favipiravir is substantially larger at h than at h ( fig. b and c) . this observation may be in agreement with a larger accumulation of mutations during larger number of replication cycles taking place during h. serial passage of usuv in the presence of each ribonucleoside drug led to sustained decreases in virus titers but not to complete viral extinction (fig. a) . virus lineages treated with ribonucleosides showed significantly lower titers ( to log on average) than during passages in decitabine or in the absence of drug. the lowest values were observed in the presence of -fluorouracil, further suggesting that this compound is most effective against usuv during serial passage in cell culture (fig. a) . the apparent lower susceptibility to nucleoside analogues in usuv than in zikv could be related to generally larger virus yields in infections with usuv. previous studies on unrelated foot-and-mouth disease virus (fmdv) suggested that the efficacy of lethal mutagenesis can be affected by the viral load; hence, extinction will be favored when the infection is initiated with a lower viral dose ( ) . thus, we decided to investigate whether passages at a lower infectious dose may also facilitate usuv extinction. we found that all three ribonucleoside analogues ( m) can reproducibly drive usuv to extinction in four independent replicas when a -fold dilution was applied before each transfer (fig. b ). viral sample dilution during sequential passages in the absence of drugs or in the presence of decitabine did not affect infectivity (virus titers in the range of to % tissue culture infective dose [tcid ] per ml; fig. b ). when drugs were used at a lower concentration ( m), the complete elimination of usuv was only observed in some sporadic cases. both ribavirin and -fluorouracil caused usuv extinction in two out of three lineages, while favipiravir never led to the complete loss of infectivity in any of three series analyzed (not shown). further analysis revealed that treatment with -fluorouracil resulted in significantly larger decreases in virus titers than passages in the presence of other drugs at m ( fig. c ; at passage , p Ͻ . for ribavirin versus -fluorouracil, and p Ͻ . for ribavirin versus favipiravir, -way analysis of variance [anova]). differences in sensitivities to nucleoside analogues in usuv and zikv are associated with alterations in their mutational patterns. to investigate whether the antiviral activities observed during treatment with favipiravir, ribavirin, and -fluorouracil are connected to their predicted mutagenic activity, we analyzed the mutation frequencies of both zikv and usuv rescued after passages in the presence of these compounds. since treatment at high concentrations ( and m) resulted in a rapid loss of zikv infectivity, we isolated and analyzed viral rna from the supernatant of infected cells after consecutive passages during exposure to drugs at m (fig. and ) . both favipiravir-and ribavirin-treated virus populations displayed significantly higher mutation frequencies (p Ͻ . ; mann-whitney test for the distribution of mutations per clone) than those untreated or isolated after treatment with -fluorouracil or decitabine. these results are in agreement with the relative antiviral efficacy of each nucleoside drug ( fig. and a ). the highest mutation frequencies were observed in the ribavirin-and favipiravir-treated zikv population ( -and -fold higher than in untreated virus, respectively). this positively links antiviral activity with mutagenesis, as the largest decreases in zikv titers observed during serial passages in the and e and a). in the -fluorouracil-treated population, we only found a modest ( -fold) increase in the mutation frequency (fig. a) , reflecting the milder antiviral behavior of this compound. treatment with decitabine did not significantly alter the mutation frequency (fig. a) , further suggesting that this compound is not affecting rna replication. similarly, we found that the largest mutation frequencies in usuv were observed in populations collected after serial passage in the presence of -fluorouracil (fig. . and b). viral populations recovered after passages in the presence of -fluorouracil ( m) showed -fold larger mutation frequencies than usuv passaged in the absence of drug. ribavirin and favipiravir led to modest increases in the mutational loads, also in agreement with their milder antiviral activities against usuv ( and -fold, respectively; fig. and b). further analysis revealed the expected transition biases typically found in viruses treated with the same drugs ( , , , , ( ) ( ) ( ) ( ) . thus, we observed slightly higher rates of g-to-a and c-to-u transitions in viruses treated with favipiravir, while the opposite changes (a-to-g and u-to-c) were identified in -fluorouracil-treated viruses (tables and ). the only exception to these typical transition mutational patterns was obtained in zikv populations treated with ribavirin (table ) , with higher frequencies in a-to-g and u-to-c nucleotide substitutions (when the [a-to-g ϩ u-to-c]/[g-to-a ϩ c-to-u] ratio is ). in contrast, usuv treated with ribavirin exhibited the expected mutational bias, with larger numbers of g-to-a and c-to-u transitions ( table ) . lethal mutagenesis has been posited as an alternative strategy to the current therapies based on classical antiviral drugs. several lines of evidence sustain that the antiviral properties of ribavirin and favipiravir in vivo can be, at least in part, connected to their mutagenic activity ( , , ) . these data encourage further study on the development of antiviral compounds with reduced toxicity, improved pharmacokinetics, and higher specificity for the viral polymerases ( , , ) . in this study, we have investigated the sensitivity to mutagenesis of two flaviviruses that have recently emerged as serious threats to public health, zikv and usuv. both pathogens were highly sensitive to the exposure of three mutagenic nucleosides, i.e., -fluorouracil, favipiravir, and ribavirin, although they exhibited different degrees of susceptibility to them. zikv was inhibited more efficiently by ribavirin and favipiravir, while usuv replication was affected to a greater extent by -fluorouracil. these inhibition profiles correlate with the relative increase in the mutation frequencies observed in populations rescued after treatment, supporting the idea that their antiviral efficacy is closely associated with their mutagenic activity. a possible explanation for the different sensitivities of usuv and zikv to mutagenic drugs is that the molecular determinants that regulate nucleotide recognition and discrimination in their respective polymerases vary. as for other rna viruses, flavivirus genome replication is an error-prone process catalyzed by the viral polymerase contained in the nonstructural protein (ns ) ( , ( ) ( ) ( ) ( ) ( ) . even though further analysis is needed to confirm this possibility, our data suggest that zikv is more prone to misincorporate purine analogues, such as ribavirin and favipiravir, while usuv shows a preference for pyrimidine substrates, like -fluorouracil. if certain, this information could be instrumental in the rational screening of nucleoside analogues to effectively control each flavivirus. many recent studies have contributed to the better understanding of the molecular biology of zikv replication, including the structural and biochemical characterization of the viral polymerase ns ( ) ( ) ( ) ( ) ( ) . however, there is limited knowledge on usuv replication, with no molecular data on its viral polymerase. future studies are thus needed to elucidate the molecular determinants responsible for the different sensitivities of these flaviviruses to mutagenic nucleosides. additional evidence hinting at molecular differences in zikv and usuv polymerase fidelity can be inferred from the mutation patterns found in viral populations after treatment with ribavirin (tables and ) . we observed the typical dominance of g-to-a and c-to-u transitions in usuv and an unexpected prevalence of the opposite transition types in zikv (the [g-to-a ϩ c-to-u]/[a-to-g ϩ u-to-c] ratios were and . for usuv and zikv, respectively). it has been suggested that the larger proportion of g-to-a and c-to-u transitions in viruses treated with ribavirin (included usuv) is possibly connected to the additive effect of mutagenesis by incorporation of ribavirintriphosphate into viral rna and depleted intracellular gtp pools as a consequence of inosine- =-monophosphate dehydrogenase (impdh) inhibition by ribavirin ( , , ) . although ribavirin-triphosphate is efficiently incorporated during viral rna synthesis opposite to u and c, in such a low intracellular gtp concentration scenario, it may be preferentially incorporated opposite to c, hence leading to those biases ( ) . the dominance of a-to-g and u-to-c mutations in ribavirin-treated zikv populations suggests that its polymerase may be favoring a different base pairing behavior of ribavirin than in other viruses with independence on intracellular nucleotide pools. other plausible scenarios may include differences in host cell rearrangements that indirectly affect virus mutability by the same drugs (e.g., alterations in the expression of cellular proteins associated with nucleotide uptake and its metabolism). further investigations are needed to clarify the mechanism underlying these differences. we deem that this study can stimulate an additional investigation on the therapeutic value of mutagenic drugs against flaviviruses and, in particular, for the treatment of persistent flaviviral disease ( , ( ) ( ) ( ) . mutagenic drugs seem to be especially effective against persistent infections, with major evidences of lethal mutagenesis obtained during treatment of chronically infected hosts, i.e., hcv-infected patients treated with ribavirin, and mice persistently infected with norovirus and treated with favipiravir ( , ) . a tentative explanation for the better efficacy of mutagenic drugs in this context may be linked to potentially larger accumulation of mutations during longer periods of exposure to drugs ( , ) . a vast range of flavivirus-associated disorders have been connected with persistent infection, including zikv ( , ( ) ( ) ( ) ) . zikv infects a broad range of cell types and tissues, including the reproductive tract and the central nervous system ( , , ) . persistent replication in these tissues has been connected to diverse severe conditions, such as end-organ disease, platelet-related illness, and potentially blinding uveitis ( ) . persistent zikv in the reproductive tissue is possibly responsible for a growing number of cases of sexually transmitted disease, with some studies predicting that this may become a major route of infection in the future ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . vaginal mucosa infection has also been related to fetal infection, further highlighting the impact of zikv persistence in the genital tract ( , ) . it is therefore tempting to investigate the potential therapeutic value of lethal mutagenesis in the treatment of persistent infection in the reproductive tract mucosa. it remains unclear whether ribavirin and favipiravir will elicit efficient antiviral mutagenesis in the central nervous system (cns), with recent data showing different efficacies of these drugs in neuron-derived cell culture systems. while some studies demonstrate that zikv replication remains unaffected by these drugs in stem cellderived neurons, other investigations have proven their inhibitory activity during the treatment of neural progenitor cell lines ( , ) . the different efficacies of these molecules could be linked to possible divergences in the cellular uptake or metabolism of nucleoside drugs in these cell lines. thus, for the control of neurotropic disease, it would be desirable to identify mutagenic compounds with improved pharmacological properties in the cns. alternatively, a combination of mutagenic drugs that are effective in controlling the infection outside the cns (favipiravir, ribavirin, and -fluorouracil), together with effective inhibitors in the neuronal tissue ( , ( ) ( ) ( ) , could lead to improved approaches to control zikv hidden in different body compartments. our study also encourages the investigation of -fluorouracil as a therapeutic drug for the control of usuv infection in an eventual epidemic spillover to humans. although severe disease in humans is rare, there is growing evidence that cases of neurologic disorders associated with usuv have been historically misdiagnosed as wnv ( , ) . the diagnosis of disease caused by usuv is further complicated by the resemblance of pathology to wnv cases and the serological cross-reactivity in diagnostic tests ( , , , - , , ) . a mouse model for usuv infection has been recently established, and it represents a valuable tool to test the in vivo antiviral effect of -fluorouracil or novel antivirals ( , ) . these studies can be extended to the potential treatment of severe disease caused by closely related flaviviruses, such as wnv and japanese encephalitis virus, for which antiviral therapies are not available. cells, viruses, and protocols for infections. we used two different zikv strains purchased from the american type cell culture (atcc). the majority of the experiments described in this paper were performed with an isolate from the asian lineage, collected during the recent epidemics in the americas (strain prvabc , puerto rico, , atcc reference number vr- ). in addition, for some experiments indicated here, we used the first zikv strain ever isolated as our reference african lineage virus (uganda, , strain mr , atcc reference number vr- ). the usuv strain ( / ) used in this study was initially isolated from infected birds in austria in and was kindly provided by giovanni savini, istituto g. caporale, italy ( ). we used african green monkey kidney epithelial cells (kindly provided by sylvie lecollinet, anses, france), namely, vero cells, for zikv and usuv propagation, titration, and viral infections in the presence or absence of mutagenic compounds. viral infections were performed as follows: on the day preceding virus inoculation, we seeded -well plates with ϫ cells per well in the presence of ml of complete medium containing % (vol/vol) fetal bovine serum (fbs; sigma), units/ml penicillin-streptomycin (thermo fisher), and mm hepes in high-glucose dulbecco's modified eagle medium (dmem; thermo fisher). cells were incubated overnight at °c with a % co concentration. on the following day, the supernatant was removed from each plate and replaced with l of fresh medium containing % fbs. then, l of virus sample (zikv or usuv) was applied to the cell monolayer at the multiplicity of infection (moi) indicated, and virus adsorption was allowed for h at °c with % co . the inoculum was removed and the cells washed with complete medium to eliminate unattached virus. cells were then covered in ml of medium containing % fbs, and supernatants from infected cultures collected at different time points. virus titration. virus samples were titrated by % tissue culture infectious dose (tcid ) assays. to this aim, vero cells in l were seeded in -well plates in the presence of medium containing % fbs. on the following day, l of -fold serial dilutions of each sample was applied to each well, reaching a final volume of l. the virus titers were determined by scoring the number of infected wells showing apparent cytopathic effect at to days postinfection, and using the spearman & kärber algorithm ( ) . cell culture infections in the presence of antiviral compounds. for infections in the presence of drugs, cell culture supernatants were removed, and l of % fbs complete medium containing to m decitabine ( -aza- =-deoxycytidine; selleckchem), -fluorouracil ( , -dihydroxy- -fluoropyrimidine; sigma-aldrich), ribavirin [ -(␤-d-ribofuranosyl)- h- , , -triazole- -carboxamide; sigma-aldrich], or favipiravir ( -fluoro- -hydroxy- -pyrazinecarboxamide; atomax) was added to each well. cells were then inoculated with l of virus at the moi indicated for each experiment in the corresponding section and incubated for h at °c and % co . supernatants were removed, cells were washed to eliminate unattached virus, and ml of fresh medium ( % fbs) containing each drug at the desired concentration was added. cell culture supernatants were collected at to h postinfection for subsequent analyses. for experiments involving serial passage of viruses in the presence of nucleoside analogues, the first infection with zikv or usuv was carried out at an moi of . tcid /cell. in sequential passages, l of neat virus from the supernatant of the previous passage (which represents / of the total volume collected) was applied to a new monolayer of cells. for experiments involving transfers of diluted usuv, we used in each transfer l of a preparation containing a -fold dilution of the viral sample collected in the previous passage, as described in previous work ( ) . viral rna extraction, reverse transcription-pcr amplification, and mutation frequency analysis. viral rna was extracted from l of viral sample supernatants using the genejet rna purification kit (thermo fisher). for the calculation of mutation frequency values, we followed previously described protocols ( , , ) . briefly, l of purified rna was reverse transcribed in l final volume using superscript iii (roche), as indicated by the manufacturer. three microliters of cdna was then pcr amplified using high-fidelity kod polymerase (toyobo). to this aim, we used primers spanning genomic positions to and to in usuv and to and to in zikv. pcr products were gel purified using the purelink quick gel extraction kit (invitrogen) and then ligated into plasmid pcr blunt using the zero blunt pcr cloning kit (thermo fisher). positive e. coli colonies were identified by pcr screening with primers flanking the vector-cloning site and dreamtaq dna polymerase (thermo fisher). the resulting pcr products, corresponding to individual zikv or usuv cdna clones, were sanger sequenced and the mutation frequency in each population calculated. quantitative pcr analysis of virus populations. the number of zikv genomic rna molecules in different biological samples was quantified using primers and a fam-tamra ( -carboxyfluorescein- carboxytetramethylrhodamine) probe targeting the zikv e gene (positions to ), following protocols previously described ( ) . for the amplification protocol, we used taqman fast virus -step master mix (thermo fisher) and one-step reaction reverse transcription-pcr (rt-pcr) amplification conditions, with a reverse transcription step ( min at °c), followed by min of incubation at °c and amplification cycles of s at °c and min at °c. for the quantification of usuv rna, we used a fam-tamra probe targeting the ns gene (positions to ) and primers previously described ( ) . the same rt-pcr cycle amplification protocol described above for the detection of zikv was used for usuv. statistical analysis. statistical significance was assessed using graphpad prism , as specified in the figure legends. for the statistical analysis of mutation frequencies, we employed a mann-whitney test that compares the ranked scores of the number of mutations found in individual clones grouped by population, as previously described ( ) . we are indebted to lars larsen (dtu vet) and his group for support in establishing our laboratory. we especially value hue thi thanh tran for helping with multiple technical challenges during our study. thomas bruun rasmussen (dtu vet) provided us with expertise in establishing qpcr methods for the detection of usuv and with constructive feedback on our research. we also thank antonio mas (uclm) and marta sanz-ramos for their critical reading and suggestions on the manuscript. this work is supported by funding procured by independent research fund denmark (technology and production sciences, dff-ftp) to a.a., application number - a. the funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. epidemiology of dengue: past, present and future prospects the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact emerging and reemerging arboviral diseases in southeast asia potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study zika virus zika virus in the female genital tract persistence of zika virus in body fluids-preliminary report zika virus 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infection and induction of flavivirus cross-protective immunity a recombinant dna vaccine protects mice deficient in the alpha/beta interferon receptor against lethal challenge with usutu virus emergence of usutu virus, an african mosquito-borne flavivirus of the japanese encephalitis virus group virus isolation and quantitation isolation of fidelity variants of rna viruses and characterization of virus mutation frequency norovirus polymerase fidelity contributes to viral transmission in vivo a rapid and specific real-time rt-pcr assay to identify usutu virus in human plasma, serum, and cerebrospinal fluid coxsackievirus b mutator strains are attenuated in vivo key: cord- - qku ml authors: billington, john; deschamps, isabelle; erck, stanley c.; gerberding, julie l.; hanon, emmanuel; ivol, sabrina; shiver, john w.; spencer, julia a.; van hoof, johan title: developing vaccines for sars-cov- and future epidemics and pandemics: applying lessons from past outbreaks date: - - journal: health secur doi: . /hs. . sha: doc_id: cord_uid: qku ml the covid- pandemic is a stark reminder of the heavy toll that emerging infectious diseases (eids) with epidemic and pandemic potential can inflict. vaccine development, scale-up, and commercialization is a long, expensive, and risky enterprise that requires substantial upfront planning and offers no guarantee of success. eids are a particularly challenging target for global health preparedness, including for vaccine development. insufficient attention has been given to challenges, lessons learned, and potential solutions to support and sustain vaccine industry engagement in vaccine development for eids. drawing from lessons from the most recent ebola epidemic in the democratic republic of the congo, as well as the h n influenza, - ebola, and - zika outbreaks preceding it, we offer our perspective on challenges facing eid vaccine development and recommend additional solutions to prioritize in the near term. the recommendations focus on reducing vaccine development timelines and increasing business certainty to reduce risks for companies. the global health security community has an opportunity to build on the current momentum to design a sustainable model for eid vaccines. up call for the global health security community, showing that even outbreaks in remote villages can have worldwide impact. indeed, the unpredictable, heterogeneous, and fastpaced nature of some highly transmissible eids makes them a particularly challenging target for global health preparedness, including for vaccine development. unfortunately, today's vaccines cannot be developed ''on demand'' in response to a surprise threat. under ideal circumstances, it will take at least to months to bring a safe and efficacious vaccine for sars-cov- to market. vaccine development, scale-up, and commercialization is a long, expensive, and risky enterprise that requires substantial upfront planning and offers no guarantee of success. , nevertheless, there is reason for optimism. the licensure of the world's first ebola virus vaccine by european, us, and some african regulatory authorities in late , along with prequalification by the world health organization (who) in record time, marked a groundbreaking milestone for global preparedness. , regulatory authorities have made extraordinary efforts to help bring safe and effective vaccines to market faster in response to emergencies. the who launched a new health emergencies division that includes eid monitoring response, and it drafted a research and development (r&d) blueprint that prioritizes pathogens with epidemic potential. gavi, the vaccine alliance, adapted its model to include stockpiling of ebola vaccines. at the same time, national and regional government programs like the us biomedical advanced research and development authority (barda) and the european union's innovative medicines initiative (imi) have sustained their commitment to r&d for eid response. finally, new entities like the coalition for epidemic preparedness innovations (cepi) are galvanizing global pandemic preparedness and response for sars-cov- and future epidemics and pandemics, using innovative approaches, including investments in novel platform technologies. the vaccine industry has the experience, capabilities, and capacities required to develop and help deliver critical vaccines to the people who need them under accelerated timelines. a new ''golden age'' in vaccinology opens doors to greater speed, versatility, and more efficient vaccine platforms as well as more nimble manufacturing capabilities for vaccine development. , vaccine manufacturers have a demonstrated track record of responding to eids when called upon. however, responding to immediate requests to develop vaccines for emergencies often requires manufacturers to put other essential, lifesaving programs on hold. it also carries considerable business, reputational, and liability risks, including both direct and indirect financial impacts. much has been written about how we might be better prepared for the next epidemic or pandemic. however, insufficient attention has been given to challenges, lessons learned, and potential solutions to support and sustain vaccine industry engagement in vaccine development for eids. drawing from lessons from the most recent ebola epidemic in kivu, democratic republic of the congo (drc), as well as the h n influenza, - ebola, and - zika outbreaks preceding it, we offer our perspective on challenges facing eid vaccine development and recommend additional solutions to prioritize in the near term. a complete solution set must also include considerations for manufacturing, scale-up, vaccine distribution, and access, which are equally if not more challenging for eid response. however, these important aspects of eid preparedness and response are beyond the scope of this article. in this section, we draw on prior experience to show how critical factors work against vaccine development in an emergency: time and risk. in the event of a widespread epidemic or pandemic, a delay in the availability of a vaccine can result in substantial human and economic loss. in past outbreaks, vaccines came too late to have a significant impact on the epidemic curve despite extraordinary measures taken to accelerate the responses ( figure ). when the h n influenza pandemic emerged, the vaccine industry had a head start because the seasonal influenza vaccine business and the requisite manufacturing infrastructure were well established. notwithstanding these advantages, and despite strong multisectoral collaboration and communication involving companies, vaccines were not available in time (the delay was in part due to the wait for the bsl- reassorted virus, import permits, and a -month production cycle). although vaccines based on the new strain were approved by the us food and drug administration (fda) and shipped globally just months after who had declared h n a public health emergency of international concern (pheic), they came too late and in insufficient quantities to prevent the estimated , to , deaths that occurred worldwide in the first year of the pandemic. while seasonal influenza has a predictable and sufficient annual market volume to motivate sustained company involvement, it has been nearly impossible to predict the need or demand for eid vaccines. in , when the ebola epidemic erupted from a remote village in west africa, there were no approved or late-stage ebola vaccines available to use. ebola was a familiar pathogen, but the incidence and geographic spread of this epidemic was unprecedented, demonstrating that even the most isolated outbreaks cannot be ignored. within months, the epidemic had spread from guinea to neighboring liberia, nigeria, and sierra leone, marking the largest ebola outbreak to date. when who declared a pheic in august , several companies acted to expedite the development of early-stage vaccine candidates that were financed in part by public research funding, especially from canada, the united states, and the european pan american health organization and world health organization. zika -epidemiological update. union. companies advanced vaccine candidates through coordinated phase safety trials starting in september over several sites across africa, europe, and north america at unprecedented speed with help from who and other partners. but the epidemic began to wane before most could complete phase efficacy trials. one company completed positive interim analyses of phase efficacy trials in guinea just prior to the end of the pheic in december . by this time, more than , people were infected and , had died. like the ebola virus, the zika virus was discovered decades ago but was thought to cause only sporadic, mild, and self-limited symptoms in africa and asia. a vaccine was not considered a public health priority. in the well-documented outbreak in , it was the rapid rise of microcephaly in babies born to zika-infected mothers in brazil that brought this disease to global prominence. as the virus spread, fear of this previously unknown complication of infection motivated who to declare a pheic in february . again, vaccine researchers and developers responded to the call, leveraging what scientists knew about zika from work they were already doing with dengue virus, another flavivirus transmitted by the same species of mosquito that transmits zika virus. one company partnered with the us government to advance the first zika virus vaccine candidate to a phase clinical trial in just months. shortly thereafter, a second company began the development of its candidate with support from barda, and other companies started development programs at their own expense. in a story that repeats itself, however, vaccines could not be developed in time to reduce the disease burden before who ended the pheic in november of that year. the disease became endemic-acute disease declined as populations were inoculated by the disease. by then, more than , babies, most of them in brazil, had been diagnosed with irreversible microcephaly or other neurological disorders. although several zika virus vaccine programs continue today, the low transmission rates and high population-level immunity pose significant obstacles for clinical development. vaccine development involves a substantial investment and a high risk of failure. typical vaccine development programs from discovery to licensure can cost companies upwards of a billion dollars, can take over a decade to complete, and on average have a % chance of failure. , while responding to global infectious disease emergencies is central to the vaccine industry's public health mission, business leaders may not always act, and shareholders may not always approve the necessary investments, because the business risks of eid vaccine development tend to outweigh any return on investment. companies have capacities but tend to be sized for the capacity they need to support their in-line portfolio and their pipeline, running at capacity across the value chain. as such, companies that respond to an outbreak have had to divert resources from core business lines, often for many years, to develop a vaccine that tends to lack a commercial market and may only be needed in limited supply and during limited periods of time. this diversion can cripple all but the largest and most well-supported efforts and creates a major disincentive. the ebola and zika experiences illustrate problems with how governments, philanthropic donors, and nongovernment organizations (ngos) currently contribute funding for eid vaccine development. first, there are only limited public financial resources to support development in advance of outbreak. the us national institutes of health (nih) funds research on diseases that pose a potential us threat, such as zika virus, but funding dedicated to accelerating product development tends to be made available only after who has declared a pheic. similarly, other government-led initiatives like barda in the united states and the imi in europe played a substantial funding role in response to recent eids like ebola virus and zika virus, but this funding is cyclical (eg, dependent on us annual appropriations) and subject to political influence and competing priorities. the creation of cepi, as discussed further below, is a promising step forward in this regard. second, the funding tends to cover only a fraction of the direct vaccine development costs and little to none of the opportunity costs. importantly, opportunity costs that result from delayed commercially viable programs, diverted manufacturing lines, and diversion of scientists, regulatory team members, and other resources can quickly outweigh direct costs. finally, funding tends to be short-term or redirected after the emergency is over. these decisions are made without consideration for the ongoing costs companies will incur in completing development, securing licensure, fulfilling post-market regulatory obligations, and scaling manufacturing appropriately to meet uncertain demand. this short-term funding may also favor firstcomer candidates over second-and third-line innovations that may ultimately provide greater value to those at risk. companies also face legal and reputational risks when responding to emergencies. for example, during all outbreaks described above, there were concerns about liability risks during clinical stages and emergency use. manufacturers are often asked to make the vaccine candidates available on preliminary safety data based on the principle that the benefits of expediting clinical development typically outweigh the risks. however, this principle is not uniformly reflected in regulatory policy and public opinion. and under the best circumstances will take at least to months, and there is a need to appropriately inform public expectations. epidemic fluctuations also may make it impossible to demonstrate vaccine efficacy before approval and implementation. despite this uncertainty, companies must continue development without a clear regulatory pathway. these factors negatively affect company risk/ benefit assessments for engagement and increase uncertainty for managers and investors, potentially discouraging companies from responding to the next eid. to ensure we are prepared to meet the challenge of eids, the world needs sustainable solutions that consider the substantial complexity, public health burden, and business risks posed by these unpredictable pathogens. experience with h n , ebola, and zika exposed critical flaws in the vaccine development ecosystem for eids but also triggered new initiatives that may provide a more coordinated global response framework for the future. here, we offer recommendations for the near term that will improve the global preparedness ecosystem in advance of the next pandemic (table ) . despite good faith efforts by industry, governments, and ngos to expedite vaccine development during prior out-breaks, a recurring lesson was that ''faster-than-ever'' is still not fast enough. the experience with recent eids suggests opportunities to speed things up: reduce regulatory timelines and complexity, improve company and partner coordination, and accelerate manufacturing. define and streamline the eid/pandemic regulatory pathway. despite considerable progress, there is still only licensed ebola virus vaccine and no licensed zika virus vaccines. after the ebola virus disease pheic in , both stringent regulatory authorities and local regulators expedited review and approval of the clinical trial protocols for candidate vaccines. however, they did not address other structural gaps that slowed progress, including the lack of an explicit pathway for local regulators to leverage recommendations from stringent regulatory authorities like the fda. such a pathway would enable local regulators to benefit from the stringent regulatory authority expertise while reducing review and approval times at the country level. ideally, having pandemic vaccines licensed and inspected for quality by key regional stringent regulatory authorities would help facilitate the process. in addition, other methods to infer likelihood of field efficacy (eg, animal models, observational studies) need to be developed, included in guidelines, and accepted with stakeholder support. in addition, the virus outbreak underscored the importance of pre-aligned protocols and data requirements for phase trials to accelerate clinical studies. in a pheic setting, conducting clinical trials is challenging for reasons: ( ) nontraditional (ie, not placebo-controlled) trial designs may be favored by local authorities, despite ethical and regulatory controversies; and ( ) access to participants and clinical sites is limited. for the ebola virus outbreak, some regulators from nonaffected countries required placebo-controlled design, whereas regulators in affected countries prohibited this on ethical grounds. over time, who and other key stakeholders developed a protocol with the manufacturer of the leading vaccine candidate, which could be tested without a placebo. , in an encouraging development, the who solidarity vaccine trial protocol for sars-cov- vaccines would create a multisite, randomized, adaptive trial, designed to accelerate the evaluation period. this important innovation could help speed up initiation of trials, enable comparisons between multiple sites and products, streamline data collection and processing, establish surrogate endpoints, and improve integration for regulatory submissions. finally, consideration is needed to ensure that nationallevel access and benefit-sharing laws do not delay timely access to virus strains and other genetic resources needed for rapid testing and vaccine development. the critical need for timely data sharing was demonstrated most recently in china with the sars-cov- strain. build partnership models that enable more cooperation and collaboration, end-to-end. existing partnership models such as the tb drug accelerator and the collaboration for aids vaccines discovery (cavd) demonstrate the potential value of establishing legally secure constructs that foster company collaboration, data-sharing, and sharing of know-how to tackle public health product development challenges. mechanisms through which companies can align with each other and with other partners on actionable information-sharing plans outside of outbreak and epidemic periods might help reduce redundancy and improve efficiency, thus enabling manufacturers to leverage all existing data. this collaborative approach carries both potential benefits for eid preparedness and some potential risks to the companies' core businesses. the approach also may require differentiated application of competition law, tailored specifically to the eid context. to encourage company participation, all partners involved must agree to, respect, and value the intellectual property that individual companies bring to the partnerships. ''germ'' games and other simulations like the event tabletop exercise are one promising way to continue this dialogue and find collaborative solutions. invest in rapid-response platforms and continue to evaluate and fund next-generation approaches. retooling existing manufacturing facilities or building new capacity to respond to eid demand for vaccines is a significant driver of cost and time. the bill and melinda gates foundation and other public and private organizations are investing through cepi and path and directly in novel manufacturing technologies and platforms that support rapid change over relatively small batches at comparatively low cost without disruption to other vaccines. as demonstrated by current efforts to rapidly develop sars-cov- vaccines, eid presents a potentially ideal application for these technologies, most of which are still at early stages. vaccine technology platforms that prove successful as eid rapidresponse platforms could also be used for more commercially viable disease targets. efforts to define regulatory expectations and pathways will be needed, in addition to buy-in from regulators. in the meantime, shared financial commitments from industry, governments, and ngos are needed to maximize the utility of existing production and stockpiling capacity. epidemic and pandemic preparedness is a high-risk business with uncertain returns. steps to reduce perceived and actual risk and ensure sustainability could increase company engagement. ensure proactive, predictable, and sustainable vaccine development and lifecycle funding. the business case for eid vaccine development can be improved by changing the way government and other funders finance these programs. funding must be proactive, early, and based on clear criteria for prioritization. the who research and development blueprint for eid is an important step forward for pathogen prioritization: prior work on sars and mers coronavirus vaccines (both listed in the blueprint) has proven beneficial in the race for a sars-cov- vaccine. however, the existence of many different (and long) lists illustrates the difficulty of defining and anticipating which pathogens need a vaccine first and for which population. , the launch of cepi in marked a watershed moment in the history of global pandemic preparedness. for the first time, there is a multisectoral, global organization dedicated to funding and managing eid early-phase vaccine development for prioritized pathogens drawn from the who blueprint. cepi has secured substantial investments from a global coalition of donors and is accessing innovative financing mechanisms to accelerate grant making. industry partners played an active role in the conceptualization and design of cepi, fully supportive of the vision for a stronger, more nimble, well-resourced publicprivate partnership. this vision is now materializing as cepi responds to sars-cov- with several vaccine programs in development. although cepi is currently resourced only to advance candidates to phase b, it has the ambition and potential to eventually support end-toend solutions, including manufacturing and scale-up. to fully realize this goal, however, it is important that the vaccine industry retain an equal seat at the table with other global stakeholders to set strategy and deliver on these promises. improve forecasting for demand and manufacturing needs. supply and demand forecasting are challenges for routinely used vaccines and are far more complex for eid vaccines because of the highly unpredictable demand, across unpredictable markets, over time. lessons from the h n influenza pandemic and ebola virus outbreaks highlight the importance of establishing priority pathogen lists and terms for business transactions between industry and vaccine purchasers before there is an emergency. for vaccine manufacturers, it is difficult to anticipate potential demand and align those forecasts with manufacturing plans. for h n influenza, this risk was compounded by hastily negotiated supply contracts, many of which went unfulfilled when the early waves of the pandemic waned. manufacturing capacity is planned during the development process, since the facilities-not just the vaccine itself-are a component of the licensure. any additional capacity that is needed must be built and approved by all countries receiving vaccine before it can be used. in , gavi, the vaccine alliance, announced an advanced purchase agreement to help secure a stockpile of an ebola virus vaccine candidate that had not yet been licensed. even though this procurement underestimated the volume needed for the drc epidemic, lessons learned from this construct could inform how large-scale, permanent solutions for other future eid vaccines might be designed. in response to sars-cov- , gavi is exploring the use of its innovative financing mechanisms to incentivize r&d and reduce manufacturing risk. create a global indemnification mechanism. liability exposure is a serious risk that can deter investment in pandemic vaccines. without protection from some types of broad liability claims, companies may not be willing to make these vaccines available without complete development data, as these claims could lead to a substantial confidence loss, business loss, and even bankruptcy. global legal mechanisms are needed to indemnify companies during emergencies when regulatory requirements are followed. who, through consultations facilitated by the world economic forum with cepi and harvard global health institute, recently developed an insurance-based indemnification model to facilitate emergency deployment of experimental vaccines. this is a promising step forward and should be expanded beyond the clinic, in collaboration with industry and other stakeholders. as inventors and producers of vaccines that prevent diseases, vaccine companies are essential partners in all efforts to prepare for and better respond to epidemics, pandemics, and emerging infectious diseases. this work is expensive, risky, and time and labor intensive, and it incurs substantial opportunity costs. a deeper understanding of the ongoing technical, policy, manufacturing, and field-level challenges is needed to inform policy, funding, and program decisions. critically, the solution must include support for manufacturing scale-up, new regulatory models, and strong end-to-end collaboration with all partners sharing risks and benefits. the licensure and roll-out of the first ebola virus vaccine is a testament to progress made over the past decade. while the trajectory of sars-cov- remains uncertain, the mobilization of governments, industry, and funders to develop vaccines and therapies demonstrates a common, global commitment to respond to pandemics. the global community has an opportunity to build on this momentum to design a sustainable model for eid vaccines. rolling recessions are the likely economic impact of new coronavirus and covid- innovation for pandemics the complexity and cost of vaccine manufacturing-an overview the complex journey of a vaccine: the steps behind developing a new vaccine. geneva: ifpma food and drug administration. first fda-approved vaccine for the prevention of ebola virus disease, marking a critical milestone in public health preparedness and response first-fda-approved-vaccineprevention-ebola-virus-disease-marking-critical-milestonepublic-health a research and development blueprint for action to prevent epidemics: funding and coordination models for preparedness and response. geneva: who gavi, the vaccine alliance. ebola vaccine purchasing commitment from gavi to prepare for future outbreaks department of health and human services. hhs funds development of two filovirus vaccines for biodefense innovative medicines initiative. ebola +: ebola and other filoviral haemorrhagic fevers novel vaccine technologies: essential components of an adequate response to emerging viral diseases platform technologies for modern vaccine manufacturing the economic and social burden of the ebola outbreak in west africa update: novel influenza a (h n ) virus infections-worldwide ebola outbreak in west africa epidemic curves pan american health organization; world health organization regional office for the americas. zika-epidemiological update unpublished ah n pdm case study first global estimates of h n pandemic mortality released by cdc-led collaboration efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola c xa suffit!) centers for disease control and prevention. - ebola outbreak in west africa. last reviewed march unpublished zika case study zika situation report: zika virus, microcephaly, guillain-barré syndrome zika vaccine development-current progress and challenges for the future risk in vaccine research and development quantified estimating the cost of vaccine development against epidemic infectious diseases: a cost minimisation study who will answer the call in the next outbreak? drug makers feel burned by string of vaccine pleas unpublished ebola case study improving vaccine trials in infectious disease emergencies implementation of an ebola virus disease vaccine clinical trial during the ebola epidemic in liberia: design, procedures, and challenges ebodac consortium organizes symposium on community engagement, communications and technology in ebola clinical trials- / world health organization. an international randomised trial of candidate vaccines against covid- regulatory policy for research and development of vaccines for public health emergencies infectious disease threats in the twenty-first century: strengthening the global response novel coronavirus -ncov exposes a flaw in the nagoya protocol cooperative development of antimicrobials: looking back to look ahead unprecedented pace and partnerships: the story of and lessons learned from one ebola vaccine program the pandemic pipeline vaccine platforms: state of the field and looming challenges national institute of allergy and infectious diseases. niaid emerging infectious diseases/pathogens. last reviewed coalition for epidemic preparedness innovations coalition for epidemic preparedness innovation turns to iffim to accelerate funding for new vaccine development response to covid- french move fuels fears for flu vaccine sales. reuters gavi board calls for bold engagement to respond to covid- the authors thank the following for their helpful com- key: cord- -i mpov k authors: houldcroft, charlotte j.; beale, mathew a.; breuer, judith title: clinical and biological insights from viral genome sequencing date: - - journal: nat rev microbiol doi: . /nrmicro. . sha: doc_id: cord_uid: i mpov k whole-genome sequencing (wgs) of pathogens is becoming increasingly important not only for basic research but also for clinical science and practice. in virology, wgs is important for the development of novel treatments and vaccines, and for increasing the power of molecular epidemiology and evolutionary genomics. in this opinion article, we suggest that wgs of viruses in a clinical setting will become increasingly important for patient care. we give an overview of different wgs methods that are used in virology and summarize their advantages and disadvantages. although there are only partially addressed technical, financial and ethical issues in regard to the clinical application of viral wgs, this technique provides important insights into virus transmission, evolution and pathogenesis. supplementary information: the online version of this article (doi: . /nrmicro. . ) contains supplementary material, which is available to authorized users. since the publication of the first shotgunsequenced genome (cauliflower mosaic virus ), the draft human genome and the first bacterial genomes (haemophilus influenzae and mycoplasma genitalium ) , and enabled by the rapidly decreasing cost of high-throughput sequencing , genomics has changed our understanding of human and pathogen biology. several large projects that aim to systematically analyse microbial genomes have recently been completed or are ongoing (for example, sequencing thousands of microbiomes and fungal genomes , ); these projects are shaping our knowledge of the genetic variation that is present in pathogen populations, the genetic changes that underlie disease and the diversity of microorganisms with which we share our environment. the methods and data from whole-genome sequencing (wgs), which have been developed through basic scientific research, are increasingly being applied to clinical medicine, involving both humans and pathogens. for example, wgs has been used to identify new routes of transmission of mycobacterium abscessus in healthcare facilities (nosocomial transmission) and to understand neisseria meningitidis epidemics in africa , whereas the sequencing of (table ) , and the importance of deeply sequencing some viral pathogens. we will also explore two areas in which viral wgs has recently proven its clinical utility: metagenomic sequencing to identify viruses that cause encephalitis (box ) ; and the role of wgs in molecular epidemiology and public health management of the pan-american zika virus outbreak (box ) . finally, we will briefly consider the ethical and data analysis challenges that clinical viral wgs presents. for small viruses, such as hiv, influenza virus, hepatitis b virus (hbv) and hepatitis c virus (hcv), the sequencing of partial genomes has been widely used for research, but it also has important clinical applications. one of the main applications and reasons for sequencing viruses is the detection of drug resistance. for example, the management of highly active antiretroviral therapy (haart) for hiv relies on viral sequencing for the detection of drug-resistant variants. haart has substantially improved the survival of patients who have hiv, but successful therapy requires long-term suppression of viral replication with antiretroviral drugs, which may be prevented by impaired host immunity, suboptimal drug penetration in certain tissue compartments and incomplete adherence to therapy . when viral replication continues despite treatment, the high mutation rate of hiv enables resistant variants to develop. it has become standard practice in many parts of the world to sequence the hiv pol gene, which encodes the main viral enzymes, to detect variants that confer resistance to inhibitors of reverse transcriptase, integrase or protease , particularly when patients are first diagnosed and when viral loads indicate treatment failure. sequencing resistant variants has enabled targeted changes in treatment, which has resulted in greater reductions in viral loads than with standard care (undetectable hiv load in % versus % of patients after six months) , . thus, sequencing resistant variants to guide hiv treatment improves disease outcomes. similar approaches have been used to identify resistant variants of hcv , hbv and influenza virus . partial genomes has been used to detect drug resistance in rna viruses, such as influenza virus , and dna viruses, such as human cytomegalovirus (hcmv) . viral genome sequencing is becoming ever more important, especially in clinical research and epidemiology. wgs of pathogens has the advantage of detecting all known drug-resistant variants in a single test, whereas deep sequencing (that is, sequencing at high coverage) can identify low levels of drug-resistant variants to enable intervention before resistance becomes clinically apparent , . whole genomes also provide good data with which to identify linked infections for public health and infection control purposes , . however, progress in using viral wgs for clinical practice has been slow. by contrast, wgs of bacteria is now well accepted, particularly for tracking outbreaks and for the management of nosocomial transmission of antimicrobial-resistant bacteria , . in this opinion article, we will address the challenges and opportunities for making wgs, using modern next-generation sequencing (ngs) methods, standard practice in clinical virology. we will discuss the strengths, weaknesses and technical challenges of different viral wgs methods why sequence whole genomes? limited sequencing of the small number of genes that encode targets of antiviral agents, such as hiv pol, has been the norm in clinical practice. for the detection of a limited number of antiviral-resistant variants, wgs has been too costly and labour-intensive to use compared with sequencing only the specific genes that are targeted by the drugs. however, the increasing number of resistance genes that are located across viral genomes, together with decreasing costs of sequencing and the use of sequence data for transmission studies, are driving a reappraisal of the need for wgs. for example, antiviral treatment for hcv now targets four gene products (ns , ns a, ns a and ns b), and these pcr reactions (which increases the chance of failure), requires more starting material, is more labour intensive and generally less tractable for diagnostic use . sequencing the whole genome simultaneously captures all resistant variants and removes the need to design and optimize pcr assays for the detection of resistance to new drugs. a good example of this is hcmv, for which wgs can simultaneously capture the genes that encode targets of licensed therapies, such as ul (unknown function), ul (dna polymerase) and ul (serine/ threonine protein kinase), and of newer drugs, such as letermovir, which targets ul (terminase complex). this enables comprehensive antiviral-resistance testing in a single test . in addition, wgs can provide information on antigenic epitopes, virus evolution in a patient over time , and evidence of recombination between hcmv strains . wgs can also detect putative novel drug-resistant variants and predict changes to epitopes, although phenotypic testing of variants is required to confirm clinical resistance and to map epitope changes . as pre-existing resistance to antiviral drugs increases (for example, hcv that is resistant to protease inhibitors and hbv that is resistant to nucleoside analogue reverse-transcriptase inhibitors ), wgs will provide the comprehensive resistance data that are required for selecting appropriate treatment. the complete knowledge of all resistant variants can also support novel decisions in clinical management; for example, the identification of extensive genome-wide hcmv drug resistance in a patient supported the decision to treat the individual with autologous cytomegalovirus-specific t cells instead of antiviral drugs . wgs may also better identify transmission events and outbreaks, which is not always possible with sequences of subgenomic fragments. for example, wgs of respiratory syncytial virus (rsv) identified variation outside of the gene that is traditionally used for genotyping, and such information could be used to track outbreaks in households when the genetic variability in single genes is too low for transmission studies . the numerous phylogenetically informative variant sites that can be obtained from full-length or near full-length genomes removes the need for high-quality sequences, which enabled the robust linking of cases of ebola virus infection and public health interventions in real time during the epidemic . genes encompass more than % of the viral genome . individually sequencing each of these genes can be as expensive and time-consuming as wgs . partial-genome sequencing is particularly problematic for large viral genomes, in particular those of the herpesviruses hcmv , varicella zoster virus (vzv) , herpes simplex virus (hsv- ) and hsv- (ref. ). these viruses have traditionally been treated with drugs that target the viral thymidine or serine/threonine protein kinases and dna polymerase. however, the increasing number of drugs in development that interact with different proteins that are encoded by viral genes scattered across the genome, means that targeted sequencing for resistance testing is costly, involves more . the routine use of pathogen wgs for diagnostic purposes is likely to have wider clinical and research benefits. for example, zika virus sequences that were generated for epidemiological purposes inform public health decisions . in addition, hiv genomes that were sequenced to identify antiviral-resistant variants have also been used to study virus evolution and viral genetic association with disease, including genotype-phenotype association studies and genome-to-genome association studies, which look for associations between viral genetic variants, host genetic variants and outcomes of infection, such as viral load set point in hiv infection , . why do we need deep sequencing? modern methods, which use massively parallel sequencing, enable better examination of diversity and the analysis of virus populations that contain nucleotide variants or haplotypes at low frequencies (less than % of the consensus sequence). minority variant analysis is particularly powerful for rna viruses, reversetranscribing dna viruses and retroviruses, because they typically show high diversity, even in a single host. hiv is the classic example; the reverse transcriptase of hiv is error-prone and introduces mutations of x or r x genotypes is predictive of maraviroc treatment failure . sub-consensus frequencies of x -tropic or r x -tropic hiv are also important for the success and failure of bone marrow transplants from ccr -deficient (ccr -Δ ) donors, and this information may influence the decision to stop antiviral therapy in these patients . minority variants and the identification of haplotypes can also be used to detect mixed infections. infections with different hcmv genotypes or super-infections are associated with poor clinical outcomes, and the detection of such mixed infections by wgs might justify more aggressive treatment. sanger sequencing of a virus population can detect minority variants at frequencies between % and % , whereas ngs can sequence those same pcr amplicons to a much greater depth , and, consequently, capture more of the variability that is present. sensitivity and specificity are specific for the analysed virus and the sequencing method. many studies of drug resistance in hiv that use deep-sequencing of pcr amplicons require minority variants to be present at > % to decrease the possibility of false-positive results , . this may miss drug-resistance mutations at frequencies of . - % and lead to poor treatment outcome . although a - % frequency threshold (or lower) may be clinically relevant for the detection of drug resistance in hiv, it is less clear whether the same degree of sensitivity is required for monitoring vaccine escape in hbv or drug resistance in herpesviruses (discussed below). large cohorts of patients need to be tested before, during and after treatment , to establish thresholds for minority drug-resistant variants and vaccine-escape variants that are clinically relevant for each virus. direct deep sequencing of clinical material, either by shotgun methods or rna-seq methods (so called metagenomic methods), also enables the unbiased detection and diagnosis of pathogens, and provides an alternative to culture, electron microscopy and quantitative pcr (qpcr; see below). sequencing viral nucleic acids, whether from cultures or directly from clinical specimens, is complicated by the presence of contaminating host dna . by contrast, most bacterial sequencing is currently carried out on clinical isolates that are cultured; thus, sample preparation is comparatively straightforward (table and at an extremely high rate ( . ± . × − per base per cell) . many closely related, but subtly different, viral variants exist in a single patient. these variants are sometimes described as a quasispecies or a cloud of intra-host virus diversity. the presence of a mixed population of viruses introduces problems for the determination of the true consensus 'majority' sequence, but these minority (non-consensus) variants may also change the clinical phenotype of the virus, and can be used to predict changes in genotype, tropism or drug resistance. for example, a minor variant that confers drug resistance in hiv that is present in only . % of sequencing reads in a patient at baseline can rapidly become the majority (consensus) variant under the selective pressure of drug treatment . similar changes in the frequency of resistance-associated alleles during treatment have been observed for hbv , hcv , hcmv and influenza virus . deep sequencing of viruses is not only required to detect drug resistance, it is also key for genotypically predicting the receptor tropism of hiv, which has treatment implications. hiv can be grouped by its use of cellular co-receptor into r (uses cc-chemokine receptor (ccr )), x (uses cxc chemokine receptor (cxcr )) or r x (dual tropism). maraviroc is a ccr antagonist that blocks infection of r -tropic hiv, but not of x -tropic and r x -tropic hiv. just a % frequency for cases of encephalitis of unknown origin, metagenomic techniques are promising diagnostic tools. there are various protocols in use, but the main methods that are used are rna sequencing (rna-seq) and metagenomics. for rna-seq, the total rna, or a subset of rna, is extracted from a sample (for example, cerebrospinal fluid or a brain biopsy), converted to complementary dna (cdna) and sequenced. metagenomics generally describes the same procedure for dna, but may also include simultaneous sequencing of dna and rna through the incorporation of a cdna-synthesis step. rna-seq may improve the detection of pathogenic viruses, as many viruses have rna genomes and viral mrnas in the cerebrospinal fluid (csf) or brain indicate both the presence of the virus and which viral genes are being transcribed. however, dna viruses, which experience low-level transcription, may be poorly detected using rna-seq, and read numbers for dna viruses may be higher in metagenomic datasets . both methods have successfully identified new or known viral pathogens in cases of encephalitis of unknown origin. metagenomics has been used to aid the diagnosis and characterization of enterovirus d in cases of acute flaccid paralysis . metagenomics identified herpesviruses in the csf of four patients who had suspected viral meningoencephalitis . rna-seq also identified herpes simplex virus (hsv- ) in a patient with encephalitis, although the use of a dnase i digestion (which was intended to decrease the amount of host nucleic acid) decreased the number of hsv- reads . mumps vaccine virus has also been detected in a patient with chronic encephalitis using rna-seq . rna-seq has been very successful in the identification of encephalitis caused by astroviruses , and coronaviruses . the deaths of three squirrel breeders from encephalitis were linked to a novel squirrel bornavirus, which was identified by separate metagenomic sequencing of dna and rna . metagenomics provides more information about the virus in a sample than pcr alone, which may be important for molecular epidemiology, whereas rna-seq can identify viral sequences and viral gene expression. reviewed in ref. ). currently, genome sequencing of viruses can be achieved by ultra-deep sequencing or through the enrichment for viral nucleic acids before sequencing, either directly or by concentrating virus particles. all of these approaches have their own costs and complexities. three main methods are currently used for viral genome sequencing: metagenomic sequencing, pcr amplicon sequencing and target enrichment sequencing (fig. ) . metagenomic approaches have been used extensively for pathogen discovery and for the characterization of microbial diversity in environmental and clinical samples , . total dna and/or rna, including from the host, bacteria, viruses, fungi and other pathogens, are extracted from a sample, and a library is prepared and sequenced by shotgun sequencing or rna sequencing (rna-seq). box explores the diagnostic applications for metagenomics and rna-seq; for example, in encephalitis of unknown aetiology [ ] [ ] [ ] , for which conventional methods such as pcr are often not diagnostic, metagenomics and rna-seq have detected viral infections - and other dna (cdna)-amplified fragment length polymorphism (aflp), abbreviated to vidisca), filtration, ultracentrifugation and the depletion of free nucleic acids, which mostly come from the host, have all been tried [ ] [ ] [ ] [ ] ; however, these methods may also decrease the total amount of viral nucleic acids so that it is insufficient for preparing a sequencing library. non-specific amplification methods, such as multiple displacement amplification (mda), which make use of random primers and Φ polymerases, can increase the dna yield. however, these approaches are time consuming, costly, and may increase the risk of biases, errors and contamination, without necessarily improving sensitivity , . moreover, the proportion of host reads often remains high . when metagenomic methods are used for pathogen discovery or diagnosis, it is crucial to use appropriate bioinformatic tools and databases that can evaluate whether detected pathogen sequences are likely to be the cause of infection, incidental findings or contaminants. bioinformatic analyses of large metagenomic datasets require high-performance computational resources. the fact that metagenomics requires no prior knowledge of the viral genome, can be considered an advantage as it enables novel viruses to be sequenced without the need for primer or probe design and synthesis. this is particularly relevant for rapid responses to emerging threats, such as zika virus . for virus-associated cancers, metagenomics can inform clinical care, provide information on cancer evolution and generate high-coverage data of integrated virus genomes . however, incidental findings, both in host and microbial sequences, may also present ethical and even diagnostic dilemmas for clinical metagenomics (see below). a recent example involved a cluster of cases of acute flaccid myelitis that were associated with enterovirus d (ref. ). the metagenomic data from samples taken from patients showed the presence of alternative pathogens, some of which are treatable, and was debated in formal and informal scientific channels (see omicsomics blogspot article). regulation and reporting frameworks will be important to resolve future issues of this kind. an alternative to metagenomic approaches is to enrich the specific viral genome before sequencing. pcr amplification of viral genetic material using primers that are complementary to causes of encephalitis. in addition, these methods have been used to sequence the whole genome of some viruses, including epstein-barr virus (ebv) and hcv . however, in clinical specimens, the presence of contaminating nucleic acids from the host and commensal microorganisms (table ) decreases sensitivity. the proportion of reads that match the target virus genome from metagenomic wgs is often low; for example, . % for ebv in the blood of a healthy adult , . % for lassa virus in clinical samples and . % for zika virus in a sample that was enriched for virus particles through filtration and centrifugation . the read depth is often inadequate to detect resistance and the cost is high. thus, metagenomic sequencing has typically only been carried out on a small number of samples for research purposes , . the concentration of virus particles (see the zika virus example above ), depletion of host material and/or sequencing to high read depth can increase the amount of virus sequence, but all of these methods add to the cost. the concentration of virus particles from clinical specimens by antibody-mediated pull-down (for example, virus discovery based on complementary whole-genome sequencing (wgs) of zika virus can help to understand the epidemiology of the recent outbreak in south america, including the origin and spread of the virus, and the connection between the virus and microcephaly. it also informs control measures, such as stopping importation of zika cases or disrupting transmission from a reservoir, and blood safety measures in hospitals. for flaviviruses, such as zika virus, wgs, or at least near whole-genome sequencing, is required to provide molecular epidemiology studies sufficient power . wgs, phylogenetic analysis and molecular clock dating, combined with other epidemiological data, were useful to study the introduction of zika virus to south america . for example, the most recent common ancestor of strains that are circulating in brazil pre-dates the football world cup, which makes it highly unlikely that this event was responsible for the introduction of the asian-lineage zika virus to south america . wgs is also central for understanding the pathogenesis of zika virus; for example, by trying to identify sequence changes that are associated with microcephaly, as it is currently unclear which genome regions determine pathogenesis. it is likely that numerous whole-genome sequences of zika virus from around the world and from individuals with microcephaly and asymptomatic infection are required to link particular mutations to birth defects. so far, no changes in the zika virus genome have been unambiguously associated with microcephaly , , . wgs and fragment sequencing were used to identify a case of zika virus transmission through platelet transfusion . this case suggested that asymptomatic donors can transmit the virus to immunocompromised individuals. pcr-based testing had already established the presence of zika virus in the blood supply in a previous outbreak, but no infection was detected in recipients of blood products . based on this new evidence, blood products may need to be screened routinely for zika virus . finally, wgs of zika virus isolates has identified sequence polymorphisms in primer binding sites , which may make pcr-based diagnosis and the quantification of viral load more difficult. this highlights the need to characterize population-level diversity, especially in epidemics in which the locally circulating virus may have diverged from viruses from other locations or time periods. several projects are underway to determine population-level diversity, including the zika in brazil real time analysis (zibra) mobile laboratory project , which uses portable metagenomic sequencing of zika virus and real-time reporting of results . a known nucleotide sequence has been the most common approach for enriching small viral genomes, such as hiv and influenza virus. recent examples of pcr amplicon enrichment followed by wgs include phylogenetic analysis of a measles virus outbreak at the winter olympics and tracking the recent ebola virus and zika virus (box ) epidemics. pcr amplicon wgs of norovirus, which has a genome size of . kb, has been used to understand virus transmission in community and hospital settings, which revealed both independent introductions of the pathogen to the hospital and nosocomial transmission despite measures to control infection . other pcr-based deep-sequencing studies have generated several whole genomes of influenza virus (~ . kb), dengue virus (~ kb) and hcv ( . kb). this was feasible because these viruses all have relatively small genomes that require only a few pcr amplicons to assemble whole-genome sequences. however, the pcr products, respectively , . for clinical applications this is problematic because of the high laboratory workload that is associated with numerous discrete pcr reactions, the necessity for individually normalizing concentrations of different pcr amplicons before pooling, the increasing probability of reaction failure due to primer mismatch (particularly for highly variable viruses), and the high costs of labour and consumables . therefore, although pcr-based sequencing of viruses as large as kb is technically possible, the proportional relationship between genome size and technical complexity make pcr-based sequencing of viral genomes that are more than - kb impractical with current technologies, particularly for large multi-sample studies or routine diagnostics. another consideration is that increasing numbers of pcr reactions require a corresponding increase in sample amount, and this is not always possible as clinical specimens are limited. improvements in microfluidic technologies may help to overcome some of these barriers; for example, fluidigm, raindance and other 'droplet' sequencing technologies. microfluidics-based pcr and the pooling of multiple amplicons have been used successfully to sequence several antimicrobial-resistance loci (for example, from the microbiome of pigs) and can also be used for viral genomes, potentially down to the single-genome level . highly variable pathogens, particularly those that have widely divergent genetic lineages or genotypes, such as hcv and norovirus, cause problems for pcr amplification, such as primer amplification , and primer mismatches . careful primer design may help to mitigate these problems, but novel variants remain problematic. target enrichment. methods of target enrichment (also known as pull-down, capture or specific enrichment methods) can be used to sequence whole viral genomes directly from clinical samples without the need for prior culture or pcr - . these methods typically involve small rna or dna probes that are complementary to the pathogen reference sequence (or a panel of reference sequences). unlike specific pcr amplicon-based methods, the reaction can be carried out in a single tube that contains overlapping probes that cover the whole genome. in a hybridization reaction, the probes, which are bound to a solid phase (for example, streptavidin-labelled heterogeneity of rna viruses, such as hcv , norovirus , rabies virus and rsv , may necessitate the use of multiple overlapping sets of primers to ensure the amplification of all genotypes. pcr amplicon sequencing is more successful for wgs from samples that have low virus concentrations than metagenomic methods , although other methods such as target enrichment of viral sequences may work equally well in such samples, as shown for norovirus samples . overlapping pcrs combined with ngs have been used to sequence the whole genomes of larger viruses, such as hcmv , but this method has limited scalability, as many primers and a relatively large amount of starting dna are required . this limits the number of suitable samples that are available and also the genomes that can be studied using this method. for example, - pcr products were required to amplify the genome of ebola virus , and two studies of norovirus needed and and hhv (ref. ). the reaction is carried out in a single well and, similarly to microfluidics-based pcr, is amenable to high-throughput automation . the lack of a culture step means that the sequences that are obtained are more representative of original virus rather than cultured virus isolates, and there are fewer mutations than in pcr-amplified templates , . the success of this method depends on the available reference sequences for the virus of interest; specificity increases when probes are designed against a larger panel of reference sequences, as this leads to better capture concentrations , . with metagenomics, the proportion of sequencing data that map to the pathogen from unenriched clinical samples is small. target enrichment can increase the percentage of on-target viral reads from . % to % or more . the improvement in quality and depth of sequence that results enables more samples to be sequenced per run than unenriched metagenomic libraries for equivalent on-target sequencing performance. this improvement also decreases the price of sequencing, although the cost of library preparation is increased. there are alternative approaches for the enrichment of viral reads, including pulsed-field gel electrophoresis (pfge) , which separates large viral genomes from smaller fragments of host dna. enrichment techniques that use degenerate rna or dna probes to capture hundreds of viral species have also been developed; for example, virome capture sequencing (vircapseq) . this method is designed for the detection of both known and novel viruses, although its performance remains to be evaluated. to date, there has been very little direct comparison between the three methods for viral genome sequencing in clinical practice, with only one paper evaluating relative performance for the sequencing of hcv . results from this study, in which three different enrichment protocols, two metagenomic methods and one overlapping pcr method were evaluated, showed that metagenomic methods were the least sensitive, yielded the lowest genome coverage for comparable sequencing effort and were more prone to result in incomplete genome assemblies. the pcr method required repeated amplification and was the most likely to miss mixed infections, but when reactions were successful it resulted in the most consistent read depth, whereas read depth was proportional to virus copy number in metagenomics and target enrichment. pcr generated more incomplete sequences for some hcv genotypes (particularly genotype ) than metagenomics and target enrichment. target enrichment was the most consistent method to result in full genomes and identical consensus sequences. the ease of library preparation for metagenomic and target enrichment sequencing of hcv was considered a major advantage for clinical applications, but pcr may still be appropriate for samples that have very low viral loads. of the diversity in and between samples. target enrichment is possible despite small mismatches between template and probe; however, whereas pcr amplification requires only knowledge of flanking regions of a target region, target enrichment requires knowledge of the internal sequence to design probes. however, if one probe fails, internal and overlapping regions may still be captured by other probes , . target enrichment is not suitable for the characterization of novel viruses that have low homology to known viruses for which metagenomics, and, in some cases, pcr using degenerate primers, which are a mix of similar but variable primers, may be more appropriate. as with all methods, the technique is constrained by the starting virus concentration. although viruses could be sequenced from samples with viral loads as low as , international units (iu) ml - (for hcv) or , iu ml - (for hcmv), there was a reduced depth of coverage in sequencing data at lower virus figure | methods for sequencing viral genomes from clinical specimens. all specimens originally comprise a mix of host (in blue) and pathogen (in red) dna sequences. for pathogens that have rna genomes, rna in the sample is converted into complementary dna (cdna) before pcr and library preparation. direct metagenomic sequencing provides an accurate representation of the sequences in the sample, although at high sequencing and data analysis and storage costs. pcr amplicon sequencing uses many discrete pcr reactions to enrich the viral genome, which increases the workload for large genomes substantially but decreases the costs. target enrichment sequencing uses virus-specific nucleotide probes that are bound to a solid phase, such as beads, to enrich the viral genome in a single reaction, which reduces workload but increases the cost of library preparation compared with pcr. similar results were achieved in a study that compared pcr amplicon sequencing and target enrichment sequencing of norovirus . with target enrichment sequencing, the whole viral genome could be sequenced in all samples, whereas pcr-based capsid sequencing was only possible in out of the samples, owing to low virus titres and pcr primer mismatches, which suggests that target enrichment is more sensitive than pcr for sequencing norovirus and better accommodates between-strain sequence heterogeneity . target enrichment has also been used for samples that have low viral loads and incomplete genome coverage in metagenomic sequencing . both metagenomic and target enrichment sequencing can be used for pathogen genomes of all sizes, whereas pcr-based methods are less suitable for large viral genomes or for non-viral (that is, bacterial, fungal and parasite) genomes. direct comparisons of different methods , will be important for determining when each method should be used, based on sensitivity and specificity, as well as factors such as cost, scalability and turn-around time, which are particularly important in clinical applications (table ) . beyond the technical challenges of viral wgs that are mentioned above, there are several other roadblocks that may slow the advance of wgs in the clinic. they may be considered in three groups: ethical issues, including incidental host and microbiological findings; regulatory issues, such as the establishment of standards, good laboratory practice, and sensitivity and specificity thresholds for sequencing; and analytical issues regarding data interpretation and the numerous choices of analysis options. in many clinical tests (for example, magnetic resonance imaging (mri) scans and sequencing of the genomes of patients), there is a risk of detecting a disease association that is not part of the original investigation but might be of clinical importance for the individual or their family. these incidental findings remain a topic of intense medical ethical debate . the risk of incidental findings in pathogen sequencing (for example, the discovery of hiv infection during metagenomic sequencing for other pathogens) is not novel and the solution the issues of sensitivity and contamination are especially important in wgs, because of the risk of both false-negative and false-positive detection of pathogens. highly sensitive sequencing (whether metagenomic, pcr-based or target enrichment-based) may detect low-level contaminating viral nucleic acids , . for example, murine leukaemia virus , and parvovirus-like sequences , are just two of many contaminants that can come from common laboratory reagents, such as nucleic acid extraction columns . as with other highly sensitive technologies, robust laboratory practices and protocols are required to minimize contamination. it is also important to remember that the detection of viral nucleic acid does not necessarily identify the cause of illness, and it is good practice when using ngs methods for the diagnosis of viral infections to confirm the findings with alternative independent methods that do not rely on testing for nucleic acids. for example, in cases of encephalitis of unknown origin, positive ngs findings can be confirmed through immunohistochemical analysis of the affected tissue , , or identification of the virus by electron microscopy or tissue culture . the standardization of methods, including bioinformatics, will be key to the success of ngs and wgs in clinical virology. software packages that use a graphical user interface (gui) are preferable to tools that require command-line expertise. strict version control of software and analysis pipelines is required to ensure that results are reproducible, to make best practices easily shareable, and to enable the accreditation of analysis software. however, best-practice analysis methods are continually evolving and the premature standardization of best practices in an overly rigid manner may inhibit innovation. commercialization and regulation may help, as they provide financial and regulatory incentives to ensure that analysis tools and technologies meet clinical needs. finally, the development of well-curated databases that show which variants are truly indicative of drug resistance will be crucial for accurate clinical interpretation. such databases have already been created for hiv , hbv , and hcv , but without recognition of their value by funding agencies and corresponding centralized funding to ensure their continued maintenance and upkeep, these databases and associated tools may become swiftly outdated or unusable. in clinical virology laboratories that use multiplex pcrs is to suppress results that have not been requested (j.b., unpublished observations). in the united kingdom, the clinical virologist who interprets the test results is part of the team that manages the patient, and, as such, may decide to discuss an unexpected result with the physician in charge. incidental host genetic findings (for example, the detection of variants that predispose to cancer development) in a pathogen metagenomic analysis are not reported to the individual in the united kingdom, because this is only permissible with the consent of the patient. in regard to both host and virus incidental findings, target enrichment and pcr have the advantage of only providing results about the pathogen of interest. the ethical and privacy concerns that are associated with the presence of host genetic data in publically available metagenomic datasets have been well reviewed and represent a separate challenge. regulatory challenges. regulation, as well as helping to address some of the ethical concerns, is also important in standardizing wgs of viruses. the framework that is required to make viral wgs sufficiently robust and reproducible in clinical practice will come from several areas. the framework of laboratory accreditation and benchmark testing that are already available (for example, clinical laboratory improvement amendments of (clia) regulations in the usa, or accreditation according to medical laboratory quality and competence standardization criteria for iso ) will support the development of viral wgs standards, provided that there is sufficient need and pressure to implement clinical viral wgs. lessons learned from the use of pcr in diagnostics may be useful here, starting with ensuring good clinical laboratory and molecular practices , . this will mean including negative samples in every sequencing run to assess contamination thresholds, spiking samples with a known virus to provide a sensitivity threshold, and including positive controls and controls for batch-to-batch variation , all of which will increase sequencing costs and are likely to deter the adoption of pathogen genome sequencing by laboratories that are sequencing only small batches of samples. the centralization of virus wgs can help to ensure the maintenance of adequate standards, the processing of large batches of samples and reducing costs. although there are good reasons for sequencing whole genomes and, in general, for using ngs, if diagnostic or hospital-based laboratories are to be persuaded to transition away from sequencing subgenomic fragments, they need to see the benefit of the additional information for patient care and the practical feasibility of wgs. this includes wgs workflows that are as scalable and automatable as subgenomic fragment sequencing, a suitable regulatory framework and a price for sequencing whole genomes that is competitive with sequencing fragments. currently, the cost of sequencing viral genomes, despite their small size, remains higher than the cost of sequencing subgenomic resistance genes. the cost difference between sequencing a target region and the whole virus genome is largely governed by the size of the genome versus the size and number of target loci. in addition, whole-genome information may provide important additional knowledge, as discussed above. what does the future hold? current ngs technologies that are based around illumina, , ion torrent or sanger methodologies all generate short-read data, which presents challenges for haplotype phasing; that is, determining whether genetic variants (whether inter-host or intra-host) occur on the same genetic background (single viral genome or clonal) or on related, highly similar but different genetic backgrounds in the same population (sometimes called a viral swarm or cloud). furthermore, repetitive regions and recombination are more difficult to resolve using short reads owing to problems such as mapping ambiguities. the clinical implications of understanding whether, for example, multidrug-resistant variants occur together on a single viral genome or are distributed between a mixed population of viruses, each with different drug-resistance profiles, are currently unclear. although there are computational tools to help resolve these issues, new technologies can generate longer reads. newer, single-molecule sequencers, such as pacbio (pacific biosciences) and minion (oxford nanopore), are capable of extremely long-read sequencing, and whole viral genomes (for example, viruses that have genomes less than kb in size, such as ebola virus, norovirus and influenza a virus) could theoretically be obtained from and selective depletion of dna with a certain methylation pattern), no similar methods exist, so far, for viral sequencing. viral wgs is of increasing clinical importance for diagnosis, disease management, molecular epidemiology and infection control. there are several methods that are available to achieve wgs of viruses from clinical samples; amplicon sequencing, target enrichment or metagenomics. currently, the choice of method is specific to both the virus and the clinical question. metagenomic sequencing is most appropriate for diagnostic sequencing of unknown or poorly characterized viruses, pcr amplicon sequencing works well for short viral genomes and low diversity in primer binding sites, and target enrichment works for all pathogen sizes but is particularly advantageous for large viruses and for viruses that have diverse but wellcharacterized genomes. two obvious areas of innovation currently exist: methods that can effectively deplete host dna without affecting viral dna, and the further development of long-read technologies to achieve the flexibility and competitive pricing of short-read technologies. new technologies are required to unite the strengths of these different methods and enable healthcare providers to invest in a single technology that is suitable for all viral wgs applications. single reads. minion also has the advantage of being very fast, taking as little as four hours to go from sample receipt to reporting of analysed data . results from the better-established pacbio technology are more promising, including a recent report of a mean read length of , bp for pseudorabies virus , which has a double-stranded dna genome that is around kb in length. . kb reads have been achieved using pacbio for hcv, although . kb of the . kb genome had been pre-amplified by pcr . a drawback of both ngs and single-molecule sequencing is the need for high coverage to minimize the effect of sequencing errors. this is particularly problematic for studies of drug resistance, as drug resistance most frequently results from single-nucleotide mutations or small deletions ( - bases), especially in lower-fidelity rna viruses . achieving the high coverage that is necessary to ensure accurate variant typing is challenging when there is a lot of host dna compared with viral sequences, and when the error profile of a technology makes point mutations particularly hard to detect . at the time of writing, minion sequencing (r pore chemistry) has raw high quality (so called ' d reads') read error rates of around % (j. quick, personal communication), which compares unfavourably with the error rates of other technologies (illumina (< . %), ion torrent (~ %), but not pacbio ( % single pass)) , although accuracy can been improved using circular consensus read sequencing , . however, combining these long-read technologies with target enrichment provides a 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microbiology of health and disease rationale and uses of a public hiv drugresistance database hepseq: international public health repository for hepatitis b hepatitis b virus reverse transcriptase sequence variant database for sequence analysis and mutation discovery the los alamos hepatitis c sequence database base-seq: a method for obtaining long viral haplotypes from short sequence reads identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing bacterial and viral identification and differentiation by amplicon sequencing on the minion nanopore sequencer enrichment of long dna fragments from mixed samples for nanopore sequencing rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis the authors thank j. brown the authors declare no competing interests. omicsomics blogspot article: http://omicsomics.blogspot. co.uk/ / /leaky-clinical-metagenomics-pipelines.html key: cord- -th da bb authors: gardy, jennifer l.; loman, nicholas j. title: towards a genomics-informed, real-time, global pathogen surveillance system date: - - journal: nat rev genet doi: . /nrg. . sha: doc_id: cord_uid: th da bb the recent ebola and zika epidemics demonstrate the need for the continuous surveillance, rapid diagnosis and real-time tracking of emerging infectious diseases. fast, affordable sequencing of pathogen genomes — now a staple of the public health microbiology laboratory in well-resourced settings — can affect each of these areas. coupling genomic diagnostics and epidemiology to innovative digital disease detection platforms raises the possibility of an open, global, digital pathogen surveillance system. when informed by a one health approach, in which human, animal and environmental health are considered together, such a genomics-based system has profound potential to improve public health in settings lacking robust laboratory capacity. supplementary information: the online version of this article (doi: . /nrg. . ) contains supplementary material, which is available to authorized users. in late and early , a lethal haemorrhagic fever spread throughout forested guinea (guinée forestière), undiagnosed for months. by the time it was reported to be ebola, the virus had spread to three countries and was likely past the point at which case-level control measures, such as isolation and infection control, could have contained the nascent outbreak. in , a new dengue-like illness was implicated in a dramatic increase in brazil's microcephaly cases; one year later, analyses revealed that the zika virus had been sweeping through the americas, unnoticed by existing surveillance systems, since late . although public health surveillance systems have evolved to meet the changing needs of our global popu lation, we continue to dramatically underestimate our vulnerability to pathogens, both old and new . indeed, the recent events in west africa and brazil highlight the gaps in existing infectious disease surveillance systems, particularly when dealing with novel pathogens or pathogens whose geographic range has extended into a new region. despite the lessons learned from previous outbreaks , such as the severe acute respiratory syndrome (sars) epidemic in - and the influenza pandemic -particularly the need for enhanced national surveillance and diagnostic capacity -infectious threats continue to surprise and sometimes overwhelm the global health response. the cost of these epidemics demands that we take action: with fewer than , cases, the ebola outbreak ultimately resulted in over , deaths, left nearly , children without parents and caused cumulative gross domestic product losses of more than % . as with prior crises, in the wake of ebola, multiple commissions have offered suggestions for essential reforms , . most focus on systems-level change, such as funding research and development or creating a centralized pandemic preparedness and response agency. however, they also call for enhanced molecular diagnostic and surveillance capacity coupled to data-sharing frameworks. this hints at an emerging paradigm for rapid outbreak response, one that employs new tools for pathogen genome sequencing and epidemiological analysis (fig. ) and that can be deployed anywhere. in this model, portable, in-country genomic diagnostics are targeted to key settings for routine human, animal and environ mental surveillance or rapidly deployed to a setting with a nascent outbreak. within our increasingly digital landscape, wherein a clinical sample can be transformed into a stream of data for rapid analysis and dissemination in a matter of hours, we face a tremendous opportunity to more proactively respond to disease events. however, the potential benefits of such a system are not guaranteed, and many obstacles remain. here, we review recent advances in genomicsinformed outbreak response, including the role of real-time sequencing in both diagnostics and epidemiology. we outline the opportunities for integrating sequencing with the one health and digital epidemiology fields, and we examine the ethical, legal the systematic collection, analysis and dissemination of health-related data to support planning, implementation and evaluation of public health practices and response. outbreaks and epidemics are both defined as increases in the number of cases of a particular disease beyond what is expected in a given setting. in outbreaks, the affected settings are smaller geographic regions; epidemics can span larger areas. clinical metagenomics. with its untargeted approach to sequencing, clinical metagenomics can cross disciplines in a way that clinical microbiology struggles to -identifying viral, bacterial, fungal and other eukaryotic pathogens in a single assay and coupling pathogen detection to pathogen discovery. given the current high cost of the technique -conservatively estimated at several thousand dollars -it is most often used when dealing with potentially lethal infections that fail the conventional diagnostic paradigm, such as the recent diagnosis of an unusual case of meningoencephalitis caused by the amoeboid parasite balamuthia man drillaris or the diagnosis and treatment of neuroleptospirosis in a critically unwell teenager . in the latter case, despite a high index of suspicion for infection, leptospira santa rosai was not detected by culture or pcr, as the diagnostic primer sequences were eventually found to be a poor match to the genome of the pathogen. intravenous antibiotic therapy resulted in rapid recovery. in such an example, the costs are easily justified, particularly when offset against the cost of a stay in an intensive treatment unit. however, routine diagnostic metagenomics is currently limited to a handful of clinical research laboratories worldwide; it is therefore regarded as a 'test of last resort' and kept in reserve for vexing diagnostic conundrums. substantial practical challenges hinder the adoption of metagenomics for diagnostics (fig. ) (reviewed in depth in ref. ) . chief among these is analytic sensitivity, which depends on pathogen factors (for example, genome size, ease of lysis and life cycle); analytic factors (for example, the completeness of reference databases and the potential to mistake a target for a close genetic relative); and sample factors (for example, pathogen abundance within a sample and contaminating background dna). as an example of a problematic sample, during zika surveillance, attempts to perform un targeted metagenomics sequencing on blood yielded few, or in some cases zero, reads owing to low viral titres . targetenrichment technologies (reviewed in ref. ) such as bait probes can be employed, but even these were unsuccessful at recovering whole zika genomes, necessitating pcr enrichment . in addition to sensitivity, universal pathogen detection through clinical metagenomics is complicated by specificity issues arising from misclassification or contaminated reagents, the challenge of reproducing results from a complex clinical workflow, nucleic acid stability under varying assay conditions, ever-changing bioinformatics workflows and cost. given these issues, could metagenomics replace conventional microbiological and molecular tests for infection? recent studies have used metagenomics in common presentations, including sepsis , pneumonia , urinary tract infections and eye infections . these have generally yielded promising results, albeit typically at a lower sensitivity than conventional tests and at a much greater cost. despite these problems, two factors will drive sequencing to eventually become routine clinical practice. first, the ever-decreasing cost of sequencing coupled with the potential for cost savings achieved by using a single diagnostic modality versus tens or hundreds of different diagnostic assays -each potentially requiring specific instrumentation, reagents, validation and labour -is attractive from a laboratory operations perspective. second, and perhaps most compelling, is the additional information afforded by genomics, including the ability to predict virulence or drug resistance phenotypes, the ability to detect polymicrobial infections and phylogenetic reconstruction for outbreak analysis. novel technologies: portable sequencing. given that outbreaks of emerging infectious diseases (eids) most often occur in settings with minimal laboratory capacity, where routine culture and bench-top sequencing are simply not feasible, the need for a portable diagnostic platform capable of in situ clinical metagenomics and outbreak surveillance is evident. a trend towards smaller and less expensive bench-top sequencing instruments was seen with the genome sequencer junior system (which has since been discontinued), the ion torrent personal genome machine (pgm) system and the illumina miseq system, which were released in close succession . each of these instruments costs <$ , and puts ngs capability into the hands of smaller laboratories, including clinical settings. in , the minion from oxford nanopore technologies was released to early access users , heralding the potential nature reviews | genetics outbreak response portable genome sequencing digital epidemiology one health figure | a genomics-informed surveillance and outbreak response model. portable genome sequencing technology and digital epidemiology platforms form the foundation for both real-time pathogen and disease surveillance systems and outbreak response efforts, all of which exist within the one health context, in which surveillance, outbreak detection and response span the human, animal and environmental health domains. the event through which a pathogen is transferred from one entity to another. transmission can be person-to-person, as in the case of ebola, vector-to-person, as with zika, or environment-to-person via routes including food, water and contact with a contaminated object or surface. the use of genome sequencing to understand infectious disease transmission and epidemiology. see fig. . for highly portable 'lab-in-a-suitcase' sequencing. the minion is pocket-sized and is controlled and powered through a laptop usb connection. it is provided under a model whereby the hardware is free but the consumer pays a premium for the reagent and flow cell consumables. compared with bench-top instruments, the absence of a rolling service contract or regular engineer visits makes it theoretically possible to scale this platform out to potentially unlimited numbers of labora tories. importantly, the minion has been used in field situations, including in diagnostic tent labora tories during the ebola epidemic , and in a roving busbased mobile laboratory in brazil as part of the zibra project , . others have taken the minion to more extreme environments where even the smallest traditional bench-top sequencer could not go, including the arctic and antarctic , a deep mine and zero gravity aboard the reduced-gravity aircraft (nicknamed the 'vomit comet') and the international space station . however, this technology is not yet a panacea; remaining challenges include high dna or rna input requirements (currently hundreds of nanograms), which often necessitate pcr-based amplification approaches; a flow cell cost of $ , keeping the cost per sample high despite multiplexing approaches; and high error rates, which require that genomes are sequenced to high coverage for single nucleotide polymorphism-based analysis and analysed at the signal level. moreover, although the long reads produced by the minion overcome a number of challenges in assembling eukaryotic microbial pathogen genomes, such as the presence of discrete chromosomes or long repetitive regions, the upstream nucleic acid extraction steps required to obtain genomic dna vary across microbial domains and might necessitate reagents and equipment far less portable than the minion. from transmission to epidemic dynamics. genomics is capable of informing not just pathogen diagnostics but also epidemiology. pathogen sequencing has been used for decades to understand transmission in viral outbreaks, from early studies of hantavirus in the united states of america to human immunodeficiency virus (hiv) in the united kingdom ; more recently, the approach has been successfully extended to include bacterial pathogens (reviewed in ref. ) and has come to be known as genomic epidemiology, a term encompassing everything from population dynamics to the reconstruction of individual transmission events within outbreaks . most transmission-focused investigations to date have been retrospective, with only a subset unfolding in real time, as cases are diagnosed [ ] [ ] [ ] [ ] [ ] . in transmission-focused investigations, genetic variants are used to identify person-to-person transmission figure | challenges to in-field clinical metagenomics for rapid diagnosis and outbreak response. a mobile medical unit deploying a portable clinical metagenomics platform has been established at the epicentre of an infectious disease outbreak, but the team faces challenges throughout the diagnostic process and epidemiological response. for example, in the case of zika virus, samples, such as blood, with low viral titres, a small genome of < kb and transient viraemia combine to complicate detection of viral nucleic acid by use of a strictly metagenomic approach. furthermore, obtaining a sufficient amount of viral nucleic acids for genome sequencing beyond simple diagnostics requires a tiling pcr and amplicon sequencing approach . other challenges include, for example, access to a reliable internet connection, the ability to collect sample metadata and translating genomic findings into real-time, actionable recommendations. the average number of secondary cases of an infectious disease produced by a single infectious case, given a completely susceptible population. a term describing infectious diseases that typically exist in an animal reservoir but that can be transmitted to humans. the transmission of an infectious disease, such as ebola, from a survivor of that disease who has recovered from their symptoms. a term describing infectious diseases that are transmitted to humans through contact with a non-human species, particularly those diseases spread through insect bites. an example is the zika virus, which is carried by mosquitos. geographical settings where a variety of factors converge to create the social and environmental conditions that promote disease transmission. the process by which an infectious disease changes from existing exclusively in animals to being able to infect, then transmit between, humans. see fig. . events (fig. ) , either through manual interpretation of the variants shared between outbreak cases or via modelbased approaches , with the result being a transmission network. epidemic investigations are very different -only a subset of the epidemic cases are sequenced. thus, the goal is to use the population structure of the pathogen to understand the overall dynamics of the epidemic. here, phylodynamic approaches are used to infer epidemiological parameters of interest. first conceptualized in by grenfell et al. as a union of "immunodynamics, epidemiology, and evolutionary biology" (ref. ), phylodynamics captures both epidemiological and evolutionary information from measurably evolving pathogens -those viruses and bacteria for which high mutation rates and/or a range of sampling dates contribute to a meaningful amount of genetic variation between sequences , -in other words, enough genetic diversity to be able to infer an evolutionary history for a pathogen of interest, even if that history is only over the short time frame of an outbreak or epidemic. this is possible for most pathogens, particularly single-stranded dna viruses, rna viruses and many bacterial species , , but there are certain species for which the lack of a strict molecular clock and/or frequent recombination complicate both phylodynamics studies and attempts to infer transmission events . phylodynamics relies on tools such as bayesian evolutionary analysis sampling trees (beast) , in which sequence data are used to build a time-labelled phylogenetic tree using a specific evolutionary process as a guide -often variations on a theme of coalescent theory . from the tree, one can infer epidemiological parameters, including the basic reproductive number r (ref. ). while the insights that can be gained from genomic data alone are exciting, the utility of phylodynamic approaches is greatly extended when additional data are integrated into the models (reviewed in ref. ). genomic epidemiology in action: ebola. the many genomic epidemiology studies from the ebola outbreak (reviewed in ref. ) used bench-top and portable sequencing platforms to reveal outbreak-level events and epidemic-level trends. real-time analyses published around the peak of the epidemic suggested the following: the outbreak probably arose from a single introduction into humans and not repeated zoonotic introductions , ; sexual transmission had a previously unrecognized role in maintaining transmission chains ; and survivor transmission -another un recognized phenomenon -contributed to disease flare-ups later in the outbreak . the first sequencing efforts, all of which had an effect on the epidemiological response in real time, unfolded months into the epidemic. had they been deployed earlier, we can only speculate as to their potential impact. arguably, the most compelling use of early sequencing would have been to provide a definitive ebola diagnosis in this previously unaffected region of west africa. however, even after the outbreak was underway, sequencing could have benefited the public health response. for example, ruling out bush meat as a source of repeated viral introductions could have changed public health messaging campaigns from avoiding bush meat to the importance of hygiene and safe funeral practices , potentially averting some cases. portable sequencing and phylodynamic approaches are currently being deployed in the ongoing zika epidemic; whether the real-time reporting of genomic findings is able to alter the course of a vector-borne epidemic remains to be seen. retrospective phylodynamic investigations are also useful for pandemic preparedness planning. a recent analysis of , ebola virus genomes -approximately % of all cases -reconstructs the movement of the virus across west africa and reveals drivers for its spread . the authors deduce that ebola importation was more likely to occur between regions of a country than across international borders and that both population size and distance to a nearby large urban centre were associated with local expansion of the virus. these findings may affect decision-making around border closures in future ebola outbreaks and point to the need to develop surveillance, diagnostic and treatment capacity in urban centres. the role of the environment in deploying genomics for surveillance, diagnostics and epidemiological investigation, a key question remains: where? many regions lack the diagnostic laboratory capacity to carry out basic surveillance, but continuous genomic surveillance in all of these settings would be impossible. numerous projects have attempted to describe the pool of geographic hot spots and candidate pathogens from which the next epidemic or pandemic will arise. determining these factors is key to predicting and preventing spillover events (fig. ) ). they report an increasing number of events each decade, generally located in hot spots defined by specific environmental, ecological and socio-economic characteristics. most eids are zoonotic in origin, with the highest risk of spillover in regions with high wildlife diversity that have experienced recent demographic change and/or recent increases in farming activity . a global biogeographic analysis of human infectious disease further supports the use of biodiversity as a proxy for eid hot spots , and reviews focused on systems-level, rather than ecological, factors identify the breakdown of local public health systems as drivers of outbreaks, suggesting that surveillance ought to be targeted to settings where bio diversity and changing demographics meet inadequate sanitation and hygiene, lack of a public health infra structure for deliver ing interventions and no or limited resources for control of zoonoses and vector-borne diseases . these analyses provide a shortlist of regions, including parts of eastern and southeastern asia, india and equatorial africa, on which genomic and other surveillance activities should be focused , . within these regions, sewer systems and wastewater treatment plants could be important foci for sample collection, providing a single point of entry to biological readouts from an entire community. indeed, proof-of-concept metagenomics studies have revealed the presence of antibiotic resistance genes , human-specific viruses . most were zoonotic in origin, and over one-quarter had been detected in non-human species many years before being identified as human pathogens. a later review reiterates this observation, noting that recent agents of concern -ebola, zika and chikungunya -had been identified decades before they achieved pandemic magnitude . as a result of ngs technology, the pace of novel virus discovery is accelerating, with recent large-scale studies revealing new viruses sampled from macaque faeces in a single geographic location and , new viruses discovered from rna transcriptomic analyses of multiple invertebrate species . however, understanding which of these new entities might pose a threat requires a new approach. one health. the emergence of a zoonotic pathogen proceeds in stages (fig. ) ; in an effort to better anticipate these transitions and more proactively respond to emerging threats, the one health movement was launched in . recognizing that human, domestic animal and wildlife health and disease are linked to each other and that changing land-use patterns contribute to disease spread, one health aims to develop systems-minded, forward-thinking approaches to disease surveillance, control and prevention . by investing in infrastructure for human and animal health surveillance, committing to timely information sharing and establishing collaborations across multiple sectors and disciplines, the goal of the one health community is an integrated system incorporating human, animal and environmental surveillance -a goal in which genomics can have an important role. the one health approach has been implemented through the predict project, which is part of the emerging pandemic threats (ept) programme of the us agency for international development (usaid). predict explores the spillover of selected viral zoonoses from particular wildlife taxa , and early efforts have focused on developing non-invasive sampling techniques for wildlife , estimating the breadth of mammalian viral diversity across nine viral families and at least , undiscovered species and demonstrating that viral community diversity is at least a partially deterministic process, suggesting that forecasting community changes, which potentially signal spillover, is a possibility . although the goal of using integrated surveillance information to predict an outbreak is still many years away, one health studies are already leveraging the tools and techniques of genomic epidemiology to understand current outbreaks. combining genomic data with data streams from enhanced one health surveillance platforms presents an opportunity to detect the population expansions nature reviews | genetics figure | inferring transmission events from genomic data. genomic approaches to identifying transmission events typically involve four steps. in the first step, outbreak isolates, and often non-outbreak control isolates, are sequenced and their genomes either assembled de novo or mapped against a reference genome. next, the genomic differences between the sequences are identified -depending on the pathogen and the scale of the outbreak, these may include features such as genetic variants, insertions and deletions or the presence or absence of specific genes or mobile genetic elements. in the third step, these features are examined to infer the relationships between the isolates from whence they came -a variant common to a subset of isolates, for example, suggests that those cases are epidemiologically linked. finally, the genomic evidence for epidemiological linkages is reviewed in the context of known epidemiological information, such as social contact between two cases or a common location or other exposure. recently, automated methods for inferring potential epidemiological linkages from genomic data alone have been developed, greatly facilitating large-scale genomic epidemiological investigations . and/or cross-species transmissions that may precede a human health event. for example, genome sequences from a raccoon-associated variant of rabies virus (rrv), when paired with fine-scale geographic information and data from canadian and us wildlife rabies vaccination programmes, demonstrated that multiple cross-border incursions were responsible for the expansion of rrv into canada and sustained outbreaks in several provinces ; this finding led to renewed concern about and action against rabies on the part of public health authorities . one of the first studies coupling detailed wildlife and livestock movement data with phylodynamic analysis of a bacterial pathogen revealed that crossspecies jumps from an elk reservoir were the source of increasing rates of brucella abortus infections in nearby livestock ; as the most common zoonosis of humans, brucellosis control programmes will benefit substantially from this sort of one health approach . this model, in which diagnostic testing in reference laboratories triggers genomic follow-up, represents an effective near-term solution for integrating genomics into one health surveillance efforts as the community explores solutions to the many challenges facing in situ clinical metagenomics surveillance of animal populations (reviewed in ref. ). initial forays into this area have been successful; for example, metagenomics analysis of human diarrhoeal specimens and stools from nearby pigs revealed potential zoonotic transmission of rotavirus . however, metagenomic sequencing across a range of animal species and environments yields more questions than answers. what is an early signal of patho gen emergence versus background microbial noise ? which emerging agents are capable of crossing the species barrier and causing human disease ? what degree of sampling is required to capture potential spillovers ? ultimately, a more efficient use of metagenomics in a one health surveillance strategy might be scanning for zoonotic 'jumps' in selected sentinel human populations rather than a sweeping animal surveillance strategy , with sentinels chosen according to eid hotspot maps and other factors and interesting genomic signals triggering follow-up sequencing in the relevant animal reser voirs. by combining genomic data generated through these targeted surveillance efforts with phylodynamic approaches, it will be possible to take simple presence or absence signals and derive useful epidemiological insights: signals of population expansion; evidence of transmission within and between animal reservoirs and humans; and epidemiological analysis of a pathogen's early expansion. most modern surveillance systems use human, animal, environmental and other data to carry out disease-specific surveillance, in which a single disease is monitored through one or more data streams, such as positive laboratory test results or reportable communicable disease notifications. despite marked advances over the preceding decades, testimony from multiple expert groups has repeatedly emphasized the need for improved surveillance capacity , , including the use of syndromic surveillance, a more pathogen-agnostic approach aimed at early detection of emerging disease , . syndromic surveillance systems might leverage unique data streams such as school or employee absenteeism, grocery store or pharmacy purchases of specific items or calls to a nursing hotline as signals of illness in a population. increasingly, digital streams are being used as an input to these systems, be they participatory epidemiology projects such as flu near you , the automated analysis of trending words or phrases on social media sites, such as twitter , , or internet search queries [ ] [ ] [ ] . this new approach to surveillance is known as digital epidemiology and is also referred to as digital disease detection . in digital epidemiology, information is first retrieved from a range of sources, including digital media, newswires, official reports and crowd sourcing; second, translated and processed, which includes extracting disease events and ensuring reports are not duplicated; third, analysed for trends; and fourth, disseminated to the community through media, including websites, email lists and mobile alerts in spillover, a pathogen previously restricted to animals gradually begins to move into the human population. during stage one (pre-emergence), as a result of changing demographics and/or land use, a pathogen undergoes a population expansion, extends its host range or moves into a new geographic region. during stage two (localized emergence), contact with animals or animal products results in spillover of the pathogen from its natural reservoir(s) into humans but with little to no onward person-to-person transmission. during stage three (pandemic emergence), the pathogen is able to sustain long transmission chains, that is, a series of disease transmission events, such as a sequential series of person-to-person transmissions, and its movement across borders is facilitated by human travel patterns . epidemiology platforms are currently operating , and their flexible nature and cost-effective, real-time reporting make them effective tools for gathering epidemic intelligence, particularly in settings lacking traditional disease surveillance systems. the fields of one health and digital epidemiology are increasingly overlapping. in the predict consortium, the healthmap system and local media surveillance were combined to identify health events in five countries over a -week period . predict also suggested a role for digital epidemiology in not just event detection but also the identification of changing eid drivers. eids are driven by multiple factors, many of which have digital outputs and represent novel sources of surveillance data . for example, human movement can be revealed by mobile phone data or by the patterns of lighted cities at night, hunting data collected by states can reveal interactions between humans and wildlife, and social media and digital news sources can reveal early signals of famine, war and other social unrest. a major challenge is that the number of digital data sets available for each driver varies substantially, from hundreds for surveying land use changes -many based on remote sensing data -to mere handfuls around social inequalities and human susceptibility to infection, with most data biased towards north america and europe. the digital and genomic epidemiology domains are also starting to overlap. in the ebola outbreak, digital epidemiology revealed that drivers of infection risk included settings where households lacked a radio, with high rainfall and with urban land cover , echoing the evidence from a genomic study suggesting that sites at which urban and rural populations mix contribute to disease . during the zika epidemic, majumder et al. used healthmap and google trends to estimate the basic reproductive number r to be . - . ; phylo dynamic estimates from brazilian genomic data gave similar ranges ( . - . ) , indicating that both types of data streams can be leveraged in calculating epi demiological parameters that help shape the public health response. a digital pathogen surveillance era recent reports have called for the integration of genomic data with digital epidemiology streams , . when informed by a one health approach, the epidemiological potential of this digital pathogen surveillance system is profound. imagine parallel networks of portable patho gen sequencers deployed to laboratories and communities in eid hot spots -regions that are traditionally underserved with respect to laboratory and surveillance capacity -and processing samples collected from targeted sentinel wildlife species, insect vectors and humans (fig. ) . samples would be pooled for routine surveillance -either through targeted diagnostics or, if the issue of analytical sensitivity can be overcome, through metagenomics -with a full genomic work-up of individual samples should a pathogenic signal be detected. at the same time, existing internet-based platforms such as healthmap and new local participatory epidemiology efforts would be collecting data to both identify potential hotspot regions and detect eid events, enabling both prospective and rapid-response deployment of additional sequencers. genome sequencing data coupled with rich metadata would then be released in real time to web-based platforms, such as virological for colla borative analysis and nextstrain for analysis and visualization . these sites -already used in the ebola and zika responses -would act as the nexus for a global network of interested parties contributing to real-time phylo dynamic and epidemiological analyses and looking for signals of spillover, pathogen population expansion and sustained human-to-human transmission. results would be immediately shared with the one health frontlineepidemiologists, veterinarians and community health workers -who would then implement evidence-based interventions to mitigate further spread. the pathway to such a reality is not without its roadblocks. apart from technical and implementation challenges, a series of larger concerns surrounds the rollout of genomics-based rapid outbreak response, ranging from the uptake of a new, disruptive technology to effecting systems-level change on a global scale. sequencing-based diagnostics, particularly clinical metagenomics approaches, are still straddling the boundary between research and clinical use. in this realm, uncertainty is a certainty, be it uncertainty inherent to the technology itself or informational uncertainty, such as how accurate, complete and reliable results actually are . early adopters of genomics in the academic domain are used to uncertainty, often acknowledging and appraising it, but routine clinical use requires meeting the evidentiary thresholds mandated by a range of stakeholders, from regulators to the laboratories implementing new sequencing-based tests. decision criteria that influence whether a new genomic test is adopted include the ability of the assay to differentiate pathogens from commensals, the correlation of pathogen presence with disease, the sensitivity and specificity of the test, its reproducibility and robustness across sample types and settings and a cost comparable to that of existing platforms . validation -defining the conditions needed to obtain reliable results from an assay, evaluating the performance of the assay under said conditions and specifying how the results should be interpreted, including outlining limitations -is also critical. much can be learned from the domain of microbial forensics, where sequencing is playing a large part . budowle et al. review validation considerations for ngs , noting that this technology requires validating sample preparation protocols, including extraction, enrichment and library preparation steps, sequencing protocols, and downstream bioinformatics analyses, including alignment and assembly, variant calling, the underlying reference databases and software tools and the interpretation of the data. complete validation of a sequencing assay may not always be possible, particularly for emerging patho gens. therefore, just as the west african ebola virus outbreak triggered a review of the ethical context for trialling new therapeutics and vaccines , the scale-up of ngs in emerging epidemics will engender similar conversations. rather than wait for this to happen, an anticipatory approach is best, outlining the exceptional circumstances under which unvalidated approaches might be used, selecting the appropriate approach and examining the benefits of a potentially untested approach in light of individual and societal interests. if the social landscape surrounding the introduction of a new technology is not considered, prior experience suggests that the road to implementation will be difficult, with hurdles ranging from public mistrust to moratoria on research . the enthusiasm of the scientific community for new technology must not lead to inflated claims of clinical utility and poor downstream decisions around the deployment of that technology. howard et al. outline several principles for successfully integrating genomics into the public health system, and as we pilot digital pathogen surveillance, the community would do well to keep many of them in mind: ensuring that the instruments and processes used are reliable and that reporting is standardized and readily interpretable by end users; that the technology is used to address important health problems; that the advantages of the approach outweigh the disadvantages; and that economic evaluation suggests savings to the health care system and society . it is also important to reconsider the role of the diagnostic reference laboratory in the new genomic landscape. as their mandates expand to include enhanced surveillance and closer collaboration with field epidemiologists, laboratory directors will face new challenges, from managing exploratory work alongside routine clinical care to hiring a new sort of technologist, one with basic genomics and epidemiology training. the ethical, social and legal implications of digital pathogen surveillance are an emerging area of research (reviewed in ref. ). chief among the issues that geller et al. identify is the tension that exists when a new technology has the power to identify a problem but there is limited or no capacity to address the issue. balancing the benefits and harms to both individuals and populations is challenging when the predictive insight offered by a genomic technology is variable -for example, using genomics to identify an individual as a 'super spreader' has important implications for quarantine and isolation, but that label may be predicated on a tenuous prediction. the problem is further compounded by the fact that many infectious disease diagnoses carry with them a certain amount of stigma and that an individual's right to privacy might be superseded by the need to protect the larger population . data sharing and integration. a critical need for successful digital pathogen surveillance is the capacity for rapid, barrier-free data sharing, and arguments for such sharing are frequently rehashed after outbreaks and epidemics. genomic epidemiology was born largely in the academic sphere, with early papers coming from laboratories with nature reviews | genetics in one such region, the syndromic surveillance system reports higher-than-average sales of a common medication used to relieve fever. spatial analysis of the data from the pharmacies in the region suggests that the trend is unique to a particular district; a follow-up geographic information system (gis) analysis using satellite data reveals that this area borders a forest and is increasingly being used for the commercial production of bat guano. an alert is triggered, and the field response team meets with citizens in the area. nasopharyngeal swabs are taken from humans and livestock with fever as well as from guano and bat tissue collected in the area. the samples are immediately analysed using a portable dna sequencer coupled to a smartphone. an app on the phone reports the clinical metagenomic results in real time, revealing that in many of the ill humans and animals, a novel coronavirus makes up the bulk of the microbial nucleic acid fraction. the sequencing data are immediately uploaded to a public repository as they are generated, tagged with metadata about the host, sample type and location and stored according to a pathogen surveillance ontology. the data release triggers an announcement via social media of a novel sequence, and within minutes, interested virologists have created a shared online workspace and open lab notebook to collect their analyses of the new pathogen. extensive histories in microbial genomics and bioinformatics. for this community, open access to genome sequences, software and, more recently, publications has tended to be the rule rather than the exception. indeed, a national research council report described "the culture of genomics" as "unique in its evolution into a global web of tools and information" (ref. ). the same report includes a series of recommendations on access to pathogen genome data, including the statement that "rapid, unrestricted public access to primary genome sequence data, annotations of genome data, genome databases, and internet-based tools for genome analysis should be encouraged" (ref. ). as genomics has moved into the domain of clinical and public health practice, the notion of free and im mediate access to genomic surveillance data has encountered several barriers: the siloing of critical metadata across multiple public health databases with no interoperability; balancing openness and transparency with patient privacy and safety; variable data quality, particularly in resource-limited settings; concerns over data reuse by third parties; a lack of standards and ontologies to capture metadata; and career advancement disincentives to releasing data [ ] [ ] [ ] . despite these challenges, the spirit of open access and open data remains strong in the community, with over public health leaders from around the world recently signing a joint statement on data sharing for public health surveillance . the ebola and zika responses in particular highlight the role of realtime sharing of data and samples, be it through the use of chat groups and a labkey server to disseminate zika data or github to share ebola data . in the wake of ebola, yozwiak et al. and chretien et al. outline additional issues facing data sharing, from differing cultures and academic norms to complicated consent procedures and technical limitations. they note that we as a community must agree on standards and practices promoting cooperation -a conversation that could begin by examining how the global alliance for genomics and health (ga gh) framework for responsible sharing of genomic and health-related data (box ) could be adapted for the digital pathogen surveillance community. the future: the sequencing singularity? transformative change to public and global health is profoundly difficult. complicating the existence of a rapid, open, transparent response is the fact that no matter the setting, there are often conflicting interests at work. in an outbreak scenario, conflict may result from governments wishing to keep an outbreak quiet and/or from the tension between lower-income and middle-income countries with few resources for generating and using data and the researchers or response teams from better-resourced settings . indeed, the conflicting values in outbreak responses meet the definition of a 'wicked' problem, where issues resist simple resolution and span multiple jurisdictions and where each stakeholder has a different perspective on the solution. even the international health regulations (ihr), which ostensibly provide a legal instrument for global health security, fail to effect a basic surveillance and outbreak response. as of the most recent self-reporting, only % of the member countries of the ihr are in compliance, meeting the prescribed minimum public health core capacities . in these settings, digital pathogen surveillance must be within the purview of the larger global health community and its diverse group of non-state actors rather than being solely the responsibility of nations themselves . this raises an important issue: if nations are willing to cede a certain amount of surveillance and diag nostic control box | the global alliance for genomics and health (ga gh) framework for genomic data sharing in the universal declaration of human rights, article outlines the right of every individual "to share in scientific advancement and its benefit". in this spirit, the global alliance for genomics and health (ga gh) data-sharing framework , which covers data donors, producers and users, is guided by the principles of privacy, fairness and non-discrimination and has as its goal the promotion of health and well-being and the fair distribution of benefits arising from genomic research. the core elements of the framework include the following: • transparency: knowing how the data will be handled, accessed and exchanged • accountability: tracking of data access and mechanisms for addressing misuse • engagement: involving citizens and facilitating dialogue and deliberation around the societal implications of data sharing • quality and security: mitigating unauthorized access and implementing an unbiased approach to storing and processing data • privacy, data protection and confidentiality: complying with the relevant regulations at every stage • risk-benefit analysis: weighing benefits (including new knowledge, efficiencies and informed decision making) against risks (including invasion of privacy and breaches of confidentiality), minimizing harm and maximizing benefit at the individual and societal levels • recognition and attribution: ensuring recognition is meaningful to participants, providing due credit to all who shared data and ensuring credit is given for both primary and secondary data use • sustainability: implementing systems for archiving and retrieval • education and training: advancing data sharing, improving data quality, educating people on why data sharing matters, and building capacity • accessibility and dissemination: maximizing accessibility, promoting collaboration and using publication and digital dissemination to share results to the global health community, the notion of reciprocity suggests that they should derive some corresponding local benefit. the 'trickle-down' effects of global genomic surveillance have yet to be fully articulated, but they are likely to be realized first in the zoonotic domain, where global surveillance efforts will feed back into improved animal health at a local level, in turn benefiting local farmers. outbreaks occur at the intersection of risk perception, governance, policy and economics , and outbreak response is often based on political instinct rather than data . building a resilient and responsive public health system is therefore more than just enhancing surveillance and coupling it to novel technology -it is about engagement, trust, cooperation and building local capacity , as well as a focus on pandemic prevention through development rather than pandemic response via disaster relief mechanisms . expert panels convened by harvard and the london school of hygiene and tropical medicine and by the national academy of medicine have called for a central pandemic preparedness and response agency and also underscored the need for deeper partnerships between formal and informal surveillance, epidemiology and academic 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aspects of the transmission of pathogenic bacteria between wildlife and food animals: a thematic analysis of published research knowledge epidemiology: molecular mapping of zika spread framework for responsible sharing of genomic and health-related data literature review of zika virus using genomics data to reconstruct transmission trees during disease outbreaks smith foundation for health research programmes. both authors contributed equally to all aspects of the article. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -qnp m o authors: taylor, robert b. title: medical words linked to places date: - - journal: the amazing language of medicine doi: . / - - - - _ sha: doc_id: cord_uid: qnp m o many medical terms come from places: towns, rivers, islands, forests, mountains, valleys, countries, and continents. these toponymous diseases, syndromes, descriptors, and other entities bring us colorful names that help us recall some of their history. today we have zika virus, its name coming from the zika forest in ghana. caucasian comes from the caucasus mountains, lesbian from the island of lesbos, and epsom salts from a mineral spring in epsom, surrey, england. chapter . / - - - - _ tells the stories behind these place-named diseases and how many of them affect us today. future disease nomenclature based on places will be one more loss of the richness of our amazing medical language. in this chapter, i will begin with the current major concerns-the viral diseases causing today's outbreaks, epidemics, and pandemics. i will then present disease names linked to a variety of places, ending with those in europe and america. in , indian automobile manufacturer tata motors decided not to call its new hatchback car by its planned name zica, derived from "zippy car." this is just one more reaction to the epidemic of zika virus infections that the world health organization has declared a global emergency (fig. . ). in addition to causing fever and malaise, when the patient is pregnant, the zika virus may also cause birth defects, notably microcephaly (from greek words meaning "small" and "head"). some adult zika virus patients go on to develop the guillain-barré syndrome. the zika virus has been a dark cloud over the fragile economy of brazil in many ways, including its adverse impact on attendance at the rio de janeiro summer olympic games. the zika virus is, for most of the world, a newcomer. on new year's day , for example, hardly anyone had heard of the infection. now the disease is well known, but where did it arise and how did it get its unusual name? in the s, researchers identified a transmissible agent in the blood removed earlier from a rhesus macaque laboratory monkey sick with a fever. the monkey had come from a mosquito-infested jungle in uganda called the zika forest, the name coming from the word for "overgrown" in the luganda language of uganda. from here, and only in the past few years, the virus has migrated across asia and the pacific to central and south america, reaching pandemic proportions in some areas. in addition to mosquito-borne infection, we now have discovered sexually transmitted zika virus disease and continue to learn more each year. one might assume that the west nile virus came from egypt, but in fact, the organism was first discovered in the west nile district of uganda in . this mosquitotransmitted arbovirus was considered a problem only for birds and horses until the s, when human infections began to be reported. most west nile viral infections are subclinical, but a few are complicated by meningoencephalitis and a flaccid paralysis reminiscent of polio. the west nile virus is a member of the family flaviviridae, from the latin flavus, meaning "yellow." the family was named for the yellow fever virus, which tends to cause liver damage, giving its victims a yellow jaundiced appearance ( fig. . ). viruses of the flaviviridae family have turned up in various places and have often acquired the names of those locations. japanese encephalitis is a disease of domestic pigs and birds, notably herons, that can be spread to humans by mosquitoes. it is the chief cause of viral encephalitis in asia. another member of the flaviviridae family is mansonia pseudotitillans, the cause of saint louis encephalitis. the mosquito-borne disease harkens to in st. louis, missouri, when more than a thousand cases were reported. la crosse encephalitis is caused by a virus from the family bunyaviridae, the same family that causes crimean-congo hemorrhagic fever. discovered in the early s in la crosse, wisconsin, the mosquito-transmitted disease is found chiefly in the midwestern and appalachian regions of the united states. several families of rna virus can cause hemorrhagic fevers. the hantavirus takes its name from the place where the virus was first isolated in the late s: the hantaan river valley in south korea. rodents, such as the cotton rat, spread the hantavirus, and humans acquire the virus through contact with rodent feces, urine, or saliva. the hantavirus belongs to the family bunyaviridae. lassa virus, a member of the arenaviridae family, causes lassa hemorrhagic fever. the disease was initially described in in lassa, a town in borno state, nigeria. a zoonotic disease, the reservoir is rodents, notably multimammate mice (mastomys natalensis), whose excreta-feces and urine-can be aerosolized, and the disease can be spread by inhalation of these tiny particles. two members of the family filoviridae are the ebola virus and the marburg virus. ebola hemorrhagic fever, spread among humans by direct contact with body fluids, such as blood, semen, or breast milk, of an infected individual, causes death by internal and external bleeding in approximately half of infected persons. the name ebola comes from the ebola river, which flows near the village of yambuku in the democratic republic of the congo (previously zaire), where the disease was identified in ( fig. . ). the name marburg virus did not originate in africa, but in germany. in the s, several monkeys were sent from uganda to europe for use in laboratory experiments. unknown to the scientists involved, these monkeys carried a filoviridae virus, resulting in infections in several dozen researchers in three cities, including the university town of marburg, giving the organism the name it carries today. one of the disease names specifically criticized by the who is the middle east respiratory syndrome (mers). the term "middle east" may not have the exotic flavor of a river in south korea or a forest in uganda, but it does give a good indication of where the disease began, and a hint as to why it was first noted in this part of the world. mers, first reported in saudi arabia in , is a viral disease of the respiratory tract causing cough, fever, dyspnea, and sometimes death. only a few cases have been reported in the united states, and, so far, these have been contracted during travel in the middle east and imported by returning travelers. other cases have been reported in more than two dozen countries including great britain, germany, greece, korea, china, malaysia, and the philippines. the intriguing aspect of mers is the likely connection to camels. the causative virus (mers-cov) has been found in camels, and some patients with the disease have told of contact with camels. the who warns against drinking raw camel milk or camel urine or eating undercooked camel meat. so perhaps the "middle east" part of the disease name is on target. where else but the middle east do humans live in close communion with camels? and drinking camel urine? yes, some in the region believe that drinking camel urine has medicinal value. what could be the connection between the vitamins essential to our health, a polish biochemist, and camel dung in the middle east? the story goes back more than three millennia. as early as the eighteenth dynasty (c. - bce and the time of king tutankhamun), the early egyptians worshiped the god amun, also called amun-ra or ammon, as the leading god of the empire, later to be identified with the greek god zeus (fig. . ). in what is now libya, they built a temple to ammon where egyptians came to worship their god; while they did so, their camels fertilized the nearby sand with urine and feces. it was from this sand that sal ammoniac, the "salt of ammon," was first derived. in fact, the ancient greek word for "sand" is ammos, probably related to the name of the god (shipley, p. ). from sal ammoniac comes ammonia (nh ), a pungent-smelling, colorless gas, so named in by swedish chemist torbern bergman ( - ), and from ammonia comes our word amine. amines are derived from ammonia by chemical substitution of one or more of the hydrogen atoms with other radicals. the next etymologic landmark in the story of the word vitamin came in when polish biochemist casimir funk ( - ) introduced the concept of amines as being vital to life. he postulated that there were at least four necessary amines and that without them patients would develop beriberi, scurvy, rickets, and/ or pellagra. to form his word, funk combined -amine with the latin word for life, vita (recall the fellini movie, la dolce vita-the "sweet life"). funk's original word vitamine was later shortened to vitamin when it was learned that not all vitamins contained amines [ ] . casimir funk was nominated for a nobel prize in , , , and . he never received the award. today we have vitamins a, b (a number of these), c, d, e, and k, the latter so designated because of its action in coagulation (originally named koagulationsvitamin in german). there are also vitamin wannabes, such as the bogus cancer drug amygdalin (laetrile), a cyanogenic substance found in apricot kernels and dubbed vitamin b , perhaps alluding to the world war ii flying fortress. we have come a long way from camel urine to drugstore shelves full of colorful vitamin bottles and a few misbegotten imitators. in chap. , i told of prometheus chained by zeus for giving humans the gift of fire; the location where he was restrained was the caucasus mountains near what are now iran and turkey ( fig. . ). it was in these mountains that german scientist johann friedrich blumenbach ( - ) discovered a skull. blumenbach, the skull from the mountains considered the father of physical anthropology, suggested that the various human subspecies ("races") could be classified by the study of their skulls. based on his anthropometric comparisons, in , he proposed five families of humans: yellow (mongolians), black (ethiopians), red (american indians), brown (malaysians), and white. for the name of this last category, he used the location of the finest skull of all, the caucasus mountains, and from that moment on, white persons became caucasian (gershen, p. ). it started with the greek lyric poet sappho (ca. - bce). she was born on the island of lesbos, located in the aegean sea off the west coast of turkey and a center of civilization even before the golden age of greece in the fifth century bce. she was the most renowned poetess of her day, and her lines had a distinctive "sapphic meter." sappho shared her poetry with a cohort of young women, and probably because of the erotic passion of some of her poems, the tale has evolved regarding homosexual relationships between the poetess and her students. thus female homosexuality came to be named for the island, lesbos, and a female homosexual, wherever she lived, became a lesbian. a word less often used to describe women who love other women is sapphism, a synonym for lesbianism. in fact, according to hendrickson (p. ), sappho was probably married and had a son, although many will argue that this would not be solid evidence of her sexual preferences. the crimean war was fought in the mid- s between russia and an alliance of the united kingdom, france, and turkey. it was the war that gave us florence nightingale and the charge of the light brigade. during this war british medical officers on the island of malta noticed a disease characterized by sweating, joint and muscle pains, and a fluctuating fever. a logical name for the mysterious malady was malta fever. in , british medical officer jeffrey allen marston ( - ) described his personal experience with the disease, and in , scottish microbiologist david bruce ( - ) linked the disease to an organism that came to be called brucella abortus. the "bruce" in the name of the genus honored bruce; the species designation abortus reflects the tendency of the disease to cause abortions in cattle. the disease was briefly called bang's disease, after danish veterinarian bernhard bang identified b. abortus as the agent causing cattle to abort. because of the wavelike nature of the fever, the disease acquired the name undulant fever. other names enjoyed popularity in various settings: scottish delight, milk sickness, goat fever, cyprus fever, gibraltar fever, and mountain fever. in the end, the medical and scientific communities have come to favor the name brucellosis, honoring the man who discerned the cause of the disease. this story begins in the town of magnesia, named for an ancient greek tribe, the magnetes. the town lies in the thessaly district of greece, an area conquered by the romans following their victory in the battle of magnesia in bce, ending the roman-seleucid war. in this conquered area, the romans discovered a white mineral that, applying the name of the town, they called magnesia. when a different substance with a dark color was found, the white substance became magnesia alba, from the latin word for "white," and the darker substance became magnesia nigra, latin for "black." in , french chemist antoine bussy ( - ) found that the magnesia alba yielded an element, which he named magnesium. magnesia nigra came to be called manganese. there is another twist to the magnesium story. in the early seventeenth century, in the town of epsom in surrey, england, a farmer offered his cows water from a nearby well. but the cows refused to drink. the farmer learned why when he tasted the bitter water. he also observed, however, the water seemed to heal skin abrasions and sores. the bitter-tasting substance in water came to be identified as hydrated magnesium sulfate, better known as epsom salt. epsom wells became a destination spa, visited by diarist and member of parliament samuel pepys; nell gwyn, mistress of king charles ii of england; and other seventeenth-century notables (evans, p. ). in the united states, there is a magnesia temple at the town of sharon springs in schoharie county, new york (fig. . ). in addition to epsom salt, magnesium has other medical uses: a water solution of magnesium oxide is milk of magnesia, popular as a laxative. magnesium has had a role in treating ventricular arrhythmias of the heart and preeclampsia/eclampsia. it is sometimes prescribed for management of migraine or of the restless leg syndrome. and the white powder that gymnasts and weight lifters use to improve their grips is magnesium carbonate. returning to the town of magnesia, in this region was found an iron oxide stone that had the apparently magical quality of attracting iron to itself. it was called the "magnesian stone" and served as the source of our word magnet (haubrich, p. ). the guinea worm is a parasitic nematode that is spread when a person drinks water containing the guinea worm larvae. the cause of the disease in humans is dracunculus medinensis, and the disease is properly called dracunculiasis. approximately a year following ingestion of the larvae, the female guinea worm finds its way to the skin, where it forms a blister. a little later the blister breaks and the worm begins to emerge. an adult female guinea worm can be two to three feet long and as thick as a strand of spaghetti. affected persons sometimes facilitate extraction of the worm by winding it around a small stick. the name guinea worm arose when european explorers first encountered the disease on the guinea coast of west africa in the seventeenth century. in latin dracunculus means "little dragon; dracunculus medinensis means the "little dragon from medina," so named because the disease was once rampant in the muslim holy city of medina in saudi arabia. dracunculiasis is no longer endemic in either location. the prevention of dracunculiasis requires nothing more than drinking filtered water, and the disease is on the threshold of being exterminated. the source of the drug colchicine is the autumn crocus, colchicum autumnale, so named because it was first discovered in the colchicum region of the republic of georgia on the black sea. the plant's nickname "naked lady" refers to the appearance of the flower without surrounding leaves. the extract of the plant can be highly toxic, and severe accidental poisonings have occurred, several involving cases in which the autumn crocus was mistaken for wild garlic (fig. . ) . use of the drug to treat joint pains can be traced to early egyptian writings, circa bce, found in the ebers papyrus. therapeutic use of colchicum extract is mentioned in the writings of persian physician avicenna ( - ) and french surgeon ambroise paré ( - ). american statesman benjamin franklin ( - ) brought the colchicum root to america from france and used it to treat his own attacks of gout. in addition to its use in the treatment of gout, colchicine is sometimes used to treat behçet disease, pericarditis, and familial mediterranean fever; the latter is a hereditary disorder also known as armenian disease. the word "plaster" in plaster of paris came from greek emplastron, to latin emplastrum, to french plastre. but what about paris? throughout history, various products had been used to bind wounds. french surgeon guy de chauliac ( - ), author of the seven-volume chirurgia magna in , introduced the use of egg white to stiffen bandages [ ] . however, the "paris connection" came in when dutch army surgeon antonius mathijsen ( - ) began incorporating gypsum into dressings used to immobilize fractures ( fig. . ) . mathijsen was not french, but the gypsum was quarried in the montmartre section of paris, hence the name plaster of paris. although the word is probably never seen today in medical records, clap remains a vulgar term for gonorrhea. the common old french term for brothel was clapier. the term technically translated to "rabbit burrow," and perhaps this image had something to do with the "red light" section paris, where a number of brothels were located, being called, in the middle ages, le clapier. as language moved across the english channel in the sixteenth century, clapier in french became clapper in english. eventually the word was shortened to clap, generally expressed with an article as "the clap" [ ] . the french had another word for this distressingly common malady, chaudepisse, describing a frequent manifestation of gonorrhea. chaude means "hot" in french. and pisse? is translation really needed? it was first called bramble disease by a norwegian physician who reported a cluster of cases of "acute muscular rheumatism" occurring in the village of bramble in norway. but alas, the original report and a few others that followed using the term "bramble disease" were published only in norwegian, and the name failed to catch on. then in , doctoral candidate ejnar sylvest ( - ) published his thesis describing a disease outbreak on the picturesque danish island of bornholm, in the baltic sea, that he termed "bornholm disease-myalgia epidemica." today we know this as bornholm disease, as well as epidemic pleurodynia, epidemic myalgia, devil's grip, and the grasp of the phantom. the disease causes the usual viral symptoms of fever and headache. an additional and distinguishing feature is severe pain in the lower chest, giving rise to the more colorful names of the disease. fortunately for those with the devil's grip, the disease is self-limited and rarely fatal. it was an american, not a german, who gave rubella its everyday name: german measles. the disease has been well known to health-care professionals and parents since first described by german physician friedrich hoffmann ( - ) in . it was given the name rubella, from the latin word meaning "little red," by english military surgeon henry veale in his description of an outbreak in india. american physician j. louis smith coined the term "german measles" in . working at new york's bellevue hospital, smith described an outbreak of the disease and, reading of similar outbreaks in germany, he named the disease "german measles" (bordley and harvey, p. ) (fig. . ) . then, amid the anti-german fervor of world war i, rubella was briefly renamed liberty measles (fortuine, p. ). this chapter is about diseases named for places. is a trench a "place"? it certainly was to the world war i soldiers who spent weeks and months slogging shoulder to shoulder in deep ditches filled with icy cold water and filth. from these barely habitable ditches came the "trench" in the names of three different diseases. the first is trench fever, with an estimated one million cases occurring in western europe during the first world war. it was first noted in an infantry private in , and by , the allied general headquarters stated: "trench fever is a matter of national importance… and it merits the attention of every physician and pathologist who has the opportunity of working among the troops" [ ] . trench fever, caused by bartonella quintana and spread by the human body louse, causes fever, prostration, a macular rash, and bone pain. despite these manifestations, some doughboys welcomed the infection because a stay in the medical facility offered respite from the trenches. trench fever has also been called five-day fever, shin bone fever, and meuse fever; the latter is a reference to the wwi meuse-argonne battle of . today, we no longer have trench warfare, but we do have urban trench fever occurring in demented, homeless, and alcoholic persons. from the muddy ditches of wwi, we also get the disease name trench foot, describing damage to the tissues that occurs with prolonged exposure of the extremity to moist cold (fig. . ). dominique jean larrey ( - ), a surgeon in napoleon's grande armée, noted the prevalence of what we now call trench foot in the french troops during their ill-fated invasion of russia in . trench foot was seen not only in wwi but also in world war ii and in the vietnam war; it was called immersion foot or paddy foot in the latter jungle-based campaign. then there is trench mouth, also known as acute necrotizing ulcerative gingivitis, caused by a mixed bacterial infection. recognized in greek soldiers in the fourth century bce, the disease was described by scottish surgeon john hunter ( - ) in . at the pasteur institute in paris in , french physician jean hyacinthe vincent ( - ) identified the fusospirochetal cause; one of the other names for acute necrotizing ulcerative gingivitis is vincent angina [ ] . in this term, angina reflects its true latin meaning, "infection of the throat." the disease did not acquire the name trench mouth until world war i, when it occurred in men spending weeks and months in trenches under physical and psychological stress, receiving a poor diet, and with scant options for oral hygiene. nantucket is a small island off cape cod, massachusetts, usa. in april of , the cape cod times, in a story titled "cape cod a hot spot for babesiosis from ticks," reported health officials calling for "hospitals to screen blood transfusion products for babesiosis or nantucket fever." in the article, dr. al demaria, state epidemiologist with the massachusetts department of public health, declared nantucket fever [ ] . what is this toponymous disease that many health professionals have never heard of? nantucket fever, caused by a tick-borne protozoan parasite called babesia microti, causes manifestations not unlike malaria, including fever, arthralgia, lymphadenopathy, and hemolytic anemia. the formal name for the febrile illness is babesiosis, first described by romanian scientist victor babeş ( - ) as a disease of cattle and sheep. the first human case of babesiosis was reported in . in cattle, babesiosis is sometimes called texas cattle fever, tick fever, or redwater fever, the latter referring to the appearance of blood in the urine. in the united states, human babesiosis has been reported chiefly in northeastern and midwestern states and occurs most often during warm weather. if a young physician today were asked to identify a disease named for a place, the answer might well be lyme disease, also called lyme borreliosis. although the disease had been recognized in europe since the eighteenth century and documented the dangers of the deer tick in wisconsin in , the full spectrum of the disease was not recognized until , in connection with a series of cases in southeastern connecticut, including the small towns of lyme and old lyme. the disease is caused by borrelia bacteria, notably borrelia burgdorferi, and is spread by the same vector as nantucket fever/babesiosis: the ixodes tick, also called the deer tick. a curious, perhaps pathognomonic, feature of the disease is the early appearance of a "bulls-eye" rash, called erythema migrans, although this helpful diagnostic clue is not seen in all patients (fig. . ). later manifestations may include fatigue, neurocognitive manifestations, disorders of heart rhythm, polyneuropathy, and arthritis. lyme disease is the most commonly occurring tick-borne disease in europe and north america. in the united states, the areas of greatest prevalence are the northeast and middle atlantic states and western wisconsin. in these locations, woodsmen and woodswomen, beware of the deer tick. another malady, along with lyme disease, well known for its geographic label is rocky mountain spotted fever. also sometimes called tick typhus or blue disease, rocky mountain spotted fever was first recognized in in the snake river valley in the rocky mountains of the western united states. the disease is spread by ticks carrying the causative organism, rickettsia rickettsii, a name that seems unnecessarily repetitive, like the name of the common black rat, rattus rattus; the latter is an animal reservoir for fleas carrying the bacteria causing bubonic plague. the name of the organism redundantly honors american pathologist howard taylor ricketts ( - , who first isolated the pathogen that causes the disease. a potentially fatal disease, rocky mountain spotted fever is the most commonly reported rickettsial disease in the united states. curiously, because of the epidemiologic distribution of the disease in america, you and i are at more risk of contracting rocky mountain spotted fever in north carolina than we are in colorado. first found in in a -year-old boy living on tangier island in virginia's chesapeake bay, tangier disease is also known as familial alpha-lipoprotein deficiency. it is a congenital disorder causing a severe deficiency of high-density lipoprotein (hdl) in the blood. in addition to abnormal serum lipids found on laboratory analysis, patients with tangier disease may have splenomegaly, hepatomegaly, neuropathy, atherosclerosis, and cloudiness of the corneas of the eyes (fig. . ) . there is a long list of other medical entities that came from cities, countries, and even continents. in , scientists working in the city of philadelphia, pennsylvania, discovered the philadelphia chromosome, an abnormality of chromosome associated with chronic myelogenous leukemia. milltown, new jersey, originally named for a local gristmill, was the manufacturing site of the anxiolytic drug meprobamate (miltown). in the s, wallace laboratories, in an effort to maintain secrecy about their new drug, applied the code name miltown, after the new jersey borough. when the drug went to market in , the company decided to keep the town name as the trade name. named originally for the peoples of the nation of mongolia, the mongolian spot is a melanocytic birthmark that may appear blue, or some shade of gray, black, or brown. the term was coined in by german anthropologist erwin bälz ( - ), who was actually working in japan and not in mongolia. in mexico, the birthmark is called rabo verde, "green butt," and in spanish the term sometimes used is mancha ("stain") de baelz (bälz), eponymously honoring the man who first described it. mongolian spots generally disappear during later childhood. the influenza pandemic of - is sometimes called the spanish flu. caused by the h n virus, the disease affected million people worldwide and resulted in more than million deaths (fig. . ). it seems ironic that spain has had its name attached to a flu that has been traced to a single index case in a wholly different country: a us army cook at fort riley, kansas. situated on the hepatitis b virus (hbv) is the surface antigen, a.k.a. the australia antigen (hbsag). its presence upon laboratory testing indicates current infection with the hbv. the name australia antigen was suggested when american physician baruch blumberg ( - ) discovered its presence in the blood of a member of the australian aboriginal population. who issues best practices for naming new human infectious diseases casimir funk: his discovery of the vitamins and their deficiency disorders catechism in medical history medical history in medical terminology trench fever: the british medical response in the great war the relation of peri-dental gingivitis to vincent's angina cape cod a hot spot for babesiosis from ticks. cape cod times key: cord- -k s v fs authors: imperiale, michael j.; casadevall, arturo title: zika virus focuses the gain-of-function debate date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: k s v fs nan the task of researching the various approaches to performing a risk-benefit analysis was contracted to gryphon scientific, a company that consults on biosecurity and other life science-related policy issues. their report, which weighed in at over , pages, begins with a thorough summary of the gof landscape and subsequently presents various risk and benefit scenarios for mers, sars, and three categories of influenza, seasonal, pandemic, and avian ( ) . we encourage the reader to peruse the executive summary for more detail, but it is our impression that this report is both balanced and comprehensive. this report, along with a paper discussing the ethical considerations of gof research that was also commissioned by the nsabb ( ) , was discussed at a meeting at the nih at the beginning of january . the board heard from a variety of individuals, some of whom have been involved in this debate for many years and others of whom provided fresh voices. both sides found parts of the gryphon report that they liked and parts that they disliked. to us, the fact that neither the pro-nor the anti-gof advocates are completely satisfied with the report means that it struck a good balance. we have previously noted that the major difficulty in resolving this debate is that it is fundamentally a conflict between different philosophies, risk assessments, and value systems and thus cannot be settled on the basis of some objective criteria. the voluminous aspect of the gryphon report seems to reflect a desire to cover all those subjects for which there are some data that can be analyzed with existing risk-benefit tools. however, the areas that separate pro-and anti-gof advocates fall into areas of judgment and belief, and these differences cannot be adjudicated by risk-benefit analysis. apart from providing an assessment of the situation by a disinterested third party, the gryphon analysis was also helpful in that it bought time for passions to settle and perhaps allow for a more cool-headed conversation that was difficult years ago, when both sides engaged in an acrimonious debate in both the scientific and the general media ( ). as we approach a time of decision, it is critically important that the nsabb makes clear, sensible recommendations to the u.s. government. while there were relatively few new ideas presented at the january meeting other than those from ethicists, many of the speakers implored the board to be precise. for example, the board was asked to provide specific examples of experiments that should or should not be performed, and the members appeared to be receptive to these pleas to provide more clarity than current guidance documents present. in addition, three of the more vocal critics of gof research with ppp recently proposed six specific policy options for consideration at the second nas meeting ( ) , which was held in early march . a question in our minds is how long it will take the government to incorporate those recommendations, as it sees fit of course, into policy. there is reason for concern. we note that the original nsabb proposed framework for the oversight of dual use life sciences research ( ) was approved by the board in june , yet no policy was forthcoming until the h n publications forced the issue almost years later. during that time, no experiments were on hold, as is the case with the current moratorium. will the fact that we are in a presidential election year mean a de facto pause in the policy-making process? the longer the moratorium persists, the more likely it is that important work will not be performed, or perhaps it will simply move overseas with funding from non-u.s. agencies as researchers tire of endless waiting for clear guidance. in fact, gof work is occurring outside the united states, and several publications describing experiments that are likely to be prohibited by the current moratorium have appeared (e.g., see references to ). hence, the u.s. moratorium is not preventing this type of work. our concern about moratoriums and the continuing controversy is that it will discourage the best and brightest from working on dangerous pathogens that threaten humanity. in this regard, there is preliminary evidence that the controversy is being closely followed by younger scientists and that it may be affecting their choice of research careers ( ) . as the great gof debate lumbers on without resolution, a new infectious threat has suddenly appeared in the form of zika virus. zika virus was discovered in a sentinel monkey in in the zika forest in uganda. over the next decades, it caused only a few documented human cases. however, in recent years, it spread to the pacific islands, and we are now in the midst of a serious epidemic in south america. although zika virus infection appears to cause a self-limited disease in most individuals, there are ominous reports of an association with microcephaly in babies of affected mothers and neurological disorders, such as guillain-barre, in some affected adults. at the time of this writing, these associations have not been proven causative, and it is unclear whether these complications are new facets of zika virus disease as a result of increased pathogenicity by passage in the human population or a new realization of rare complications as the virus infects millions. the zika virus provides a new lens with which to view the great gof debate. this serves as a reminder to us of why proponents of gof argue that work on dangerous pathogens should continue and why opponents of gof argue for a halt because of the threat of laboratory-derived pandemics. gof types of experiments could be used to ascertain whether the potential for causing neuropathology and microcephaly is a potential characteristic of this virus that has finally emerged by recent passage in human populations. for example, comparison of earlier and current zika virus isolates might reveal differences that could be associated with new symptoms. showing that the virus can acquire those new properties through gof-type experiments would help establish causality and might provide new insights for therapy and vaccines. as we have noted, gof-type experiments are epistemologically rich and can provide unambiguous answers to problems of causality ( ) . hence, we have argued that gof experiments are powerful tools of human inquiry that should have a role in studying problems of virulence and transmissibility, provided that these studies can be performed safely. on the other hand, the zika virus outbreak is an example of what concerns the anti-gof proponents, the rapid spread of a new virus in human populations with the potential to cause devastating damage in those populations. fortunately, it does not appear that zika virus can spread by a respiratory route, which undoubtedly would make control much more difficult to achieve. although the zika virus outbreak does not help to resolve the gof debate, it does provide a lens that helps to refine the resolution of both pro-and anti-gof arguments. the threat of viral pandemics and other infectious diseases is ever-present. it is obvious that our best defense against this threat is advancing our knowledge such that we understand the biology of the pathogens and what determines their virulence in human and animal hosts. these studies, when undertaken in a safe and responsible manner, will inform the development of diagnostics, vaccines, and therapeutics. it is essential that the moratorium end as soon as possible, before nature outguns us. gain-of-function research: unknown risks rethinking biosafety in research on potential pandemic pathogens doing diligence to assess the risks and benefits of life sciences gain-of-function research risks and benefits of gain-offunction experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion potential risks and benefits of gain-of-function research framework for conducting risk and benefit assessments of gain-of-function research risk and benefit analysis of gain of function research gain-of-function research: ethical analysis six policy options for conducting gain-of-function research proposed framework for the oversight of dual use life sciences research: strategies for minimizing the potential misuse of research information influenza a virus acquires enhanced pathogenicity and transmissibility after serial passages in swine novel avian-origin human influenza a(h n ) can be transmitted between ferrets via respiratory droplets h n hybrid viruses bearing /h n virus genes transmit in guinea pigs by respiratory droplet infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? mbio an epistemological perspective on the value of gain-of-function experiments involving pathogens with pandemic potential key: cord- -gmw gl r authors: saiz, juan-carlos; de oya, nereida jiménez; blázquez, ana-belén; escribano-romero, estela; martín-acebes, miguel a. title: host-directed antivirals: a realistic alternative to fight zika virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gmw gl r zika virus (zikv), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the americas in , where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. currently, no specific antiviral therapy against zikv is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. nevertheless, lately, search for antivirals has been a major aim in zikv investigations. to do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. here, we present an updated review of current knowledge about anti-zikv strategies, focusing on host-directed antivirals as a realistic alternative to combat zikv infection. since the beginning of the st century, a number of infectious disease threats have emerged that demand a global response. among them, severe acute respiratory syndrome virus, avian influenza in humans, pandemic influenza a (h n ), middle east respiratory syndrome coronavirus, chikungunya virus, and ebola virus have been the most threatening ones. nonetheless, the emergency of a vector-borne virus, zika virus (zikv), which is responsible for congenital malformations and other neurological and ophthalmological disorders, was hard to predict. zikv is a mosquito-borne virus belonging to the spondweni serocomplex in the genus flavivirus of the family flaviviridae [ ] . the virus has been isolated from various mosquito species, although it seems that the natural transmission vectors are mosquitoes of the genus aedes [ , ] . besides mosquito bites, viral direct human-to-human transmission can occur perinatally, sexually, and through breastfeeding and blood transfusion [ ] . the zikv genome is a single-stranded rna molecule (≈ . kb) of positive polarity encoding a single open reading frame (orf) flanked by two untranslated regions at the and ends [ ] . zikv was first isolated from the serum of a monkey in , and one year later from aedes africanus mosquitoes caught in the same area, the zika forest [ ] . until it was detected in asia in the s, the virus had been confined to africa. later on, human outbreaks were reported in the pacific islands, micronesia in and, then, in french polynesia in [ ] . the natural course of zikv infection was usually asymptomatic or produce a relatively mild illness and an uneventful recovery [ ] , hence, the virus was considered an almost neglected pathogen until its recent introduction into the americas in , when it became a threat to global health, showing increased virulence, rapid spread, since the recent outbreak in in the americas, a quite high number of possible antiviral candidates are being tested in vitro and in vivo. however, until now, no specific therapy has been approved against any flavivirus [ ] , including zikv [ ] , and, thus, current treatments are mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration [ ] . nevertheless, it should be noted that some commonly used drugs, such as acetylsalicylic acid, are contraindicated in zikv-infected patients, since they increase the risk of internal bleeding, and other arboviruses (dengue or chikungunya viruses) that can co-infect the patients may produce hemorrhages [ ] . due to the natural course of zikv infection, which is usually asymptomatic or produce a relatively mild illness and an uneventful recovery, when facing anti-zikv strategies, a very important point to take into account is the main target population that would benefit from it, namely immunocompromised patients and pregnant women and their fetuses [ ] . in this sense, only for some of the tested drugs their safety profiles are known [ ] . however, in cases of food and drug administration (fda) (https://www.drugs.com/) category b compounds (animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women), or even in those of category c (animal reproduction studies have shown an adverse effect on the fetus and there are no adequate and well-controlled studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), or d (there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), their use in pregnancy can be contemplated if the potential benefit outweighs the risks. even more, some of the assayed compounds cross the placenta and, thus, can also benefit the fetus. nonetheless, if used, this should be done in an individualized way, conditioning dosage and timings, and always under a clinician's control where the patient is informed of the pros and cons. current search for zikv antivirals is being conducted with different approaches: by screening of compounds libraries; by the repurposing of drugs of known active efficacy against other diseases now in use in clinical practice, many of which display broad-spectrum activity; and by testing natural products. two different strategies can be applied when pursuing for antivirals, those searching for compounds directed to viral targets (direct-acting antivirals) and those aimed to target cellular components needed for the viral life cycle (host-directed antivirals). among the virus-directed drugs tested [ , ] are those acting against the viral rna-dependent rna polymerase (non-structural protein (ns )) catalytic domain, including nucleoside analogs and polymerase inhibitors; the methyltransferase catalytic domain of the ns responsible for transferring the mrna cap; the ns b-ns trypsin-like serine protease needed for proper processing of the viral polyprotein; and the ns helicase. the crystal structures of all these proteins have already been resolved and will certainly help to find new antivirals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the same way, structures from other viral proteins are also available that could help to design zikv therapeutic alternatives, such as those of the capsid c protein [ ], whose destabilization may impair zikv multiplication, the ns [ , ], an immuno-modulator, or the envelope glycoprotein [ - ], which mediates cell binding and endosomal fusion, constitutes a major target for neutralizing antibodies, and could be also the target for virucidal compounds [ ] . on the other hand, it has also been reported that passive transfer of neutralizing antibodies to pregnant mice suppresses zikv multiplication, inhibits cell death, reduces the number of progenitor neuronal cells, and prevents microcephaly [ , ] . likewise, administration of monoclonal antibodies (mabs) recognizing the domain iii of the zikv-e protein protect mice of lethal zikv challenge [ , ] and other mabs are able to bind and neutralize zikv, including those directed against the e dimer epitope [ ] . human polyclonal antibodies produced in transchromosomal bovines also protect mice from zikv lethal infection, eliminated zikv induced tissue damage in the brain and testes, and protected against testicular atrophy [ ] . thus, administration of therapeutic antibodies seems to also be a potential strategy against zikv. nevertheless, it should be noted that, although still controvertial in the case of zikv infection [ ] , the well-known antibody dependent enhancement effect (ade) [ ] , of which dengue virus (denv) is the prototypic model, may potentiate the risk of disease exacerbation. flaviviruses have small rna genomes (around . kb in length) and thus require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently [ ] . this offers an opportunity to search for host targets as therapeutic tools that, in many instances, as they are shared by different members of the flaviviridae family, can be envisaged as pan-flaviviral antivirals [ ] [ ] [ ] . this strategy can be directed to host factors implicated in infection, pathogenesis, and in the immune response, as it has been shown for denv and the west nile virus (wnv) [ ] . in addition, their effect would be less prone to the emergence of mutants that will escape their action, as often occurs with drugs targeting viral components. consequently, this kind of approach could ideally lead to the discovery of broad spectrum antivirals that could provide low cost but effective tools for the control of flaviviral threats. different approaches are being used to identify potential host factors as therapeutic targets against flaviviruses including the analyses of transcript levels (e.g., next generation rna sequencing) for altered expression patterns during infection, proteome changes, kinases activities variations, and protein-rna interactions (e.g., two-hybrid screenings and affinity chromatography). likewise, functional analysis can be applied by overexpressing cdnas or by rnai-mediated loss of function screens using dsrna, sirna, or shrna libraries, although it should be noted that in some cases downregulation is inefficient and some genes have redundant functions [ ] . replicons may also be used to specifically assay replication activity [ , ] . theoretically, host-acting antivirals can be directed to any molecule or pathway implicated in the different steps of the viral life cycle, from early events (binding, entry, and fusion), to the formation of the replication complex, and the viral maturation and egress. the first step of zikv infection is its binding to the cellular receptor ( figure ). several molecules have been proposed as a zikv receptor (members of the tyro /axl/mer (tam) family of receptor tyrosine kinases, t-cell immunoglobulin and mucin domain (tim) and dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)) that are expressed in different neuronal and non-neuronal permissive cell types. these molecules are also receptors for other viruses, including flaviviruses such as denv and wnv, regulate several cellular activities (adhesion, migration, proliferation, and survival, release of inflammatory cytokines, antigen uptake, and signaling), and play important roles in the host's response to infection [ ] . however, elimination of a known receptor does not necessarily result in complete protection from viral infection, since flaviviruses use different receptors and, thus, there is always redundancy and alternatives. for instances, inhibiting, downregulating, knocking-down, or ablating axl, although in some cases they reduce zikv infection, they do not completely abolish it, pointing to the use of different cell surface receptors on different cell types [ ] [ ] [ ] [ ] . different molecules have been shown to inhibit zikv infection at the entry step ( figure ). r (an axl kinase inhibitor) and myd (an axl decoy receptor) compromises, but do not completely abolish, zikv infection of glial cells [ ] . r , as well as cabozantinib, an inhibitor of axl phosphorylation, that are currently in clinical trials for anticancer activities, significantly impairs zikv infection of human endothelial cells in a dose-dependent manner by affecting a post-binding step [ ] . likewise, curcumin, a widely used food additive and herbal supplement, reduces zikv infection in cell culture inhibiting cell binding while maintaining viral rna integrity [ ] , as does suramin, an anti-parasitic that interferes with attachment to host cells and with virion biogenesis by affecting glycosylation and maturation [ , ] . once zikv binds to the cell receptor, like other flaviviruses, it is internalized through clathrin-mediated endocytosis and transported to the endosomes with the involvement of cellular actin and microtubules to establish a productive infection ( figure ) [ ] . after internalization, to start translation and replication, the viral genome is released inside the cytoplasm by fusing the viral envelope with the membranes of the cellular endosomes, a process triggered by acidic ph inside them [ , ] . nanchangmycin, an insecticide and antibacterial polyether, inhibits zikv multiplication and, although the exact mechanism of action has not been completely elucidated, it probably targets axl and blocks clathrin-mediated endocytosis [ ] . acid endosomal ph triggers rapid conformational changes on viral envelope protein that result in its fusion with endosomal membrane in a ph-dependent manner, thus allowing nucleocapsid release to the cytoplasm for genome uncoating ( figure ). the optimal ph for conformational rearrangements and viral fusion is . - . , and these processes are likely dependent on the presence of cholesterol and specific lipids in the target membrane [ ] . these processes can be potentially druggable, and in fact, arbidol, a broad-spectrum antiviral and immunomodulatory use for human influenza a and b infections, inhibits zikv multiplication in cell culture probably because it intercalates into membrane lipids leading to the inhibition of membrane fusion between virus particles and plasma membranes, and between virus particles and the membranes of endosomes [ ] . chlorpromazine, an antipsychotic drug that also inhibits clathrin-mediated endocytosis, reduced zikv infection, confirming the requirement for clathrin-mediated endocytosis of zikv [ ] . in addition, -hydroxycholesterol ( hc) is increased in zikv-infected human embryonic cells and brain organoids, and reduces viremia and viral loads without affecting viral binding, but blocking internalization and suppressing viral and cell membranes fusion [ ] . even more, hc reduces mortality and prevents microcephaly in zikv-infected mice, and also decreases viral loads in the urine and serum of treated non-human infected primates [ ] . daptomycin, a lipopeptide antibiotic that inserts into cell membranes rich in phosphatidylglycerol, which suggests an effect on late endosomal membranes enriched in this lipid, has also been described as a zikv inhibitor [ ] . the dependence on endosomal acidification for zikv infection also provides a host target suitable for antiviral intervention. for instance, obatoclax (or gx - ), an anti-neoplastic and pro-apoptotic inhibitor of the bcl- that targets cellular mcl- , impairs zikv endocytic uptake by reducing the ph of the endosomal vesicles in cell culture, and thereby most likely inhibits viral fusion [ , ] . however, obatoclax, which presents a low solubility, has not produced satisfactory results in clinical trials for hematological and myeloid diseases. saliphenylhalamide (saliphe), which targets vacuolar adenosine triphosphatase enzyme (atpase) and blocks the acidification of endosomes, inhibits zikv multiplication in human retinal pigment epithelial cells [ ] that are natural targets for zikv infection [ ] . similar results were found by adcock et al. ( ) with saliphe using a different screening [ ] ; however, they reported that, contrary to that described by others [ ] , other compounds that interfere with the endocytic pathway, such as dynasore, that blocks clathrin-mediated endocytosis, or monensin, a cation transporter, were either toxic for the cells used or did not show any anti-zikv activity, as neither did chloroquine (cq). these contradictory results are probably explained by the different methodologies, cell types, and, to a lower extent, viral strains used to analyze the antiviral activities of the compounds and suggest that compounds showing different activities should be carefully evaluated before going further with investigations. in this line, and contrary to above mentioned report [ ] , cq, an fda-approved anti-inflammatory -aminoquinoline and an autophagy inhibitor widely used as an anti-malaria drug that is administered to pregnant women at risk of exposure to plasmodium parasites, was shown to have anti-zikv activity in different cell types (vero cells, human brain microvascular endothelial cells (hbmecs), and human neural stem cells (nscs)), affecting early stages of the viral life cycle, possibly by raising the endosomal ph and inhibiting the fusion of the envelope protein to the endosomal membrane [ , ] . cq has been shown to reduce placental and fetal zikv infection [ ] , and also attenuate zikv-associated morbidity and mortality in mice and protect the fetus from microcephaly [ ] . even more, cq attenuated vertical transmission in zikv-infected pregnant interferon signaling-competent swiss jim lambert (sjl) mice, significantly reducing fetal brain viral loads [ ] . similarly, cq, and other lysosomotropic agents (ammonium chloride, bafilomycin a , quinacrine, mefloquine, and n-tert-butyl isoquine (gsk )) that neutralize the acidic ph of endosomal compartments, block infection of a human fibroblast cell line and vero cells [ , ] . additionally, by medicinal chemistry-driven approaches, a series of new , -bis(trifluoromethyl)quinoline and n-( -(arylmethylimino)ethyl)- -chloroquinolin- -amine derivatives have been proved to inhibit zikv replication in vitro with a higher potency than chloroquine or mefloquine [ , ] . more recently, by screening fda-approved drugs using a cell-based assay, it has been shown that amodiaquine, another antimalarial drug, also has anti-zikv activity in cell culture by targeting early events of the viral replication cycle [ ] . niclosamide, a category b antihelmintic drug approved by fda, was capable of inhibiting zikv infection, and although its antiflaviviral effect has been associated to its ability to neutralize endolysosomal ph and interfere with ph-dependent membrane fusion, in the case of zikv, it seems that it was affecting other post-entry steps [ ] . in addition, recently, it has been reported that niclosamide decreases zikv production, partially restores differentiation, and prevents apoptosis in human induced nscs; even more, it can partially rescue zikv-induced microcephaly and attenuate infection in a developed humanized zikv-infected embryo model in vivo [ ] . likewise, tenovin- , which represses cell growth and induces apoptosis in cells expressing p by inhibiting the protein-deacetylating activities of sirt and sirt and, thus, affects endosome functions, potently inhibits zikv infection in primary placental fibroblast cells [ ] . iron salt ferric ammonium citrate (fac) also inhibits zikv infection through inducing viral fusion and blocking endosomal viral release by promoting liposome aggregation and intracellular vesicle fusion [ ] . overall, these studies evidence the potential of targeting viral entry to combat zikv. once zikv-rna is released from the endosomes in the cytoplasm, it acts as mrna to synthesize the negative-strand viral rna that directs positive-strand rna synthesis (figure ) [ ] . silvestrol, a natural compound isolated from the plant aglaia foveolata that it is known to inhibit the asp-glu-ala-asp (dead)-box rna helicase eukaryotic initiation factor- a (eif a) required to unwind structured -untranslated regions and thus impairing rna translation, exerts a significant inhibition of zikv replication in a cells and primary human hepatocytes [ ] . n-( -hydroxyphenyl) retinamide (fenretinide or -hpr), an activator of retinoid receptors that inhibits the proliferation of cancer cells and can induce apoptosis, inhibits zikv in cell culture and significantly reduces both serum viremia and brain viral burden in mice by decreasing the rate of viral rna synthesis, though not via direct inhibition of the activity of the viral replicase [ ] . zikv relies on polyamines for both translation and transcription [ ] , so that, drugs targeting the polyamine biosynthetic pathway, such as difluoromethylornithine (dfmo or eflornithine), an fda-approved drug that is used to treat trypanosomiasis, hirsutism, and some cancers, as well as diethylnorspermine (denspm) limit viral replication in bhk- cells [ ] . zikv replication and particle morphogenesis take place associated with a virus-induced organelle-like structure derived from the membrane of the endoplasmic reticulum (er) (figure ) [ ] . de novo synthesized positive strand-rna, once packaged, form enveloped immature virions in the er, enter the secretory pathway and, then, in the trans-golgi network, the prm is cleaved before the virus is released from the infected cell ( figure ) [ , ] . er-membrane multiprotein complexes, such as the oligosaccharyltransferase (ost) complex, have been reported to be critical host factors for flavivirus multiplication. in this regard, it has been shown that the n-linked glycosylation inhibitor- (ngi- ) chemical modulator of the ost complex blocks zikv-rna replication in different cell types [ ] . similarly, the host er-associated signal peptidase (spase) is an essential, membrane-bound serine protease complex involved in cleavage of the signal peptides of newly synthesized secretory and membrane proteins at the er and also for processing of the flavivirus prm and e structural proteins [ ] . it has also been reported that cavinafungin, an alaninal-containing lipopeptide of fungal origin, potently inhibits growth of zikv-infected cells [ ] . nitazoxanide, a broad-spectrum antiviral agent approved by the fda as an antiprotozoan and with potential activity against several viruses in clinical trials (rotavirus and norovirus gastroenteritis, chronic hepatitis b, chronic hepatitis c, and influenza), also inhibits virus infection targeting a post-attachment step, most likely virus genome replication [ ] . likewise, brefeldin a, a penicillium sp. product that inhibits protein transport from the er to the golgi apparatus, inhibits zikv multiplication [ ] , as does emetine, an anti-protozoal agent that inhibits both zikv ns polymerase activity and disrupts lysosomal function [ ] . zikv infection leads to cell-death by inducing host caspase- and neuronal apoptosis during its propagation [ ] . thereby, bithionol, a caspase inhibitor, inhibits zikv strains of different geographical origin in vero cells and human astrocytes [ ] . similarly, by using a drug repurposing screening of over molecules, it was found that emricasan, a pan-caspase inhibitor that restrains zikv-induced increases in caspase- activity and is currently in phase clinical trials in chronic hepatitis c virus (hcv)-infected patients, protected human cortical neural progenitor cells (npc) in both monolayer and three-dimensional organoid cultures, showing neuroprotective activity without suppression of viral replication [ ] . additionally, bortezomib, a dipeptide boronate proteasome inhibitor approved for treatment of multiple myeloma and mantle cell non-hodgkin's lymphoma that regulates the bcl- family of proteins, has also been described as a zikv inhibitor [ ] . similarly, different cyclin-dependent kinase (cdk) inhibitors, such as (alphas)- -(acetylamino)-alpha-methyl-n-( -( -methylethyl)- -thiazolyl)benzeneacetamide (pha- ), reduced zikv-infection and propagation [ ] . however, cdk inhibitors should not be suitable for the treatment of pregnant women but could be useful for the treatment of other non-pregnant patients, preventing the complications associated with zikv infection. the need for specific host lipids for flavivirus replication and particle envelopment make lipid metabolism a potential target for an antiviral search [ , ] , and, even though manipulating a major metabolic pathway such as lipid biosynthesis can be envisaged as a dangerous antiviral approach due to the undesirable effects that could be detrimental for the host, current use of drugs such as ibuprofen and aspirin (cyclooxygenase- (cox- ) inhibitors) or statins ( -hidroxi- -metil-glutaril-coa (hmg-coa) reductase inhibitors) highlights the feasibility of lipid-based therapeutics [ , ] . accordingly, inhibition of key enzymes involved in fatty acid synthesis, such as acetyl-coa carboxylase (acc) [ ] , and fatty acid synthase (fasn) [ ] [ ] [ ] , are potential targets for anti-zikv therapy. in this line, we have reported that nordihydroguaiaretic acid (ndga) and its derivative tetra-o-methyl nordihydroguaiaretic (m n or terameprocol), two compounds that disturb the lipid metabolism probably by interfering with the sterol regulatory element-binding proteins (srebp) pathway, inhibit the infection of zikv and wnv, likely by impairing viral replication, as did other structurally unrelated inhibitors of the srebp pathway, such as -[(diethylamino)methyl]-n-[ -( -methoxyphenyl)ethyl]-n-( r)- -pyrrolidinyl-benzamide dihydrochloride (pf- ) and fatostatin [ ] . in the same way, the dependence on cholesterol for different processes during flavivirus infection also provides a suitable target for antiviral strategies. as mentioned above, hc reduces viremia and viral loads in vitro, and also reduces mortality and prevent microcephaly in mice, and decreases viral loads in the urine and serum in non-human infected primates [ ] . lovastatin and mevastatin are hypolipidemic agents (hmg-coa inhibitors) belonging to the family of statins that are widely used for lowering cholesterol in patients with hypercholesterolemia and have been previously shown to present antiviral activity against dengue and hepatitis c viruses. both agents have been proposed as therapeutic candidates against zikv [ ] . in fact, lovastatin attenuates nervous injury in animal models of gbs [ ] . likewise, imipramine, an fda-approved antidepressant, inhibits zikv-rna replication and virion production in human skin fibroblasts, probably by interfering with intracellular cholesterol transport [ ] . regarding sphingolipid metabolism, which has been involved in flavivirus infection [ ] , treatment with the neutral sphingomyelinase inhibitor gw reduced zikv production by affecting viral morphogenesis [ ] as described for other flaviviruses [ ] . finally, since adenosine monophosphate-activated protein kinase (ampk) is a master regulator of lipid metabolism, its activation by pf- or metformin reduced zikv infection by impairing viral replication [ , ] . thus, targeting lipid metabolism could provide therapeutic alternatives for the discovery of host-directed antivirals against zikv. the ns protein is the viral rna-dependent rna polymerase responsible for the rna synthesis that also inhibits interferon (ifn) signaling by acting over the signal transducer and activator of transcription (stat ) protein [ ] , being, thus, a major target for antiviral design. besides the proven antiviral activities of different nucleosides analogs and inhibitors of the zikv-ns [ ] , several inhibitors of the biosynthesis of nucleosides (purines and pyrimidines) also impair zikv replication (figure ). ribavirin is an inhibitor of the inosine monophosphate dehydrogenase (impdh) with antiviral activity to several rna viruses [ ] , but its mechanism of action is not entirely clear. it may act as a guanosine synthesis inhibitor, a viral cap synthesis inhibitor, a viral rna mutagen, and as an inducer of lethal mutagenesis [ ] [ ] [ ] . by using a cell-based assay, no antiviral activity of the drug was initially observed [ ] but, later on, it was reported that although no activity against zikv was detected in vero cells, the drug did inhibit virus multiplication in human cell lines, including liver huh- and rhabdomyosarcoma (rd) cells [ ] . further studies have confirmed an inhibitory activity of ribavirin against zikv strains of different geographical origin in various types of cells, such as human neural progenitor cells (hnpcs), human dermal fibroblasts (hdfs), human lung adenocarcinoma cells (a ), and even in vero cells [ ] [ ] [ ] . still more, the drug was shown to abrogate viremia in zikv-infected stat- -deficient mice [ ] , which lack type i ifn signaling, are highly sensitive to zikv infection, and exhibit a lethal outcome. two other inhibitors of impdh, merimepodib (mmpd or vx- ) [ ] and mycophenolic acid (mpa) [ , , ] also inhibit zikv-rna replication in different cell types, including huh- cells, human cervical placental cells, and neural stem and primary amnion cells. however, other authors [ ] have described that mpa have little effect on zikv replication and showed significant cell toxicity. likewise, azathioprine, another inhibitor of purine synthesis and immunosuppressant, impaired zikv replication in hela and jeg cells [ ] ; nonetheless, its use in pregnant women is not recommended. the above described contradictory results stress again the differences that drug treatments may have as a consequence of the different viral strains, cell types, and methodologies used to assess them. as with the inhibitors of purine biosynthesis, compounds inhibiting the synthesis of pyrimidines have also effect on zikv replication (figure ). so that, the virus was highly susceptible to brequinar and cid treatments in cell culture [ ] . however, it should be noted that it has been reported that brequinar, as well as dd , antiviral activity may not be due to pyrimidine deprivation, but rather to the induction of the cellular immune response [ , ] . similarly, other inhibitors of the pyrimidine synthesis, such as gemcitabine, an activator of cellular caspases [ , ] , and, although with a lower efficiency probably due to its lower solubility, -azauridine and finasteride, a -azasteroid analog of testosterone that inhibit type ii and type iii α-reductase and is being tested for benign prostatic hyperplasia and male pattern baldness, reduce zikv replication [ , ] . several other compounds have been shown to have anti-zikv activity by inhibiting viral entry and/or rna synthesis, although their mechanisms of action have not yet been fully elucidated. among them are antiparasitics such as ivermectin (used mainly against worms infections) and pyrimethamine (a folic acid antagonist that inhibits the dihydrofolate reductase and, thus, dna and rna synthesis, is classified as a pregnancy category c, and was initially used to treat malaria and now toxoplasmosis and cystoisosporiasis) [ ] ; antibiotics such as azithromycin that prevents infection, replication, and virus-mediated cell dead [ ] , and kitasamycin (a natural product from streptomyces narbonensis that inhibits protein biosynthesis) [ ] ; drugs used to prevent chemotherapy-induced nausea and vomiting as palonosetron (a fda-approved -ht antagonist) [ ] ; antidepressants like sertraline (a selective serotonin reuptake inhibitor) [ ] and cyclosporine (that is also use for rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome, and in organ transplants, is believed to lower the activity of t-cells, and is currently in clinical trials for tis possible use in ameliorate neuronal cellular damage) [ ] . similarly, after chemical screening, it was found that hippeastrine hydrobromide (hh), an active component of traditional chinese medicine, and amodiaquine dihydrochloride dihydrate (aq), an fda-approved drug for treatment of malaria, inhibit zikv infection of human pluripotent stem cell-derived cortical npcs and in adult mouse brain in vivo even when the infection was already ongoing but, again, their mechanisms of action are not known [ ] . besides drugs that act against host targets directly implicated in the viral cycle, there are compounds that can prevent undesirable effects of zikv infection. in this regard, zikv infection leads to massive neuronal damage, especially of neural progenitor cells, and neurodegeneration [ ] [ ] [ ] , via both direct replication in neuronal cells and possibly through increased excitotoxicity via over activation of n-methyl-d-aspartate receptor (nmdar)-dependent neuronal excitotoxicity in nearby cells. memantine, a pregnancy category b fda-approved drug widely used to treat patients with alzheimer's disease, as well as other nmdar blockers (dizocilpine, agmatine sulfate, or ifenprodil), prevents neuronal damage and death and intraocular pressure increase induced by zikv infection in infected mice, but it does not affect virus replication, pointing to its possible use to prevent or minimize zikv-related microcephaly during pregnancy [ ] . ebselen (ebs), an antioxidant that reduces oxidative stress and improves histopathological features in a testicular injury study model and is currently in clinical trials for various diseases, showed minor effects in reducing zikv progeny production and viral e protein expression and on overall survival and viremia level of challenged ag mice; however, it should be noted that ebs reduced some zikv-induced effects, such as testicular oxidative stress, leucocyte infiltration, and production of pro-inflammatory response, whereas, in a model of male-to-female mouse sperm transfer, the drug improved testicular pathology and prevented the sexual transmission of zikv [ ] . ifns play a key role in the elimination of pathogens and they are release upon the activation of the innate immune response by infecting viruses. in this way, zikv infection induces ifn signaling pathways and further activates cytoplasmic retinoic acid inducible gene protein (rig )-like receptors (rlrs) and several type i and iii ifn-stimulated genes, driving to the subsequent activation of the janus kinase (jak)/stat innate immune pathway that confer resistance to zikv infection [ ] . different studies showed that ifn-α, ifn-β, and ifn-γ inhibit zikv replication in cell culture [ , , ] , and that treatment of pregnant mice with ifn-λ reduced zikv infection [ ] . in addition, ifitm and ifitm , which are interferon-induced transmembrane proteins, impair early stages of zikv infection. even more, ifitm prevents zikv-induced cell death [ ] . likewise, it has been reported that an interferon-activating small molecule ( -( -fluorophenyl)- -( -isopropyl- , , -thiadiazol- -yl)- , -ihydrochromeno [ , -c] pyrrole- , -dione (avc) strongly inhibits replication of zikv in cell culture [ ] . however, it is also known that the virus is capable of evading type i ifn responses by acting over the jak/stat signaling pathway [ , [ ] [ ] [ ] , and that type i ifns might be mediators of pregnancy complications, including spontaneous abortions and growth restriction [ ] . in any case, use of ifn against zikv, alone or in combination with other antivirals, deserve further studies. by screening a library of known human micrornas (mirnas), small, noncoding rnas (sncrnas) that modulate gene expression post-transcriptionally and regulate a broad range of cellular processes, several mirnas were found to inhibit zikv by increasing the capability of infected cells to respond to infection through the interferon-based innate immune pathway [ ] . another alternative is intervening over epigenetic regulation by using epigenetics modulators. for instance, histone h k methyltransferases (ezh and ezh ) suppress gene transcription and it has been shown that inhibitors such as -[( s)-butan- -yl]-n-[( , -dimethyl- -oxo- h-pyridin- -yl)methyl]- -methyl- -( -piperazin- -ylpyridin- -yl)indole- -carboxamide (gsk- ) reduce zikv multiplication in cell culture through the activation of cellular antiviral and immune responses [ ] . in any case, further studies are needed to evaluate the potential therapeutic capability of these immunomodulators against zikv infection. a great effort is being lately made to find compounds to fight zikv infection by applying different approaches, from repurposing of drugs with known antiviral activity to the screening of bioactive molecules from different libraries, as well as natural products. however, most of the already tested drugs have been found to inhibit viral replication in vitro, and only a few have been tested in vivo. hence, since, in many instances, the results will be difficult to extrapolate to humans, it would be hard for most of the tested antivirals to complete the entire drug development pipeline. in addition, it should be remarked that many drugs could have untoward effects and, thus, careful evaluation should be conducted before using them in clinical practice, as the main target populations for anti-zikv therapy will be pregnant women and patients with other medical complications. many of the already tested drugs are directed against viral structural and enzymatic proteins, including, for instance, anticancer and anti-inflammatory molecules, antibiotics, and antiparasitics; however, it is well known that this approach can easily lead to the appearance of resistance. since flaviviruses require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently, this offers an opportunity to search for host targets as therapeutic tools that, in many instances, can be broad spectrum agents, and which effect would be less prone to the emergence of mutants that will escape their action. because of that, and even though manipulating host metabolic pathways can be seen as dangerous due to the undesirable effects that could be detrimental for the host, its success for other diseases make of them a realistic option for the treatment of zikv infection. phylogeny of the genus flavivirus potential of selected senegalese aedes spp. mosquitoes (diptera: culicidae) to transmit zika virus zika virus zika virus: the latest newcomer full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus. i. isolations and serological specificity zika virus outbreak on yap island, federated states of micronesia neurological manifestations of zika virus infection the history of zika virus detection of zika virus in urine zika virus tissue and blood compartmentalization in acute infection of rhesus macaques zika virus pathogenesis and tissue tropism zika virus infection damages the testes in mice zika virus infection of rhesus macaques leads to viral persistence in multiple tissues zika 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small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells antiviral effects of ferric ammonium citrate inhibition of zika virus replication by silvestrol antiviral activity of n-( -hydroxyphenyl) retinamide ( -hpr) against zika virus interferon-induced spermidine-spermine acetyltransferase and polyamine depletion restrict zika and chikungunya viruses inhibition of polyamine biosynthesis is a broad-spectrum strategy against rna viruses a structural perspective of the flavivirus life cycle post-translational regulation and modifications of flavivirus structural proteins a small-molecule oligosaccharyltransferase inhibitor with pan-flaviviral activity a crispr screen defines a signal peptide processing pathway required by flaviviruses the natural product cavinafungin selectively interferes with zika and dengue virus replication by inhibition of the host signal peptidase pediatric drug nitazoxanide: a potential choice for control of zika identification of novel antiviral of fungus-derived brefeldin a against dengue viruses emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry zika virus infects human cortical neural progenitors and attenuates their growth bithionol blocks pathogenicity of bacterial toxins, ricin, and zika virus targeting host lipid synthesis and metabolism to inhibit dengue and hepatitis c viruses the structure of mammalian cyclooxygenases present status of statin therapy modification of the host cell lipid metabolism induced by hypolipidemic drugs targeting the acetyl coenzyme a carboxylase impairs west nile virus replication dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis west nile virus replication 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flavivirus infection through modification of host cell lipid metabolism suppression of zika virus infection and replication in endothelial cells and astrocytes by pka inhibitor pki - zika virus targets human stat to inhibit type i interferon signaling ribavirin-current status of a broad spectrum antiviral agent broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx- : a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon rna virus error catastrophe: direct molecular test by using ribavirin extinction of hepatitis c virus by ribavirin in hepatoma cells involves lethal mutagenesis efficacy of the broad-spectrum antiviral compound bcx against zika virus in cell culture and in a mouse model in vitro susceptibility of geographically and temporally distinct zika viruses to favipiravir and ribavirin ribavirin inhibits zika virus (zikv) replication in vitro and suppresses viremia in zikv-infected stat -deficient mice favipiravir and 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alleviates testicular pathology in mice with zika virus infection and prevents its sexual transmission zika virus infectious cell culture system and the in vitro prophylactic effect of interferons type iii interferons produced by human placental trophoblasts confer protection against zika virus infection gestational stage and ifn-lambda signaling regulate zikv infection in utero the ifitms inhibit zika virus replication a novel agonist of the trif pathway induces a cellular state refractory to replication of zika, chikungunya, and dengue viruses zika virus inhibits type-i interferon production and downstream signaling zika virus antagonizes type i interferon responses during infection of human dendritic cells axl promotes zika virus infection in astrocytes by antagonizing type i interferon signalling type i interferons instigate fetal demise after zika virus infection a microrna screen identifies the wnt signaling pathway as a regulator of the interferon response during flavivirus infection inhibitors of the histone methyltransferases ezh / induce a potent antiviral state and suppress infection by diverse viral pathogens key: cord- -r qtoqu authors: hellmich, luisa; rongisch, robert; rasokat, heinrich; von stebut, esther; fabri, mario title: exantheme nach auslandsreisen date: - - journal: hautarzt doi: . /s - - -y sha: doc_id: cord_uid: r qtoqu in view of globalization and the associated transport of goods as well as rising travel activity, imported infections with subtropical and tropical pathogens are increasing in germany. in returning travelers presenting with fever, general symptoms and skin rash, a number of diseases need to be considered. the clinical appearance of the skin rash, accurate travel history and epidemiological information on country-specific risks are helpful in making the correct diagnosis. in this article we provide an overview of the most common exanthemas in travelers who have returned, associated symptoms, diagnostic methods, therapies, as well as prevention strategies. abb. -jähriger patient mit a und b makulopapulösem exanthem und c enanthem nach rückkehr aus mittelund südamerika zwar unwahrscheinlich, aufgrund des klimawandels und steigender sommerlicher temperaturen aber potenziell denkbar [ ] . das zika-virus gehört zur familie der flaviviren. die Übertragung auf den menschen erfolgt meistens durch mücken (s. oben) ist aber auch sexuell, pränatal und durch muttermilch möglich. seit oktober sind in deutschland mehr als zika-virus-infektionen bei reiserückkehrer*innen (beispielsweise brasilien, südostasien, florida und von karibikinseln wie st. barth) diagnostiziert worden [ , , ] . in einem fall wurde eine sexuelle Übertragung des zika-virus beobachtet [ ] . um die erkrankung besser überwachen zu können, gibt es seit mai eine gesetzliche meldepflicht in deutschland. auch wenn die jährlich übermittelten zika-virus-infektionen insgesamt zurückgehen ( : fälle, : fälle, : fälle), ist aufgrund der häufig symptomlosen infektion von einer beträchtlichen dunkelziffer auszugehen (rki [robert koch-institut]) [ ] . eine zika-virus-infektion verläuft nur in etwa % der fälle symptomatisch. es kommt dann nach einer inkubationszeit von bis tagen zunächst zu einer influenza-ähnlichen symptomatik mit (eher leichtem) fieber. in ca. % der fälle tritt ein makulopapulöses, nur leicht juckendes exanthem im median tag (spanne von bis tage) nach der allgemeinsymptomatik auf und ist typischerweise an stamm, hals und im gesicht, in wenigen fällen zusätzlich palmoplantar lokalisiert. bei < % der betroffenen zeigen sich hämorrhagien (petechien, blutende ulzerationen, gaumenbluten, spontane hämatome). typisch für eine zika-virus-infektion ist eine meist gleichzeitig mit dem exanthem auftretende konjunktivitis, die im schnitt etwas länger als das exanthem anhält (median bis tage) [ ] . nur selten kommt es zu neurologischen komplikationen, etwa dem guillain-barré-syndrom [ ] . der zusammenhang zwischen pränataler zika-virus-Übertragung und embryopathien wie neuralrohrdefekten und mikrozephalie wurde erstmals im september bekannt, als sich in süd-und mittelamerika eine neue zika-epidemie auszubreiten begann [ , ] . prospektive kohortenstudien an schwangeren mit bestätigter, in der schwangerschaft erworbener zika-virus-infektion zeigten dann eine prävalenz von - % für das kongenitale zika-syndrom [ , , , , ] . dabei ist die vulnerabilität bei einer infektion im ersten trimenon besonders hoch [ , ] . vom dengue-virus, das wie das zika-und das gelbfieber-virus zur familie der flaviviren gehört und über mücken der gattung aedes (s. oben) übertragen wird, existieren serotypen (denv- bis denv- ), die weltweit in den tropen und subtropen verbreitet sind. das dengue-fieber ist das dengue-fieber ist die sich weltweit am schnellsten ausbreitende arbovirusinfektion cme abb. makulopapulöses exanthem mit "islands of white in a sea of red" a am rumpf und b am arm eines patienten mit klassischem dengue-fieber die sich weltweit am schnellsten ausbreitende arbovirusinfektion [ ] . in deutschland hat es mit bis fällen pro jahr [ ] die malariarate bereits überholt. bei der erstinfektion mit einer der denv-typen kann sich nach einer inkubationszeit von bis , maximal tagen ein klassisches dengue-fieber mit plötzlichem (häufig biphasischem) fieber (> °c), einem hämmernden retrobulbärschmerz, starken muskuloskelettalen schmerzen ("break-bone-fever") und konjunktivitis entwickeln. dabei zeigt die erkrankung besonders bei reiserückkehrern meist eher milde verläufe, bei denen der "knochenbrecheraspekt" ausbleibt. an der haut kann es bis tage nach fieberbeginn zu einem an den hand-und fußrücken beginnenden, sich dann auf die proximalen extremitäten und den rumpf ausbreitenden makulopapulösen, nicht juckenden exanthem mit palmoplantarer aussparung kommen ( [ ] ; . abb. ). besonders charakteristisch sind dabei nappes-claires-artige aussparungen nichtbetroffener haut ("islands of white in a sea of red") (. abb. ). ausgedehntere einblutungen der haut oder (oft ein frühes warnzeichen) der konjunktival-und bindehaut sind hinweis auf den Übergang zum lebensbedrohlichen hämorrhagischen dengue-fieber bzw. zum dengue-schocksyndrom. diese treten in der regel nur bei einer späteren zweitinfektion mit einem anderen denv-serotyp auf, wobei für die oft erhebliche foudroyanz vermutlich die bildung infektionsverstärkender antikörper im vorsensibilisierten organismus verantwortlich ist. nach bis tagen kommt es zu einer akuten verschlechterung mit sugillationen, generalisierten Ödemen, zeichen der leber-und zns(zentralnervensystem)-beteiligung sowie einem hämatokritanstieg und thrombozytenabfall. die letalität liegt unbehandelt bei %, kann aber durch rechtzeitige intensivmedizinisch-supportive therapie auf - % gesenkt werden [ ] . diagnostisch ist an den krankheitstagen bis der nachweis von virus-rna mittels pcr oder von dengue-ns -antigen mittels elisa ("enzyme-linked immunosorbent assay") im blut möglich. ab tag bis zeigt sich auch die serologie positiv, wobei niedrige anfangstiter auch besonders charakteristisch sind nappes-claires-artige aussparungen nichtbetroffener haut ab tag bis zeigt sich die serologie positiv cme zeichen einer durchgemachten anderen flavivirusinfektion oder der gelbfieberimpfung sein können. die behandlung erfolgt supportiv-symptomatisch. die wirksamkeit bereits verfügbarer medikamente (z. b. antimalariamittel) gegen dengue wird derzeit erforscht [ ] . prophylaktisch ist in erster linie der mückenschutz zu nennen. zur prävention schwerer verläufe wurde im oktober von der europäischen arzneimittelbehörde ein tetravalenter attenuierter lebendimpfstoff für personen von bis jahren zugelassen, die in einem endemiegebiet leben und bereits eine (laborbestätigte) infektion durchgemacht haben [ ] . die herstellung und prüfung weiterer impfstoffe ist derzeit noch in der entwicklung [ , ] . epidemiologie im jahr wurden in deutschland laut robert koch-institut (rki) importierte chikungunya-virus-erkrankungen übermittelt. außer einer in italien erworbenen infektion lagen alle infektionsländer außerhalb europas (v. a. indien, brasilien) [ ] . im jahr kam es in italien erstmals zu autochthon-europäischen chikungunya-fällen mit > betroffenen personen. das virus war durch einen fernreisenden aus indien eingeschleppt worden und hatte sich dann in einer in der toskana heimischen aedes-albopictus-population ausgebreitet [ ] . nach diesem verbreitungsmodell sind in den vergangenen jahren immer wieder kleinere und größere chikungunya-, aber auch dengue-ausbrüche in den albopictus-endemiegebieten des mittelmeerraums aufgetreten [ , ] . die . abb. zeigt die von der europäischen gesundheitsbehörde ecdc (european centre for disease prevention and control) herausgegebene aktuelle verbreitungskarte der vektormücken [ ] . bei der infektion mit dem zu den togaviridae gehörenden rna-virus kommt es nach einer inkubationszeit von bis tagen zu einem plötzlichen fieberanstieg, starken kopfschmerzen und sehr heftigen bilateralen arthralgien (v. a. der extremitäten; "chikungunya" heißt "der gekrümmt gehende"). im unterschied zur klassischen differenzialdiagnose einer dengue-infektion breitet sich das in - % der fälle auftretende makulopapulöse exanthem zentripetal aus und kann auch eine palmoplantare beteiligung bieten [ , , ] . es bleibt ca. eine woche bestehen. rötungen der ohrmuschel sind typisch, und eine augenbeteiligung (konjunktivitis, uveitis) ist möglich [ ] . hämorrhagische verläufe mit multiorganversagen und neurologische komplikationen sind sehr selten. gefürchtete langzeitkomplikationen sind persistierende oder rezidivierende arthralgien, die eine rheumatoide arthritis imitieren können [ , ] . in den ersten tagen kann der virusnachweis mittels pcr aus dem serum erfolgen, während ig(immunglobulin)m ab tag bis und igg ab tag bis positiv werden. antivirale medikamente wie ribavirin oder interferon-α wurden bisher nur in vitro getestet [ ] . die prophylaxe konzentriert sich auch hier auf mückenschutz. ein impfstoff existiert bisher nicht [ , ] . die gramnegativen obligat intrazellulären rickettsien nutzen arthropoden als vektor. die von ihnen verursachten fieberhaften erkrankungen werden nach antigener und genetischer verwandtschaft in große gruppen aufgeteilt (. tab. , verändert nach [ ] ). auch wenn das tsutsugamushi-fieber in der gegenwärtig gültigen taxonomie aufgrund der phylogenetischen unterschiede als eigenständige krankheitsentität betrachtet wird [ ] , wird es hier aufgrund der den rickettsiosen ähnlichen klinik mit aufgeführt. alle rickettsiosen können mit einem exanthem einhergehen und viele zeckenstichfieber sowie das tsutsugamushi-fieber sind mit einem kutanen eschar (syn. "tache noir") assoziiert, der eine nekrotisierende vaskulitis an der einstichstelle darstellt und klinisch wie ein schlecht heilender insektenstich aussieht (. abb. ). in endemiegebieten (afrika, karibik) sind bis zu % der zecken mit rickettsien durchseucht, und bis zu % aller safaritouristen im südlichen afrika entwickeln das sog. "african tick bite fever" (atbf) [ ] . nach einer inkubationszeit von bis tagen kommt es -häufig auch vor der ausbildung des eschars -zu einer allgemeinsymptomatik mit mehrere tage anhaltendem fieber, starken kopfschmerzen, arthralgien und myalgien. etwa bis tage nach dem ersten fieber entwickelt sich in bis zu % der fälle ein makulopapulöses, häufig petechiales exanthem mit möglicher palmoplantarer beteiligung [ , ] . multiple vaskulitiden der inneren organe können v. a. beim "rocky mountain spotted fever" und selten beim mediterranen zeckenstichfieber vorkommen und zu lungenödem, hirnödem, myokarditis, nierenversagen und disseminierter intravasaler gerinnung führen. weitere (seltene) komplikationen sind subakute neuropathien, die nach monaten z. b. zu parästhesien und hörverlust führen können. fleckfieberrickettsiosen sind insgesamt sehr selten ( bis fälle pro jahr in deutschland [ ] den goldstandard stellt eine ab dem ende der ersten krankheitswoche positiv werdende serologie dar. allerdings kann aufgrund der starken kreuzreaktivität zwischen den mehr als humanpathogenen spezies in der "spotted fever group" nicht unterschieden werden, sodass zum erregernachweis auf speziesebene eine pcr aus einer biopsie vom rand des eschars notwendig ist [ ] . die kreuzreaktivität zu den fleckfieber verursachenden rickettsien ist gering. orientia tsutsugamushi ist mittels pcr auch im edta-blut nachweisbar. bei passender anamnese und klinik sollte sehr frühzeitig eine kalkulierte therapie mit doxycyclin ( mg erstdosis, dann mg -mal täglich für tage bzw. bis tage nach sistieren der symptome) begonnen werden, alternativ kommt eine therapie mit azithromycin infrage. präventive maßnahmen zum schutz vor zeckenstichen (lange, helle kleidung, repellenzien) sollten durchgeführt werden. während eines auslandsaufenthaltes haben bis zu % aller reisenden gelegenheitssex mit einem neuen partner [ ] , sodass syphilis (. abb. ) und eine akute hiv("human immunodeficiency zurück zu unserem patienten (s. falldarstellung in der einleitung): aufgrund des abrupt einsetzenden hohen fiebers, des makulopapulösen, leicht petechialen exanthems und der konjunktivitis stellten wir in zusammenhang mit der entsprechenden reise nach mittel-und südamerika primär die verdachtsdiagnose einer arbovirose. nach ausschluss einer malaria ergaben sich folgende untersuchungsbefunde: blutkulturen, tppa (treponema-pallidum-partikelagglutinationsassay) und hiv-suchtest negativ, leptospiren-serologie negativ, masern-, ebv-, cmv-serologie: jeweils igm negativ, igg positiv, dengue-ns- -antigen: negativ, chikungunya-virus-pcr: negativ, dengueund chikungunya-virus-serologie: negativ, zika-virus-rna im urin und serum: positiv, zika-virus-serologie: igm positiv, igg negativ. in zusammenschau der befunde konnte bei dem patienten eine akute zika-virus-infektion diagnostiziert werden. es erfolgte eine symptomatische behandlung mit antipyretika, unter der sich der patient innerhalb woche vollständig erholte. dem patienten wurde empfohlen, für mindestens monate mit kondomen zu verhüten. einige der genannten erkrankungen sind nach dem infektionsschutzgesetz (ifsg) meldepflichtig. namentlich zu melden sind der verdacht einer erkrankung, die erkrankung sowie der tod in bezug auf arbovirosen und masern (ifsg § ) sowie der direkte oder indirekte nachweis, sofern er auf eine akute infektion hinweist, von rickettsia prowazekii, masernvirus, salmonella typhi, eine malaria muss bei fieber nach aufenthalt in endemiegebieten immer ausgeschlossen werden cme humanpathogene leptospira spp. nicht namentlich zu melden ist der direkte oder indirekte nachweis, sofern er auf eine akute infektion hinweist, von hiv, treponema pallidum und plasmodium spp. (ifsg § ). keine meldung erfolgt bei sandfliegenfieber, zeckenübertragenen rickettsiosen, tsutsugamushi-fieber, ebv und cmv. laut einschlägiger empfehlungen des robert koch-instituts (https://www.rki.de/de/content/ infekt/krankenhaushygiene/kommission/downloads/uebersinfektionserkrmassn_ .pdf?__ blob=publicationfile; letzter abruf . . ) sollen in einrichtungen des gesundheitswesens an masern erkrankte nach auftreten des exanthems für tage -also bis zum . exanthemtag -in isolierzimmern mit vorraum untergebracht werden; diese maßnahme ist bei immunsupprimierten patienten über die gesamte dauer der symptomatik hin beizubehalten. ebenso sind an varizellen erkrankte personen bis zum eintrocknen aller virusbläschen in isolierzimmern mit vorraum unterzubringen. personen mit akuter ebv-infektion sollen in bereichen mit hochgradig immunsupprimierten patienten in standardeinzelzimmern untergebracht werden. an typhus abdominalis (erreger: salmonella typhi) erkrankte sollten bis zum vorliegen von insgesamt negativen stuhluntersuchungen (erste stuhlprobe frühestens h nach abschluss der antimikrobiellen therapie, abstand der proben bis tage) mit schleuse isoliert werden. sfb-förderung | land nordrhein-westfalen, rückkehrerprogramm. -honorar (an uniklinik köln) und reisekosten von abbvie für vortrag | honorar von novartis für vortrag bei advisory board (s. unten) | reisekosten von almirall für vortrag bei seminar. -honorar von novartis für vortrag bei advisory board (s. oben) die vollständige erklärung zum interessenkonflikt der wissenschaftlichen leitung finden sie am kurs der zertifizierten fortbildung auf www der verlag erklärt, dass für die publikation dieser cme-fortbildung keine sponsorengelder an den verlag fließen für diesen beitrag wurden von den autoren keine studien an menschen oder tieren durchgeführt. für die aufgeführten studien gelten die jeweils dort angegebenen ethischen richtlinien. für bildmaterial oder anderweitige angaben innerhalb des manuskripts, über die patienten zu identifizieren sind, liegt von ihnen und/oder ihren gesetzlichen vertretern eine schriftliche einwilligung vor the asian tiger mosquito aedes albopictus (diptera: culicidae) in central germany: surveillance in its northernmost distribution area european centre for disease prevention and control areas with high hazard potential for autochthonous transmission of aedes albopictus rk-i. infektionsepidemiologisches jahrbuch meldepflichtiger krankheiten für zikavirus-infektionen: tropische krankheit mit relevanz für deutschland zika virus in germany: case report and possible routes of transmission sexual transmission of zika virus in germany antworten auf häufig gestellte fragen zum zikavirus mucocutaneous features of zika-a review zikavirusandthe guillain-barré syndrome-case series from seven countries possible association between zika virus infection and microcephaly-brazil microcephaly in brazil: how to interpret reported numbers maternal-fetal transmission and adverse perinatal outcomes in pregnant women infected with zikavirus: prospectivecohortstudy in french guiana pregnancy outcomes after maternal zika virus infection in a non-endemic region: prospective cohort study zika virus infection in pregnant women in rio de janeiro new york city department of health and mental hygiene zika working group ( ) first months of life for infants in new york city pregnancy outcomes after zikv infection in french territories in the americas zika virus sexually acquired zika virus: a systematic review duration of the presence of infectious zika virus in semen and serum the current and future global distribution and population at risk of dengue dengue fever virale hämorrhagische fieber recent update on antidengue drug discovery antworten auf häufig gestellte fragen zu dengue und zur impfung immunogenicity and safety of one versus two doses of tetravalent dengue vaccine in healthy children aged - years in asia and latin america: -month interim data from a phase , randomised, placebo-controlled study dengue vaccine: global development update infection with chikungunya virus in italy: an outbreak in a temperate region transmission dynamics of the ongoing chikungunya outbreak in central italy: from coastal areas to the metropolitan city of rome, summer chikungunya and dengue autochthonous cases in europe chikungunya virus and the global spread ofamosquito-bornedisease chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen inflammation of the external ear in acute chikungunya infection: experience from the outbreak in treatment of chikungunya musculoskeletal disorders: a systematic review development of vaccines for chikungunya fever chikungunya as a paradigm for emerging viral diseases: evaluating disease impact and hurdles to vaccine development measles virus and the nervous system implications of the human immunodeficiency virus epidemic for control and eradication of measles rickettsioses: cutaneous findings frequently lead to diagnosis-a review afrikanisches zeckenbissfieber -papulovesikuläres exanthem mit fieber nach südafrika-aufenthalt african tick bite fever in travelers to rural sub-equatorialafrica update on tick-borne bacterial diseases in travelers foreign travel, casual sex, and sexually transmitted infections: systematic review and metaanalysis key: cord- -i ewz authors: chidiac, c.; ferry, t. title: agents infectieux émergents date: - - journal: transfus clin biol doi: . /j.tracli. . . sha: doc_id: cord_uid: i ewz emergence of many emerging or re-emerging infectious diseases have occurred over the past decade, some of which are major public threat. sars, mers-cov, highly pathogenic avian influenza a(h n ), ebola virus disease have raised concerns because of their virulence, their mortality, and/or their modality of transmission, or their impact on maternofoetal transmission (zika virus). the witness of these emergences have conducted health authorities to have policies and plans and to imagine new organizations for health systems in order to identify any case of highly communicable virulent disease for immediate isolation, and adequate management. en , william stewart, surgeon général des États-unis aurait déclaré la « fin de la guerre contre la pestilence et la fermeture du grand livre des maladies infectieuses » ; dans le même temps, se propageait l'épidémie de sida [ ] . depuis, de nombreux autres pathogènes ont émergés, dont le vhc, le syndrome respiratoire aigu sévère (sras), l'infection à virus west nile, le chikungunya, la grippe aviaire a(h n ), le mers-cov, la * auteur correspondant. adresse e-mail : christian.chidiac@chu-lyon.fr (c. chidiac). grippe pandémique a(h n )pdm , la maladie à virus ebola (mve), l'infection à virus zika. . . selon la définition retenue par le hcsp, une maladie infectieuse émergente (mie) est une maladie infectieuse -ou présumée infectieuse -inattendue (en référence aux propriétés intrinsèques ou à la connaissance que l'on a de la biologie de son agent responsable), touchant l'homme, l'animal ou les deux [ ] . il peut s'agir : (i) d'une entité clinique d'origine infectieuse nouvellement apparue ou identifiée, ou (ii) d'une maladie infectieuse connue, dont l'incidence augmente ou dont les caractéristiques (cliniques, évolutives. . .) se modifient dans un espace ou dans un groupe de population donné. http le but de cette revue limitée est de décrire certaines maladies infectieuses émergentes et les réponses apportées. cependant, l'évolution permanente des connaissances, en particulier pour les émergences, invite les lecteurs à se reporter aux publications les plus récentes postérieures à cette revue non exhaustive. l'année a été marquée par l'émergence du syndrome respiratoire aigu sévère (sras), maladie liée au virus sars-cov de la famille des coronavirus, qui se caractérise par une symptomatologie principalement respiratoire [ , ] . l'épidémie a rapidement pris une dimension internationale, faisant plus de malades et morts dans une trentaine de pays [ ] . en février , un médecin chinois de la province du guangdong qui séjourne à l'hôtel métropole de hong kong a contaminé une quinzaine de clients et visiteurs de cet hôtel, lesquelles ont été à l'origine des différents foyers dans le monde [ ] . l'alerte oms a été déclenchée le mars . une transmission du germe de personne à personne par contact rapproché a été suspectée en raison du nombre élevé de cas parmi les personnels soignants [ ] et les modalités de transmission décrites ont celles d'une transmission par les sécrétions, puis envisagée par aérosol. la transmission principalement aérienne du virus couplée à des voyages de plus en plus nombreux ont permis la propagation du virus [ ] . l'évaluation du risque a été faite rapidement sur la gravité des cas, la diffusion rapide et l'atteinte plus particulière des personnels soignants [ , ] . l'existence de formes cliniques paucisymptomatiques, voire asymptomatiques, de sras est possible (tableaux et ). les signes cliniques débutent le plus souvent après une période d'incubation de à jours ( jours en moyenne). le tableau comporte les éléments suivants : fièvre ( à % des cas), tableau fièvre frissons a a toux myalgies malaise - rhinorrée pharyngite dyspnée - -diarrhée céphalées d'après lee et al. [ ] , peiris et al. [ ] , donnely et al. [ ] , booth [ ] . a frissons. syndrome pseudo-grippal ( à %), signes respiratoires ( à %), signes digestifs ( à %). À la phase précoce, l'examen physique est souvent pauvre, avec parfois une auscultation pulmonaire anormale. l'évolution est marquée par le risque de détresse respiratoire au cours de la deuxième semaine, avec % de séjour en réanimation et environ % de mortalité surtout fonction des comorbidités [ , ] . le diagnostic de sras repose d'abord sur le contexte épidémiologique. la biologie sanguine non spécifique peut révéler l'existence d'une lymphopénie, d'une thrombopénie, d'une élévation des transaminases, d'une augmentation des ldh et de la cpk. la radiographie pulmonaire peut être normale initialement. les anomalies sont à prédominance interstitielle, focalisées ou diffuses. le diagnostic de certitude repose sur l'identification du virus dans les sécrétions respiratoires ou dans les selles ou sur la sérologie, le meilleur examen étant la détection par rt-pcr [ ] . les autres étiologies de pneumopathie doivent être systématiquement éliminées. les mesures ayant permis le contrôle de la maladie sont : • la détection précoce des cas suspects et probables ; • les mesures strictes d'isolement respiratoire des cas probables ; • les mesures de protection du personnel soignant et d'un isolement pendant jours (quarantaine) des personnes ayant été en contact avec des cas probables ; • l'information du public et recommandations quant à la restriction de voyager dans les zones de transmission active du sras ; • les mesures de contrôle aux frontières ; • la mise en place d'un système d'alerte et de surveillance national et international [ ] . le virus a(h n ) apparu à hong kong en a diffusé par la suite à de nombreux pays, marquant le début de la propagation de l'épizootie qui s'est étendue de l'asie, à l'europe puis l'afrique (situation épidémiologique internationale) et au moyen-orient. des cas humains de grippe aviaire ont été décrits, notamment à hong kong en ( cas humains, décès) et en janvier , puis dans les pays ayant signalé des foyers animaux de grippe aviaire. l'oms rapporte pour la période - , cas dont décès dans pays [ ] . le virus de la grippe aviaire a(h n ) peut se transmettre à l'homme lors de contacts fréquents et intensifs avec des sécrétions respiratoires et des déjections d'animaux infectés. bien que quelques clusters de taille réduite aient été rapportés, incluant des cas chez des soignants, les données épidémiologiques et virologiques suggèrent que le virus n'a pas acquis la compétence d'une transmission inter humaine [ , ] . les manifestations cliniques sont celles d'une infection respiratoire aiguë sévère, d'évolution souvent fatale. les caractéristiques décrites à partir de près de cas figurent au niveau du tableau . est considéré comme cas possible selon l'invs [ ] : le diagnostic biologique est réservé à des laboratoires spécialisés dont les laboratoires de référence pour l'organisation mondiale de la santé (oms) [ ] . un nouveau coronavirus (mers-cov), proche de celui du sras, a été identifié en septembre chez des patients présentant des pneumonies sévères en arabie saoudite et au qatar. depuis cette date, l'oms rapporte cas confirmés, décès, et pays atteints [ ] . la majorité des cas (plus de %) a été rapportée dans la péninsule arabique et la majorité des cas hors péninsule arabique ont été exportés de cette région. l'épidémie survenue récemment en corée est la plus importante hors le moyen-orient. en france, depuis le er janvier , signalements ont été enregistrés dont cas possibles, testés puis exclus [ ] . la principale hypothèse est celle d'une transmission des chauves-souris aux camélidés, lesquels constituent le principal réservoir et la source de la transmission à l'homme. la consommation de produits dérivés des animaux (incluant viande et lait) crus ou insuffisamment cuits est à risque de transmission [ ] . de multiple foyers d'infection à mers-cov ont impliqué des établissements de santé [ ] [ ] [ ] [ ] [ ] , y compris en corée du sud le tableau clinique typique associe fièvre, toux, dyspnée (tableau ). la pneumonie est fréquente mais non constante. des signes digestifs, en particulier diarrhée, ont été rapportés. la mortalité rapportée est de l'ordre %. l'existence de comorbidités, telles que le diabète, l'insuffisance rénale, les maladies pulmonaires chroniques, les immunodépressions sont des facteurs de risque de maladies sévères à mers-cov [ , ] . est considéré comme cas possible toute personne ayant voyagé ou séjourné dans un des pays listés, qui, au cours des jours après son retour, a présenté : (i) des signes cliniques et/ou radiologiques de détresse respiratoire aiguë (sdra) ou d'infection du parenchyme pulmonaire, avec une fièvre ≥ • c et de la toux, sans autre étiologie identifiée pouvant expliquer la pathologie ; (ii) tout contact (ex. : famille, soignants) d'un cas possible ou confirmé, ayant présenté une infection respiratoire le diagnostic repose sur l'imagerie pulmonaire et la mise en évidence du virus par rt-pcr. seuls les prélèvements des voies aériennes basses profonds sont formellement informatifs. certains prélèvements trop précoces (moins de jours après l'apparition des symptômes) peuvent être faussement négatifs. l'évolution clinique rapidement favorable des patients classés cas possibles est un critère de jugement utile pour exclure une infection à mers-cov. d'autres prélèvements sont possibles : selles (ou écouvillonnage rectal) en cas de diarrhée, sanguins, en respectant strictement l'application des mesures d'hygiène de type contact (virémie démontrée chez certains patients) [ ] . les mesures reposent sur la surveillance, la détection, le diagnostic, les mesures d'isolement des cas suspects et possibles jusqu'à exclusion, et a fortiori des cas confirmés, et des mesures locales vis-à-vis de la manipulation et la consommation d'aliments dérivés des camélidés, qui ne doivent pas être exclus car nourrissants, mais bien cuits. de manière importante, le risque de transmission nosocomiale souligne la nécessité d'observer les mesures de prévention et de lutte contre les infections respiratoires pour éviter la propagation du mers-cov dans les établissements de soins [ ] . il n'existe pas de vaccin ou de thérapeutique spécifique. le la transmission vectorielle est réalisée par des moustiques, principalement a. aegypti et a. albopictus (transmission urbaine). le moustique hématophage s'infecte lors d'un repas sanguin et re-transmet le virus lors de repas sanguins ultérieurs [ ] . une transmission autochtone chez cas a été décrite récemment en floride [ ] . un premier cas de transmission sexuelle a été décrite aux États-unis [ ] . depuis d'autres cas ont confirmé cette transmission, et une transmission de la femme à l'homme a été décrite récemment [ ] . en , cas de transmission sexuelle ont été rapportés aux États-unis et dans d'autres pays [ ] . le plus long délai décrit par rapport au début des symptômes atteint - jours [ ] . l'arn du virus zika a été détecté dans le sperme jusqu'à jours après le début des symptômes [ ] et plus récemment dans le fluide vaginal et la glaire cervicale et jours après le début des symptômes respectivement [ , ] . une transmission par transfusion sanguine, et périnatale par le lait maternel n'a jamais été mise en évidence, mais le risque ne peut être écarté [ , ] . les symptômes ( à % asymptomatiques) non spécifiques se caractérisent par une éruption cutanée (exanthème maculo-papuleux), éventuellement accompagnée de démangeaisons, avec ou sans fièvre, et d'autres symptômes tels que conjonctivite, fatigue, douleurs musculaires et articulaires, maux de tête et douleurs rétro-orbitaires. ces symptômes durent quelques jours et disparaissent spontanément. le diagnostic de certitude est difficile, notamment lorsque coexistent dans la zone concernée d'autres arboviroses telles que la dengue ou le chikungunya. deux types principaux de complications sont décrits : des complications neurologiques, principalement des syndromes de guillain-barré et des complications embryofoetales, notamment des microcéphalies (taille anormalement petite du cerveau) et des anomalies du développement cérébral intra-utérin. le lien entre l'infection et ces malformations est avéré [ ] . il repose sur la mise en évidence du génome (arn) du virus dans le sang, les urines et d'autres liquides biologiques (examen direct par rt-pcr), ou la sérologie sur un prélèvement de sang (détection des anticorps spécifiques de la maladie zika, igm et igg anti-zika). la virémie et la virurie sont précoces et transitoire, les anticorps apparaissent secondairement igm puis igm (fig. ) . la stratégie diagnostique impose une recherche simultanée d'infection par les virus de la dengue, du chikungunya et zika [ ] . deux points sont à considérer : la stratégie repose sur : (i) la surveillance des cas importés et les cas groupés autochtone, (ii) le signalement des cas suspects d'infection par le virus zika à la plateforme régionale de veille et d'urgences sanitaires de l'ars, (iii) la prise en charge des patients en transposant les connaissances de la pec du chikungunya y compris les mesures pour la prévention de la dissémination à l'entourage (isolement de tout malade présentant une maladie à virus zika suspectée ou confirmée pendant la période fébrile sous moustiquaire ou dans un local avec fenêtres fermées, afin d'éviter la contamination de nouveaux moustiques vecteurs) [ , ] . le dispositif de surveillance renforcée du chikungunya et de la dengue mis en oeuvre chaque année de mai à novembre dans les départements où a. albopictus est implanté fait partie intégrante du « guide relatif aux modalités de mise en oeuvre du plan anti-dissémination du chikungunya et de la dengue en métropole » piloté par le ministère chargé de la santé. de plus, les recommandations ont été diffusées dans le cadre de la prévention de la transmission sexuelle, de la prévention chez les femmes enceintes, les femmes ayant un projet de grossesse ou les femmes en âge de procréer, et chez les voyageurs se rendant dans une zone de circulation du zika [ ] [ ] [ ] . l'infection se propage d'homme à homme : • par contact direct avec tout fluide corporel de personnes infectées (sang, larmes, salive, lait maternel, sperme, sueur, selles, vomissements) ; • par exposition directe à des objets contaminés par les sécrétions de patients ; • possiblement par voie aéroportée notamment en cas d'atteinte pulmonaire et de manoeuvres de soins générant des aérosols, une transmission par voie aérienne a été documentée sur modèles animaux [ ] . les personnels de santé et de laboratoire sont particulièrement à risque. un nombre infime de particules virales suffit pour infecter un individu. un patient asymptomatique n'est pas contagieux. le début de la contagiosité est lié à la virémie et à l'apparition des premiers symptômes. plus la maladie évolue, plus le patient est contagieux. la disparition des symptômes chez les survivants est corrélée à la disparition du risque de contagion. le cas du sperme est mieux documenté et des mesures de protection lors de rapports sexuels sont préconisées durant quelques mois aux convalescents [ ] . le temps d'incubation est en moyenne de jours ( à jours). les signes sont peu spécifiques avant la phase hémorragique inconstante. il existe des formes frustres et des infections inapparentes. dans la forme habituelle, la maladie débute brutalement par un syndrome pseudo-grippal (fièvre, myalgies, arthralgies, céphalées) et une profonde asthénie psychomotrice. en - jours, apparaissent d'autres signes cliniques cutanéomuqueux (conjonctivite, exanthème maculeux ou maculopapuleux, dysphagie) et digestifs (diarrhée, vomissements). la phase terminale est marquée par des signes neurologiques d'encéphalite (de l'obnubilation au coma, agitation, épilepsie) et des signes hémorragiques (principalement saignements aux points de ponction, gingivorragies, hématémèse, mélaena, selles sanglantes ; plus rarement épistaxis, hémoptysie, hémorragie génitale ou hématome). on peut observer plus inconstamment hoquet, paresthésies, acouphènes, trismus, hépatomégalie, splénomégalie, pancréatite, uvéite, parotidite, orchite, et douleur thoracique. dans les formes hémorragiques, le décès survient dans % des cas en moyenne jours après l'apparition de la fièvre. sinon la guérison est sans séquelle mais la convalescence est longue avec une asthénie prolongée pendant plusieurs semaines et des arthralgies fluctuantes et migratrices. les anomalies biologiques associent lymphopénie initiale ( - premiers jours), suivie d'une hyperleucocytose à polynucléaires neutrophiles, thrombopénie, coagulation intravasculaire disséminée (civd), augmentation des transaminases (parfois considérable, portant plus sur les sgot que les sgpt), de l'amylase, de la bilirubine, et des ldh. le manque de spécificité des signes cliniques, surtout à la phase initiale, et l'accès limité aux examens biologiques simples, nécessitent de considérer de très nombreuses pathologies tropicales endémiques (paludisme notamment) et maladies cosmopolites à évoquer au retour de voyage en pays tropical [ ] . selon l'invs [ ] , un cas suspect est défini par toute personne présentant, dans un délai de jours après son retour de la zone à risque, une fièvre supérieure ou égale à , • c. un cas possible est défini (i) comme toute personne présentant une fièvre supérieure ou égale à , • c et pour laquelle une exposition à risque avérée a pu être établie dans un délai de jours avant le début des symptômes, ou (ii) qui présente une forme clinique grave compatible avec une fièvre hémorragique virale à virus ebola sans évaluation possible des expositions à risque. les expositions à risque sont définies comme suit : • contact avec le sang ou un autre fluide corporel d'un patient infecté, ou suspecté d'être infecté par le virus ebola ; • contact direct avec une personne présentant un syndrome hémorragique ou avec le corps d'un défunt, dans la zone à risque ; • travail dans un laboratoire qui détient des souches de virus ebola ou des échantillons contenant le virus ebola ; • travail dans un laboratoire qui détient des chauves-souris, des rongeurs ou des primates non humains originaires d'une zone d'épidémie d'ebola ; • contact direct avec une chauve-souris, des rongeurs, des primates non humains ou d'autres animaux sauvages dans la zone à risque, ou en provenance de la zone à risque ; • manipulation ou consommation de viande issue de la chasse, crue ou peu cuite, dans la zone à risque ; • rapports sexuels avec un cas d'ebola confirmé, dans les semaines suivant le début des symptômes du cas ; • prise en charge pour une autre pathologie ou visite dans un hôpital ayant reçu des patients infectés par le virus ebola. le diagnostic de certitude repose sur la détection du matériel génétique du virus ebola par pcr : cette technique peut être utilisée dès les premiers stades de l'infection. le risque rare d'une négativité de la rt-pcr au début de la phase symptomatique, notamment chez les patients paucisymptomatiques, on ne peut exclure un cas sur la base d'une rt-pcr négative dans les heures suivant l'apparition des symptômes. le déploiement de kits de diagnostic rapide par rt-pcr permet de réduire le délai du diagnostic dans tous les esrh. une confirmation en urgence par le cnr est impérative en cas de positivité. la détection des anticorps igm ou igg spécifiques par des méthodes sérologiques de type elisa est beaucoup moins performante, et non utilisable dans un contexte d'urgence. [ ] l'incubation est de à jours ( - jours). les signes associent une fièvre élevée d'apparition brutale accompagnée d'arthralgies pouvant être intenses, touchant principalement les petites articulations des extrémités (poignets, chevilles, phalanges), de myalgies, de céphalées, d'une éruption maculopapuleuse. l'évolution est le plus souvent favorable. l'infection peut aussi évoluer vers une phase chronique marquée par des arthralgies persistantes. dans à % des cas l'infection est asymptomatique. il repose sur la détection du virus ou de son génome ou la détection d'anticorps (fig. ) . un diagnostic précoce (dans la semaine qui suit le début des symptômes) peut être obtenu par amplification génique (rt-pcr). les igm peuvent être identifiées à partir du cinquième jour après l'apparition des signes cliniques et persistent en moyenne à mois. les igg apparaissent quelques jours après les igm et persistent toute la vie. ainsi, la démarche diagnostique recommandée est la suivante : • jusqu'à jours après le début des signes (j ) : rt-pcr ; • entre j et j : rt-pcr et sérologie ; • après j : sérologie uniquement (igg et igm) avec un second prélèvement de confirmation au plus tôt jours après le premier prélèvement. la prévention individuelle repose essentiellement sur les moyens de protection contre les piqûres de moustiques (répulsifs en sprays ou crèmes, serpentins, diffuseurs électriques, vêtements longs, moustiquaires). le moustique vecteur pique la journée, essentiellement à l'extérieur des maisons, avec une activité plus importante en début de matinée et en fin de journée [ ] . les mesures de lutte contre les moustiques utilisables pour prévenir la diffusion du chikungunya en france métropolitaine repose sur (i) la lutte antivectorielle à l'échelle de territoires, réalisée par des opérateurs publics de démoustication, (ii) la lutte communautaire, de la responsabilité de tous, réalisée par la destruction des gîtes larvaires potentiels autour des habitations (eau stagnante dans les soucoupes, gouttières, vases, seaux, détritus. . .) pour priver les moustiques des sites où leurs larves peuvent se développer, (iii) la protection individuelle contre les piqûres de moustique [ , , ] . les mie ou ré-émergentes survenues au cours des dernières décades posent des problèmes différents, selon la virulence de l'agent infectieux, la mortalité imputée, les modalités de transmission, l'impact sur la transmission maternofoetale (zika). chaque praticien doit être conscient de possibles émergences, savoir repérer une pathologie inhabituelle, et savoir appliquer les plans et des recommandations établis par les autorités de tutelle, de manière à identifier le plus vite possible tout cas de maladie infectieuse émergente hautement transmissible virulente pour isolement immédiat et prise en charge adéquate. les auteurs déclarent ne pas avoir de liens d'intérêts. les maladies infectieuses émergentes : état de la situation et perspectives. la documentation française koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome syndrome respiratoire aigu sévère. l'épidémie de sras en en france ; rapport sur la gestion épidémiologique du sras par l'invs severe acute respiratory syndrome (sars) -multi-country outbreak-update epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong sars transmission among hospital workers in hong kong sars in three categories of hospital workers virus pneumotropes communautaires hors grippe including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) influenza at the human-animal interface dossier thématique grippe : virus a(h n ) hors france et a(h ) en france probable person-to-person transmission of avian influenza a (h n ) surveillance et investigation des cas de grippe aviaire a(h n ) et a(h n ) hors france et a(h ) en france who. middle east respiratory syndrome coronavirus bilan mensuel au juillet middle east respiratory syndrome coronavirus (mers-cov) fact sheet n o middle east respiratory syndrome middle east respiratory syndrome coronavirus: update for clinicians middle east respiratory syndrome coronavirus: implications for health care facilities hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus in healthcare settings epidemiologic investigation of mers-cov spread in a single hospital in south korea mers-cov outbreak in jeddah -a link to health care facilities avis relatif à la définition et au classement des cas possibles et confirmés d'infection à mers-cov ainsi qu'aux précautions à mettre en oeuvre lors de la prise en charge de ces patients emerging infectious diseases: focus on infection control issues for novel coronaviruses (severe acute respiratory syndrome-cov and middle east respiratory syndrome-cov), hemorrhagic fever viruses (lassa and ebola), and highly pathogenic avian influenza viruses, a(h n ) and a(h n ) avis relatif à la prise en charge des patients suspects d'infections dues au nouveau coronavirus (hcov-emc) surveillance des infections liées au mers-cov. définition des cas et signalement dengue et zika -données de la surveillance renforcée en france métropolitaine en prise en charge médicale des personnes atteintes par le virus zika florida investigation links four recent zika cases to local mosquito-borne virus transmission probable non-vector-borne transmission of zika virus suspected female-to-male sexual transmission of zika virus prevention of sexual transmission of zika virus: interim guidance update late sexual transmission of zika virus related to persistence in the semen zika virus in semen of a patient returning from a non-epidemic area zika virus in the female genital tract cdc update: interim guidance for prevention of sexual transmission of zika virus -united states evidence of perinatal transmission of zika virus point sur les connaissances epidémie de zika : recommandations pour les femmes enceintes avis relatif à la transmission du virus zika par voie sexuelle ebola virus disease avis relatif à la conduite à tenir autour des cas suspects de maladie ebola invs. maladie à virus ebola. définition de cas au avril avis relatif à la stratégie de classement des patients cas suspects de maladie à virus ebola avis relatif à la conduite à tenir face au risque de résurgence virale chez des patients considérés guéris de maladie à virus ebola (mve) et à la prise en charge de leurs contacts dengue et zika -données de la surveillance renforcée en france métropolitaine en état des connaissances dispositif de lutte contre la dissémination du moustique aedes albopictus dans le sud de la france dispositif de lutte contre la dissémination du moustique dgs/ri / / du avril mettant à jour le guide relatif aux modalités de mise en oeuvre du plan anti-dissémination du chikungunya et de la dengue en métropole a major outbreak of severe acute respiratory syndrome in hong kong coronavirus as a possible cause of severe acute respiratory syndrome epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the writing committee of the world health organization (who) consultation on human influenza a/h epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study key: cord- -j i xfs authors: kraemer, m. u. g.; cummings, d. a. t.; funk, s.; reiner, r. c.; faria, n. r.; pybus, o. g.; cauchemez, s. title: reconstruction and prediction of viral disease epidemics date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: j i xfs a growing number of infectious pathogens are spreading among geographic regions. some pathogens that were previously not considered to pose a general threat to human health have emerged at regional and global scales, such as zika and ebola virus disease. other pathogens, such as yellow fever virus, were previously thought to be under control but have recently re-emerged, causing new challenges to public health organisations. a wide array of new modelling techniques, aided by increased computing capabilities, novel diagnostic tools, and the increased speed and availability of genomic sequencing allow researchers to identify new pathogens more rapidly, assess the likelihood of geographic spread, and quantify the speed of human-to-human transmission. despite some initial successes in predicting the spread of acute viral infections, the practicalities and sustainability of such approaches will need to be evaluated in the context of public health responses. infectious disease outbreaks pose a significant threat to human health. the frequency of such outbreaks is thought to have increased over the past decade. for example, quickly after an epidemic of ebola virus affected guinea, sierra leone, and liberia in - [ ] , chikungunya virus (chikv) caused an extensive international epidemic in the americas and beyond, and was quickly followed by zika virus (zikv) emergence. to date, there have been more than confirmed or probable cases of zikv but the true number of cases remains unknown [ ] . yellow fever (yf), a vaccine-preventable disease, recently posed major public health problems. in - , the largest yf outbreak since the s was observed in angola and the democratic republic of the congo, causing confirmed cases and deaths [ ] . yf also poses an ongoing public health risk to large, urban and under-vaccinated populations in the coastal areas of southern brazil, a country that successfully eradicated yf in the s and s [ ] [ ] [ ] . examples of other emerging pathogens that have caused international health security concerns include the severe acute respiratory syndrome (sars) virus and the middle east respiratory syndrome coronavirus (mers-cov) [ ] [ ] [ ] [ ] . this list extends to other pathogens such as influenza, nipah and henipaviral diseases, and lassa fever [ ] . these examples show the continued risks that infectious diseases pose and highlight the challenges of large international outbreaks to epidemic planning and response. during emerging infectious disease outbreaks, empirical information and mathematical modelling techniques are now commonly used to characterise and predict the spatio-temporal dynamics of the spread of pathogens. such analyses may help policymakers to evaluate the threat to public health, determine the resources required to reduce disease burden, and guide disease surveillance efforts and the deployment of interventions. in the last decade, our ability to perform such assessments has been improved by advances in a number of disciplines, including digital disease surveillance [ ] , environmental modelling [ , ] , genomics [ ] and mathematical modelling [ ] . for example, environmental variables such as rainfall and precipitation [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] can be used to better understand the landscape within which the disease may be transmitted, and detailed transmission data from a small sampled population can be extrapolated to larger, un-surveyed areas [ ] . attempts have been made to illustrate the spatial structure of epidemics mainly using human movement data [ ] [ ] [ ] [ ] , to provide mechanistic insights in how the disease may disperse locally [ , , ] or how effective reactive vaccination campaigns may be [ ] [ ] [ ] . there are continued efforts to reconstruct epidemic dynamics using information derived from pathogen genomic data, which contain unique information about the history of transmission [ , [ ] [ ] [ ] . although each of these disciplines has an established relationship to disease prevention and control, the benefits of integrating them into a unified framework have yet to be fully achieved. here we describe the common applications and models used to predict acute viral diseases and discuss the current challenges and limitations. we then outline the advantages of integrating disparate data sources to advance our understanding of epidemic spread. we discuss how such research has been used in recent outbreaks and outline shortcomings that may be addressed in the future. phylogenetic and phylodynamic tools are increasingly being used to infer a range of outbreak properties [ ] . common spatiotemporal analyses of pathogen genomes focus on mapping and predicting virus lineage exchange among locations, with the underlying aim of reconstructing the pathways of disease introduction and spread, albeit at a coarse spatial resolution, and often retrospectively [ , , , , , ] . an additional feature that can be inferred from genomic data is the timing of individual founder introductions [ ] . blue arrows in fig. indicate the time when the first report was published inferring the likely geographic origin of four major international infectious disease outbreaks. phylogenetic tools can help to characterise the number of introductions that lead to disease transmission in a new location [ ] , quantify the risk of cross-species transmission [ ] , and infer ecological drivers of transmission [ , ] . genome-derived estimates have been compared qualitatively to those from epidemiological data, but formal model-based integration of both data sources are rare [ , ] . in principle, pairing genomic information with epidemiological inference should enable us to quantify the number of cases missed in each location and help to estimate parameters such as the basic reproductive number and doubling time of the epidemic, as done for zikv at the tail end of the epidemic (fig. a ) [ ] [ ] [ ] . a common limitation when genetic data are used is the absence of a rigorous and formal sampling scheme. in many instances, genomic sampling is affected by convenience and expedience and may not reflect underlying incidence, although this can be improved post-hoc in large data sets via sub-sampling using, for example continuous phylogenetic inference [ ] [ ] [ ] . strong sampling biases may affect estimates of the arrival time of a pathogen and its pathways of dissemination among locations [ ] . static disease mapping is a powerful tool to visualise and defines the landscape within which transmission occurs, based on ecological drivers of transmission [ , , ] . when combined with global data on human travel and mobility, it can be used to understand the global dynamic risk surface of infectious disease, especially when there are strong ecological determinants of transmission, as there are for the vector-borne diseases zika, dengue, chikungunya and yf [ , ] . publication of reports that estimate geographic spread for the diseases in fig. are indicated by green arrows. the global epidemic history of zika, for example, remains poorly understood. the challenge to accurately reconstruct the epidemic pathway of the virus is further complicated by its relatively unspecific clinical presentation. this may explain why the initial studies that aimed to understand the geographic origin of the zika epidemic in the americas were published relatively late into the epidemic (> year, fig. a ). for the other major outbreaks highlighted in fig. , estimates of the geographic origin were documented between and months after the first reports of human cases ( fig. b-d ; table ). however, given the underlying ecological determinants of transmission that restrict the reproduction of the virus in the mosquito vector species, large areas can be excluded from the risk of local virus transmission. when overlaying information on the reported presence of zika cases vs. the underlying ecological risk map, surveillance gaps may be identified [ , ] . areas where there is a mismatch in the predicted presence and reported presence (i.e. cases detected) should be targeted for active surveillance. the spatial spread process of new pathogens, however, is not only determined by the underlying ecological determinants in each location but also by the dynamic nature of importation, often driven by human movements [ ] . spatial models that take into account the patterns of human spread and mobility may, therefore, improve our ability to characterise and anticipate spatial expansion. different models have been proposed to predict the geographic spread of epidemics but rarely have they been used in real time during the course of an epidemic [ , ] (fig. ) . for example, during the yf outbreak in angola and the democratic republic of the congo, estimates of geographic spread to provinces outside luanda, the capital of angola, were published > months after the last cases were reported (fig. c) . such information could guide public health institutions to decide where and when to implement surveillance and control programs [ ] . more work, however, is needed to dynamically map the spread of infectious diseases and to extract meaningful and interpretable quantities for public health practitioners. in parallel to these efforts to model the spread of pathogens at a meta-population level (e.g. between cities, regions, countries or continents), we also need to better understand transmission dynamics at a much more granular level and assess the characteristics of the inter-human transmission. while historically, the potential for inter-human transmission has often been summarised with a single statistic; the reproduction number r (i.e. the average number of secondary infections generated by a case). however, it has long been recognised that it is also essential to assess heterogeneities in individual r values, since the presence of super-spreading events may have a major impact on the risk of emergence and our ability to control outbreaks [ ] . this was exemplified in a large mers-cov outbreak in south korea in in which only a small number of cases were responsible for the majority of infections [ , ] . other factors that may drive the spatial differences in the reproductive number are ecological (population density, climatic factors, or others) and can now be readily incorporated in transmission models [ ] . ideally, these assessments should be performed on detailed data documenting chains of transmission, as such data can provide precise quantification of the transmission potential and the impact of targeted interventions in different settings and over time, and allow testing specific hypotheses about the transmission process (e.g. what is the contribution of re-introductions to the overall dynamic?) [ ] . however, such data are rarely available as it is difficult to identify the source of infection for most pathogens. as a result, sophisticated statistical techniques are often required to reconstruct chains of transmission and estimate transmission parameters from more limited data that may include: (i) in the context of zoonoses, the size of human clusters [ ] [ ] [ ] or the proportion of surveillance cases that reported a contact with the natural reservoir [ ] , (ii) the growth rate in the case count [ ] [ ] [ ] [ ] , (iii) partial data on chains of transmission [ ] , or (iv) outbreak data where the timing of symptom onsets and location of cases are recorded in small communities such as households [ ] [ ] [ ] [ ] , schools [ ] or villages [ ] . in cases of high-density sampling, genomic data can help to reconstruct transmission chains [ ] . mechanistic models of infectious disease dynamics can be used to make predictions about the future course of an outbreak within a given location [ ] . increasingly, such models are being used in real time, such that predictions are updated every time a new data point becomes available [ , ] . some other applications track pathogen evolution over time as data become available [ ] . however, the perceived ability of such models to successfully or unsuccessfully make 'correct' predictions can generate considerable controversy [ , ] . there are few studies that systematically investigate forecasting accuracy and its relationship to the length of time that is being predicted and to the quantity and quality of data available [ , ] . other examples are forecasting challenges for ongoing epidemics such as chikv in the americas (https://www.darpa.mil/news-events/ - - ), evd in west africa [ ] and seasonal influenza [ , ] , designed and initiated by funding agencies and public health governments. this is an important area for future research. there are clear benefits to combining information from different data sources in order to better predict viral epidemic spread. previous work most commonly presents estimates from different sources side-by-side, for example, estimates of the epidemic reproductive number derived from genomic vs. epidemiological data [ ] . such comparisons are important to assess the consistency of data sources and may help to derive new hypotheses. spurred by technological innovation such as portable sequencing using the minion device (oxford nanopore technologies, oxford, uk) [ ] and by interdisciplinary collaborations during disease outbreaks, researchers have started to work to combine three types of transmission data: spatial, genomic and epidemiological which have now been published for three of the four major outbreaks we considered here (fig. , red arrows) [ , , ] . for example, such interdisciplinary work helped to identify the introduction of zika into the americas [ ] , investigated the main drivers of transmission of zikv through climatic suitability of its mosquito vectors [ ] and tried to extrapolate how many people had been infected with the virus [ , , ] . in the context of phylogenetic analyses, environmental and other spatial data may be helpful in reconstructing the drivers of transmission and spread using, for example, information on the reservoir or host movements [ , ] . in turn, phylogenetic information may complement epidemiological analysis by providing more evidence on the transmission routes that are common in an outbreak [ ] . this may be particularly useful for diseases that have a highly structured transmission dynamic, such as mers or sars, where a small number of people are responsible for the majority of secondary cases [ , ] , transmission from the animal reservoir is frequent, or importation drives locally observed epidemics [ ] . one common assumption in many epidemiological models is that it is equally likely for people to meet and infect others living in the same location and that population immunity is proportional to the demographic structure [ ] . hence, observed cases are often assumed to arise from other cases that are reported locally as long as they are consistent with the generation time of the disease. however, a wellconnected location can, in principle, accrue a large number of incident cases through recurring introductions from elsewhere, rather than via local transmission [ ] . these results can have large implications for surveillance and control, as different competing strategies (e.g. limiting importations or eradicating the disease locally) may be considered. while analytical approaches of various degrees of complexity have been proposed to probabilistically reconstruct transmission trees from incomplete outbreak data [ , , ] , contact tracing, which can be very labour intensive [ ] , remains a gold standard information source. this may allow us to is to determine the true distribution of cluster sizes (i.e. the number of subsequent cases resulting from each introduction) but is often only available for a small number of locations. however, using genomic data can help refine the understanding of heterogeneity in transmission but such framework does not yet allow to exactly quantify the fraction of observed cases that are attributable to local transmission versus introduction from elsewhere, or to determine how many importations are responsible for the local incidence, despite its crucial importance for eradication campaigns [ , , ] . in the context of the zika outbreak in florida, combining genomic data from the outbreak with epidemiological analysis revealed that the outbreak was driven by a large number of introductions rather than by persistent local transmission. in the recent yellow fever outbreak in southern brazil, linking epidemiological, spatial and genomic data and techniques could provide insights into the transmission potential and risk of urban transmission [ ] . one dataset and analysis alone would have not been strong enough to make such conclusions [ ] . inferences about epidemic processes made using mathematical models rely on a number of assumptions. geographic modelling approaches, mostly informed by spatial ecology, attempt to fill gaps where no data has been observed, hence inferences may be uncertain, as the underlying ecological process may be poorly understood and dynamical aspects of the invasion process are ignored. these deficiencies can be ameliorated, in part, by adding virus genome data that contain information about past transmission and invasion patterns [ ] . however, due to incomplete and poor sampling (as discussed above), genomic data alone may provide an incomplete picture of the timing of viral introduction and spread among locations. this, in turn, can be supported by the addition of epidemiological time series of reported cases and serological information about population immunity [ , ] . despite this, building a joint inference framework that combines all available data sources and which characterises observation and sampling processes correctly is a daunting task. however, we are entering a period where the data for this task are becoming available in a timely fashion but need to ensure that results are communicated as soon as they are generated in order to avoid delays. initial successes have already led to important advancements in epidemic control and should progress to a tool-kit for guiding public health, hopefully available in real time for future epidemics. author orcids. m. u. g. kraemer http://orcid.org/ - - - ebola virus disease in west africathe first months of the epidemic and forward projections zika virus in the americas: early epidemiological and genetic findings spread of yellow fever virus outbreak in angola and the democratic republic of the congo - : a modelling study the elimination of urban yellow fever in the americas through the eradication of aedes aegypti the hidden geometry of complex, network-driven contagion phenomena emergence and pandemic potential of 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types: mapping the year history understanding herd immunity use of serological surveys to generate key insights into the changing global landscape of infectious disease key: cord- -y o e k authors: elachola, habida; gozzer, ernesto; zhuo, jiatong; memish, ziad a title: a crucial time for public health preparedness: zika virus and the olympics, umrah, and hajj date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: y o e k nan groups. furthermore, the long-term adherence to a daily injection therapy for patients with non-alcoholic steatohepatitis remains to be determined. most importantly, short-term histological outcomes are used to assess the effi cacy of treatment. unlike in viral hepatitis, histological outcomes are not known to be valid surrogate outcomes in the assessment of non-alcoholic steatohepatitis. after all, the ultimate aim is not to change histology but to prevent the development of cirrhosis and hepatocellular carcinoma. in this respect, results of recent longitudinal studies , showed that fi brosis, but not other histological features of non-alcoholic steatohepatitis, correlated with overall and disease-specifi c mortality. fibrosis progression can also occur in patients with nafld who do not have steatohepatitis, albeit at a slower rate. , these outcomes highlight the urgent need to defi ne reliable surrogate outcomes for the disorder. until the time comes when there are robust surrogate outcomes for the treatment of non-alcoholic steatohepatitis, the follow-up of treated patients for long-term clinical outcomes will be very important. the lean study has introduced liraglutide as a new potential treatment option for patients with non-alcoholic steatohepatitis. the drug should be tested further in large studies with a long duration of follow-up. this study has also raised issues pertinent to drug development in this area. in the meantime, keeping lean remains the most important aspect of management of non-alcoholic steatohepatitis. department of medicine and therapeutics, the chinese university of hong kong, hong kong; and state key laboratory of digestive disease, the chinese university of hong kong, hong kong wongv@cuhk.edu.hk vw-sw has served as an advisory board member for gilead and janssen, as a consultant for abbvie, merck, and novomedica, and has received paid lecture fees from abbvie, echosens, gilead, and roche. gl-hw has served as an advisory board member for gilead and has received paid lecture fees from abbvie, bristol-myers squibb, echosens, gilead, janssen, and roche. the spread of the arbovirus to more than countries, zika virus could be following the geographical spread of dengue and chikungunya, all of which are transmitted by the aedes aegypti mosquito. , the potential role of scheduled international mass gatherings in could exacerbate the spread of zika virus beyond the americas. in brazil, the rio carnival on feb - attracts more than visitors, and on aug - more than million visitors are expected to go to the summer olympics followed by paralympic games on sep - . meanwhile, saudi arabia expects to host more than million pilgrims from over countries for the umrah, between june and september, and the hajj pilgrimage on sept - . , saudi arabia receives about pilgrims from latin america annually. since the rio carnival participants are largely domestic, and the spread of zika virus is already extensive, it will be challenging to assess if there was excess transmission related to the carnival. although winter temperatures mean that mosquito density is expected to be low in brazil at the time of the olympics, given the summer time mosquito density in the northern hemisphere, including in saudi arabia, the introduction of a few infections to the mosquito population might be suffi cient to cause outbreaks of zika virus in other countries. , in brazil, cases of dengue are more frequent from february to may. in the regions of saudi arabia frequented by pilgrims (jeddah, mecca, medina), aedes aegypti larvae are present throughout the year, nearly two thirds in indoor habitats. larvae density is, however, variable and decreases in the months before october. in these regions, where rainfall is rare and unpredictable, reports have suggested all year risk for dengue fever, with dengue seroprevalence ranging from % to % among general patients seeking medical consultations. , although the olympics and the hajj are very diff erent events, each of them might favour transmission of zika virus. the olympics attracts mostly young healthy adults from middle and upper-middle income groups who live in developed countries. such visitors are less likely to have been exposed to arbovirus infections and less familiar with mosquito bite prevention than hajj pilgrims. sexual transmission of zika virus from commercial sex workers with asymptomatic infection might also be a possibility for those who attend the olympics. by contrast, the hajj and umrah participants are more likely to be older adults, many of whom have pre-existing health problems, and about two thirds of them originate from low-income countries and the tropics where personal habits of mosquito bite prevention can be suboptimum. also, umrah and hajj pilgrims' immersion in religious rituals could reduce personal uptake of mosquito avoidance measures. for saudi arabian authorities, it is now a standard procedure to convene international public health consul tations each year before the pilgrimage season to develop disease-specifi c recommendations. brazilian authorities in collaboration with the organizing committee for the olympic and paralympic games have already outlined vector control measures in the olympics vicinity. although both countries may have robust vector control eff orts, no single approach is adequate to prevent mosquito bites and non-vector modes of zika virus transmission; a combination of measures is needed at personal, community, and policy levels. with the emergence of chikungunya and dengue, hajj authorities have been proactive in vector control measures. given that pilgrim fl ow to saudi arabia is continuous, these eff orts will help minimise current transmission of zika virus as well. one important issue is the targeted promotion of options for personal mosquito bite protection-eg, the use of insect repellents, protective clothing, including long-sleeved shirts and trousers, insecticide-treated mosquito nets, and air conditioning in residences. despite the uncertainty about sexual transmission of zika virus, , the promotion of safe sex and provision of condoms is benefi cial from a broader health perspective. health-care providers can be encouraged to use travel health visits as an opportunity to emphasise the need for personal protection against mosquito bites and sexual transmission. health advice for individuals can be provided during predeparture health visits that are usually routine for pilgrims travelling to saudi arabia. additionally, by training athletic coaches on prevention of zika virus transmission, their frequent contacts with athletes can be used to remind athletes about the need for compliance with public health advisories. public health agencies in the home countries of travellers to brazil and saudi arabia can partner with travel agencies and transport services, including airlines, to engage in communication about risks of disease transmission. advice on personal protection can be reinforced at points of departure and arrival in home and host countries. increasing the availability and distribution points of methods to prevent mosquito bites is also crucial. similar approaches were part of prevention eff orts for pandemic infl uenza a h n in and middle east respiratory syndrome (mers) in during the hajj. , , methods for prevention of mosquito bites can be provided to each traveller at the arrival port before immigration control. given that brazil is facing a shortage of supply of insect repellents, global eff orts will be needed to procure and distribute them in adequate quantity. in the absence of commercially available rapid test kits and the asymptomatic nature of most zika virus infections, it is premature to consider mandatory entry or exit screening and restrictions. although there are confl icting reports on the value of exit and entry temperature screening, it can help the detection of a few individuals with symptoms and might persuade some people with febrile illness to avoid travel and can help reinforce health advisories. these mass gatherings provide an additional opportunity to undertake research on the transmission and prevention of zika virus. preparedness has been the key to success of recent hajj mass gatherings held amid known risks, such as pandemic infl uenza a h n , mers, and ebola outbreaks. , lessons from saudi arabia's success with hosting hajj during declared pandemics can be helpful to brazil and the olympics organisers. the next months will be a crucial period for both brazilian and saudi authorities to review emerging research fi ndings on the natural history of zika virus through expert consultations. international stakeholders can facilitate the needed advocacy and support. with proactive planning and preparedness, the eff ect of zika virus infection on mass gatherings participants and their home and host countries can be minimised and the events can be held with a sense of confi dence among organisers, participants, and the global community. by doing so, available global and country resources can be used to address the unanticipated course of the zika virus threat. habida elachola, ernesto gozzer, jiatong zhuo, *ziad a memish incidence of non-alcoholic fatty liver disease in hong kong: a population study with paired proton-magnetic resonance spectroscopy the natural history of nonalcoholic fatty liver disease with advanced fi brosis or cirrhosis: an international collaborative study nonalcoholic fatty liver disease: a feature of the metabolic syndrome liraglutide safety and effi cacy in patients with non-alcoholic steatohepatitis (lean): a multicentre, double-blind, randomised, placebo-controlled phase study liraglutide versus glimepiride monotherapy for type diabetes (lead- mono): a randomised, -week, phase iii, double-blind, parallel-treatment trial liraglutide once a day versus exenatide twice a day for type diabetes: a -week randomised, parallel-group, multinational, open-label trial (lead- ) weight loss through lifestyle modifi cation signifi cantly reduces features of nonalcoholic steatohepatitis orlistat for overweight subjects with nonalcoholic steatohepatitis: a randomized, prospective trial vitamin e, or placebo for nonalcoholic steatohepatitis farnesoid x nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (flint): a multicentre, randomised, placebo-controlled trial surrogate end points and long-term outcome in patients with chronic hepatitis b liver fi brosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease fibrosis stage is the strongest predictor for disease-specifi c mortality in nafld after up to years of follow-up disease progression of non-alcoholic fatty liver disease: a prospective study with paired liver biopsies at years fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies guangxi centers for disease control and prevention eg is director of peru's instituto nacional de salud. jz is deputy director for guangxi centers for disease control and prevention. we declare no competing interests executive board on zika situation interim guidelines for pregnant women during a zika virus outbreak-united states ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations mass gathering medicine: hajj and umra preparation as a leading example kingdom of saudi arabia. hajj -health regulations zika virus transmission from french polynesia to brazil household survey of container-breeding mosquitoes and climatic factors infl uencing the prevalence of aedes aegypti (diptera: culicidae) in makkah city, saudi arabia monitoramento dos casos de dengue, febre de chikungunya e febre pelo vírus zika até a semana epidemiológica the epidemiology of dengue fever in saudi arabia: a systematic review potential sexual transmission of zika virus olympics will inspect for water to help prevent zika pandemic h n and the hajj pandemic h n infl uenza at the hajj: understanding the unexpectedly low h n burden international travels and fever screening during epidemics: a literature review on the eff ectiveness and potential use of non-contact infrared thermometers key: cord- - smnl i authors: chan, jasper f.w.; choi, garnet k.y.; yip, cyril c.y.; cheng, vincent c.c.; yuen, kwok-yung title: zika fever and congenital zika syndrome: an unexpected emerging arboviral disease date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: smnl i unlike its mosquito-borne relatives, such as dengue, west nile, and japanese encephalitis viruses, which can cause severe human diseases, zika virus (zikv) has emerged from obscurity by its association with a suspected “congenital zika syndrome”, while causing asymptomatic or mild exanthematous febrile infections which are dengue- or rubella-like in infected individuals. despite having been discovered in uganda for almost years, < human cases were reported before . the massive epidemics in the pacific islands associated with the zikv asian lineage in and were followed by explosive outbreaks in latin america in . although increased mosquito breeding associated with the el niño effect superimposed on global warming is suspected, genetic changes in its rna virus genome may have led to better adaptation to mosquitoes, other animal reservoirs, and human. we reviewed the epidemiology, clinical manifestation, virology, pathogenesis, laboratory diagnosis, management, and prevention of this emerging infection. laboratory diagnosis can be confounded by cross-reactivity with other circulating flaviviruses. besides mosquito bite and transplacental transmission, the risk of other potential routes of transmission by transfusion, transplantation, sexual activity, breastfeeding, respiratory droplet, and animal bite is discussed. epidemic control requires adequate clearance of mosquito breeding grounds, personal protection against mosquito bite, and hopefully a safe and effective vaccine. globalisation and urbanisation with increasingly frequent and large-scale movements of humans, animals, and commodities by aviation and water transport has led to the spread of previously geographically-restricted microbes and vectors to distant and isolated places. recent examples of emerging viruses that have spilled over to other continents from their original localities via exportation of travelrelated cases include coronaviruses (severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus), influenza viruses, and ebola virus. e moreover, global warming and climate changes have redefined the geographical distributions of important vectors of arthropod-borne viruses (arboviruses), such as the aedes mosquitoes, and facilitated the global spread of these viruses. dengue virus (denv), west nile virus (wnv), and chikungunya virus (chikv), have been introduced (wnv in and chikv in ) and/or spread rapidly in the western hemisphere in the past two decades. zika virus (zikv) is an arbovirus that was little known before it caused a large outbreak on yap island of the federated states of micronesia in . even then, zikv was not considered as an important emerging pathogen because clinical disease was generally mild. the recent report of a possible association between zikv infection and an epidemic of microcephaly among neonates in brazil has attracted global attention. the rapid spread of zikv beyond africa and asia to the americas and europe, and the potentially novel "congenital zika syndrome" outbreak have led the world health organisation (who) to declare the zikv epidemic as a global public health emergency on february . it would therefore be important to review the current knowledge on the epidemiology, virology, clinical manifestations, and laboratory diagnosis of zikv infection, and most importantly, to formulate clinical management options with special reference to perinatal care and control measures based on comparisons made with other mosquito-borne arboviruses. important historical and epidemiological events zikv (strain mr ) was first isolated from the blood of a febrile sentinel rhesus monkey (macaca mulatta), rhesus , during a study on yellow fever virus (yfv) in zika forest of uganda in april (table ) . in , zikv was isolated from aedes africanus mosquitoes caught in zika forest, suggesting that the virus might be mosquito-borne. in , zikv was isolated from the serum of a -year-old nigerian girl who had fever and headache, implying its role as a possible human pathogen. further virological and/or serological evidence of human zikv infection was reported in african (uganda, tanzania, egypt, central african republic, sierra leone, and gabon) and asian (india, malaysia, the philippines, thailand, vietnam, and indonesia) countries. e zikv infection remained relatively restricted geographically with less than sporadic cases reported in these areas in the first years after its discovery. , in , zikv emerged outside africa and asia for the first time and caused a major outbreak on yap island of the federated state of micronesia. over % of the yap residents who were ! years were infected within months. the attack rate of zikv infection in this outbreak was . per residents (range, . e . per residents). subsequently, another major outbreak was reported in french polynesia in october . an estimated , humans (> % of the french polynesian population) were infected by zikv. zikv infection then spread from french polynesia to other pacific islands including new caledonia, cook islands, vanuatu, and solomon islands. e the first cases of human zikv infection in the western hemisphere occurred on easter island, chile, in february , possibly originating from french polynesia during the annual tapati festival. phylogenetic analysis revealed that the ns gene sequence of the chilean strains had ! . % nucleotide and % amino acid identity to the french polynesian strains. the epidemic continued to expand rapidly and autochthonous human cases were reported in many latin american countries in the ensuing years. brazil stands out as the hardest hit latin american country with an estimated , e , , cases of zikv infection since march . based on the close phylogenetic relationship between the south american strains and asian and oceanic strains of zikv, the virus might have been introduced into brazil by asian travellers during the world cup or participants from the oceanic countries of the va'a world sprint championship canoe race in the summer of . , e the climate changes associated with el niño in north and eastern south america in on the background trend of global warming might have facilitated the rapid spread of aedes mosquitoes and zikv. currently, > countries in africa, asia, south america, oceania, and micronesia have reported autochthonous cases of human zikv infection. travel-related cases from endemic and epidemic regions were also reported in europe, north america, australia, and japan. e more worryingly, the brazil health ministry reported the detection of an unusual increase in the number of cases of neonates with microcephaly in northeastern brazil in october , coinciding with the expanding zikv infection epidemic. over suspected cases including some fatal cases were reported during the second half of alone. this represented a > -fold increase in the rate of microcephaly as compared to previous years. on november , the french polynesia health authorities also reported an unusual increase in the number of foetal and neonatal central nervous system malformations in and . like other flaviviruses, zikv is mainly transmitted by mosquitoes. in addition to the sylvatic (enzootic) transmission cycle between the haematophagous mosquito vectors and susceptible primary vertebrate hosts, the recent large-scale epidemics suggest that zikv is also adapting to an urban transmission cycle. , among the various mosquito species, aedes (stegomyia) mosquitoes appear to be the most important vector for zikv transmission, although some anopheles, culex, eretmapodites, and mansonia species have also been proposed as possible vectors (table ) . , , e the animal reservoirs of zikv are unclear. non-human primates including m. mulatta, cercopithecus aethiops, c. ascanius schmidti, c. mona denti, c. albigena johnstoni, chlorocebus sabaeus, colobus abyssinicus, erythrocebus patas, and pongo pygmaeus, and other mammals including zebras, elephants, and rodents, have been suggested as possible vertebrate hosts of zikv in africa and asia, based on virological and/or serological evidence of infection. , , , , e ae. africanus is the first mosquito species from which zikv was isolated, and is likely an important vector in the sylvatic transmission cycle of zikv. , inoculation of unfiltered supernatant of zikv-infected ae. africanus into mice and rhesus macaques led to clinical disease and/or neutralising antibody response. , ae. hensilli is the most commonly found mosquito species on yap island, but no virus isolate was made from field-collected mosquitoes to ascertain its role as a vector for zikv transmission during the outbreak. ae. aegypti and ae. albopictus, which have much wider geographical distributions than other aedes mosquitoes, are considered to be more important vectors in the urban transmission cycle of zikv. these aedes mosquitoes are highly susceptible to zikv infection in vitro with potential for further transmission after an extrinsic incubation period of e days. , they bite both indoors and outdoors, and mostly during daytime. non-vector-borne transmission routes of zikv have been proposed (table ) . like other arboviruses, blood transfusion-related transmission of zikv is possible, especially in endemic regions or where blood products obtained from infected travellers immediately returning from endemic regions are used. zikv rna was detected in the blood of . % of the donors in french polynesia during the epidemic. sexual transmission of zikv appears highly probable, especially in patients presenting with haematospermia with infectious viral particles and rna in semen. , notably, no other arboviruses have been associated with haematospermia or isolated from human semen. this might further complicate the control of the zikv epidemic, since most infected patients are asymptomatic. inadvertent sexual transmission of zikv to the female partner may then lead to virus transmission to the foetus, which may be potentially associated with severe congenital anomalies. besides transplacental transmission, perinatal transmission of zikv may also occur during delivery, via breastfeeding, and/or close contact after birth via exchange of saliva and other bodily fluids. zikv rna could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated , perinatal transmission of other arboviruses, including denv, chikv, wnv, and yfv, has also been reported. e other suspected routes of transmission of zivk infection are those reported for other flaviviruses. these include mucocutaneous exposure to the virus in infected blood or via monkey bite, haemodialysis, or organ transplantation. e particularly, as zikv may be shed in the urine of infected patients for more than days, the risk to recipients of donated kidneys from donors at or returning from endemic areas has to be considered. , , , , it is unknown whether zikv could be transmitted via respiratory droplets as viral rna could occasionally be detected in nasopharyngeal swab and saliva samples. , , , virology and pathogenesis zikv is an enveloped, positive-sense, single-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. it is closely related to spondweni virus and the viruses represent the only members of their clade within the mosquito-borne cluster of flaviviruses ( fig. ) . , phylogenetic analysis suggests that zikv has likely emerged between and in uganda. the two major lineages of zikv are the african (subdivided into west and east african) and asian lineages, which are responsible for causing the majority of infections in africa and asia (as well as the pacific and americas), respectively. , , the single-stranded rna genome of zikv has a size of , nucleotides encoding amino acids, with flanking untranslated regions ( and utrs) and a single long open reading frame encoding a polyprotein, which is cleaved into capsid (c), precursor of membrane (prm), envelope (e), and non-structural (ns) proteins , reverse transcription-polymerase chain reaction (rt-pcr) using primers targeting the e or ns gene is a key laboratory diagnostic tool for zikv infection in the recent outbreaks. , , the e protein is a major virion surface protein that is involved in receptor binding and membrane fusion. the domain iii of e protein contains a panel of antigenic epitopes that are important targets of serological assays, neutralising antibodies, and vaccines. , loss of the n glycosylation site in the e protein may be associated with adaptation to mosquito vectors and thus facilitate transmission. a single amino acid mutation in the e protein (e -a v) of chikv has been reported to be associated with increased fitness of the virus in ae. albopictus and allows chikv to disseminate in regions lacking the typical ae. aegypti vector. the recent spread of the asian lineage of zikv to oceania and the americas may be associated with significant ns codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titres. mutations in the e and ns genes should be detected in zikv strains causing the current epidemic. when an infected aedes mosquito bites an infected patient, it ingests a blood meal containing zikv. as in other flaviviruses, zikv likely replicates in the midgut epithelial cells and subsequently the salivary gland cells. after an extrinsic incubation period of e days, zikv can be found in the mosquito's saliva which can then infect human. , moreover, the virus can likely be vertically transmitted transovarially as other flaviviruses. when the mosquito's saliva containing zikv is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the subcutaneous layer, and the langerhans cells. the keratinocytes and fibroblasts contain axl, tyro , and tim- , which can serve as attachment factors or receptors for zikv. the langerhans cells contain dc-sign, which can also serve as a receptor for virus entry. zikv infection of primary skin fibroblasts is associated with the upregulation with tlr mrna expression, and enhanced transcription of rig-i and mda , which are known innate immune responses to rna virus infection. this is followed by enhanced expression of interferon-alpha and -beta, and their downstream pathways of immune activation. both types i and ii interferons can suppress the viral load of infected cells. moreover, zikv is capable of increasing its replication by the induction of autophagy in host cells. thus, autophagy inhibitors can decrease the viral load of infected cells. infected cells of human skin explant exhibits cytoplasmic vacuolation, pyknotic nuclei, and oedema in the stratum granulosum. after replication in endemic/epidemic areas: + universal nucleic acid testing of blood donors. + temporary discontinuation of blood donation (importation of blood products from blood blank centres in non-endemic regions). non-endemic/epidemic areas: + pre-donation questionnaire to identify donors with recent travel history to endemic/epidemic areas. + deferral of blood donors who have travelled to endemic areas within the preceding ! days. + self-reporting of symptoms after blood donation ( these local tissue cells and the regional lymph nodes, zikv may then disseminate from the lymphatics and bloodstream to reach other organs/tissues, including the central nervous system, the skeletal muscles, myocardium, and perhaps transplacentally to the foetus. zikv was highly neurotropic in infected suckling mice. the brains of infected suckling mice show neuronal degeneration, cellular infiltration, and softening in the brain with virus replication in astroglial cells and neurons on histopathological examination. , , moreover, evidence of inflammation in skeletal muscles and myocardium has also been demonstrated in infected suckling mice. axl and tyro are members of the tam family of receptor tyrosine kinases (rtks). they are also present in neurons and under the influence of gonadotropin releasing hormone (grh), which in turn may affect neuronal survival and migration. furthermore, flaviviruses such as yfv may persist for up to days after intracerebral inoculation in rhesus macaques. the neurotropism and persistence of zikv may therefore partially explain microcephaly and predominantly neurological complications and foetal anomalies in this suspected entity of congenital zikv infection. most patients with zikv infection are asymptomatic. in the outbreak of zikv infection on yap island, only % of cases were estimated to be symptomatic. the incubation period of zikv infection is unclear, but is estimated to be similar to other mosquito-borne flaviviruses ( e days). , the clinical syndromes of symptomatic zikv infection can be broadly divided into zika fever and congenital infection ("congenital zika syndrome") ( table ). zika fever is an acute "dengue fever-like" illness characterized by low-grade fever ( . e . c), rash, retroorbital headache, bilateral non-purulent conjunctivitis, myalgia, and arthritis/arthralgia with periarticular oedema of the small joints of hands and feet. the rash in zika fever is typically described as a generalized, erythematous, maculopapular rash that spreads downward from the face to the limbs. less commonly, some patients may have more prominent systemic symptoms including high-grade fever, chills, rigours, sore throat, hypotension, and cervical, submandibular, axillary, and/or inguinal lymphadenopathies. digestive tract symptoms including nausea, vomiting, diarrhoea, constipation, abdominal pain, and aphthous ulcers may also be present. , , patients with genitourinary symptoms including haematuria, dysuria, perineal pain, and haematospermia often have detectable viral rna or infectious virus particles in urine and/or semen. , haematological and biochemical laboratory parameters are usually normal. however, some patients may have transient and mild leucopenia, neutropenia, lymphopenia or activated lymphocytes, monocytosis, thrombocytopaenia, and elevated serum levels of lactate dehydrogenase, aspartate aminotransferase, g-glutamyl transferase, fibrinogen, ferritin, c-reactive protein, and erythrocyte sedimentation rate during the viraemic phase. associated with restoration of normal number of peripheral immune cells and normal function of antigen-presenting cells. notably, the clinical manifestations of zika fever are non-specific and may mimic those seen in infectious diseases caused by other arthropod-borne pathogens, especially denv and chikv. some suggest that zika fever may be distinguished from dengue fever and chikungunya fever by more prominent oedema of the extremities, less severe headache and malaise, and milder degree of thrombocytopaenia seen in the former. , moreover, haemorrhagic complications seen in dengue fever have not been reported in zika fever, and arthralgia in zika fever is less severe than that in chikungunya fever. however, none of these features are pathognomonic and laboratory confirmation is required to exclude co-infections with these arboviruses and other causes of acute febrile illness in returned travellers from endemic regions, such as malaria. zika fever is usually self-limiting with most clinical manifestations resolving completely within e days. , , no death, hospitalisation, or haemorrhagic complication was reported during the outbreak on yap island. however, some patients may experience more protracted symptoms and other non-haemorrhagic complications. zika fever-related rash usually resolve within the first week, but may last for up to days and may be pruritic. other exanthematous diseases, such as denv, chikv, rubella virus, measles virus, parvovirus b , adenovirus, enterovirus, and rickettsial infection, should be excluded. the median duration of arthralgia is . days, but some patients may develop persistent or recurrent arthralgia for more than a month after symptom onset, mimicking the post-infectious chronic arthritis seen in chikungunya fever and lyme disease. , lymphadenopathies may be present for weeks after symptom onset, and alternative diagnoses such as infectious mononucleosis-like syndrome, streptococcus pyogenes infection, and toxoplasmosis should be considered in refractory cases. a post-infection asthenia appears to be frequent and further investigations may be necessary to determine possible association between zikv infection and chronic fatigue syndrome. , , immune-thrombocytopenic purpura and cardiac complication have also been reported in a few cases. jaundice was observed in patients with virological and/or serological evidence of zikv infection in eastern nigeria in the s who had co-infections (malaria and microfilaraemia) and a patient with sickle cell anaemia. , a possible association between zikv infection and severe neurological complications has been proposed during the recent epidemics in oceania and south america, during which the incidence of guillainebarré syndrome has increased by e times in french polynesia. , / ( . %) patients with suspected zikv infection in the french polynesia outbreak developed neurological syndromes after presenting with a zika fever-like illness. forty-two of these ( . %) patients were diagnosed with guillainebarré syndrome. , , similarly, guil-lainebarré syndrome has been reported among patients with zika fever-like illness in south america. , other neurological complications potentially linked to zikv infection include encephalitis, meningoencephalitis, myelitis, paraesthesia, vertigo, facial paralysis, and , , , , e suspected fatalities due to zikv-related guillainebarré syndrome have been reported. while the neurotropism of zikv may partially explain these neurological manifestations, more details and serial studies on their cerebrospinal fluid and magnetic resonance images by case-control studies are required to ascertain their association. zika fever-related death appears to be extremely rare but a number of probable cases have been reported, especially among immunocompromised patients and neonates with suspected congenital zikv infection. , , a small number of patients with coinfection with denv or hiv did not appear to have more severe disease. , further studies should be conducted to identify patients who are at risk of severe disease or death. microcephaly (head circumference ! standard deviations below the mean for sex and gestational age at birth) is the most prominent and commonly reported clinical feature of suspected congenital zika syndrome. , besides microcephaly, neonates and foetuses with suspected congenital zikv infection also had other malformations (table ). general features included low birth-weight, redundant scalp skin, anasarca, polyhydramnios, and arthrogryposis. neurological abnormalities included cerebral lesions, polymalformative syndromes, brainstem dysfunction, and absence of swallowing. ophthalmological defects included cataract, asymmetrical eye sizes, intraocular calcifications, macular atrophy (well-defined macular neuroretinal atrophy and/or macular pigment mottling and foveal reflex loss), optic nerve hypoplasia, iris coloboma, and lens subluxation. , , notably, other features characteristic of intrauterine infections, such as hepatosplenomegaly, rash, and chorioretinitis have not been reported. ultrasonographic examination revealed cerebral atrophy, intracranial calcifications especially over the white matter of frontal lobes, caudate, lentostriatal vessels, cerebellum, or around the lateral and fourth ventricles, dysgenesis of corpus callosum, vermia, and thalami, enlarged cisterna magna, asymmetrical cerebral hemispheres, severe unilateral ventriculomegaly, displacement of the midline, and thinning of the parenchyma on the dilated side, pons and brainstem. , , , zikv particles and rna may be detected by electron microscopy and rt-pcr, respectively, in autopsied samples. two important questions concerning congenital zikv infection remain unanswered. the first question is whether zikv is indeed the cause of microcephaly and other congenital anomalies in these patients. severe consequences have been reported for materno-foetal transmission of other arboviruses, such as dengue virus (preterm delivery, foetal death, low birth-weight, prematurity, acute foetal distress during labour), wnv (chorioretinitis and focal cerebral destruction), and chikv (encephalopathy and haemorrhagic fever). , , preliminary analysis in the current epidemic of microcephaly has not yet completely excluded other infectious or environmental aetiologies. moreover, there is some virological evidence to support the association between congenital zikv infection and these anomalies. zikv rna has been detected by rt-pcr in the amniotic fluid of pregnant women whose foetuses had ultrasonographic evidence of microcephaly, in the blood and foetal tissues of a neonate with microcephaly and other congenital anomalies who died within the first min of birth, and in the neonatal brain tissues of a few cases of full-term miscarriages and neonates with microcephaly. , , , however, there is still no large-scale prospective cohort or caseecontrol study to demonstrate a causal link between the presence of zikv in the foetus and the congenital anomalies after exclusion of other infectious and toxic causes. some have suggested that the apparent microcephaly surge might be attributable to the intense search for cases due to the heightened awareness of a possible association with the zikv outbreak or the use of larvicide. furthermore, detailed investigations for exclusion of other pathogens associated with congenital malformations have only been reported in a small number of cases. , microcephaly is well reported in congenital cytomegalovirus, rubella virus, and varicella zoster virus infection. chorioretinitis and intracranial calcifications are common in congenital cytomegalovirus infection and toxoplasmosis, but the latter is more commonly associated with hydrocephalus. cataract and cardiac anomalies are characteristic of congenital rubella syndrome, although cataract can also be found in congenital herpes simplex virus infection. thus, the diagnosis of congenital zika syndrome would depend on the exclusion of these "torch" infections in future studies using clinical criteria, histopathological findings, and serological, molecular and conventional cell culture techniques. if zikv is eventually confirmed to be the cause of these congenital anomalies, the second key question would be whether congenital zika syndrome actually comprises a wider spectrum of varying clinical severities than that seen in the reported cases. as with other congenital infections, it is possible that the reported cases of microcephaly represent only the tip the iceberg, focussing on the more severely affected patients, and that the timing of infection is likely to be important in determining the severity and outcome of the affected foetus. early infection during the first or even second trimester may be associated with congenital anomalies or even intrauterine death. , , indeed, preliminary data suggested that the greatest risk of microcephaly or congenital anomalies in the affected neonates appears to be associated with zikv infection in the first trimester of pregnancy. of mothers with infants born with microcephaly, % and % had a rash during the first and second trimester of pregnancy, respectively. besides neurological defects, cardiac and muscular abnormalities should also be excluded, as suckling mice infected with zikv developed evidence of central nervous system infection, myositis and myocarditis. some suspected cases of congenital zika syndrome developed severe arthrogryposis. , , , it is possible that intrauterine zikv infections that occur at a later stage of the pregnancy may present differently, either with less severe manifestations, such as mental retardation, sensorineural deafness, and/or ophthalmological lesions, or as full-term miscarriages. neonates with probable perinatal transmission of zikv infection appear to have mild disease and favourable outcome. further investigations should be conducted to better define the spectrum of manifestations in different gestational stages of congenital zikv infection. definitive diagnosis of zikv infection requires laboratory confirmation as there are no pathognomonic clinical, biochemical, or radiological features that reliably distinguish zika fever from other arboviruses, and congenital zikv infection from other infective, toxic, or genetic causes of congenital anomalies. successful isolation of zikv in viral culture, the gold-standard of laboratory diagnosis of viral infections, mainly depends on the timing of specimen collection and viral loads in the specimens. zikv has been isolated in vero and vero e cells inoculated with infected patients' serum, urine, and/or semen samples (table ) . , , however, infectious virus particles were not recovered by culture in most specimens with low viral loads. a positive serum immunoglobulin (ig) m or -fold rise in the titre of neutralising antibodies in paired serum samples collected approximately weeks apart also establishes the diagnosis of zikv infection. igm may be detected by enzyme-linked immunoassay on as early as day of symptom onset and may last for over months. , igm antibodies to denv and wnv usually persist for months and months, respectively. e the major limitation of these serological tests is possible cross-reactivity with other flaviviruses. neutralising antibodies detected by plaque-reduction neutralisation test may be more specific than igm detection by elisa for primary zikv infection, but may also have indeterminate results for secondary infection, including patients with previous vaccination against or exposed to other flaviviruses. , , , this is especially problematic in areas where there is cocirculation of multiple flaviviruses with the same aedes mosquito vectors. , , , patients with primary zikv infection and past denv infection are more likely to have higher titre (usually ! -fold) of igm and/or neutralising antibodies against zikv than against denv or other flaviviruses. , a positive serum denv ns antigen test without serial increase in igm or the combination of a positive igm response to denv and lack of an igg seroconversion in the convalescent-phase serum sample should prompt the clinician to investigate for another flavivirus such as zikv. moreover, co-infections with other mosquito-borne arboviruses, such as denv, chikv, wnv, and japanese encephalitis virus, are always possible and should be excluded by more extensive laboratory testing if clinically indicated. rapid and accurate diagnosis of zikv infection during the recent epidemics has mainly been achieved by the application of rt-pcr using primers that target the e or ns gene of zikv. , , , , alternatively, rt-pcr sequencing using universal primers that target the conserved regions in the genomes, such as the ns gene, of multiple flaviviruses, may allow simultaneous detection of > different flaviviruses. serum samples should be collected in the early phase of the disease, because viraemia is usually shortlived (usually days, rarely up to days) and may be low-level ( copies/ml). , alternatively, urine and semen samples may have higher viral rna loads table advantages, limitations, and uses of different diagnostic tests and types of specimens for laboratory diagnosis of zikv infection. , , , , , e , , , , , , , e , may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. other tissues brain, liver, spleen, and pooled visceral (kidney, lung, and heart) tissues were positive in a fatal case (an adult male with co-morbidities and immunosuppressive treatment). may be useful to exclude concomitant infections in patients with unusually severe or fatal infection. abbreviations: rt-pcr, reverse transcription-polymerase chain reaction; zikv, zika virus. (> copies/ml) than serum samples, and may be persistently positive for > days and ! days after symptom onset, respectively. , in a few cases, zikv rna has also been detected in saliva and nasopharyngeal swab samples of patients whose serum samples tested negative for zikv. these samples should therefore also be collected in suspected cases of zikv infection. , , , collection of amniotic fluid should be considered in pregnant women with positive zikv test result or if the foetuses show ultrasonographic evidence suggestive of congenital zikv infection. , , cerebrospinal fluid, placental, and/or umbilical cord tissues from neonates with suspected congenital zikv infection should be sent for virological and/or histopathological examinations to establish the diagnosis. , , zikv rna may also be detected in organ tissues in the rare cases of suspected zikv-related deaths. future studies should aim to better stratify the clinical use of these tests and to develop point-of-care tests (eg: antigen tests) that can be widely used in less developed regions without the facilities and expertise for molecular or serological tests. treatment is usually not required for patients with asymptomatic or uncomplicated zika fever. the mainstay of treatment is supportive as there are no specific anti-zikv antiviral agents. acetaminophen may be used to relieve fever and arthralgia. anti-histamines may help to control pruritus. adequate rehydration for fluid loss through sweating, vomiting, and insensible losses should be encouraged. aspirin should be avoided due to the risks of bleeding in those with thrombocytopaenia and developing reye's syndrome in children less than years of age. nonsteroidal antiinflammatory drugs are also contraindicated in cases where denv and chikv infections cannot be confidently excluded in order to avoid haemorrhagic complications. potential neurological complications, especially guillainebarré syndrome, should be diagnosed promptly to allow early use of intravenous immunoglobulins and/or plasmapheresis. the risk of immune enhancement should also be considered if convalescent-phase plasma therapy with neutralising antibodies against zikv is used for treatment of severe cases. virological testing and foetal ultrasound to exclude zikv infection and foetal microcephaly or intracranial calcifications should be offered to pregnant women who develop zika fever-like symptoms during or within weeks of travel to areas with zikv transmission. besides collecting the appropriate specimens for virological tests, serial foetal ultrasound examinations should be performed every e weeks to monitor foetal anatomy and growth in suspected cases of congenital zikv infection. foetal ultrasound and/ or aminocentesis should also be offered to asymptomatic and seropositive pregnant women with history of travel to affected areas. after delivery, serum should be collected either from the umbilical cord or directly from the neonate within days of birth for rt-pcr, igm and/ or neutralising antibodies against zikv. comprehensive physical examination including measurement of the occipitofrontal circumference, length, and weight, evaluation for neurological abnormalities, dysmorphic features, hepatosplenomegaly, rash, ophthalmological lesions, and auditory defects, and laboratory testing for torch screening should be performed. the affected child and the family should be managed and counselled by a multidisciplinary team consisting of paediatric neurologist, clinical geneticist or dysmorphologist, infectious disease specialist, medical social worker, and other relevant specialists. long-term follow-up to monitor physical, intellectual, and functional progress of the child should be offered. both vector control and personal preventive measures are important for interrupting the transmission of zikv. systematic mosquito surveillance and control programs should be established and coordinated by health authorities. mass sanitation campaigns to eliminate mosquito breeding sites in household and high-risk areas such as garbage collection points, construction sites, illegal dumping grounds, and invalid car fields should be organised. mosquitoes should be removed with a radius of at least m around areas with high population densities, such as schools, transport terminals, churches, and healthcare facilities. in areas where autochthonous or imported cases of zikv are detected, the use of adulticide through spraying to remove infected adult mosquitoes should be considered. residents in or travellers to affected areas should stay indoor with air conditioning, window and door screens if possible, wear long sleeves and pants, use permethrintreated clothing and gear, and use insect repellents when outdoor. most environmental protection agency (epa)registered insect repellents, including n,n-diethyl-mtoluamide (deet), should be safe for pregnant and lactating women ( % deet), and children ( % deet) aged > months. individuals returning from affected areas to non-affected regions should continue to use insect repellents for at least an additional days to prevent local non-infected mosquitoes from the acquisition of virus from the asymptomatically infected returned travellers. this will serve to interrupt the mosquito-human-mosquito transmission chain. hospitalised laboratory-confirmed cases should be managed in designated wards to avoid mosquito bites. the effects of other novel mosquito-control measures, such as the wolbachia biological control approach, should be evaluated. other animals such as rodents should also be investigated as potential animal reservoirs and controlled as findings indicate. non-vector-borne transmission of zikv may be prevented by specific measures (table ) . concerning blood transfusion, universal nucleic acid testing of blood donors is recommended. the use of universal primers that can simultaneously detect multiple arboviruses such as denv and zikv should be considered. temporary discontinuation of blood donation should be considered during an outbreak situation. in non-endemic areas, pre-donation questionnaire to identify donors with recent travel history to regions with reported cases of zikv infection and deferral of blood donation from these donors until at least days after returning from affected regions should be implemented. most transfusion-related transmissions of arboviruses are associated with asymptomatic infections, and symptomatic donors who were rt-pcr-positive for zikv usually developed symptoms between and days after blood donation. newer pathogen reduction technologies for blood products should be considered. similarly, donated organs, especially kidneys, from individuals with travel history to affected areas should be tested for zikv as the virus may persist in the genitourinary tract for an undetermined period. , , , , barrier methods should be used to prevent sexual transmission through infected semen. male returned travellers should continue the use of condom with pregnant sex partner throughout the whole duration of pregnancy. future studies should evaluate the duration of virus shedding in semen and the infectiousness of rnapositive semen samples, in order to determine how long barrier methods should be used by men returning to nonendemic regions. some regional authorities have advised women to avoid pregnancy until the epidemic is over. pregnant women or those planning for pregnancy should defer travelling to regions with reported cases of zikv infection. if such travel was unavoidable, they should strictly comply with personal protective measures to avoid mosquito bites. further studies are needed to determine the risk of zikv transmission by breast milk and saliva. other less common transmission routes, including mucocutaneous exposure to infected bodily fluid during laboratory and patient-care procedures, and bites by infected primates should be avoided with strict compliance to infection control measures. 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approaches a review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available arthropod-borne viral infections of man in nigeria dengue and other arboviral diseases in south-east asia zika virus, french polynesia, south pacific rapid spread of emerging zika virus in the pacific area first report of autochthonous transmission of zika virus in brazil zika virus spreads across americas as concerns mount over birth defects zika virus-related hypertensive iridocyclitis laboratory infection with zika virus after vaccination against yellow fever the study was partly supported by the consultancy service for enhancing laboratory surveillance of emerging infectious disease of the department of health, the food and health bureau, hong kong special administrative region, china; and by the donations of hui hoy and chow sin lan charity fund limited and mr. larry chi-kin yung. the authors declare no conflict of interest. key: cord- -e ic pnc authors: yang, jiancheng; carey, patrick; ren, fan; lobo, brian c.; gebhard, michael; leon, marino e.; lin, jenshan; pearton, s.j. title: nanosensor networks for health-care applications date: - - journal: nanosensors for smart cities doi: . /b - - - - . - sha: doc_id: cord_uid: e ic pnc functionalized transistors provide effective sensors for a variety of viruses (zika, severe acute respiratory syndrome), toxins (botulinum), cancers (breast and prostate), and disease or injury biomarkers (troponin, cerebrospinal fluid). a hallmark of this approach is high specificity, rapid response (< minutes), and ability to be integrated with wireless data transmission capabilities. the ultimate goal is hand-held point-of-care detection that can streamline patient diagnosis. there is a need for continued development of selective, cost-effective hand-held biosensors with rapid response and detection sensitivities compared to existing lab assay methods. these could play a significant role in speeding patient diagnosis and, in some cases, reducing emergency room overcrowding. for biological and medical sensing applications, disease diagnosis by detecting specific biomarkers (functional or structural abnormal enzymes, low molecular weight proteins, or antigen) in blood, urine, saliva, or tissue samples has been established using a number of approaches, such as enzyme-linked immunosorbent assay (elisa), particle-based flow cytometric assays, electrochemical techniques based on impedance and capacitance, electrical measurement of micro-cantilever resonant frequency change, and conductance measurement of semiconductor nanostructures [ À ] . for some of these techniques, here are some drawbacks related to assay time and throughput. elisa allows only one analyte measurement at a time. particle-based assays allow for multiple detections by using multiple beads, but the whole detection process is generally longer than hours, which is not practical for in-office or bedside detection. electrochemical devices are low cost, but improvements in sensitivities are still needed for clinical samples. microcantilevers are capable of detecting concentrations as low as pg/ml but suffer from an undesirable resonant frequency change due to the viscosity of the medium and cantilever damping in the solution environment. nanomaterial devices have provided an excellent option toward fast, label-free, sensitive, selective, and multiple detections for both preclinical and clinical applications. in this chapter, we describe the use of semiconductor transistor-based systems in which specific functional layers are placed directly on the gate region of the transistor or connected to it from disposable glass or plastic slides to provide a sensor capable of fast response and excellent detection sensitivity. examples are given of detection of various viruses, cancers, or disease biomarkers using this approach, as well as the integration with wireless data transmission capability. networked systems like this are attractive in health-care applications. severe acute respiratory syndrome (sars) outbreaks are capable of causing a large number of fatalities and severe economic disruption [ ] . the sars coronavirus is the cause of the condition. the virus replication occurs through a coronavirus protein, nucleocapsid protein (n protein), and encapsulating the coronavirus genomic rna. sars-n protein is a nucleic acid-binding agent capable of interacting with rna and dna [ À ]. functionalized algan/gan high electron mobility transistors (hemts) have been utilized as an assay for sars and to examine the binding between double-stranded dna and the sars coronavirus nucleocapsid protein [ , ] . these hemts have been widely used as biosensors, as summarized in table . . by functionalizing the gate region with appropriate layers, highly specific sensing of antigens or other biomarkers can be achieved. in the case of sars, the binding was detected through a change in current in the hemt and allowed extraction of the dissociation constants of the nucleotideÀprotein interaction. this is a good example of the use of hemts not only as sensors but also for determining the binding affinity of ligandÀreceptor complexes [ ] . investigating the nucleotideÀsars-n protein interaction can assist in constructing a genome packaging model. in the past, electrophoretic mobility shift assay and filter-binding assays have been used to study proteinÀnucleic acid interactions [ À ]. these methods require labeling of fluorescent probes or isotope elements on nucleic acids to provide detection. the assay cost is high and the labeling may alter the binding affinity of molecules. the number of binding sites and the dissociation constants of a receptor are related to the ratio of the number of ligand-bound receptors to the total number of receptors on the sensor and can be obtained in either one-binding or multiple-binding site models [ , ] . the use of hemts allows rapid determination of these parameters. similar studies have been performed for the binding affinity of nonnucleoside reverse transcriptase inhibitors (nnrtis) to the reverse transcriptase (rt) of hiv- for determining the efficiency of the drug performance. the hiv- rt immobilized hemts were used to find the dissociation constant of nnrtis [ ] . the zika virus (zikv) is a flavivirus, primarily transmitted via the aedes mosquito [ À ] and leads to abnormal brain development in fetuses [ À ]. zikv can be detected using rna in human urine, serum, and saliva using the reverse transcription polymerase chain reaction [ À ] . however, the degradation of rna in saliva may occur during the saliva collection, storage, and processing [ ] . reverse transcription loop-mediated isothermal amplification was also used to detect zikv rna in unprocessed biological samples, such as urine, plasma, and zika-infected mosquito carcasses, with a detection limit of . pfu [ ] . these methods are time consuming and require a well-trained technician to perform the tests. we have shown that functionalized hemts may be used for zikv detection. fig. . shows the schematic of the sensor, consisting of an antibody-functionalized cover glass and an algan/ gan hemt. pulsed biasing of the electrode fabricated on the cover glass and functionalized with zika antibody was used for detection [ ] . the target zika antigen (recombinant zikv ns ) solutions were diluted with bovine serum albumin (bsa) in pf . pbs solution with . , , , or ng/ml concentration. the reversible antigen and antibody binding through active sites on these two protein molecules and the hemt drain current changes could fit with the langmuir extension model [ ] . in addition, the hookean spring model was employed to simulate the relaxation portion of the time-dependent drain current. since the drain current is proportional to the gate voltage of the hemt or the stretched distance of the antibody and antigen molecules, the solution for the stretched distance is directly proportional to the drain current (i d ) as where c* is the ratio of antibody bound with antigen to the total available antibody on the functionalized contact window, τ and τ are the relaxation time constants of antibody and antigen molecules, respectively, a, b, and e are constants. fig. . shows the modeled drain current for pbs solution without antigen and pbs solutions with different antigen concentrations. the ratio of antibody bound with antigen to the total available antibody on the functionalized contact window increased from . at . ng/ml to . at ng/ml, with the ratio scaling faster than concentration due to increased interaction probability. a wide range of zika antigen . À ng/ml was detected. clostridium botulinum neurotoxins are deadly toxins, with a lethal dose in unvaccinated humans of only ng/kg [ À ]. conventional methods of detection involve the use of high-performance liquid chromatography, mass spectrometry, and colorimetric elisa assays; but these methods must be carried out at centralized locations and are too slow to be used in the field. antibody-immobilized algan/gan hemts have been used to detect botulinum toxin type-a with a limit of detection (lod) below ng/ml [ , ] . the antibody was anchored to the gate area through immobilized thioglycolic acid and the toxin detected through bonding to the botulinum antibody and the signal detected by changes in the hemt drain current [ , ] . the sensor saturated above ng/ml of the toxin, with a lod below ng/ml in pbs buffer solution. the sourceÀdrain current change was nonlinearly proportional to botulinum toxin concentration. the long-term stability was tested by storing the sensor stored in pbs at c and periodically testing over months at room temperature. the sensitivity losses were %, %, and % after , , and months, respectively [ ] . the toxicity of heavy metal ions, including mercury(ii) hg , lead(ii) pb , copper(ii) cu , and zinc(ii) zn , is a chronic environmental problem [ À ]. mercury is released into the environment through the combustion of fossil fuels, mining, volcanic emissions, and solid waste incineration. mercury and lead impact on wildlife ecology and human health. some types of bacteria convert inorganic hg ions into neurotoxic organic mercury compounds, which bioaccumulate through plants and the food chain. bare au-gated and thioglycolic acid-functionalized au-gated algan/gan hemts can detect mercury(ii) and copper(ii) ions [ À ]. fast detection of less than seconds was achieved for thioglycolic acid-functionalized sensors. the thioglycolic acid-functionalization increased the sensitivity for detection of mercury by . over the bare au-gated surface. the limit of mercury(ii) ion detection was m and s selectivity of more than for detecting hg over na or mg ions. the sensors could be recycled using a de-ionized (di) water rinse, as shown in fig. . . mortality in breast cancer patients can be reduced by increasing the screening frequency [ À ]. most patients are screened by mammography, which is invasive (radiation) and limits the frequency of screening. a % survival rate is predicted to be achievable if patients could be screened every months, but this would require low cost, point-of-care technologies that can screen more frequently and noninvasively [ , ] . salivary testing for markers of breast cancer may be used in conjunction with mammography [ À ]. saliva-based diagnostics for the protein c-erbb- , a prognostic breast marker assayed in tissue biopsies of women diagnosed with malignant tumors, shows potential. soluble fragments of the c-erbb- oncogene and the cancer antigen À were found to be significantly higher in the saliva of women who had breast cancer than in those patients with benign tumors. to fully realize the potential of salivary biomarkers, technologies are needed that will enable facile, sensitive, and specific detection of breast cancer at home with concomitant wireless data transmission into the clinic. if cheap technologies that can wirelessly detect breast cancer are developed, early diagnosis will significantly lower mortality. antibody-functionalized, au-gated algan/gan hemts were used to detect c-erbb- , a breast cancer marker [ ] . the antibody was anchored to the gate area through immobilized thioglycolic acid. the sensor showed a response of less than seconds when target c-erbb- antigen in a buffer at clinically relevant concentrations from . to . μg/ml was added to the antibody-immobilized surface. this electronic detection of biomolecules is a significant step toward a compact sensor chip, which can be integrated with a commercially available hand-held wireless transmitter to realize a portable, fast response, and high sensitivity breast cancer detector. prostate cancer is the second most common cause of cancer death among men in the united states and the most common form of cancer among men, other than skin cancer [ À ]. the most commonly used serum marker for diagnosis is prostate-specific antigen (psa). one in six men will be diagnosed with prostate cancer during their lifetime [ À ]. the american cancer society recommends health-care professionals offer the psa blood test and digital rectal exam yearly for men above the age of . psa is made by cells in the prostate gland and is found in semen and in the blood. when prostate cancer develops, the psa level usually goes up above ng/ml. about % men with a psa below will have prostate cancer on biopsy. if the patient's psa level is between and , their chance of having prostate cancer is b %. if the patient's psa level is above , there is more than % chance of prostate cancer. psa testing approaches are costly, time consuming, and need sample transportation. antibody-functionalized au-gated algan/gan hemts were used to detect psa in saline solutions [ ] . the psa antibody was anchored to the gate area through the formation of carboxylate succinimdyl ester bonds with immobilized thioglycolic acid. the hemt drainÀsource current showed a response time of less than seconds when target psa in a buffer at clinical concentrations was added to the antibody-immobilized surface. the devices could detect a range of concentrations from μg/ml to pg/ml, two orders of magnitude lower than the cutoff value of psa measurements for clinical detection of prostate cancer. fig. . shows the real-time psa detection in pbs buffer solution using the source and drain current change with constant bias of . v. no current change can be seen with the addition of buffer solution or nonspecific bsa, but there was a rapid change when ng/ml psa was added to the surface. the ultimate detection limit appears to be a few pg/ml [ ] . cerebrospinal fluid (csf) is a physiologically critical extracellular liquid secreted from the choroid plexus in the cerebral ventricles [ À ]. csf covers the brain and spinal cord, held in the central nervous system by the meninges. in addition to acting as a physiological buffer solution, providing nutritional and waste transport, it also helps to maintain intracranial pressure and acts as a physical shock absorber, cushioning the brain in the case of sudden movement or force. csf is constantly replenished. in a normal human adult, there is À ml of csf at one time, which is replenished every hours, so approximately À ml of csf is produced daily. leakage of csf leak is a serious complication that can result traumatically, iatrogenically, or spontaneously. while imaging studies can often elucidate the site of a leak, the standard for detection of csf is an assay for beta transferrin (b ) in nasal secretions or other drainage [ À ]. the primary methods of detection for b are immunofixation electrophoresis (ife) and elisa. consistent ife results down to μg/ml can be obtained in patient samples but require a . hour testing period, which is not expedient for real-time feedback during ent surgeries. to achieve good sensitivity and handle the inherently low concentration of β transferrin in csf, laboratory procedures have required samples to be concentrated by as much as -fold or the sample to be run in duplicate to ensure accurate detection. these tests are performed only at limited sites throughout the country due to cost and expertise, resulting in real-life return times on the order of days to a week. to alleviate the slow turn-around times of hospital laboratories and limited lower lods, there has been interest in electronic detection methods using biologically functionalized transistors, which provide an electronic readout and are readily integrated with wireless transmission of data. we developed a disposable testing slide externally integrated with a transistor to detect b at concentrations, from . ng/ml to μg/ml. a disposable testing slide was externally integrated with a si mosfet to detect csf from . ng/ml to μg/ml. a si mosfet pcb was designed to simplify the testing setup. human pooled csf was diluted in ph . pbs and wt% bsa with . ng/ml to μg/ml. we recognize that the concentration of csf is not the concentration of β t, it is actually much lower since it is only a minor constituent of csf. to test the csf sensor, diluted csf solution was applied to the sensing electrode and allowed to bind for minutes before measurement. time-dependent detection of csf dilutions from . to ng/ml is shown in fig. . (left) , while the response as a function of concentrations is shown in fig. . (right). cardiac troponin i (ctni) and the complex involving ctni, cardiac troponin t, and cardiac troponin c in the cardiac muscle tissue are standard clinical biomarkers for acute myocardial infarction (ami) and diseases that produce cardiac muscle damage [ , ] . their concentrations in the blood rise quickly following the onset of ami as they are released from myocardial cells following cell death. elevated troponin concentrations can be detected in the blood within a few hours up to several days following the onset of angina (where myocardial cells suffer reversible damage) to ami where myocardial cells die [ , ] . the time dependence of concentration of these species is commonly detected by radioimmunoassay, elisa fluorimetric, luminometric, colorimetric, and electrochemical methods, many of which are time consuming and require trained personnel to perform tests [ À ]. the measurement of blood troponin concentrations can decide whether ami has occurred or that chest pain and other symptoms are due to other causes. inexpensive techniques to provide rapid, accurate blood troponin concentrations would be welcome in managing treatment of emergency room patients. sarangadharan et al. [ , ] reported an electrical double-layer gated high-field algan/gan hemt biosensor in which the gating mechanism overcomes charge screening effects that are prevalent in traditional field effect transistor (fet)-based biosensors, allowing detection of target proteins in physiological solutions. they were able to detect troponin i in blood samples in the range . À ng/ml, using antibody or aptamer functionalization [ , ] . the cover glass approach leads to an increase in the pulsed current, as shown in fig. . [ ] . in this configuration, the receptor immobilization produces a decrease in total capacitance of the solution plus dielectric capacitance and thus a decrease in effective gate voltage and an increase in current. the electronic double-layer hemt designs enhance the current gain of the sensor in high ionic strength solutions, resulting in increased sensitivity and specificity in detection of troponin i. the ability to use a simple, functionalized glass slide as the active sensing area opens up the possibility of inexpensive cartridge sensors. radio frequency (rf) communication circuits can be integrated with sensors, enabling robust wireless sensors which transmit their data to a central location [ ] . a simple wireless sensor network is therefore needed to manage the collection and process of data from multiple sensors. the rf transceiver should be simple to reduce size and power consumption. radio frequency identification (rfid) tags can be monolithically integrated on a chip. the device consumes little power and can operate with a small battery or without a battery. in the latter case, the tag device is powered by the external interrogation signal from the base station. its low data bandwidth and low power operation make it suitable to integrate with sensors for wireless sensing, since the data bandwidth and operating power of sensor are also very low. once the detected sensor data are digitized, it can be merged with the id code and transmitted together. the rfid part of the integrated wireless sensor functions as a simple rf transceiver which receives base station signal to turn on the transmitter and sends back the sensor data. this type of wireless sensor is suitable for short-range operation similar to personal area network. the network consists of multiple sensors of the same function or different functions. each sensor is connected to an rfid tag with a unique id code that identifies its sensing function and location. in active operation mode, it sends out data to a central monitor station whenever the sensor detection is positive and triggers the rfid tag transmitter [ ] . in passive operation mode, the sensor is activated by the central monitor interrogation signal and responds with the data. fig. . shows the block diagram of the integrated wireless sensor with rfid. the sensor can be powered by the external interrogation rf energy from base station or an integrated battery. the sensor data are merged with rfid code, which modulates the antenna load and the reflected signal from base station. wireless sensor array using rfid is similar to the earlier except that multiple wireless sensors are integrated together on one substrate. for applications where size and power consumption are not critical, but operating distance is a major concern, the wireless sensor architecture in fig. . can be used. the device consists of multiple sensors, a memory stores its id code, a data processor that collects sensor data and merges with id code, and transmitter with power amplifier and antenna array for long-range transmission. the beam-forming antenna array can be preprogrammed to point the antenna beam to the central station [ À ] . for wireless transmission of data, an instrumentation amplifier can be used for the detection circuit to sense the change of current due to antigen detection. the current variation, embodied as a change in the output voltage of the detection circuit, will be fed into a microcontroller. the microcontroller will calculate the corresponding current change and control a zigbee transceiver which will transmit data to a wireless network server. the block diagram of the sensor module and wireless network server is shown in fig. . . the transceiver module is completely turned off for most of the time and is turned on to transmit data in extremely short intervals. when the sensor module is turned on, it is programmed to power up for the first seconds. following the initialization process, the detection circuit is periodically powered down for seconds and powered up again for another second, achieving a . % duty cycle. the zigbee transceiver is enabled for . ms to transmit the data only at the end of every cycle. this gives an rf duty cycle of only . %. data acquisition and transmission, if continuous, consumes most of the power in a wireless sensing system. since the sensor only needs to acquire and transmit data for a few seconds in every minutes during normal operation, the average power consumption can be significantly reduced by using low duty cycle operation. fig. . illustrates the package sensors mounted on a circuit board containing the detection circuit and microcontroller and the wireless transmitter for data collection. the sensor module is fully integrated on an fr pc board and is packaged with battery. the dimension of the sensor module package is . v . v v. the maximum line of sight range between the sensor module and the base station is m. the base station of the wireless sensor network server is also integrated in a single module ( . v . v . v) and is ready to be connected to a laptop by a usb cable. the base station draws its power from the laptop's usb interface and thus does not require any battery or wall ac transformer, which reduces its form factor. in detection of medical biomarkers, many different methods have been employed. there is also a need for small, handheld sensors with wireless connectivity, which have fast response. for medical sensing applications, disease diagnosis by detecting specific biomarkers (functional or structural abnormal enzymes, low molecular weight proteins, or antigen) in blood, urine, saliva, or tissue samples has been established. most of the techniques mentioned earlier, such as elisa, possess a major limitation in that only one analyte is measured at a time. particle-based assays allow for multiple detection by using multiple beads but the whole detection process is generally longer than hours, which is not practical for in-office or bedside detection. semiconductor-based sensors can be fabricated using mature techniques from the si chip industry and/or novel nanotechnology approaches. the goal is to realize real-time, portable, and inexpensive biological sensors and to use these as hand-held exhaled breath, saliva, urine, or blood monitors with wireless capability. frequent screening can catch the early development of diseases, reduce the suffering of the patients due to late diagnoses, and lower medical costs. there are still some critical issues. the sensitivity for some antigens needs to be improved to allow sensing in body fluids other than blood (urine, saliva). second, a sandwich assay allowing the detection of the same antigen using two different antibodies needs to be tested. third, integrating multiple sensors on a single chip with automated fluid handling and algorithms to analyze multiple detection signals, and fourth, a package that will result in a cheap final product is 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biosensors and the sensor array the work at uf was partially supported by a grant from the nsf i/ucrc of the multi-functional integrated system technology (mist center) iip- . the authors acknowledge the use of the nanoscale research facility (nrf) in the nanoscience institute for medical and engineering technology at the university of florida. key: cord- -mk eyceg authors: bendezu-quispe, guido; torres-roman, junior smith; salinas-ochoa, brenda; hernández-vásquez, akram title: utility of massive open online courses (moocs) concerning outbreaks of emerging and reemerging diseases date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: mk eyceg the emergence and re-emergence of infectious diseases such as ebola, chikungunya, and zika increase the necessity of knowledgeable and skilled health professionals. massive open online courses (moocs) arise as opportunities that allow people around the world to participate in higher education courses. a search was conducted on specialized mooc platforms to find courses related to outbreaks, using terms included in the list of the who disease outbreaks from january st to december (st), . we found seven courses about ebola, two about zika, three about the dynamics of epidemics and pandemics, and only one course about dengue, chikungunya, and malaria. most of the courses were conducted in english. the courses on ebola, zika and chikungunya were released after their last outbreak. moocs could be used to learn about health issues of global relevance, and with the necessity of fast divulgation of knowledge and skills. translating the courses into more languages could give these courses more traction, and allow participation of professionals in regions affected by these outbreaks. the emergence and re-emergence of infectious diseases is partly due to the climate change, more specifically due to the rise in global temperature, as well as the increased migration and unplanned urbanization . these events of great relevance to global health have turned these unknown diseases into realities many health professionals have to face daily. ebola virus causes an acute and severe disease that is usually fatal if left untreated. its last outbreak in march was the largest in history, causing a dramatic number of more than , deaths and , new infections worldwide. it affected several countries in west africa, generating much concern worldwide due to its estimated % mortality rate. similarly, diseases such as chikungunya and zika have shown several reasons to be considered serious infectious diseases. given the pandemic potential of these viral diseases, it is important to assess the knowledge and awareness of our health professionals regarding the mode of transmission of these diseases. in this era of globalization and technology, one of the main impacts of the internet and the web . have been the acceleration of the process of sharing information, allowing health professionals to have quick and easy access to the latest research in medicine and health . massive open online courses (moocs) are online classes or lectures accessible for people all around the world that want to participate in higher education courses. moocs material includes videos, slideshows, discussion boards, quizzes, audios or any combination thereof. usually, participants do not pay any fee to take a course. the topics in moocs vary widely and include science, engineering, and arts; and are usually developed by well-known figures in the study area . we found seven courses about ebola, two about zika, three about the dynamics of epidemics and pandemics, and only one course about dengue, chikungunya, and malaria. the duration of the courses ranged from to weeks. out of courses were held only in english, with the possibility to select subtitles in english or other languages; there was one in english and chinese, and only one exclusively in spanish. most courses ( out of ) originated from to usa centers including emory university, university of pittsburgh, the pennsylvania state university, harvard university and university system of maryland. the information provided with the courses included audiovisual material, papers, and self-assessments. all the courses were made for health-related professionals and presented information about epidemiology and lessons about the outbreaks and prevention activities for a possible new scenario of transmission of infectious diseases. the courses on ebola, zika and chikungunya were released after the last outbreaks of these diseases, respectively (table ) . the dataset contains information on the learning platform, institution, course length, time required per week, language and availability of subtitles. this new version adds information on the methods, mentioning that mooc aggregator platforms were used when information about the courses was not available on the platforms that offered that courses before. in the results, now there is a clarification that notes that all the courses founded about moocs and outbreaks had been made for health-related professionals. finding moocs about ebola, chikungunya, and zika after the start of their last outbreaks demonstrates the interest of institutions in offering information to the public. the vast majority of courses are offered in english, with a few having subtitles in other languages. moocs offer a recent and emerging form of education. there is a continuous increase in the number of courses offered in this format and, by , a total of million participants in . moocs were counted, with . % of these courses corresponding to health and medicine. the spread of diseases makes it necessary to invest in alternative methods of spreading knowledge, to improve the capabilities of health professionals in topics that affect people worldwide. moocs could be used to learn about health issues of global relevance, and with the necessity of fast divulgation of knowledge and skills. because the countries most affected by these diseases do not have english as the native language, promoting the translation of content into more languages could give these courses more traction, and allow participation of professionals in regions affected by these outbreaks. no competing interests were disclosed. the author(s) declared that no grants were involved in supporting this work. . . this is an interesting research note identifying moocs relating to emerging and re-emerging diseases. the work in its current form is acceptable as a research note, and the conclusion that such moocs need to be available in more languages is an important point. if the authors are to extend their work there is scope for improvement: the search window was very limited, it is possible that moocs that had completed would not be found. adding mooc aggregators (such as class central) to the search areas could have identified more courses, than those on offer in may . there are many barriers to accessing moocs, especially in developing countries, for example access to computers, connectivity, digital skills, as well as fluency in international languages. consideration of all potential barriers will be important in future work. the target audience of the moocs is not clear in this report, and there may be benefit in classifying courses that are aimed at health professionals and those aimed at the general public. then considering whether there is benefit in a direct linguistic translation for particular moocs. are all the source data underlying the results available to ensure full reproducibility? yes . about the use of mooc aggregators, the authors used class central and mooc list for both obtain the list of mooc websites and for obtaining information about moocs completed before our time frame. now, that information is included in methods: from st may to st may , we conducted a manual search on several learning platforms that offer moocs, including coursera, edx, futurelearn, udacity, miríada x, alison, fun.mooc, canvas network, and university to find courses about disease outbreaks using the terms included in the list of who disease outbreaks from january st to december st, (box ). because information about the learning platform, institution, course central and mooc list length, time required per week, language and subtitles availability for every course were collected and reported using frequencies in the case of categorical data and ranges for numerical data. . thank you for your recommendation. our study group is developing a survey to measure barriers to accessing and using moocs produced locally. . about the target population, all the courses were aimed at health professionals. considering this, we have included this information in the results section: we found seven courses about ebola, two about zika, three about the dynamics of epidemics and pandemics, and only one course about dengue, chikungunya, and malaria. the duration of the courses ranged from to weeks. out of courses were held only in english, with the possibility to select subtitles in english or other languages; there was one in english and chinese and only one exclusively in spanish. most courses ( out of ) originated from to usa centers including emory university, university of pittsburgh, the pennsylvania state university, harvard university and university system of maryland. the information provided with the courses included audiovisual material, papers, and self-assessments. all the courses were made for and presented information about epidemiology and lessons about health-related professionals the outbreaks and prevention activities for a possible new scenario of transmission of infectious diseases. the courses on ebola, zika, and chikungunya were released after the last outbreaks of these diseases, respectively. no competing interests were disclosed. public health emergency of international concern. based on that announcement, we conducted a search about moocs of zika. we expected that both public and academia concern about this disease will impact the number of moocs developed about this topic. however, the presence of outbreaks of diseases such as chikungunya and oropuche during , expanded our research scope. the who's list of diseases outbreaks included: ebola, viral hemorrhagic fevers, hemorrhagic fever syndrome, zika, chikungunya, lassa, mers-cov, middle east respiratory syndrome coronavirus, yellow fever, human infection with avian influenza, oropouche virus, rift valley fever and wild polio and vaccine derived polio. this list includes a vast number of diseases that were also included in the annual lists of outbreaks published by who before . hence, it is not expected that a significant increase in the number of moocs after increasing the list of outbreaks before . no competing interests were disclosed. the benefits of publishing with f research: your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and more the peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f .com drivers, dynamics, and control of emerging vectorborne zoonotic diseases this is a nice short report from the study conducted by bendezu-quispe g about the existence of et al. moocs with focus on emerging and re-emerging diseases. the idea is simple, and so is the methodology. however, i have concern regarding the time frame selected for the study which was limited to the year . some of the outbreaks highlighted in the background started in earlier years and were terminated in . i think that more valid and strong data could have been provided if there were no time limits thus allowing for exploration of moocs presented at the time of epidemics. otherwise, authors have properly presented their data. i believe their work would be a good start for further exploration of this area. are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. public health emergency of international concern. based on that announcement, we conducted a key: cord- - y vjhux authors: wang, qihui; yan, jinghua title: isolation of monoclonal antibodies from zika virus-infected patient samples date: - - journal: zika virus doi: . / - - - - _ sha: doc_id: cord_uid: y vjhux the combination of sorting antigen-specific memory b cells with determining immunoglobulin (ig) genes at the single-cell level enables the isolation of monoclonal antibodies (mabs) in individuals. this method requires a small amount of blood (usually ml) and is rapid (less than weeks to isolate antigen-specific mabs). due to the application of antigens as the bait to capture the specific memory b cells, the majority of isolated mabs are true binders to the antigen, which increases the isolation efficiency. here, applying this approach, we describe the characterization of mabs against zika virus from a convalescent patient sample. from ml whole blood, we sorted zika envelope (e) protein-interacting single memory b cells. the ig genes from cells were determined, and mabs were found that bind to zika e protein with varied binding affinities. zika virus has caused global concern due to the accumulating evidence suggesting that infection is associated with microcephaly and neurological complications, such as guillain-barré syndrome [ ] [ ] [ ] [ ] . however, there are currently no approved antivirals against zika infection [ ] . administration of polyclonal or purified neutralizing monoclonal antibodies (mabs) to pregnant mice helps to clear the virus and alleviates the neurological disorders in their fetuses [ , ] , providing proof of concept that neutralizing mabs can be used to treat zika virus infections. currently, multiple strategies have been reported to generate human neutralizing mabs against zika infection, including sequencing antigen-specific memory b cells [ , ] or generating epstein-barr virus-immortalized memory b cells from zika patient samples [ ] , and identifying functional mabs from phage display naïve antibody libraries [ ] . in addition, murine mabs against zika virus have also been reported [ ] . here, we introduce a method to apply zika envelope (e) glycoproteins, which play pivotal roles in virus entry and contain important neutralizing epitopes, to sort single memory b cells from a convalescent zika patient. subsequently, the immunoglobulin (ig) genes encoded by the sorted cells were determined at the single-cell level. the approach to determine the ig genes from single cells was first reported by tiller et al. [ ] . later, scheid et al. modified this approach to sequence single gp -binding memory b cells, which were isolated from human immunodeficiency virus (hiv) patients [ ] . then the mabs against human papillomavirus (hpv) or respiratory syncytial virus (rsv) were isolated from immunized or naturally infected human donors, respectively [ , ] . during the epidemic of zika virus, we and other groups also applied this strategy to isolate mabs targeting zika virus from zika patients [ , ] . from ml whole blood, we were able to pair mabs from collected single cells. as detected by surface plasmon resonance (spr), of the mabs were true binders to zika e protein [ ] . although the protocol depicted in this chapter focuses on the isolation of zika mabs from a zika convalescent patient, it could also be expanded to other viruses, and we recently reported the isolation of human neutralizing mabs against rift valley fever virus (rvfv) from a convalescent rvf patient, applying the same strategy [ ] . . primers for reverse transcription (rt) are listed in table . . primers for the first round pcr (pcra) to amplify v h are listed in table . . primers for the pcra to amplify v κ are listed in table . . primers for the pcra to amplify v λ are listed in table . . primers for the second round pcr (pcrb) to amplify v h are listed in table . . primers for the pcrb to amplify v κ are listed in table . . primers for the pcrb to amplify v λ are listed in table . . other primers used in this study are listed in table . table primers for the pcra to amplify v κ forward primer cκ-ext gag gca gtt cca gat ttc aa table primers for the pcra to amplify v λ forward primer . lb agar plates containing ampicillin: dissolve g of tryptone, . g of yeast extract, . g of nacl, and . g of agar in l of ddh o. the agar will not dissolve until autoclaved. autoclave for min and then allow it to cool until the bottle can be held with bare hands. add ml of ampicillin ( mg/ ml). invert to mix thoroughly. carefully pour out into sterile . trypsin-edta ( . %) and phenol red. table other primers used in this study . transfer buffer: dissolve . g of tris base and . g of glycine together in . l of ddh o. then add ml of methanol and mix. at last, add ddh o to a final volume of l. characterization for the mabs . biacore t system. . biacore t evaluation software, version . . . collect ml of whole blood, using a k edta tube, from a convalescent zika patient or a healthy donor with informed consent (see note ). . dilute the anticoagulated blood with ml pbs (one volume of the original blood). . invert the density gradient medium bottle several times to ensure thorough mixing. . add ml of density gradient medium to a ml centrifuge tube with a conical bottom. . carefully layer the diluted blood sample onto the density gradient medium solution (see note ). . centrifuge at  g for min at room temperature (see notes and ). . draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the pbmc layer undisturbed at the interface. aliquot ml of the upper layer per vial, which contains the plasma, and store them at À c ( fig. ). . transfer the layer of pbmcs to a sterile ml centrifuge tube using a sterile pipette (see note ). . add pbs until the total volume reaches ml ( . volumes of the original blood). resuspend the cells by gently drawing them in and out of a pipette. . centrifuge at  g for min at room temperature, and discard the supernatant. . add ml pbs to resuspend the cells. mix thoroughly and transfer μl of cell suspension in a . ml tube for cell counting. . load μl of cell suspension into a cell counting chamber. . insert the chamber into an automated cell counter and run the counting. . repeat step in this section. . quickly resuspend the cell pellet by adding freezing medium to a cell density of  /ml (see note ). . aliquot ml of the cell-medium mixture per vial, and place in a freezing container. . freeze the cells overnight at À c. . transfer vials to a liquid n tank for storage (see note ). cytometry ( . day) . samples used for staining are designed as follows: (a) zika patient pbmcs stained with zika e protein, as well as a panel of memory b-cell markers (see note ). (c) healthy donor pbmcs stained with mers-rbd protein (positive control for anti-his antibody). . transfer a vial containing the frozen pbmcs of either a convalescent zika patient or a healthy donor from the liquid n tank, and place them into a c water bath (see note ). . gently swirl the vial in the c water bath, and quickly thaw the cells (< min) until there is just a small bit of ice left in the vial. . transfer the vials into a hood. before opening, wipe the outside of the vials with % ethanol. . transfer the thawing cells into a centrifuge tube ( ml) containing ml of pre-warmed culture medium. . centrifuge the cell suspension at  g for min at room temperature. . decant the supernatant without disturbing the cell pellet. . gently resuspend the cells in facs buffer, μl per cells. . keep the mixture on ice for h. . add ml of facs buffer to the cells and pellet the cells by centrifuging at  g for min at c. decant the supernatant without disturbing the cell pellet (see note ) . . resuspend the cells with μl of facs buffer per cells (see note ). . prepare staining master mix in a . ml microcentrifuge tube (see table ) and store them at c in the dark before use. . add μl of the staining master mix to cells that prepared in step in this section. incubate them on ice for min. . add ml of facs buffer to the mixture of cells and antibodies in the last step and pellet the cells by centrifugation at  g for min at c. decant the supernatant without disturbing the pellet. . resuspend the cells with . ml facs buffer. place the tube on ice and avoid light before cell sorting. controls ( h) . label a facs tube for each of the six fluorochromes that will be used in the cell sorting. add . ml of facs buffer into each tube. . mix compensation beads by vigorously inverting at least ten times. . add μl of compensation beads into each tube. . load the zika pbmcs sample stained with zika e protein and memory b-cell markers. gate the target cells as in fig. d. . adjust the flow rate so that the event rate is approximately events/s. . mix and briefly centrifuge each component in the rt-pcr synthesis system for the first-strand cdna before use. table in a . ml tube. table ) into each well containing the collected single cells prepared in step in subheading . . . incubate the plate at c for min and then place it on ice for at least min. . prepare the following cdna synthesis mix in a . ml tube, adding each component in table in the indicated order. table ) to each well, mix gently, and collect by centrifugation at ,  g at c for s. . the rt reaction is performed at c for min and terminated at c for min. chill on ice and then store at À c until use. . prepare primers for the pcra (see tables - ). the amount for reactions is listed in table . . perform the pcra to amplify the ig genes. prepare the reaction system as indicated in table (see note ) . the cycling conditions for the pcra are shown in table . store the pcra products at À c until further use. . prepare primers for the pcrb (see tables - ). the amount listed in table is for reactions. . perform the pcrb to amplify the ig genes. prepare reaction system as indicated in table (see notes and ). the cycling conditions for the pcrb are indicated in table . . load the samples onto an agarose gel ( . %), and separate the dnas by electrophoresis. . a typical band size for v h , v κ , and v λ is approximately bp, as displayed in fig. . . perform the pcrc. prepare reaction system as indicated in table . . load the samples onto an agarose gel ( . %), and separate them by electrophoresis. . cut out the~ bp bands and extract the dna segments using a gel extraction kit according to the manufacturer's instructions. table ) is incubated at c for min, followed by cycles of c for s, c for s, and c for s, with a final incubation at c for min. lf new ( μm)  pcr buffer table reaction system for amplification of the constant regions  pcr buffer high-fidelity dna polymerase . cut out the~ bp bands for c h and~ bp for c κ and c λ . extract the dna segments using a gel extraction kit according to the manufacturer's instructions. . perform overlapping pcr to generate the expression cassette (see table ). the pcr reaction is incubated at c for min, followed by cycles of c for s, c for s, and c for min, with a final incubation at c for min. . digest the pcr products from the last step with ecori and xhoi for both the heavy and lambda chains. digest the kappa chain with kpni and xhoi (see table ). mix gently and spin down for a few seconds. incubate at c overnight. . purify the digested pcr segment using a universal dna purification kit according to the manufacturer's instruction. . ligate the expression cassette to the pcaggs vector (see table ). for both heavy and lambda chains, the vector was  pcr buffer high-fidelity dna polymerase pcaggs-z h encodes the heavy chain of the rd sorted cell). . the generation of vectors for mab expression is displayed in schematic diagram in fig. . fig. strategy to clone and express zika e-specific human mabs. the ig genes from the sorted cells were determined and cloned into the expression vectors by a reported approach with some modifications [ , ] . the ig transcripts in the collected zika e-interacting memory b cells were first reverse-transcribed into cdna using the ig gene-specific primer mix at the single-cell level. then the variable regions for heavy, kappa, and lambda chains were amplified by nested rt-pcr. the first round pcrs were performed with the forward primer mix specific for the leader region and reverse primers specific for the constant regions of heavy, kappa, and lambda chain, respectively. the second round pcrs were performed with the forward primer mix specific for framework segment fr and respective nested reverse primers specific for the heavy, kappa, and lambda constant regions. then the pcr products were separated by electrophoresis and sent for sequencing. in terms of the correct segments, another round of pcr (pcrc) was performed with the forward primer containing the signal peptide of mouse ig κ and reverse primer paired with the framework segment fr . then the resultant pcr segments were overlapped with the respective constant region to get the full expression cassettes for each chain, which were then ligated to the linearized pcaggs vector. all expression plasmids were sequenced and aligned with those in pcrb . load the samples onto a precast sds-page gel ( - %), and separate the proteins by electrophoresis. . transfer the proteins onto a nitrocellulose membrane. . perform western blotting to assess the expression of each mab using anti-higg/hpr (diluted by : ). as indicated in fig. . etbr stains dna by intercalating between the bases of dna. it will also intercalate into human dna, so wear gloves to prevent contact with it. a separate space is also recommended for performing all experiments using etbr-containing materials. do not pull the comb out too soon, as it causes the wells to collapse. it will take - min to gel. if the gel cannot be used in h, it is recommended to transfer the gel without comb into a tank containing  tae buffer. . the primers used for the amplification of ig genes are the same as reported (see tables - ) [ ] . however, after sequencing, cloning of the expression vectors was designed based on the pcaggs vector. . all primers are stored in small aliquots to avoid repeated freezing and thawing. . the blood sample of the convalescent zika patient was collected days post onset of fever, headache, and dizziness. it is reported that after immunization, antigen-specific b cells with a memory phenotype could be detected in the blood within week [ ] . in the presence of antigens, memory b cells undergo affinity maturation, and their b-cell receptors have increased affinities for the antigen. studies on memory b cells after smallpox vaccination in humans indicated that antigenspecific memory b cells initially declined postimmunization ( year) but then reached a plateau~tenfold lower than peak and were stably maintained for > years after vaccination [ ] . thus, considering the time that needed for affinity maturation, we recommend collecting the blood month- year post onset of symptoms to isolate specific mabs. . when layering the sample, do not mix the density gradient medium solution and the diluted blood sample. keep them in separate layers. . the break should be turned off at this step. for other steps using centrifuging, set the break on. . if the blood has been collected for > h, extend the centrifuging time to min. . usually, - ml of solutions containing pbmcs will be pipetted out from ml of the blood. . for example, if the cell density is determined to be  / ml at step in subheading . , the total cell number is  /ml  ml ¼ .  . thus, we need to add . ml freezing medium to resuspend the cells. . freshly prepared pbmcs exert higher efficiencies for ig gene amplification than frozen cells. thus, freshly prepared pbmcs are recommended for the isolation of mabs. in case the following cell sorting cannot be performed immediately, the methods using the freezing cells are provided in subheading . . . memory b cells express cd and cd , but not cd . in this study, we focus on the igg + memory b cells. . if fresh pbmcs are used here, please skip to step . if using frozen cells, please follow step . . before turning the tube right side up, it is recommended to aspirate the liquid left around the tube orifice. . it is difficult to decant the entire buffer. usually,~ μl of buffer will be left. thus, we add μl facs buffer for cells or μl for  cells. . there are three antibodies conjugated with pe-cy ® . they are anti-human cd /pe-cy ® , anti-human cd /pe-cy ® , and anti-human cd a/pe-cy ® . any of the three are suitable for preparation of the compensation beads for pe-cy ® . . the steps in this section are specific to, but not limited to, a bd facsaria iii cytometer. . here, we use the markers for t cells, nk cells, and platelets for negative selection, to exclude their disturbance. . here, we include fsc-a and fsc-h to exclude cell aggregates. we are not sure about the proportion of memory b cells that bind to zika e protein. thus, it is difficult to set the threshold to distinguish antigen-specific memory b cells from those targeting other antigens in the sample of zika patient pbmcs. in previous work, we studied the interaction between the receptor-binding domain (rbd) of middle east respiratory syndrome coronavirus (mers-cov) and its receptor cd , which is widely expressed on lymphocytes, including t cells. here, we used the mers-rbd, which is also tagged at its c-terminus with  his like the zika e protein, to stain pbmcs from a healthy donor. then, anti-his/pe was applied to bind to the his tag. through comparison between the results in fig. b and c, we could determine the threshold to gate the zika e-specific memory b cells, as indicated in fig. d . . the pcr reactions are performed in -well plates. . the combined primers are used to amplify ig genes in the pcrb. however, different primers in a single tube might disturb each other and reduce their annealing efficiency. thus, in terms of the single cells that yield typical bands for the kappa or lambda chain but not for heavy chain in gel electroporation, we usually repeat pcrb using the separate primers. in addition, mgcl exerts effects on the activities of dna polymerase (for sing-cell pcr). varied concentrations of mgcl , ranging from . to . mm, can also be applied to amplify the variable region of the heavy chain from single cell, whose light chains' variable regions have been sequenced. . from pmbcs isolated from ml whole blood of a convalescent patient, we finally sequenced paired mabs, which have been published previously [ ] . qihui wang and jinghua yan . here, we introduce the traditional method to double digest the pcr segments with two restriction enzymes and clone them into the same sites in the linearized pcaggs vector using t ligase. however, other methods (e.g., in-fusion reaction [ ] ) can be used. . here we displayed the results for z , z , and z . . the steps in this section are specific to, but not limited to, a biacore t system. . in addition to spr, enzyme-linked immunosorbent assay is another typical method to assess the interaction between mabs and antigens. however, for zika e-specific mabs, we found that some of them displayed relatively low response to the antigens coated on the plate (data not shown here). it is possible that zika e proteins undergo some conformational changes when adsorbing to the plate, which results in the decreased binding to certain mabs. in terms of spr experiments, the mabs were captured on the chip through interactions with protein a, which binds to the fc region. thus, the cdrs of mabs orient to the buffer flowing over the chip surface. in addition, the zika e proteins in the buffer of hbs-ep are label-free and more prone to be in its native conformation than those coated on the plate. thus, we chose spr assay to detect the interaction between mabs and antigens. . due to the different binding kinetics between an antigen and its mab, we set varied dissociation time. for example, mab z dissociates with zika e with very low rate; thus we set s for their dissociation. however, for both z and z , they are ready to dissociate with the antigen, and s was used for their dissociation (fig. s g, j, and l in the paper [ ] ). . due to the different binding kinetics between mabs and zika e protein, the data could be fit by either a steady state affinity model or : (langmuir) binding model. guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study zika virus associated with meningoencephalitis zika virus associated with microcephaly zika virus in brazil and macular atrophy in a child with microcephaly monoclonal antibodies against zika virus: therapeutics and their implications for vaccine design transfer of convalescent serum to pregnant mice prevents zika virus infection and microcephaly in offspring a single injection of human neutralizing antibody protects against zika virus infection and microcephaly in developing mouse embryos molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus recurrent potent human neutralizing antibodies to zika virus in brazil and mexico specificity, cross-reactivity and function of antibodies elicited by zika virus infection neutralization of zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain iii structural basis of zika virus-specific antibody protection efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals rapid profiling of rsv antibody repertoires from the memory b cells of naturally infected adult donors characteristics of memory b cells elicited by a highly efficacious hpv vaccine in subjects with no pre-existing immunity neutralization mechanism of human monoclonal antibodies against rift valley fever virus high-throughput isolation of immunoglobulin genes from single human b cells and expression as monoclonal antibodies early appearance of germinal center-derived memory b cells and plasma cells in blood after primary immunization cutting edge: long-term b cell memory in humans after smallpox vaccination sitespecific recombinational cloning using gateway and in-fusion cloning schemes key: cord- -stwmxpbv authors: dolai, subhashish; tabib‐azar, massood title: whole virus detection using aptamers and paper‐based sensor potentiometry date: - - journal: med devices sens doi: . /mds . sha: doc_id: cord_uid: stwmxpbv paper‐based sensors, microfluidic platforms, and electronics have attracted attention in the past couple of decades because they are flexible, can be recycled easily, environmentally friendly, and inexpensive. here we report a paper‐based potentiometric sensor to detect the whole zika virus with a minimum sensitivity of . nv/zika and a minimum detectable signal (mds) of . x ( ) zika. our paper sensor works very similar to a p‐n junction where a junction is formed between two different regions with different electrochemical potentials on the paper. these two regions with slightly different ionic contents, ionic species and concentrations, produce a potential difference given by the nernst equation. our paper sensor consists of ‐ mm x mm segments of paper with conducting silver paint contact patches on two ends. the paper is dipped in a buffer solution containing aptamers designed to bind to the capsid proteins on zika. we then added the zika (in its own buffer) to the region close to one of the silver‐paint contacts. the zika virus ( nm diameter with kda or . x (‐ ) gm weight) became immobilized in the paper’s pores and bonded with the resident aptamers creating a concentration gradient. atomic force microscopy and raman spectroscopy were carried out to verify that both the aptamer and zika become immobilized in the paper. the potential measured between the two silver paint contacts reproducibly became more negative upon adding the zika. we also showed that a liquid crystalline display (lcd) powered by the sensor can be used to read the sensor output. sensors built with paper as a substrate have short response time, are low-cost [ ] and they are flexible [ ] , [ ] . moreover, they are biodegradable and suitable for mass deployment in resource-limited areas and can be easily used by unskilled operators. paper is also a great medium for immobilization and trapping and, in some cases, for binding with biomolecules. its porous structure with large connected pores composed of cellulose fibers allows transporting liquid by means of capillary forces that result in short response time. the porous structure of paper also allows any nano and microparticles to remain immobilized in the paper structure. paper can be functionalized with certain materials such as nitrocellulose paper used for immobilizing nucleic acids for selective sensing [ ] . paper-based potentiometric sensors are reported for detecting many ions and proteins [ ] - [ ] . potentiometric paper-based sensors [ ] , [ ] - [ ] utilize gradients of ion distributions that generate open-circuit voltages (v oc ) that are measured to transduce analytes. paper-based pathogen and virus sensors are also easy to incinerate. they can be used one-time use front end in sensor systems that can be pealed-off and replaced. there are many zika sensors and detection methods reported in the literature. these include serum analysis using the antibody detection assays [ ] , [ ] , and detection of viral rna using molecular-based techniques like conventional polymerase chain reaction (pcr). real-time reverse transcription-polymerase chain reaction (rt-pcr) [ ] - [ ] zika has also been reported. these techniques involve using viral antibody or dna/rna extraction, followed by labeled detection using fluorescent probes. they provide high sensitivity and specificity but require specialized equipment, expensive procedures, and are time-consuming. the paper-based sensors are fast, inexpensive, and can be used in regions with limited resources. we show that a simple liquid crystalline display (lcd) can be used to measure the sensor output with the power generated from the sensor itself. the paper sensor reported here can be modified by replacing its aptamer with other virus aptamers, such as covid- . our work shows for the first time that a potentiometric paper sensor can reliably detect a whole virus using standard printer papers functionalized with antigens or aptamers. aptamers are single strand dna structures that are artificially synthesized using a combinatorial selection process called systematic evolution of ligands by exponential enrichment (selex) [ ] to detect biomolecules and pathogens with high specificity [ ] , [ ] . in our sensors, when zika was added to one side of the paper, it resulted in a concentration gradient with the associated electrochemical potential difference. electrical contacts to the paper sensor can be made with graphene [ ] , [ ] conducting glues and epoxies and silver paint. we also this article is protected by copyright. all rights reserved developed a proof-of-concept device ( fig. (a) ) that can be printed with silver paint contacts and show that the paper-based zika sensor can be connected to an lcd for electronic readout. the charge distribution and ionic transport of differently charged species in the paper device and the resulting open-circuit voltage can be explained using the phase boundary model formed by two different ionic species (aptamer in the background and zika added on the anode side). the total electrochemical potential difference (v oc ) (fig. a) is the sum of the phase boundary potentials [ ] between the aptamer-electrode (v oc ), the liquid junction potential [ ] , [ ] between the zika/aptamer-aptamer (v oc ) and between the zika/aptamer-electrode (v oc ): v oc =v oc + v oc +v oc . fig. .a shows the schematic of the phase boundary between electrodes and the paper and liquid junction potential at the center. the phase boundary potential between the aptamer-electrode (v oc ) and zika/aptamer-electrode (v oc ) are obtained from the nernst equation: where, is the standard potential, r is the gas constant, t is temperature, f is the faraday constant (coul./mol), z = is the valence of the aptamer, and z = is the valence of the zika. zika is a complex extended pathogen with surface proteins, dna, rna, etc. and has many oxidation/reduction potentials corresponding to more than one charge transfer that we have indicated in eq. . however, under the small voltages and chemically "mild" conditions of our paper sensors, it is reasonable to assume the single valency indicated in eq. . the liquid junction potential between (zika-aptamer) and aptamer in the middle of the device (fig. ) can be written as [ , ] : where, is the valence of the particular sample species (in our case z's for aptamers and the zika are equal to ) , denotes the activity of the ions/sample species and is the transference number and this article is protected by copyright. all rights reserved signifies the fractional conductance of the i th ion/sample species. the transference number defined by = , where u is the mobility of the i th ion, and m j is the molar concentration, with j ranging over all the ions. we note that there are space charge regions that form (fig. a) between the anode/cathode and the paper and at the boundary of the zika/aptamer-aptamer regions in the middle. in our experiments, the paper was uniformly primed with the buffer solution and aptamers. we assume that the anode-paper and cathode-paper nernst potentials (v oc and v oc in fig. a ) are approximately equal and are determined by the electrochemical potential differences between these electrodes and the primed paper. thus, v oc =v oc and the total v oc between the cathode and the anode is approximately equal to v oc (fig. a) . the space-charge region at the zika boundary ( fig. a and b) is composed of charges that are induced by the zika-aptamer-buffer on one side of this junction and aptamer-buffer on the other side ( fig. b) . the boundary is controlled by the amount of zika added and the flow of the buffer that accompanies the zika. we note that aptamers and zika viruses become immobilized in the pores of the paper. according to our experiments, the aptamer and zika had negative residual charges. each of these analytes is charge-neutral in their respective medium, but when they are added together, because of their different polarizability, they exhibit different residual charges. in the case of zika + aptamer, there is an additional charge re-distribution because of the hydrogen binding. the binding process does not happen instantly and proceeds as a function of time. in our experiments, the zika-aptamer complex provided higher v oc change than zika alone. the selectivity of the sensor to detect zika is primarily due to its preferential binding with the aptamer. when we added the zika to the anode region, v oc became negative, suggesting that the space charge region has a charge, electric field, and potential distributions schematically shown in figs. c, d, and e. this article is protected by copyright. all rights reserved c)-e) space charge, resulting in the electric field and potential at the boundary of zika in the center of the paper device. the devices used in our work ( fig. ) consisted of a sample holder with two clips and electrodes mounted on a glass slide. the devices were connected to a data acquisition system (ni-usb daq) and this article is protected by copyright. all rights reserved a computer with a custom labview program (fig. b) . we measured the open-circuit voltage (v oc ) of the paper device as a function of time as buffer, aptamer, and zika were added to the sensor. the glass slide was located inside a grounded copper enclosure to shield and improve the signal-to-noise ratio. paper strips ( . - . cm x cm) used in our devices were manually cut from a standard printer paper and were contacted with silver paint (from ted-pella). these paper devices were then coated (impregnated) with appropriate analytes such as de-ionized (di) water/buffer/aptamer, and the buffer/zika solutions were added from the anode side. this article is protected by copyright. all rights reserved the aptamer used in our experiments had a thiol end group and consisted of nucleotide-chain that folded to bind with the zika sf envelope protein [ ] . fig. c shows the schematic of the structure of the aptamer. the inactivated zika virus ( cfhi) was obtained from the zeptometrix corporation. µl of tcid_ zika stock solution was diluted with µl buffer. the number of zika viruses in the diluted µl volume was ~ x , estimated in the same manner as in [ ] . the open-circuit voltage (v oc ) of the sensor containing di water with the buffer solution introduced on the anode is shown in fig. a . the v oc , in this case, was measured immediately after the buffer was added to the anode region. v oc diminishes as a function of time (fig. b) as the buffer diffuses in the paper, and its concentration gradient diminishes. the zika virus has a small residual negative charge (fig. a ) that reduces the v oc as soon as it is added to the sensor. fig. shows the response of the sensor when zika was added. the initial rise in fig. a is due to the pipette tip approaching the sensor to deliver the zika. fig. b shows another run with the zika added in the beginning and later at . min. in both cases, the sensor output reached a steady-state value. this indicates that the concentration gradient introduced by the addition of zika did not change as a function of time, and it appears that the zika became immobilized in the paper. in the second case (fig. b) , the paper was not primed with the aptamer, and the change in v oc was due to the residual charge of the zika and buffer ions that accompany the zika as part of its solution. it is also possible that the printer paper we are using has its own impurities, which in the presence of zika + buffer became ionized. zika is spherical with nm diameter, and it is expected to get entangled in the paper pores. if zika was mobile, the this article is protected by copyright. all rights reserved resulting v oc would diminish as a function of time as the zika diffuses and reduces its concentration gradient. in separate experiments with buffer solutions alone (fig. b) , the sensor response diminished after the initial introduction of the buffer at the anode, indicating that the buffer ions could diffuse in the paper, decreasing their initial concentration gradients. in the device charge content and its gradient, are introduced by the additional zika alone that has negative residual charges. successive additions of zika (used to increase its concentration) introduced larger changes in v oc followed by smaller changes once all the resident aptamers were saturated (fig. b) , and ∆v oc became smaller at the end. the average "point" sensitivity in these devices was . nv/zika calculated at ∆v oc of ~ mv for µl zika (~ x number of zika per µl). the sensitivity was nearly the same for l zika in device p but not in device p (fig. ) . the paper-based sensor's output response had a rms noise voltage of mv, and the minimum detectable voltage was mv that resulted in the mds of . x zika. this article is protected by copyright. all rights reserved to verify the presence of aptamers and zika in the paper-based devices, we carried out two additional experiments. the first series of experiments used atomic force microscopy (afm) to image zika-coated regions of the paper-based device, as shown in fig. a . although one can see large zika complexes, individual zika viruses could not be resolved. the paper has large surface roughness in excess of m rms that prevented performing high spatial resolution scans. we then performed afm scans on aptamers and zika deposited on gold-coated glass samples [ ] . as can be seen in fig. b one could resolve individual zika viruses on gold. next, afm studies using afm probes with an aptamer-functionalized bead (fig. c) were carried out. the stiction force [ ] obtained from force-displacement curve using aptamer-functionalized afm probe (fig. c) . the afm probe has been prepared by gluing a µm diameter polyester microbead to the afm tip and subsequently coating the microbead with the aptamer ( µm) for min. it showed larger stiction forces with papers coated with aptamers than zika papers with aptamers or zika alone, as shown in fig. d . the stiction force of zika/aptamer/paper was higher due to aptamer-specific binding with zika on the aptamer/paper. this article is protected by copyright. all rights reserved the second set of experiments used raman spectroscopies on paper-based devices coated with different analytes. the raman spectra shown in fig. are from different regions of the paper coated with aptamers, zika, and zika/aptamers. a deltanu spectrometer with examiner raman unit was used in these experiments. l, m aptamer solution was used to prime the paper, and the zika concentration was . x /l, with the amount used in each application was l. the buffer solution was l of x pbs with mm mgcl . the raman peak due to zika was around cm - [ ] . this article is protected by copyright. all rights reserved buffer/paper aptamer/paper zika/paper zika+aptamer/paper cm - raman signal figure . raman spectra of buffer/paper, aptamer/paper, zika/paper, and zika/aptamer/paper. the spectra are normalized with dry paper raman spectrum. these spectra clearly show the difference between aptamers, zika, and buffer deposited on the paper. we then used printed patterns on paper, followed by adding silver paint contacts, as shown in fig. a and fig. insets. the purpose of the printed device is to clearly show the location of the zika solution (or fluids that may contain zika) on the device. fig. a shows the v oc -time response for paper-based devices (d -d ). the ∆v oc in these devices are shown in fig. b . we verified that the aptamer stayed and was resident in the paper even after rinsing the paper with di water. fig. c shows the response of adding zika on three different aptamer-primed sensors (d -d ). the v oc levels in fig. c are similar and consistent with that of the aptamer-primed device in fig. b . the actual interactions between different components in the paper matrix is quite complex, and the sensor response depends on the history of the paper, temperature, air current on the sensor, and other environmental factors [ ] . the results shown here had a standard deviation of ~ mv from run to run. uniformity of the electrical contacts can be a concern that can be addressed using -d printing. another very important source of error was in making sure that the paper sensor was not saturated with the analytes introduced at each step. when the paper was saturated, the addition of zika resulted in a thin layer of liquid above the paper sensor, and zika did not incorporate in the paper resulting in unreliable sensor output. the sensor was also very sensitive to spurious environmental electric fields generated by us and other objects nearby that could be addressed by proper grounding. we next explored the possibility of detecting the virus from the paper-based sensor in the form of direct electronic readout through an lcd instead of measuring the open-circuit voltage using a digital voltmeter. the lcd was configured to display a letter such as "a" once the paper sensor voltage reached a threshold value generated by the presence of sufficient number of zika viruses. in order to demonstrate the feasibility of such application, we used an lcd screen from a digital watch and experimentally found the turn-on voltage for a single display segment to be ~ mv. from the previous results, we observed that the addition of zika resulted in v oc ~ mv. we used paper sensors connected in series to generate ~ mv (fig. a) that was sufficient to power many readout devices, including lcds. the external lcd load did not draw more than a current to cause internal sensor resistor to become important. as can be seen in fig. b , the lcd displayed "a" upon adding zika. the lcd can be replaced by an electronic-ink display that requires much lower voltages/power eliminating the necessity of using many devices in series. (a) (b) the paper-based sensors we constructed and reported here were very reproducible as long as they were not saturated (with buffer or water) and as long as they were grounded properly. their "point" sensitivity of . nv/zika and their minimum detectable signal of . x zika are quite good given their simple structures and ease of operation. selectivity of this sensor as well as other aptamer-based sensors are limited by the selectivity of their respective aptamers that can be modified to achieve better selectivity if needed. in the case of paper-based sensors discussed here, the selectivity may also be affected by viruses becoming immobilized in the paper even if they are do not bind with the resident aptamer. these other viruses can be removed by mildly washing them away that will not affect the zika-aptamer complexes appreciably. the real-world application of zika paper sensors will involve the presence of bodily fluids such as urine or sweat or saliva. these biofluids are very complex, and proper filtering on the sensor will be used to remove their components that may affect the paper sensor operation adversely. as with any sensor development, the research and development efforts have many different stages. it usually starts with developing a transduction mechanism using modified "clean" samples and progresses toward more realistic samples in the real world. the present work describes the transduction phenomena of "sensing pure zika" virus using a paper-based potentiometric device that, to the best of our knowledge, was not done before. the innovation has two parts: a) using paper-based sensors to detect the whole virus and b) using resident aptamers in the paper to bind with zika. the sensor's selectivity comes from its accepted article resident aptamers. we will measure and report our sensors' selectivity and verify their operations in the presence of bodily fluid in the near future. the paper can also be used as an active sensing medium located over electrodes connected to the measurement apparatus. in this approach, the paper can be peeled off and replaced with a fresh paper. paper itself can be used as a filter preventing larger particles from reaching the sensitive layers in intimate contact with the electrodes. this work demonstrates, for the first time a paper-based sensor for detecting zika virus using potentiometry. the sensor had a minimum sensitivity of . nv/zika and a minimum detectable signal of . x zika. these sensors can be generalized to detect any other viruses, pathogens and bacteria by simply using appropriate aptamers. the most important and unique aspect of the potentiometric paper-based zika virus is its structural simplicity and ease of use. this study demonstrates the feasibility of detecting whole zika viruses and the use of simple electronic readout of paper-based sensors. although aptamers are known for their high specificity to capture target pathogens and viruses, more study is required with different viruses to verify and measure the cross-sensitivity of aptamers and our sensors. a low-cost paper-based inkjet-printed platform for electrochemical analyses foldable printed circuit boards on paper substrates paper electronics a paper-based potentiometric cell for decentralized monitoring of li levels in whole blood potentiometric sensing utilizing paper-based microfluidic sampling towards protein assays on paper platforms with potentiometric detection a disposable planar paper-based potentiometric ion-sensing platform paper-based potentiometric ion sensors constructed on ink-jet printed gold electrodes integrated, paper-based potentiometric electronic tongue for the analysis of beer and wine a fully integrated paper-microfluidic electrochemical device for simultaneous analysis of physiologic blood ions a paper based, all organic, reference-electrode-free ion sensing platform paper-based microfluidic sampling and separation of analytes for potentiometric ion sensing paper-based enzymatic electrode with enhanced potentiometric response for monitoring glucose in biological fluids paper-based microfluidic sampling for potentiometric accepted article this article is protected by copyright. all rights reserved determination of ions paper-based potentiometric ph sensor using carbon electrode drawn by pencil high-performance flexible potentiometric sensing devices using free-standing graphene paper zika virus: a new pandemic threat the zika challenge sensing viruses by mechanical tension of dna in responsive hydrogels prolonged detection of zika virus in vaginal secretions and whole blood detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study one-step rt-pcr for detection of zika virus detection of zika virus in urine dengue virus detection using whole blood for reverse transcriptase pcr and virus isolation detection of zika virus in saliva systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t dna polymerase aptamer-based elisa assay for highly specific and sensitive detection of zika ns protein high-resolution molecular discrimination by rna the phase-boundary potential model the liquid junction potential -the free diffusion junction calculation of liquid junction potentials base pair biotechnologies -aptamer discovery company mhz lithium niobate microbalance aptamer-coated whole zika virus sensor with hz/ng sensitivity microfabricated nano-gap tunneling current zika virus sensors with single virus detection capabilities recommendations for the use of an atomic force microscope as an in-fab stiction monitor surface-enhanced raman spectroscopy-based sandwich immunoassays for multiplexed detection of zika and dengue viral biomarkers accepted article esterowitz. this article is protected by copyright. all rights reserved accepted article key: cord- -kwzo puo authors: si, lulu; meng, yu; tian, fang; li, weihua; zou, peng; wang, qian; xu, wei; wang, yuzhu; xia, minjie; hu, jingying; jiang, shibo; lu, lu title: a peptide-based virus inactivator protects male mice against zika virus-induced damage of testicular tissue date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: kwzo puo zika virus (zikv) was a re-emerging arbovirus associated with guillain–barré syndrome in adult and congenital zika syndrome in fetus and infant. although zikv was mainly transmitted by mosquito bites, many sexual transmission cases have been reported since the outbreak in . zikv can persist in testis and semen for a long time, causing testicular tissue damage and reducing sperm quality. however, no drug has been approved for prevention or treatment of zikv infection, especially infection in male testicular tissue. previously reported peptide z could inactivate zikv, inhibiting zikv infection in vitro and in vivo. importantly, z could inhibit vertical transmission of zikv in pregnant mice, reducing zikv infection in fetus. here we showed that intraperitoneally administered z could also be distributed to testis and epididymis, resulting in the reduction of zikv rna copies in testicular tissue and protection of testis and epididymis against zikv-induced pathological damage and poor sperm quality in type i interferon receptor-deficient a mice. thus, z , a zikv inactivator, could serve as an antiviral agent for treatment of zikv infection and attenuation of zikv-induced testicular tissue damage. zika virus was a re-emerging arbovirus (gao, ; pettersson and bohlin, ) , like dengue virus (denv) and japanese encephalitis virus (jev), belonging to flavivirus genus in the flaviviridae family. since the outbreak in brazil in , zikv has rapidly spread to countries and territories (baud et al., a; gorshkov et al., ) , attracting global attention. about % of those infected with zikv presented with asymptomatic, or only mild illness (petersen e. et al., ; pierson and diamond, ) . however, zikv infection has been associated with more severe complications: guillain-barré syndrome in adult (brasil et al., ; peixoto et al., ) and congenital zika syndrome in fetus and infant (baud et al., b; gurung et al., ) . zikv is mainly transmitted through mosquito bites (petersen l.r. et al., ) , while it can also be transmitted via in utero from mother to fetus (besnard et al., ) , blood transfusion (tai et al., ) and intercourse (duggal et al., ; mead et al., ; sakkas et al., ) . it is reported that zikv was transmitted through sexual contact, perhaps up to days after the onset of symptoms (turmel et al., ) , and infective virions were still isolated from semen days after infection (arsuaga et al., ; garcia-bujalance et al., ) . the viral load in semen was , times that in the blood at weeks postinfection (mansuy et al., ) , and viral rna was still detected up to days after illness onset (barzon et al., ) . it is also reported that zikv infection caused patients to have a decreasing total sperm count in the acute phase of infection (joguet et al., ) and abnormal spermogram results year after infection (avelino-silva et al., ) , suggesting zikv was harmful to human spermatozoa production. testis explants from uninfected donors were also proven to be susceptible to zikv infection (matusali et al., ) . as determined from an in vitro human testicular organoid culture system, zikv-infected testicular organoids may lead to multiple kinds of cell death (strange et al., ) . although little was known about zikv infection in human testis and epididymis, except for semen, many murine models were used to study damage to testicular tissue. govero et al. ( ) performed a study in wild-type c bl/ mice in the presence of the anti-ifnar antibody and revealed that zikv preferentially infected spermatogonia and sertoli cells in the testis. this led to cell death and destruction of the seminiferous tubules in association with testis damage and poor sperm quality (govero et al., ) . ma et al. ( ) also established a mouse model using ifnα/β receptor-deficient mice (ifnar −/− knockout mice), and demonstrated that zikv infection induced inflammation in the testis and epididymis, leading to severe damage to testes at days post-infection. taken together, these findings suggested that zikv could persist in testicular tissue for a long time, causing severe damage to testis and epididymis and reducing sperm quality. currently, no approved drug is available to inhibit zikv infection (da silva et al., ) , especially infection in testicular tissue. ebselen (ebs), an antioxidant in clinical trials, was reported to alleviate testicular pathology in zikv-infected mice by reducing the level of oxidative stress and proinflammatory cytokines. however, it only had a weak effect on zikv directly, and its safety for pregnant women was unknown (simanjuntak et al., ) . this calls for the development of safe and effective drugs to prevent zikv-induced testicular damage. the testis is a male reproductive organ, mainly producing spermatozoa and androgen. specifically, spermatogenesis is a complex cellular event taking place in the seminiferous epithelium of seminiferous tubules and protected by sertoli cells that form the blood-testes barrier (btb) by tight junction protein (su et al., ) . the btb provides a specialized microenvironment for spermatogenesis by preventing harmful agents from entering the seminiferous tubule, but this was found to pose a major obstacle to the delivery of therapeutic drugs to the seminiferous epithelium (cheng and mruk, ) . therefore, any drug able to prevent zikvinduced damage in testicular tissue should be able to cross the btb into seminiferous tubules, or reach the testicular tissue, to inhibit zikv from entering into seminiferous tubules. the most promising anti-zikv drugs so far include small-molecule compounds (deng et al., a; chan et al., ; li et al., ) , antibodies (zhang et al., ; wang et al., wang et al., , , and peptides (yu et al., ; jackman et al., ) . compared with small-molecule compounds, peptides were safer, especially for pregnant women. as a macromolecular substance, passing through the btb was challenging for antibodies, and it is reported that the concentration of specific igg entering into the rete testis was . % of that in blood serum (knee et al., ) . therefore, the safer and cheaper peptide drugs, which consisted of dozens of amino acids, began to gain gradual acceptance. we previously demonstrated that an amphipathic peptide z , derived from the stem region of zikv e protein (figure a) , inhibited zikv infection in vivo and in vitro, suggesting its promise as an anti-zikv candidate drug. what's more, z had a protective effect in pregnant mice and their fetuses, suggesting it was able to cross the placental barrier (yu et al., ) . however, whether z could enter the seminiferous tubules and protect testicular tissue against zikv infection remained unknown. in this work, we showed that intraperitoneally injected z could be distributed in the testicular tissue. it then inhibited zikv infection, resulting in significant reduction of viral loads and protection of testis, epididymis, and sperm from zikvinduced pathological damage. these results suggest that z has the potential to be further developed as an anti-zikv agent for treatment and prevention of zikv-induced damage in testicular tissue. (sirohi et al., ) ]; di, dii, diii, stem and transmembrane domain are shown in red, yellow, blue, orange, and wheat, respectively; the fusion peptide is shown in cyan, and z ( - ) is shown in green. tm cells were infected with zikv strain sz (b), mr (c) and flr (d), treated with z at different concentration, and then viral copies in the supernatant at h were measured by qrt-pcr. data were presented as means ± sd. (e) z -treated zikv of different strain lost infectivity on tm cells irreversibly. after incubation with z at • c for h, zikv particles were separated from the unbound z by peg to measure their infectivity on tm cells. each sample was tested in triplicate and the experiments were repeated at least once. the data from two independent experiments were presented as mean ± sd. mixture f- (dmem/f , thermo fisher scientific, waltham, ma, united states) supplemented with % horse serum and . % fbs at • c and % co . zika virus (zikv) strain sz / (genbank no. ku ) was kindly provided by dr. cheng-feng qin (deng et al., b) and preserved in our laboratory. zikv strains mr (#vr ) and flr (#vr ) were obtained from atcc. zikv was propagated in vero-e cells. briefly, vero-e cells were infected with the virus at multiplicity of infection (moi) of . . the supernatants were harvested at days post-infection, centrifuged at , rpm for min to remove cellular debris, and stored at − • c as stock. peptides z (mavlgdtawdfgsvggalnslgkgihqifg aaf), z -cy and scrambled peptide (ldiiaglsagfq ggatfvdahgmvkasflggnw) were synthesized at kangbei bio, co., ltd. (ningbo, china) with % purity. peptides were solubilized in dimethyl sulfoxide (dmso) at mm and stored at − • c. virus titer was detected by plaque forming assay as shown below. bhk- cells were seeded onto a -well plate with × cells per well and cultured overnight at • c and % co . serially fold diluted virus were added to each well and incubated for h at • c. then the supernatant was replaced with ml dmem containing % low melting agarose and % fbs. after agarose solidification, the cells were cultured at • c and % co for days. then cells were fixed with % formalin and stained with % crystal violet overnight. finally, the plaque forming units were counted, and virus titer was calculated. six male a mice ( - weeks old) were randomly divided into two groups. mice in each group (n = ) were injected intraperitoneally with z -cy ( µg in µl pbs) or pbs (vehicle in µl pbs) as control. after anesthetization with pentobarbital sodium, mice were imaged by the ivis lumina k series iii in vivo imaging system from perkinelmer (waltham, ma, united states) for h. to determine the distribution of z -cy in the testicular tissue, mice were sacrificed using pentobarbital sodium, and all testes and epididymides were removed for imaging. the radiant efficiency (ps − cm − sr − ) (µw − cm − ) in mouse body and testis and epididymis was calculated by living image . . software. to determine the protection of z against zikv-induced testis damage, male a mice ( - weeks old) were randomly divided into three groups (n = ): z -treated group, vehicletreated group, and mock-infected group. mice in the z -or vehicle-treated group were intraperitoneally injected (i.p.) with pfu zikv with z ( mg/kg) or vehicle on day , followed by i.p. administration of z ( mg/kg) or vehicle once daily for six consecutive days, respectively. mice in the mock-infected group only received pbs as normal control. the body weight was monitored daily for days, and blood was collected at , , , , and days post-infection (d.p.i.) for detection of zikv copies in sera. at d.p.i., mice were euthanized by co inhalation, and the testes and epididymides were removed. the weight and size of testes were measured as previously described (de la vega et al., ) . after imaging, the left testes and epididymides were immersed in bouin's for hematoxylin-eosin (h&e) staining. the right testes and epididymides were soaked in rnaiso plus reagent at − • c for further detection of zikv copies. for safety analysis of z in testicular tissue, male balb/c mice ( - weeks old) were randomly divided into two groups. five mice in each group were i.v. administered with z at mg/kg of body weight or pbs control for days. the body weight of mice was monitored every other day for days. blood was collected at h, as well as , , and days post-injection and sera were separated from the blood samples for use in elisa to detect the concentration of testosterone and inhibin b as well as the titer of z -specific antibody. at days, mice were sacrificed, and the testes and epididymides were removed for histological examination. mature sperm in the cauda epididymis of the three groups of male a mice were collected and placed in ml pbs (preincubated at • c) immediately after euthanasia. the sperm suspension was analyzed for total sperm count and motility by computer-assisted sperm analysis (casa), as previously described (goodson et al., ) , using hamilton thorne ivos ii (beverly, ma, united states). then, after smear, desiccation and fixation, remaining sperm were stained by the papanicolaou staining method for manual morphological analysis. sperm morphology was observed in each mouse. zikv-infected mice were euthanized at days post-infection. testes and epididymides were homogenized with beads in ml rnaiso plus reagent (takara, japan) using tissuelyser- (jingxin, shanghai, china) after weighing. homogenized tissue were centrifuged for min at , rpm at • c, then total rna in tissues was extracted according to the operating manual and stored at − • c for the next step. sperm collected in pbs were placed in rnaiso to extract total rna under the same procedure. viral rna in sera samples on specific days was extracted using the easypure r viral dna/rna kit (transgen, china) and stored at − • c for the next step. zikv rna was examined by one-step real-time quantitative reverse transcription pcr (qrt-pcr) using the mastercycler r ep realplex real-time pcr system (eppendorf, germany). zikv rna copies were calculated based on the standard curve which was determined by plasmid containing specific sequence. the following primers were used: zikv-f: -ttggtcatgatactgctgattgc- ; zikv-r: -ccttccacaaagtccctattgc- ; zikv-probe: -fam-cggcatacagcatcaggtgcataggag-bhq - . the concentration of testosterone and inhibin b in the sera of balb/c mice was detected by mouse testosterone (ml , mlbio, shanghai, china) and inhibin b elisa kit (ml , mlbio, shanghai, china), respectively. according to the manual, µl standard or testing samples were added to a -well plate, which was coated with purified mouse testosterone or inhibin b antibody combined with hrp labeling. hrp-conjugate reagent was added to each well, except blank well (no sample; hrpconjugate reagent added as background). the plate was closed with closure plate membrane and incubated at • c for min. after washing, chromogen solution was added and incubated for min at • c. stop solution was added to each well, and absorbance was read at nm. peptide z was dissolved in dmso and diluted to different concentration by dmem/f . then µl of different zikv strains were incubated with z for h at • c. the mixture was added to × cells seeded into a -well plate and incubated at • c for h. after the culture supernatant was replaced by dmem/f- with % horse serum, cells were cultured for h at • c. then the culture supernatant was collected to detect zikv rna copies by qrt-pcr, as described above. the ability of z to inactivate different zikv strains was determined as follows. briefly, µl z or z -scr, at graded concentration were added to µl zikv ( × pfu/ml), followed by incubation at • c for h. then, peg- and nacl were added to the treated virus at final concentration of % and . m, respectively. after incubation on ice for h, the mixture was centrifuged at , rpm for h. the supernatants were removed, and the pellet was resuspended in µl dmem with % fbs. the infectivity of the zikv particles in the pellet was determined by cck- on bhk- cells or qrt-pcr on tm cells. the testis and epididymidis of z -or vehicle-treated zikvinfected mice and mock-infected mice were all collected post mortem. tissues were fixed in bouin's overnight, dehydrated, embedded in paraffin and sectioned. then the sections ( µm thick) were stained by h&e. subsequently, observation was made via panoramic scanner ( d histech pannoramic midi, hungary). student's unpaired two-tailed t-test was used to monitor the distribution of z in male a mouse body and testicular tissue and to analyze the difference of viral rna level in sera or tissues between z -and vehicle-treated a mice. one-way anova was used to examine the effect of z on the weight, length and width of testes, as well as sperm count, sperm motility and progressive sperm motility among the three groups. p-value was calculated by graphpad prism software, v. . , and significant difference was achieved with p-value less than . . * p < . ; * * p < . ; * * * p < . ; * * * * p< . . to determine the protective effect of z on zikv infection of testicular tissue, we tested if z could inhibit infection by different zikv strains of asian and african lineages in mouse sertoli tm cells, which are nurse-like cells that support spermatogenesis (wei et al., ) and important target cells for zikv testicular infection. zikv sz (asian lineage), flr (asian lineage), or mr (african lineage) was pretreated with z at different concentration before addition of tm cells and incubation at • c for h. after replacement of culture medium and further incubation for h, the viral copies in the supernatant were examined by qrt-pcr. as shown in figures b-d, z treatment resulted in a decrease of zikv copies in a dosedependent manner. considering that z -mediated inhibition of zikv infection is possibly attributed to its viral inactivation activity (yu et al., ) , different strains of zikv were incubated with z at different concentration for h at • c, followed by separating virions from the unbound free peptide by peg and detecting the infectivity of z -treated zikv in tm cells ( figure e ) and bhk- cells (supplementary figure s ) . we found that z -treated zikv strains lost their infectivity in a dose-dependent manner with % effective concentration (ec ) of . ± . µm (for sz ), . ± . µm (for mr ) and . ± . µm (for flr), respectively, suggesting that z inhibits infection of zikv strains of both asian and african lineages in tm and bhk- cells via inactivation of virions. to determine whether z entered testicular tissue of male mice, we employed cy -conjugated z peptide (z -cy ) to detect the distribution of z in the organs of male mice. as shown in figure a , the bodies of the z -cy -treated mice showed a strong fluorescence signal with average radiant efficiency of about . × (ps − cm − sr − ) (µw − cm − ), which is significantly higher than that in pbs-treated mice (p = . , student's two-tailed t-test; figure b) . then, the testes and epididymides were collected for examination of the fluorescence signal in the testicular tissue. as expected, both testes and epididymides of mice treated with z -cy showed a strong fluorescence signal, while those in the pbs-treated mice displayed no significant fluorescence signal (figure c) . the average radiant efficiency in testes and epididymides of z -cy -treated mice was significantly figure | distribution of z in the testicular tissue of male a mice. (a) imaging of male a mice treated with z -cy or pbs by the ivis lumina k series iii from perkinelmer. male a mice were injected intraperitoneally with µg z -cy (n = ) or pbs (n = ) as control, followed by imaging analysis. (b) the statistical analysis of fluorescence signal intensity in mouse body. data were presented as means ± sd. (c) imaging of the testes and epididymides from male a mice. (d) statistical analysis of fluorescence signal intensity in testis. each sample was tested in triplicate and the data were presented as mean ± sd. (e) statistical analysis of fluorescence signal intensity in epididymis. data were presented as means ± sd. * p < . ; * * * * p < . , student's two-tailed t-test. higher than that of pbs-treated mice (p < . , student's twotailed t-test; figures d,e) . these results suggest that z peptide can be distributed in the testis and epididymis of male mice. to determine the protective effect of z against zikv infection of male mice, pfu zikv were intraperitoneally injected into male a mice (type i interferon receptor-deficient). the infected mice were i.p. administered with z at mg/kg of body weight or vehicle, respectively, daily for days (figure a) . mice in the mock-infected group received pbs as normal control. results showed that the z -treated mice had neither weight loss ( figure b ) nor obvious clinical symptoms, consistent with mice in the mock-infected group (data not shown). however, in the vehicle-treated group, mouse body weight began to decline from the fifth day post-infection (d.p.i.) (figure b) , and some symptoms, like hunched posture and ruffled fur, appeared. we then examined viral copies in sera of z -or vehicle-treated mice at different time points by qrt-pcr. a high level of viral load was detected in sera of vehicle-treated mice, e.g., about copies/ml at d.p.i. (figure c) . however, viral load in sera of z -treated mice (figure c ) was as low as copies/ml at all time points tested, significantly lower than that of vehicle-treated mice. these results suggest that z can exert protection against zikv infection of male mice. to further evaluate the protective effect of z against zikvinduced damage of testicular tissue in male mice, all mice were sacrificed at d.p.i. and their testes were collected for analysis of weight and size. we found that testis weight in vehicle-treated mice was around mg, which was significantly lower than that in z -treated mice (∼ mg) (p < . , one-way anova, figure a ). the length and width of testes in vehicle-treated mice were both significantly decreased compared with those of testes in z -treated mice the change of mouse body weight was monitored daily for days. (c) the change of zikv rna level in mouse sera were detected by qrt-pcr at days , , , , and after zikv infection. male a mice were intraperitoneally injected with pfu zikv with z ( mg/kg) or vehicle on day , followed by daily injection of z ( mg/kg) or vehicle for six consecutive days. each sample was tested in triplicate and the data were presented as mean ± sem, * * * p < . ; * * * * p < . , student's two-tailed t-test. (p = . and p < . , one-way anova, figures b,c) . no significant difference in weight (p > . , one-way anova, figure a ), as well as length (p > . , one-way anova, figure b ), and width (p > . , one-way anova, figure c ) of testes were noted between z -treated zikvinfected mice and mock-infected mice. the representative image of testes from the three groups of mice were shown in figure d . subsequently, we examined the testes and epididymides for histopathological changes. the results of h&e staining of testes in vehicle-treated mice revealed that the normal architecture of the seminiferous tubule was seriously destroyed and replaced with an infiltrate of mixed inflammatory cells and necrotic debris, accompanied by degeneration of the spermatogenic lineage ( figure e, upper panel) . the connective tissue areas surrounding the seminiferous tubule had also been infiltrated by a large number of inflammatory cells (figure e, upper panel) . however, the architecture of the seminiferous tubule in the testes of both z -treated and mock-infected mice was intact and clear. spermatogenic cells at different stages were organized tightly and identified clearly (figure e, upper panel) . histological analysis of epididymis showed that epididymides from mice in the vehicle-treated group were also damaged. sperm in the lumen of caput epididymides decreased precipitously, only to be replaced by secretions and numerous necrotic epithelial cells (figure e, middle panel) . the lumens of cauda epididymis contained degenerating spermatozoa and a small number of normal spermatozoa, accompanied by scattered necrotic epithelial cells and inflammatory cells (figure e , lower panel). however, histological analysis of the caput epididymis and cauda epididymis showed no apparent microscopic differences between z -treated and mock-infected mice (figure e middle, lower panel). the architecture of the caput epididymis and cauda epididymis in these two groups was normal with no obvious morphological damage, suggesting that z protected testicular tissue against zikv-induced pathological damage. we used casa to evaluate the protective effect of z on the count and motility of mouse sperm. as shown in figure , the figure | z effectively attenuated damage to testes and epididymides of zikv-infected male a mice. (a) the weight, (b) length, and (c) width of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . each symbol represents one testis; all horizontal bars indicate mean, and error bars reflect sem. (d) the representative image of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . scale bar, mm. (e) histopathological analyses of testes and epididymides collected from z -or vehicle-treated zikv-infected male a mice and mock-infected mice used as a control. scale bar: µm. upper panel, testes; middle panel, caput epididymides; lower panel, cauda epididymides. * * p < . ; * * * * p < . ; one-way analysis of variance with tukey's multiple comparison post hoc tests. sperm count of z -treated mice was significantly higher than that of the vehicle-treated group (p = . , one-way anova; figure a ). meanwhile, the percentages of total ( figure b ) and progressively ( figure c ) motile sperm in z -treated mice were dramatically higher than those in the vehicle-treated group (p = . and p = . , one-way anova), but similar to that in the mock-infected mouse group (p > . , oneway anova). papanicolaou staining of morphological spermatic features revealed more noticeable teratogenesis of sperm in vehicle-treated mice compared to the other groups ( figure d) . to investigate whether the protective effect of z on testicular tissue results from the reduction of local viral load, we examined the viral copies in different testicular tissues. results showed a high level of viral rna ( - equivalents per g) detected in the testis and epididymis of vehicle-treated mice at d.p.i., much higher than that ( - equivalents per g) in z -treated mice (figures a,b) . notably, zikv rna was also detected (up to equivalents per ml) in the mature sperm collected from cauda epididymis in vehicle-treated mice, which was significantly higher than that in z -treated mice (p = . , student's twotailed t-test; figure c ). finally, to examine the safety of z for male mice, male balb/c mice were injected intravenously with z at mg/kg of body weight (n = ) or pbs (n = ). results showed that body weight change of mice was nearly consistent in the two groups ( figure a) , indicating that z peptide did not cause significant harm to the male mice. since the levels of testosterone and inhibin b reflect testicular function and sperm count, the concentration of these hormones in mouse sera at the indicated time points was measured. we found no significant difference between the z -and pbs-treated groups at all time points tested (figures b,c) , suggesting z may not affect the function of testis or sperm. we then compared the potential histopathological changes between the two groups. as shown in figure d , h&e analysis of testis and epididymis revealed no obvious pathological abnormality in mice treated with z compared with the pbs group. besides, the titer of z -specific antibody in sera of mice at and days post-injection was detected. as shown in figures e,f , no significant level of z -specific antibody was detected in the sera of mice that were intravenously injected with high doses of z peptide, consistent with the finding from our previous report for studying anti-mers-cov peptides (xia et al., ) . this result suggests that z peptide consisting of amino acids is unable to elicit a significant z -specific antibody response after it is intravenously administered in the absence of figure | z inhibited zikv replication in testes, epididymides and sperm. zikv rna copies in (a) testes, (b) epididymides, and (c) sperm of z -or vehicle-treated zikv-infected male a mice at day were detected by qrt-pcr. each symbol represents data from individual mice; all horizontal bars indicate mean, and error bars reflect sem. experiment was repeated at least twice. * * * p < . or * * * * p < . respectively, student's two-tailed t-test. figure | safety analysis of z for male balb/c mice. balb/c mice were injected with z ( mg/kg/day, i.v.) for days (n = ), and another group of mice (n = ) received pbs as a control. (a) body weight change of balb/c mice at different time points. data were presented as means ± sem. concentration of (b) testosterone and (c) inhibin b in sera before and after z injection. data were presented as means ± sem of triplicate experiments. (d) histological analysis of the testis and epididymis collected from z -or pbs-treated male balb/c mice. scale bar: µm. (e) z -specific antibody response in mice days after i.v. administration of z or pbs. (f) z -apecific antibody response in mice days after i.v. administration of z or pbs. each sample was tested in triplicate and the data were presented as mean ± sd. adjuvant. therefore, z is safe for male mice, especially for their testicular tissue. currently, many studies have reported the deleterious effects of zikv on male testicular tissue, causing severe damage of testis and epididymis, even leading to infertility uraki et al., ) . two dna vaccines were reported to reduce zikv persistence in the testicular tissue and zikv-associated pathological lesion (griffin et al., ) , or partially prevent infertility of male mice (de la vega et al., ) . however, no effective and safe antiviral agent has ever been reported to prevent or treat zikv infection in testicular tissue. our previous study has demonstrated that z peptide is highly effective in inhibiting zikv infection in vivo and in vitro (yu et al., ) . noticeably, it can penetrate the placental barrier to inhibit vertical transmission of zikv in pregnant mice. however, whether z could cross the btb and protect testicular tissue against zikv infection remained unknown. several studies have reported that sertoli cells play an important role in the entry of zikv into the seminiferous tubules and support long-term replication of zikv in the testicular tissue (siemann et al., ; kumar et al., ) . we found that z peptide possesses potent antiviral activity against zikv infection in bhk- and vero cells (yu et al., ) . in this study, we found that z was also highly effective in inhibiting infection of divergent zikv strains with asian and african lineages in tm cells, the mouse sertoli cell line. particularly, z treatment via intraperitoneal injection resulted in dramatically decreased zikv rna level in the testis of a mice, suggesting that z can protect testis against zikv infection in sertoli cells. meanwhile, we employed z -cy to examine whether z could enter seminiferous tubule, and we found that intraperitoneally injected z could be distributed in the testicular tissue of male a mice, consistent with the observation in mice intravenously administered with z (yu et al., ) . however, because of the intricate structure of capillary vessel and seminiferous tubule in mouse testis, we could not obtain sufficient evidence to prove that z crossed btb into seminiferous tubule. h&e analysis showed no obvious pathological damage in the testicular tissue of z -treated mice, but it did reveal severe pathological damage in the testis and epididymis of vehicle-treated mice, consistent with the findings of other studies (govero et al., ; ma et al., ) . when combined with evidence that viral load in mature sperm of z -treated a mice was significantly decreased, we speculate that z may, indeed, cross the btb and enter seminiferous tubule to inhibit zikv infection in the sperm. zika virus infection in the testicular tissue not only damages male testicular tissue, resulting in pathological lesion of testes and epididymides, but also produces zikv-infected semen, causing infertility. in addition, zikv in semen of an infected male can be sexually transmitted to his pregnant partner (russell et al., ; nelson et al., ) , who can further pass the virus to her fetus, causing congenital zika syndrome in the newborn (yarrington et al., ) . sexual transmission may also contribute to the spread of zikv in regions where the aedes mosquito is not endemic (rowland et al., ) . here we found that z treatment could significantly reduce viral load in sperm of zikv-infected a mice and improve the number and motility of sperm, implying that application of z can limit the damage to testicular tissue and sperm caused by zikv infection and reduce the risk of sexual transmission of zikv. conclusion z administered via intraperitoneal or intravenous injection could be distributed in mouse testicular tissue, protect the tissue against zikv infection and zikv-induced pathological damage and poor sperm quality, suggesting that z peptide has the potential to be further developed as an anti-zikv therapeutic for treatment of zikv infection and attenuation of zikv-induced damage in the testicular tissue. all datasets generated for this study are included in the manuscript/supplementary files. the animal study was reviewed and approved by shanghai public health clinical center animal welfare and ethics committee institutional laboratory animal care and use committee at fudan university. probable sexual transmission of zika virus from a vasectomised man potential effect of zika virus infection on human male fertility? 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a highly potent antibody against zika virus we are very grateful to the staff at the animal experiment department of shanghai public health clinical center for their contribution to this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -fhruiw authors: jaeger, anna s.; weiler, andrea m.; moriarty, ryan v.; rybarczyk, sierra; o'connor, shelby l.; o'connor, david h.; seelig, davis m.; fritsch, michael k.; friedrich, thomas c.; aliota, matthew t. title: spondweni virus causes fetal harm in ifnar (-/-) mice and is transmitted by aedes aegypti mosquitoes date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: fhruiw spondweni virus (sponv) is the most closely related known flavivirus to zika virus (zikv). its pathogenic potential and vector specificity have not been well defined. sponv has been found predominantly in africa, but was recently detected in a pool of culex quinquefasciatus mosquitoes in haiti. here we show that sponv can cause significant fetal harm, including demise, comparable to zikv, in a mouse model of vertical transmission. following maternal inoculation, we detected infectious sponv in placentas and fetuses, along with significant fetal and placental histopathology, together suggesting vertical transmission. to test vector competence, we exposed aedes aegypti and culex quinquefasciatus mosquitoes to sponv-infected bloodmeals. aedes aegypti could efficiently transmit sponv, whereas culex quinquefasciatus could not. our results suggest that sponv has the same features that made zikv a public health risk. zika virus (zikv) was originally isolated over seventy years ago, and was thought to cause a mild, self-limiting, febrile illness (dick et al., ; simpson, ) . not until the outbreak in the americas in and was zikv identified as a cause of significant adverse pregnancy outcomes (johansson et al., ; melo et al., ) . before the definition of congenital zika syndrome (czs) in , gestational arbovirus infection was not associated with birth defects. spondweni virus (sponv) is the closest known relative to zikv, but whether sponv is an emerging threat to pregnant women and their babies is unknown. it was previously thought that sponv was geographically confined to africa and caused only mild disease in rare human infections, reminiscent of the consensus around zikv in the decades following its discovery, but recent data suggest that it may be spreading beyond africa (white et al., ). sponv may therefore be poised to harm pregnancies in new, immunologically naive populations. to do this, sponv would need to fulfill two major criteria: it would need to be vertically transmitted and cause fetal harm, and be transmitted between humans by the urban mosquito vector aedes aegypti, which is associated with large-scale outbreaks of related arboviruses. the first identification of sponv was thought to have occurred in in south africa (theiler and downs, ; wolfe et al., ) . however, it was later recognized that sponv was in fact isolated three years earlier in nigeria, but was misidentified at the time as a strain of zikv because of serological cross-reactivity (haddow et al., ; simpson, ; draper, ) . serological cross-reactivity with zikv and other flaviviruses likely still confounds accurate diagnostics today. as a result, only six well-documented clinical cases of draper, ) . it is likely that many infections have gone unrecognized-serosurveys have detected evidence of sponv infection in countries throughout sub-saharan africa (kokernot et al., a; kokernot et al., b; brottes et al., ; ardoin et al., ; wolfe sponv caused fetal harm, similar to what is observed from zikv infection in this model. vector competence experiments showed that ae. aegypti could transmit sponv when exposed to bloodmeal titers that approximate physiological titers, while cx. quinquefasciatus nonpregnant, mixed sex -to -week-old mice lacking type i interferon signaling (ifnar -/-) ar (this is the only strain used in these studies, so it will be referred to hereafter as sponv); or pfu of the highly pathogenic african-lineage zikv strain dak ar (zikv-dak) (jaeger et al., ) . since contemporary sponv isolates from haiti do not exist, we used the only available low-passage isolate, sponv strain sa ar . this strain is . % nucleotide identical with the sponv genome recovered from mosquitoes in haiti (genbank:mg ). serum was collected at , , and days post-inoculation (dpi) to confirm infection and determine the replication kinetics of sponv in nonpregnant ifnar -/- mice. we also collected and tested serum at , , and days from mice surviving sponv inoculation, because sustained vrna loads were observed with the ifnar -blocking mab model (salazar et al., ) . sponv viral titer in the serum peaked at dpi (fig. a) , and in surviving animals there was no detectable viremia at , , or dpi. higher serum titers were observed in animals inoculated with the lowest dose of sponv ( pfu). we postulate that this could be the result of higher inoculating doses causing a rapid initial rise in viremia, which in turn induces a more robust immune response, leading to more rapid clearance of virus from the serum, but confirmation will require further studies. zikv-dak viremia also peaked at dpi and reached significantly higher titers at dpi than either pfu of sponv or pfu zikv-dak. based on our preliminary experiments with sponv in nonpregnant animals, and the results from our past studies (jaeger et al., ), we chose this dose to minimize the potential confounding impacts of maternal illness on fetal outcomes. we collected serum samples from dams at and dpi to confirm maternal infection. all dams were productively infected, with detectable viremia for all groups by dpi (fig. a) . zikv-dak replicated to significantly higher titers at dpi as compared to sponv (student's t-test p-value = . , t = . , df = ). dams were monitored daily pregnancies and with uninfected counterparts. in general, fetuses appeared either grossly at the time of necropsy, we observed high rates of resorption from both zikv-dak-and sponv-infected pregnancies. resorption rates from zikv-dak-and sponv-infected pregnancies were not significantly different (zikv-dak: . % vs. sponv: . %, fisher's exact test, p = . ). resorption rates for both sponv and zikv-dak were significantly higher than pbs-inoculated controls (p < . ). despite significantly higher maternal viremia observed at dpi with zikv-dak-infected dams, the fact that resorption rates did not significantly differ between the two groups indicates that both zikv-dak and sponv have a propensity to harm the developing fetus that is independent of the amount of replication in maternal blood. surprisingly, and in contrast to the results described by to further characterize the range of pathogenic outcomes of congenital sponv infection and to assess differences between models, we repeated experiments by treating dams with inoculation with zikv-dak or sponv (sheehan et al., ) . this model has been used previously for assessing both zikv and sponv pathogenesis during pregnancy, but does confirm infection, and all dams were productively infected with sponv or zikv-dak following treatment with either dose of mab (fig. d) . maternal viremia did not significantly differ between treatment groups (sponv/ mg vs. sponv/ mg: p= . ; zikv/ mg vs. zikv/ mg: p= . ; one-way anova with tukey's correction for multiple comparisons). zikv- dak titers, however, were significantly higher than sponv titers (sponv/ mg vs. zikv/ mg: p= . ; sponv/ mg vs. zikv/ mg: p= . ). next, adhering to our previously established experimental timeline, dams were necropsied on e . to assess and compare fetal outcomes. at the time of necropsy, we observed no significant resorption from either zikv or sponv infected pregnancies, after either dose of mab (fig. e) , consistent with the results described by salazar et al. observed after e . virus challenge and e . or e . dam sacrifice (salazar et al., ) . resorption rates from zikv-dak-and sponv-infected pregnancies were not significantly different (fisher's exact test, p> . for all comparisons). it is possible that the differences in outcomes in these two models may be due to the closely related to both zikv and sponv and it is not known to cause adverse pregnancy outcomes in humans. to examine whether maternal denv- infection is sufficient to induce fetal resorption, we s.c. inoculated pregnant dams on e . with . x pfu of denv- . prior to studies in pregnant animals we confirmed that this route and dose would result in productive infection in nonpregnant animals (fig. a) . all dams were productively infected with denv- with detectable vrna loads at and dpi (fig. a) . importantly, fetuses continued to develop as examined on e . , and rates of resorption were not significantly exact test, p = . ) (fig. b) . these observations confirm that fetal harm was specifically associated with zikv-dak and sponv infection, but because denv- infected mice do not show clinical signs, we cannot exclude the possibility that the more severe fetal outcomes to begin to understand the potential for sponv to be vertically transmitted, a subset of placentas and fetuses were collected for plaque assay at time of necropsy from all virus treatment groups. from the ifnar +/tissues, infectious virus was detected in % of zikv- dak placentas and fetuses screened (fig. c) . virus was detected in all but one sponv placenta and % of fetuses (fig. c) . viral titers were significantly higher in sponv placentas than their corresponding fetuses (one-way anova with tukey's multiple comparisons; p < . ), as were zikv-dak placenta viral titers as compared to zikv-dak fetuses (p = . ). in addition, zikv placenta and fetal viral titers were significantly higher than sponv titers (p < . ). placental tissues from dams treated with anti-ifnar mab (fig. f) . antibody dose did not affect the viral titer present in fetuses or placentas after either sponv-or zikv-dak- inoculation (p > . for all comparisons; one-way anova with tukey's multiple comparisons). in general, fetal and placenta tissue titers were significantly higher in zikv- dak challenge groups as compared to sponv challenge groups, with a more significant difference in placenta tissue titers than fetal tissue titers (fig. f) . of note, infectious sponv was detected in fetuses from both mab treatment groups, which is in contrast to the placental tissues from denv- infected pregnancies were also screened for infectious virus via plaque assay. infectious virus was not detected in any of the screened fetal and placental tissues, further suggesting the specificity of fetal harm to zikv and sponv (fig. c) . to better understand the impact of in utero sponv exposure, tissues from the developing ifnar +/placenta and fetus were evaluated microscopically. in pbs-and denv-inoculated fetal blood spaces (fig. ) . in contrast, zikv-dak-and sponv-inoculated dams displayed varying degrees of placental pathology with severe effects predominantly observed in the the labyrinth zone, including vascular injury involving maternal and/or fetal vascular spaces, infarction (obstructed blood flow), necrosis, apoptosis, and hemorrhage (fig. ) . overall, the severity of the vascular injury in the labyrinth zone was similar between zikv-dak and in the fetuses, there was no significant microscopic pathology from pbs-and denv- inoculated dams. in contrast, fetuses from zikv-dak-and sponv-inoculated dams demonstrated varying degrees of pathology. in fetuses from the sponv-inoculated dams, fetal injury was evident as mild pulmonary inflammation and mild to moderate segmental necrosis of the brain and spinal cord (fig. ) . these data provide indirect evidence that vertical transmission did occur. pathologic findings were more widespread and severe in fetuses from zikv-dak-inoculated dams and included severe necrosis and inflammation of the lung, liver, kidney, brain, and spinal cord. because sponv rna was detected in a pool of cx. quinquefasciatus in haiti, we compared the relative abilities of ae. aegypti and cx. quinquefasciatus from florida to transmit sponv in the laboratory. sponv titers in naturally infected hosts-to which feeding mosquitoes might be exposed in nature-are undefined. therefore, we conducted our experiments with blood meal titers ranging from ~ - pfu/ml. we considered these doses to be physiologically relevant based on studies with denv ( % mosquito infectious doses = . - . viral cdna copies/ml) (duong et al., ) and zikv ( % mosquito infectious doses = . - . pfu/ml) (ciota et al., ) . to assess vector competence, mosquitoes were exposed to viremic bloodmeals via water-jacketed membrane feeder maintained at table ). ae. aegypti that had been exposed to we speculate that the difference in outcomes between these two models could be due to displayed the most severe histologic phenotype that corresponded with higher placenta and fetus titers in both pregnancy models (fig. ) . sponv histopathology was more following inoculation with sponv, zikv, or pbs, mice were sacrificed at e . . tissues were carefully dissected using sterile instruments that were changed between each mouse to minimize possible cross contamination. for all mice, each organ/neonate was evaluated grossly in situ, removed with sterile instruments, placed in a sterile culture dish, and further processed to assess viral burden and tissue distribution or banked for future assays. briefly, uterus was first removed, and then dissected to remove each individual conceptus (i.e, fetus and placenta when possible). fetuses and placentas were either collected in pbs supplemented with % fbs and penicillin/streptomycin (for plaque assays) or fixed in % pfa or % neutral buffered formalin for imaging. we characterized an embryo as in the resorption process if it met the following criteria: significant growth retardation compared to litter mates and controls accompanied by clearly evident developmental delay, i.e., morphology was ill defined; or visualization of a macroscopic plaque in the uterus (flores et al., ) . tissues were fixed in % paraformaldehyde for hours and transferred into cold, sterile dpbs until alcohol processed and embedded in paraffin. paraffin sections ( μm) were stained with hematoxylin and eosin (h&e). pathologists were blinded to gross pathological findings when tissue sections were evaluated microscopically. the degree of pathology at the maternal-fetal interface was rated on a scale of - : -no lesions (normal); -mild changes ( - focal lesions or - % of zone involved); -mild to moderate changes ( - focal lesions or - % of zone involved); -moderate to severe changes ( - focal lesions or - % of zone involved); -severe (> focal lesions or > % of zone involved). the final score was dependent upon the greater of two parameters (# of lesions or % zone involved). this was an identical scoring system to what we reported previously (jaeger et al., ). the final scores were determined as a consensus score of two independent pathologists. for each zone in the placenta (myometrium, decidua, junctional zone, labyrinth, and chorionic plate/membranes) a 'general' overall score was determined, a score for the amount of 'inflammation', and a score for direct 'vascular injury'. the 'general' score was based on an interpretation of the overall histopathologic findings in each placenta, which included features of necrosis, infarction, apoptosis, hemorrhage, thrombosis, mineralization, vascular injury, and inflammation. the 'inflammation' score quantified the amount of inflammation in that layer. the 'vascular injury' score assessed vascular wall injury (fibrinoid necrosis, endothelial swelling), dilatation of the vessels or spaces, necrosis, loss of vascular lumen diameter, and intraluminal thrombi. the myometrial layer representing the uterine therefore meaningful comparisons between strains could not be assessed. the decidual layer (maternal in origin), the junctional zone composed of fetal giant cells and spongiotrophoblast, and the labyrinth layer (the critical layer for gas and nutrient exchange between the fetal and maternal vascular systems) were scored. since the percentage of injured/pathologic labyrinth zone is a predictor of poor fetal outcome, we also independently scored the labyrinth zone based only on the percentage of fetal and maternal vascular injury/loss using the following scoring system: - %- (background); - %- (mild); - %- (moderate); - %- (moderate to severe); and > %- (severe). photomicrographs were obtained using a bright light microscope olympus bx and olympus bx (olympus inc., center valley, pa) with attached olympus dp digital camera (olympus inc.) and spot flex mp camera (spot imaging), and captured using commercially available image-analysis software (cellsens dimensionr, olympus inc. and spot software . ). all mosquitoes used in this study were maintained at the university of minnesota, twin cities as described (christensen and sutherland, ) vector competence studies mosquitoes were exposed to sponv-or zikv-infected bloodmeals via water-jacketed membrane feeder maintained at . °c (rutledge et al., ) . bloodmeals consisted of defibrinated sheep blood (hemostat laboratories, inc.) and fresh virus supernatant, yielding infectious bloodmeal titers ranging from ~ - pfu/ml. bloodmeal titer was determined after feeding. infection, dissemination, and transmission rates were determined for individual mosquitoes and sample sizes were chosen using long established procedures (aliota et al., fisher's exact test was used to determine differences in rates of normal vs. abnormal concepti. virus stock sequence data have been deposited in the sequence read archive (sra) with accession codes pending. the authors declare that all other data 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arthropod- borne viruses in a population of culicine mosquitoes in tongaland type i interferons instigate fetal demise after zika virus infection. sci immunol , analyzed data and drafted the manuscript o. developed and performed the deep sequencing pipeline declaration of interests ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work the authors acknowledge the university of minnesota, twin cities bsl program for facilities and neal heuss for support. we thank natalie benett for her contribution in mosquito the authors declare no competing financial interests. collected from mice during three independent replicates at , , and/or days post inoculation and titered via plaque assay. assay limit of detection was pfu. viremia peaked at dpi for all virus groups, with zikv-dak replicating to significantly higher titers at dpi than sponv (one-way anova). ****p < . ; ***p < . ; **p < . (b) survival curves of six-to eleven-week old ifnar -/mice s.c. inoculated with pfu of sponv, pfu of sponv, pfu zikv-dak, or a pbs control. sponv : n= ; sponv : n= , key: cord- -ueh q authors: koenig, kristi l.; almadhyan, abdulmajeed; burns, michael j. title: identify-isolate-inform: a tool for initial detection and management of zika virus patients in the emergency department date: - - journal: west j emerg med doi: . /westjem. . . sha: doc_id: cord_uid: ueh q first isolated in from a monkey in the zika forest in uganda, and from mosquitoes in the same forest the following year, zika virus has gained international attention due to concerns for infection in pregnant women potentially causing fetal microcephaly. more than one million people have been infected since the appearance of the virus in brazil in . approximately % of infected patients are asymptomatic. an association with microcephaly and other birth defects as well as guillain-barre syndrome has led to a world health organization declaration of zika virus as a public health emergency of international concern in february . zika virus is a vector-borne disease transmitted primarily by the aedes aegypti mosquito. male to female sexual transmission has been reported and there is potential for transmission via blood transfusions. after an incubation period of – days, symptomatic patients develop rapid onset fever, maculopapular rash, arthralgia, and conjunctivitis, often associated with headache and myalgias. emergency department (ed) personnel must be prepared to address concerns from patients presenting with symptoms consistent with acute zika virus infection, especially those who are pregnant or planning travel to zika-endemic regions, as well as those women planning to become pregnant and their partners. the identify-isolate-inform ( i) tool, originally conceived for initial detection and management of ebola virus disease patients in the ed, and later adjusted for measles and middle east respiratory syndrome, can be adapted for real-time use for any emerging infectious disease. this paper reports a modification of the i tool for initial detection and management of patients under investigation for zika virus. following an assessment of epidemiologic risk, including travel to countries with mosquitoes that transmit zika virus, patients are further investigated if clinically indicated. if after a rapid evaluation, zika or other arthropod-borne diseases are the only concern, isolation (contact, droplet, airborne) is unnecessary. zika is a reportable disease and thus appropriate health authorities must be notified. the modified i tool will facilitate rapid analysis and triggering of appropriate actions for patients presenting to the ed at risk for zika. following closely after the ebola outbreak, due to concerns for rapid spread and a link to birth defects, the world health organization (who) declared zika virus a public health emergency of international concern (pheic) on february , , making it only the fourth time in history for such a declaration. a pheic is defined as an extraordinary koenig et al. identify-isolate-inform: zika virus detection and management affected state's national border, and . may require immediate international action. who declared prior pheics for the april h n pandemic, in may with the resurgence of polio after its near-eradication, and in august in response to the ebola outbreak in west africa. the zika declaration is unprecedented as it is the first time that a vector-borne disease, i.e. an infection that is not generally transmitted from person-to-person, has been deemed a pheic. vector-borne diseases, including malaria and dengue, account for more than % of all infectious diseases and cause more than one million deaths annually. while diseases spread by vectors are among the most complex of all infectious diseases to prevent and control, identification and management in the emergency department (ed) setting differs dramatically from preparation for other infectious diseases such as ebola, measles, and middle east respiratory syndrome (mers), each of which require immediate implementation of various types of isolation (contact, airborne, and droplet, respectively). zika virus is a rna flavivirus related to dengue, yellow fever, west nile, and japanese encephalitis viruses, but not to chikungunya, which is a togavirus. it was first isolated in from the serum of a febrile sentinel rhesus monkey kept in a cage on a wooden platform in the tree canopy of the zika forest in uganda, in which the investigators were studying yellow fever transmission. the following year it was isolated from aedes africanus mosquitoes in the same forest. sporadic human cases were reported from africa and southeast asia over the next several decades. the first recognized outbreak occurred on yap island in the federated states of micronesia in , in which an estimated % of the population greater than three years of age was infected and % of infections were subclinical. a larger outbreak then occurred in french polynesia in - affecting % of the population, with spread to other south pacific islands in . in the french polynesia outbreak, there was an associated large increase in the reported incidence of guillain-barre syndrome (gbs). a case-control study of this outbreak found a gbs attack rate of . per , zika virus infections, with gbs developing rapidly after the onset of infection with a generally favorable outcome. finally, the outbreak in the western hemisphere, which began in northeastern brazil in , with explosive spread to many countries and territories, has infected more than one million persons ( figure ). after microcephaly was reported in the zika virus epidemic in south america, a retrospective analysis of the french polynesia outbreak found that the number of microcephaly cases associated with zika virus infection was ( % ci ) per , women infected in the first trimester, which is about times higher than the baseline rate in the population. zika virus rna can be detected in amniotic fluid as well as in brain tissue of fetuses with microcephaly at a time when the maternal serum and urine are negative for rna, supporting the association between zika virus infection and microcephaly. as with all emerging infectious diseases, healthcare workers must keep up to date with information about how to detect and manage zika virus. this paper describes the adaptation of the identify-isolateinform ( i) tool (initially developed for ebola virus disease , and modified for measles and middle east respiratory syndrome (mers)) for use in the detection and management of potential zika virus patients presenting to the ed, including women who are pregnant or contemplating pregnancy, and their partners. using this novel i tool, emergency physicians will be better prepared to detect and manage patients presenting to the ed with concerns about zika virus. zika virus is of particular concern to women who are pregnant or considering becoming pregnant (and their partners) as it has been detected in fetuses, amniotic fluid, and semen. these patients, especially if they have been in, or are considering travel to, areas with documented zika transmission, may present to the ed requesting information and testing. zika infection is asymptomatic in % of cases. when symptomatic, it is typically described as a mild dengue-like illness, manifested by two or more of the following: sudden onset fever, conjunctivitis, arthralgias, and maculopapular rash ( figure ). myalgias and headache are also common. the who interim case definition describes suspected, probable and confirmed cases. unlike in dengue, death from acute zika virus infection is rare, but has been reported in a child with sickle cell disease in colombia. the incubation period for zika virus after a person is bitten by koenig et al. an infected mosquito ranges from - days, and may be up to days. this is the reason for screening for risk factors within days of symptom onset. the incidence of gbs or microcephaly appears to be similar between symptomatic and asymptomatic cases. zika virus is transmitted by the bite of the aedes aegypti mosquito and likely also aedes albopictus. mosquito bites account for the vast majority of infections, and thus prevention from bites is the principle protective measure. in addition, zika has been detected for prolonged periods in semen and documented cases of sexual transmission are increasing. , intrauterine/perinatal transmission also occurs. furthermore, laboratory exposure and other blood-borne transmissions are possible; thus, blood banks have developed screening and protection criteria to mitigate spread. theoretically, zika virus could also be contracted from infected breast milk or organ/tissue transplants. patients should be assessed for pregnancy as well as for neurological symptoms and signs indicating possible gbs. while the situation is rapidly evolving, as of march , laboratory testing is only available through the centers for disease control and prevention (cdc) and several state and territory health departments. generally, testing is not recommended for asymptomatic persons, with the exception of pregnant women to weeks after travel to areas with ongoing zika virus transmission. in symptomatic patients, serum should be obtained for polymerase chain reaction (rt-pcr), igm, and igg if within seven days of symptom onset. if more than seven days has elapsed, only igm and igg should be assessed; rt-pcr is not indicated. note that serological cross reactivity may occur between zika and other flaviviruses (e.g., dengue, yellow fever, st. louis encephalitis, japanese encephalitis, west nile). public health departments may decline to test if the clinical scenario is not suggestive of zika virus or associated complications. in particular, due to the high cross-reactivity with related flaviviruses, serologic test interpretation is complex and can be difficult, leading to false positive results. conversely, a negative zika igm or rt-pcr test result does not rule out zika virus infection. emergency clinicians should consult with public health experts to assist with interpretation of test results. the differential for zika virus infection is broad as many diseases present similarly. in patients who have traveled to, or lived in, an area with ongoing zika transmission within days, chikungunya and dengue viruses, as well as malaria, leptospirosis, and rickettsial infections, are among the other infections that should be considered. co-infection with dengue and zika has been reported. it is likely that some travelers to zika endemic areas will be found to be co-infected with other pathogens acquired from arthropod bites, especially malaria. distinguishing these infections from one another based on clinical symptoms and signs can be difficult, but malaria is not typically associated with rash or conjunctivitis. treatment for zika virus infection is entirely supportive as no specific antiviral therapy is yet available. aspirin and nonsteroidal anti-inflammatory drugs should be avoided until dengue can be ruled out, to reduce the risk of hemorrhage. as noted, the vast majority of cases (approximately %) are asymptomatic; most of the remainder of patients have self-limited illnesses, with symptom resolution within seven days. patients who develop gbs may require intensive care, including mechanical ventilation. pregnant women who test positive for zika virus should be offered ultrasound assessments every - weeks to assess for microcephaly with consideration for amniocentesis for zika virus rna testing on amniotic fluid. fetal microcephaly is often not detectable in the first trimester of pregnancy but is most easily diagnosed in the late second and early third trimester. in some cases, it can be missed on prenatal ultrasound and not detected until after birth. while under development, as of early , no vaccine against zika virus exists. avoidance of travel to zikaendemic areas or protection against mosquito bites is the best prevention. mosquitoes that spread zika bite mostly during the daytime. preventative measures include the following: . use approved insect repellents (deet and permethrin are safe and effective for pregnant women when used in accordance with the product label) . use window and door screens, or stay in airconditioned buildings . wear long-sleeved shirts and long pants . use a mosquito bed net. for patients with known or suspected zika virus, prevent spread to others by avoiding mosquito bites (as this could infect the mosquito, which could then bite and spread the virus). furthermore, infected men should take measures to avoid sexual transmission of virus to their partners by abstaining, using condoms, and refraining from anal intercourse and fellatio. only male to female sexual transmission has been reported as of early . admission criteria for patients who are at risk for zika virus are similar to those for any other patient. most patients can be managed conservatively as outpatients; however, those with complications such as gbs may require hospitalization. the identify-isolate-inform ( i) tool, initially developed for ebola virus disease and subsequently adapted for measles and mers, can be modified for the ed evaluation and management of patients under investigation for zika ( figure ). as most cases of zika virus are mild and self-limited and would not be contagious in the ed setting, the tool does not need to be immediately applied on initial patient contact (as it would be for highly contagious infectious diseases). the first step in using the zika i tool is to identify patients with a possible zika exposure within days of symptom onset. this involves taking a travel history and assessing whether the patient was bitten by mosquitoes or has had sex with a patient with zika virus. if the patient lives in, or has traveled to, areas with ongoing zika virus transmission within two weeks, assess whether two or more of the following symptoms are present: sudden fever, maculopapular rash, arthralgias, and conjunctivitis. if the epidemiologic and symptom screens are positive, or if a pregnant patient requests testing within to weeks after potential exposure, investigate for the presence of zika virus by serologic (antibody) testing. while every patient presenting to the ed should be assessed for the potential to transmit disease (the "vital sign zero" concept) , if only arboviruses, malaria or rickettsia are being considered in the differential diagnosis, isolation is not necessary. as with all patients, standard precautions should be maintained; however, contact, droplet and airborne precautions (as would be needed for selected other infectious diseases) are not required. isolation is not necessary for zika (due to its transmission characteristics, e.g. which are different from mers and ebola). the second "i" in the i tool can be considered to be "investigate" rather than "isolate" if diseases that are contagious from person-to-person are not being considered. "isolate," however, remains a useful term to remind the healthcare providers who initially assesses the patient to consider whether isolation precautions are needed, as patients may present with similar symptoms associated with other infectious diseases that are contagious from person to person. in addition, to avert acquiring disease, people should "isolate" from mosquito vectors to prevent infection and, if infected, to prevent potential transmission to others. patients presenting in high-risk areas of the u.s. should be advised to use precautions such as staying indoors in an air-conditioned environment and wearing long-sleeved shirts and long pants. on march , , the u.s. national center for atmospheric research published a public release on potential zika virus risk estimated for u.s. cities indicating that "summertime weather conditions are favorable for populations of the mosquito along the east coast as far north as new york city and across the southern tier of the country as far west as phoenix and los angeles." thus, there is a heightened concern for an increased incidence of zika virus in the u.s. the final action of the tool is to "inform." on january , , u.s. government regulations authorized cdc to receive case notifications for arboviral illnesses and zika virus, adding these entities to the list of nationally notifiable infectious diseases. in addition to notifying the hospital infection prevention and control team, emergency physicians should ensure that suspected zika cases are reported to appropriate health authorities. patients who do not meet medical criteria for admission should be informed about measures to take to limit the risk of virus transmission, e.g. to avoid sexual activity, mosquito bites, and blood donation. zika virus disease will likely have substantially less direct impact on ed operations than other emerging infectious diseases such as the h n influenza epidemic and ebola virus disease outbreak. nevertheless, due to worldwide media attention and an evolving situation, it is important for emergency personnel to be familiar with the basic principles of assessment and management of zika virus. in particular, women who are pregnant (or considering pregnancy) and have been in (or are considering travel to) areas with ongoing zika virus transmission, as well as women who have had sex with a male who has travelled to a region of endemic transmission, may present to the ed seeking testing and other advice. cdc issued an unprecedented travel advisory for pregnant women , and health authorities have recommended that women living in affected areas delay pregnancy. in addition, patients may seek information on the potential for development of microcephaly and other birth defects and the possibility of sexual transmission of zika virus. it is possible that in the future zika virus will be added to the list of "torch" infections, which include toxoplasmosis, other (syphilis, varicella-zoster, parvovirus b ), rubella, cytomegalovirus (cmv), and herpes infections, that are associated with congenital anomalies. patients requiring emergency blood transfusions will need to be counseled about the risks and what measures have been taken to prevent them from contracting zika virus. pregnant women are likely to be particularly concerned about receiving blood. finally, there is the potential for shortages to the blood supply due to a decrease in eligible donors, particularly as zika virus becomes more widespread over time and during warmer weather. as with all contagious infectious diseases, the question of when to use the public health tools of quarantine and isolation is critical. , while at least one u.s. politician has suggested that patients returning home from areas with ongoing zika virus transmission such as brazil should be quarantined, there is no scientific basis for this approach. , as noted previously, zika virus is not readily transmitted from person to person, either before or after symptom onset. caution is needed to avoid negative psychosocial consequences like stigmatization such as was seen in the ebola virus disease outbreak. , zika virus has a massive economic impact in many sectors, including healthcare. thus, there may be a shift of resources away from other critical areas of healthcare operations and research. for example, while not approved, in the u.s. alone, president obama requested . billion usd in emergency zika funding. zika is an emerging infectious disease that has gained worldwide attention in large part due to its association with fetal microcephaly and rapid global spread. as with any novel infection, it is important not only to identify and treat individual patients, but also to protect healthcare providers and the public health. the identify-isolate-inform ( i) tool is an instrument that can be used real-time on the front lines to rapidly detect and manage patients at risk for zika virus disease presenting to the ed. use of the i tool will assist emergency physicians in performing rapid and appropriate screening and management and counseling for patients concerned about zika virus. ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations who vector-borne diseases zika virus in the americas -yet another arbovirus threat guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study association between zika virus and microcephaly in french polynesia, - : a retrospective study centers for disease control and prevention: webinar-update on interim zika virus clinical guidance and recommendations the ebola virus outbreak and other emerging infectious diseases inform: a -pronged approach to management of public health emergencies ebola virus disease: essential identify-isolate-inform: zika virus detection and management public health principles for clinicians identify-isolate-inform: a tool for initial detection and management of measles patients in the emergency department identify-isolate-inform: a modified tool for initial detection and management of middle east respiratory syndrome patients in the emergency department who emergency preparedness and response. zika virus disease. interim case definition fatal zika virus infection in girl with sickle cell disease, colombia emerg infect dis potential sexual transmission of zika virus available at fda issues recommendations to reduce the risk for zika virus blood transmission in the united states update: interim guidelines for health care providers caring for pregnant women and women of reproductive age with possible zika virus exposure -united states division of vector-borne diseases memorandum on revised diagnostic testing for zika, chikungunya, and dengue viruses in us public health laboratories the emerging zika pandemic: enhancing preparedness available at: http:// journals.cambridge.org/action/displayabstract?frompage=online&aid = &fulltexttype=ac&fileid=s &specialarti cle=y potential zika virus risk estimated for u.s. cities. national center for atmospheric research public release cdc issues interim travel guidance related to zika virus for countries and territories in central and south america and the caribbean brazil warns against pregnancy due to spreading virus is there a case for quarantine? perspectives from sars to ebola. disaster medicine and public health preparedness health care worker quarantine for ebola: to eradicate the virus or alleviate fear? why we shouldn't quarantine travelers because of zika quarantine for zika virus: where's the science ifrc battling fear and stigma over ebola in west africa. available at who zika strategic response framework & joint operations plan obama asks for $ . billion in emergency zika funding. usa today the authors thank dr. susan huang, heather reasin-bodden, linda dickey, and dr. shruti gohil of the uc irvine health, epidemiology and infection prevention team for their critical review of the zika i tool. key: cord- - aakik a authors: sayres, lauren; hughes, brenna l. title: contemporary understanding of ebola and zika virus in pregnancy date: - - journal: clin perinatol doi: . /j.clp. . . sha: doc_id: cord_uid: aakik a understanding the pathophysiology, management, and prevention of emerging infectious diseases among pregnant women is imperative to achieve a successful response from the medical community. ebola and zika viruses represent infections with profound public health implications. in particular, ebola virus is associated with high case fatality and pregnancy and neonatal loss rates, while zika virus has been associated with multiple congenital anomalies; these features present critical clinical dilemmas for management of pregnant and reproductive aged women. the objective of this article is to summarize key background information and best practices for management of ebola and zika virus in pregnancy. the past decade has yielded the emergence of several infectious diseases of critical importance in pregnancy. in particular, the ebola resurgence of to resulted in infection of at least women of reproductive age and a pregnancy loss rate of nearly %. the to outbreak of zika virus captured widespread media attention as , women of childbearing age became infected, and the significant associated teratogenicity became apparent. both diseases were determined to be public health emergencies of international concern by the world health organization (who). this article seeks to review the epidemiology, transmission, pathogenicity, and management of ebola and zika viruses as among the most important emerging infectious diseases in modern history. our objective is to provide clinically relevant background information and guide best practices for obstetricians as they approach these novel infections. the natural reservoir of ebola virus is unknown, although evidence is accumulating to implicate bats as the most likely source. most outbreaks in people have been traced back to single spillover events from an infected source. human-to-human spread of the virus is via direct contact with infected blood and bodily fluids including urine, saliva, stool, vomit, and semen, or from surfaces that have been contaminated with infected fluids. ebola virus has specifically been identified in amniotic fluid, placental and fetal tissue, and breastmilk. [ ] [ ] [ ] pregnant women are not thought to be at greater risk of acquiring ebola virus. pathogenicity ebola virus effectively targets host immune cells, which allows reproduction and dissemination of the virus. the virus inhibits both the innate and adaptive immune responses via inhibition of interferons, impairment of antigen presentation, and neutralization of antibodies against viral glycoproteins. the viral glycoproteins then induce cytopathic effects by damaging endothelial cells and disrupting coagulation pathways, while the activated immune mediators induce cellular apoptosis or necrosis. in particular, these effects result in hepatic, renal, and gastrointestinal injury and coagulopathy. clinical findings are initially nonspecific and include malaise, myalgia, and fever. as the viral rna load rises, progression to asthenia, vomiting, secretory diarrhea, and hemorrhage occurs, which without adequate resuscitation can result in hypovolemic shock, multiorgan dysfunction, and death. there is poor understanding of the specific mechanisms of ebola pathogenicity in pregnancy. in pregnancy, the effects of hypovolemia and immune dysregulation become exaggerated because of a need for expanded blood volume and an already-altered immune state, respectively. hypoperfusion of the placenta may represent the mechanism by which miscarriage or intrauterine fetal demise occur. furthermore, transplacental viral transmission to the amniotic fluid and fetus can result in fetal infection and therefore interruption of normal development and growth. , diagnosis diagnosis of ebola virus can be made presumptively based on symptoms and history of exposure and should be confirmed with molecular testing. testing typically includes reverse transcriptase polymerase chain reaction (rt pcr) for the detection of viral rna in serum, although it is important to note that such tests can be negative for up to hours after symptom onset. alternatively, enzyme-linked immunoassay (elisa) testing for immunoglobulin m (igm) specific to the virus is available, although it is less commonly used, because positive results can be delayed up to weeks in acutely symptomatic patients. novel rapid diagnostic tests are currently under study but not yet available. testing strategies remain the same for pregnant women, although rapid testing should be even more imperative given the overlap of the presentation of pregnancy-related conditions and ebola virus and the potential need for acute obstetric intervention. the mainstay in management of individuals infected with ebola virus is supportive management, namely massive fluid resuscitation. additional measures include frequent monitoring for and correction of electrolyte, glucose, and acid-base imbalances, antiemetic and antidiarrheal therapy, nutritional support, vasopressor support, intubation and ventilation, and continuous renal replacement therapy in the setting of renal dysfunction. additionally, broad-spectrum antibiotics may be necessary for concomitant infections such as gastroenteritis secondary to bacterial translocation in the setting of gut inflammation or malaria, a frequent coinfection. there has not been significant research as to the specific care for ebola virus infections among pregnant women. in , the who released guidelines for the management of pregnant women with ebola virus, which represent the first comprehensive guidelines on specific strategies for their care. when possible, specifically trained providers should be available to care for pregnant women and their neonates, and care should be provided at a private, high-risk facility. shared decision making for women infected by or recovering from ebola virus is of necessity. generally, maternal rather than fetal indications should take precedent in management decisions. aggressive fluid resuscitation of up to l per day is particularly important among pregnant women given their significantly increased volume of distribution. there is no strong evidence to suggest that induced abortion, labor induction, or invasive procedures for fetal indications are of benefit to pregnant women with ebola virus. in fact, fetal monitoring is generally not recommended. there may be benefit to delaying delivery when possible because of the risk of coagulopathy in the setting of high viral load. a decision for surgical delivery should be made on a case-by-case basis, as there is unlikely to be fetal benefit, and women with severe disease may not survive an operation. fundal massage and administration of uterotonic medications should be strongly prioritized over surgical management of postpartum hemorrhage. for survivors of ebola virus who plan pregnancy continuation, comprehensive counseling regarding the risks of fetal or neonatal demise and risk of transmission from pregnancy tissues should be performed. regular prenatal care with a high-risk obstetrician is recommended for pregnant women who recover from ebola virus. expectant management is considered most appropriate. there is no current approved treatment for ebola virus. several therapeutics are undergoing phase i and ii clinical trials; these include zmapp, regn-eb , and mab (chimeric monoclonal antibodies) and remdesivir (a broad-spectrum antiviral). additional antivirals, some of which were designed to target other viruses, are under investigation for use with ebola virus but have not held as much promise as the aforementioned agents. convalescent plasma treatment has also demonstrated possible benefit in treatment. the monitored emergency use of unregistered and investigational interventions ethical framework recommends that vulnerable contemporary understanding of ebola and zika virus populations including pregnant women be offered similar treatments to the nonpregnant population when potential benefits can outweigh risks. to date, reports describe use of investigational therapeutics in pregnant women with ebola virus. a % mortality rate was noted among women who received convalescent plasma. additionally, a report details pregnant woman who received favipiravir, a broadspectrum antiviral, but subsequently died of hemorrhagic shock during labor. however, her newborn is the first reported survivor of congenitally acquired ebola after receiving zmapp and remdesivir as well as a buffy coat transfusion from an ebola virus survivor. , prevention critical to the prevention of ebola virus is interruption of community and healthcareassociated spread. this includes isolation and contact tracing of infected individuals, appropriate containment of infectious material, and protection for those in contact with infected individual. even if an individual recovers from ebola virus, a person's bodily fluids may be infectious for months or longer. in particular, pregnancy-related tissue including amniotic fluid, placenta, and fetus are highly infectious even if the pregnant woman has recovered from acute illness. handling of tissues should be avoided when possible, and potentially infectious waste should undergo proper disposal. autopsies should be avoided. personal protective equipment should include a face mask with head cover and eye protection, a gown, double gloves, and boots. minimization of sharps and invasive procedures can mitigate risk to health care providers. multiple trials are underway to study candidate vaccines against ebola virus. the most promising is the live replicating rvsv-zebov-gp vaccine, which is manufactured by merck under the trade name ervebo and has been prequalified by the who. , although no vaccine, including rvsv-zebov-gp, has been specifically evaluated in pregnant women, an analysis of available data on women who became pregnant during the rvsv-zebov-gp study time frame suggests there to be no significant increase in pregnancy loss or congenital anomalies among vaccinated women. it has been strongly recommended by multiple international public health and humanitarian organizations that pregnant women be included in vaccine clinical trials and compassionate use protocols. , after years of exclusion from vaccination, pregnant women in the democratic republic of the congo, the location of a widespread ebola outbreak, were able to start receiving the vaccine in . zika virus is a member of the family flaviviridae, which also includes dengue, west nile, yellow fever, and japanese encephalitis viruses. zika virus was first isolated in in a rhesus macaque in the zika forest of uganda. human infection was identified in . there were only cases identified over the next years, until an outbreak affecting people occurred in micronesia in . subsequent outbreaks occurred in pacific islands, with sporadic cases occurring across southeast asia. , most recently, a pandemic occurred in brazil, with virus spreading across the americas to affect greater than million individuals between and . recognition of the more serious complications of zika virus began to emerge during the pandemic in brazil. in particular, approximately cases of microcephaly were detected in infants of women infected with zika virus. it has been estimated that % to % of infected pregnant women will transmit zika virus to their fetus. , approximately % to % of cases of vertical transmission will result in fetal loss, and % to % will result in congenital zika syndrome including microcephaly. vertical transmission can occur in any trimester and regardless of maternal symptomatology. zika virus is spread by multiple species of aedes mosquito to mammalian hosts, including people. the geographic distribution of these mosquito species is expanding secondary to climate change. more recently, it has been discovered that in addition to vectorborne transmission, zika virus can be spread via sexual contact, perinatal transmission (both during pregnancy and delivery), blood transfusion, and bone marrow and organ transplantation. although breastmilk has been shown to contain zika virus, there is no conclusive evidence that the virus can be transmitted by breastfeeding. pathogenicity zika virus exhibits broad tropism in human cells, undergoing replication and widespread distribution after cell entry by endocytosis. zika virus preferentially targets neural stem cells and progenitor cells. zika inhibits the ability of interferons and other cellular signaling pathways to produce an innate immune response to the virus. in pregnancy, hofbauer macrophages and other immune cells of the placenta that usually serve as a maternal-fetal barrier provide a mechanism for the virus to infect fetal cells. zika virus has an incubation period of to days. most infected individuals are asymptomatic or display mild symptoms such as fever, rash, and myalgias lasting up to week. however, a minority of people will suffer severe neurologic symptoms including meningoencephalitis or guillain-barré syndrome because of neurotropism or postinfectious immune response. given this predilection for neural cells, congenital zika syndrome is primarily a constellation of central nervous system abnormalities. in particular, fetal brain disruption sequence results in microcephaly and cortical atrophy, intracranial calcifications, other neural and ocular abnormalities, and growth restriction. , in infants, this manifests with developmental delay, visual or hearing impairment, seizures, and movement and behavioral disorders. it is important to note that asymptomatic pregnant women can still vertically transmit zika virus to their neonates. zika virus infection is typically detected by serum and urine rt pcr nucleic acid molecular screening and/or serologic igm elisa screening. molecular testing should be performed within weeks of onset of symptoms, whereas serologic testing can be accurately used up to weeks after symptom onset. of note, serologic testing is subject to significant cross-reactivity with antibodies to other flaviviruses, in particular dengue virus, and to persistence of antibodies for many months after the resolution of the acute infection. in nonpregnant individuals with a possible exposure to zika virus yet without severe symptoms, serum and urine molecular testing are recommended. in the setting of concern for guillain-barré , molecular testing of the serum and urine as well the cerebrospinal fluid is recommended as soon as possible. if the index of suspicion for zika virus infection is high but molecular testing is negative, serum testing for igm should be performed within to weeks to evaluate for seroconversion. for pregnant women, the screening paradigm shifts. , for those with symptoms, serum and urine molecular testing and serologic testing are recommended as soon as contemporary understanding of ebola and zika virus possible. if there is a discrepancy with a negative molecular result and positive serologic result, a plaque reduction neutralization serologic test is recommended to confirm infection. pregnant women with ongoing exposure but no symptoms should be screened with molecular testing every trimester. dedicated zika virus testing is not routinely recommended as a routine part of preconception screening or in the setting of a single asymptomatic exposure for pregnant women. detailed anatomy ultrasound is recommended at to weeks of gestation for women with symptoms or recurrent exposure, and serial scans should be performed every to weeks thereafter to evaluate for development of evidence of congenital infection. should there be evidence of fetal anomalies consistent with congenital zika syndrome on ultrasound, amniotic fluid should be sent for nucleic acid testing if amniocentesis is already being performed for diagnostic purposes. however, amniocentesis is not universally recommended, as a negative result cannot rule out congenital infection caused by the transient and sometimes delayed nature of shedding of the virus into amniotic fluid. if microcephaly or other complex central nervous system abnormalities are noted on routine ultrasound in the absence of alternate explanation and with possible zika exposure, molecular and serologic testing should be undertaken. in the setting of suspected congenital zika infection, newborn or fetus and placenta should be tested after delivery. treatment of zika virus consists of supportive management including antipyretics, analgesics, and rehydration, typically with good response. severe cases involving guillain-barré syndrome may require plasma exchange or immunoglobulin. there is no approved targeted treatment for zika virus infection, although several agents are being investigated. these include both host-and virus-targeting antivirals or antibodies, either repurposed from drugs in use for other diseases or screened from compound libraries. , for example, drugs that upregulate the interferon innate immune pathway or suppress viral replication have shown therapeutic promise. as of yet, no medication has completed phase ii clinical trials. , additionally, although there is no drug that is proven to mitigate the effects of zika infection during pregnancy, studies of neutrally active compounds such as nmda blockers can potentially modulate the fetal neuronal damage that occurs with vertical transmission. prevention primary prevention strategies against zika virus include vector control. to prevent mosquito bites, high-risk outdoor areas and in particular bodies of standing water should be avoided. long sleeves and pants, mosquito repellant, and mosquito netting should be used in endemic regions. insect repellants, when used per manufacturers' guidelines, are safe for use among pregnant women. individuals who may have a zika virus exposure or have a known infection should also undertake measures to avoid mosquito bites and therefore reduce spread. standard precautions to prevent nosocomial transmission of zika virus are also important, although as of , no cases of occupational infection have been documented. travel guidelines have evolved as the zika virus epidemic progressed, and additional research is needed to appropriately inform travel restriction policy. general recommendations have been to avoid travel to high-risk regions during pregnancy or while attempting conception. , couples residing in affected areas are advised to delay pregnancy. those with a known exposure or infection should delay attempts at conception for months for female partners and months for male partners. a pregnant woman whose partner has had an exposure or infection with zika virus should practice abstinence or consistent condom use for the remainder of the pregnancy. efforts are underway to develop a vaccine against zika virus, although none has yet been approved for use. the most promising vaccine candidates have been shown to induce effective antibodies in mice, and phase i and ii clinical trials are in process. [ ] [ ] [ ] among the candidates are dna vaccines, synthetic peptide vaccines, nanoparticles, and live recombinant and purified inactivated viruses. as one faces emerging infectious diseases such as the novel coronavirus first identified in late , the details of the recent ebola and zika virus outbreaks can provide a framework for an approach to new diseases. understanding of the transmission, maternal and fetal effects, and effective treatment, vaccination, and containment methods are imperative to achieving a successful response from the public health and scientific research communities. specifically addressing emerging infections in pregnancy is important because of the potential differential impact of infections among pregnant women, the possible effects on the fetus, and the unique prophylaxis and treatment strategies necessary for efficacy and acceptability among pregnant women. attention must be paid to the successes and failures of the response to the ebola and zika outbreaks as physicians strive to provide excellent care for pregnant women who are affected by or at risk for emerging infectious diseases. what is current practice? molecular testing is standard of care for both ebola and zika viruses. supportive care including rehydration and antipyretics is necessary for individuals infected with ebola and zika virus; there are no targeted therapeutics. no vaccines are available. prevention of ebola virus includes containment of infected substances and personal protection equipment use, and prevention of zika virus entails protection against mosquito bites, avoidance of high-risk regions, and delay of childbearing. what changes in current practice are likely to improve outcomes? development of targeted therapeutics and vaccines can decrease the burden of disease and in particular reduce risk for pregnant women and their offspring. test individuals who have traveled to endemic areas or have been in contact with individuals infected with ebola or zika virus who display associated symptoms. there is a role for testing of asymptomatic individuals with recurrent zika virus exposure also. successful management of ebola virus includes aggressive rehydration and intensive care. delivery timing should be based on maternal rather than fetal indications. properly dispose of all infected substances and use necessary personal protective equipment to prevent nosocomial and community transmission. pregnant women with zika virus should undergo serial ultrasounds to evaluate for congenital zika syndrome, although vertical transmission cannot be prevented once a woman has been infected. uninfected women who are pregnant or considering childbearing should avoid travel to zika virus endemic areas or unprotected intercourse with infected or exposed partners. ebola virus is associated with high mortality and a very high pregnancy or neonatal loss rate. zika virus carries a risk of congenital zika syndrome. treatment of both is limited to management of 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other human coronavirus infections addressing the effects of established and emerging infections during pregnancy key: cord- -upsc cf authors: taylor-robinson, andrew w title: a vaccine effective against zika virus is theoretically possible but may not be delivered anytime soon date: - - journal: res rep trop med doi: . /rrtm.s sha: doc_id: cord_uid: upsc cf following the first report in may of the unexpected emergence of zika in north east brazil, there has been an explosive epidemic of this infection across latin america. the outbreak has caused alarm among social and news media as to the virulence and transmission potential of the aedes mosquito-borne virus. this debate is heightened by the proximity, both in time and distance, to the forthcoming olympic games to be held in rio de janeiro this august, provoking fears for the safety of athletes and spectators alike. the threat, real or perceived, is exacerbated by the movement between nations in the same or separate continents of persons who act unwittingly as asymptomatic carriers. pregnant females are considered at greatest risk because microcephaly in newborn infants is linked to, if not yet proven as caused by, zika infection. in february this year, the world health organization declared that further to the then unconfirmed association between the virus and the clinical manifestations of microcephaly and also guillain-barré syndrome, the zika epidemic was a “public health emergency of international concern”. no anti-zika therapy, vaccine or drug, is currently available and while the production of the former has now been prioritized by multiple funding agencies, the history of infectious disease vaccine development indicates that this may take several years to reach the market place. the fact that zika is a close relative of yellow fever and japanese encephalitis viruses, for both of which there are already effective vaccines, provides a rational basis for the fast-tracked laboratory-based preparation of a candidate vaccine. however, undertaking clinical trials on pregnant females provides ethical and practical hurdles to overcome before licensure is granted for public administration. meanwhile, public health management strategies, including mosquito control programs to reduce breeding, are needed to limit the global spread of this re-emerging disease. the aedes mosquito-transmitted viral disease of humans, zika, was originally identified in and is named after the rainforest in uganda, east africa, where it was first isolated from rhesus macaques. for close on years, the prevalence of zika infection was very low, such that prior to now it has attracted little interest apart from arbovirus and tropical medicine specialists. in the last several months, this scenario has changed dramatically, however, subsequent to a major zika epidemic in over countries in latin america and the caribbean. this includes an estimated , , clinical cases in brazil, from where the outbreak arose in early , although questions regarding the accuracy of reporting have been raised. furthermore, the world health taylor-robinson organization (who) predicts that by the end of this year up to million people across the americas may be infected with the zika virus. the impact on the vast majority of those people will be minimal, but particularly in infants the effect may be profoundly debilitating and long-lasting. such is the extent of the issue, real and predicted, and the degree of concern that it has engendered globally, that on february , the international health regulations emergency committee of the who declared the zika epidemic as "a public health emergency of international concern" and highlighted the importance of aggressive measures to reduce infection, especially of pregnant females and those of childbearing age. subsequently, the us centers for disease control and prevention moved zika to level activation, the highest available state of response. similar to the etiological agents of yellow fever, japanese encephalitis and dengue, zika is a member of the flavivirus genus of enveloped, positive sense, single-stranded rna viruses. compared to each of these close relatives, infection with which can be severely incapacitating for a person of any age, it is thought that approximately % of adults infected with zika show no clinical manifestations. hence, for several days after being bitten by an infectious mosquito, they may serve as asymptomatic carriers of infection. if a person is ill, the main symptoms may last for up to week and are similar to but less severe than other related febrile illnesses -a mild headache, fever, myalgia, arthralgia, conjunctivitis, and maculopapular rash. the principal possible consequence of zika infection, for which there is now claimed to be a causal link, occurs via congenital transmission from a pregnant female to her fetus in utero or newborn baby, , the effects of which can be severe. in brazil alone, the virus has been associated with over , cases of microcephaly, a previously uncommon condition that as a result of aberrant brain development produces babies with abnormally small heads and, in most cases, neurological impairment. in march , panama registered a baby born with microcephaly linked to the zika virus, in what is thought to be the first such case outside brazil during the current outbreak. the baby died within hours and at postmortem examination the virus envelope protein was detected by enzyme-linked immunosorbent assay in the baby's umbilical cord. rarely, zika is also associated with, but still not proven to be causally linked to, guillain-baré syndrome, a neural demyelination syndrome that is considered to be an autoimmune sequela of infectious disease. , the zika epidemic is arousing apprehension among the general public just months before the world's attention will focus on rio de janeiro for the summer olympic games. at present, the advice to pregnant females from non-endemic regions is to not travel to brazil and surrounding countries. aside from this immediate concern in latin america, the increasing media frenzy has fed misinformation with regard to the possible global spread of zika, and the impact this may have on public health in regions that are currently not affected. as a consequence of global climate change the geographical distribution of a. aegypti, the species of mosquito that is considered to act as the principal vector of zika transmission, is expanding gradually in tropical and subtropical zones. for non-endemic industrialized nations in north america, europe, and australasia, the critical issue is whether or not zika virus-permissive mosquito populations can adapt for persistence in temperate climates. a more rapid spread of the virus via the intercontinental travel of infected persons is an additional concern, although for zika to become established in a location distant to an endemic area requires local transmission of the initially imported focus of infection; this is dependent on the availability of the vector. vertical, sexual, and blood-borne transmission of zika has been suggested, , which, if confirmed, may provide auxiliary routes to enable viral persistence in regions that are not endemic for aedes mosquitoes. thus, one recommendation to prevent disease, especially microcephaly, is to practice safe sex in those territories where zika is reported as endemic. importantly, the risks should be considered in family planning until such time as the apparent association between viral infection and microcephaly is either confirmed or refuted. , another re-emerging infectious disease the zika epidemic comes hard on the heels of other notable infection outbreaks by pathogenic viruses around the world in recent years, each of which has posed a threat to human wellbeing. these include the coronaviruses that cause severe acute respiratory syndrome and middle east respiratory syndrome, the h n swine influenza, h n and h n avian influenza, and the filovirus ebola. each in turn has led epidemiologists to consider transmission cycles and zoonoses, infectious zika vaccine development disease immunologists to improve emergency measures such as pathogen detection and containment, and vaccinologists to aim to develop effective immunization regimens. , in much the same way as the upsurge of ebola cases in west africa in , zika may be considered as a re-emerging infectious disease; that is one which is established but displayed in a novel setting. while the reasons for the current zika epidemic in south america are multifactorial, inadequate vector control of an unexpected population explosion of mosquitoes is a suspected cause. this raises the question as to what guidance is appropriate to provide to combat zika, especially in industrialized nations where there has been the loudest call for anti-zika prophylaxis, such as that which could be provided by sterilizing immunization. in the short term, as anti-viral therapies are yet to be realized alternative means should be used, including low technology measures that focus on vector control such as insecticide fogging, limiting mosquito breeding, and providing protection from mosquito bites. the delivery of different interventions, singularly or in combination, should be informed by, and thereby tailored to, local settings. what should not be overlooked in the current furore over zika is that a. aegypti is also responsible for transmitting yellow fever, japanese encephalitis, dengue, and chikungunya, viruses that have arguably far more impact on global public health. the implementation of any program to limit or eliminate a. aegypti as a result of concern over the spread of zika will have the added benefit of coincidentally reducing the risk of incidence of these other important viral human pathogens. , it is also possible that a species of mosquito other than a. aegypti is the main vector fueling the current epidemic of zika in a continent where this virus has not been established previously, although this too would be susceptible to identical means of vector control. until now there has been little in the way of research into zika, both with regard to the virus and the pathologies that it causes. hence, there is a knowledge gap surrounding infection transmission and clinical manifestations of disease. since there has been no pressing demand before the present epidemic, no prophylactic vaccine or therapeutic drug is currently available with which to treat zika infection. for any infectious disease, the vaccine development pathway from candidate design, via preclinical screening, through phase i-iii clinical trials to final approval for public administration is long, demanding, and expensive. while this has now been prioritized by international funding agencies for zika, it may take several years for a vaccine to come to commercial fruition. as efficacious vaccines have been prepared against yellow fever and japanese encephalitis viruses, it is anticipated that a similar therapeutic is feasible for zika. nevertheless, in spite of ring-fenced funds to support research into developing a vaccine, a note of caution should be advised. it is feasible that the laboratory-based design and preclinical screening of a candidate vaccine may be fast-tracked in a matter of months, perhaps by the end of . this is particularly so if a dna-based construct is developed. both the indian pharmaceutical company bharat biotech and the us national institute of allergy and infectious diseases have plans to produce a vaccine. however, in all probability, it will take several years to perform any ensuing clinical trials and to attain full approval from national regulatory bodies for public administration. , although to gain ethical approval for, and to conduct, tests of vaccine safety and efficacy in humans necessitates diligence and caution at all times, the due process for any candidate vaccine that is dispensed to pregnant females is naturally subject to extremely exacting analysis. , this would relate especially to zika as, allegedly, the gravest manifestation of infection, microcephaly, affects pregnancy. the positive news is that the latest clinical trial of an experimental dengue vaccine, tv , has proved % effective, albeit a small scale volunteer challenge study of only the dengue- serotype performed under highly controlled conditions. while this is no doubt a significant breakthrough in the ongoing fight against dengue, as an offshoot of this success it is hoped that a modified version of the live attenuated construct may be developed in order to treat the related zika virus. this head start may accelerate production of a candidate zika vaccine but will not truncate its potentially arduous journey through clinical trials. during this time, scientists, clinicians, and health care professionals all should be attentive to stress the standard caveats and qualifications to the outcomes of any study, so not to augment the plethora of misinformation that concerns zika by implying, however unintentionally, that a vaccine is forthcoming anytime soon. , the recent striking emergence of cases of zika in latin america poses a threat for a worldwide outbreak of this mosquito-borne flavivirus infection. the public health challenges presented by zika will remain unless and until the taylor-robinson current deficit of knowledge relating to its epidemiology, pathology, and immunology is addressed. current measures to combat the spread of infection rely on vector control programs, which evidently require improved operation and integrated management. a specific anti-zika therapy does not exist, but in response to the ongoing epidemic research funds have been committed to develop a vaccine. while prior success in producing effective vaccines against closely related human viral pathogens provides a proof of principle for this approach, vaccine design and clinical testing can be laborious and the timescale long. in the short term, therefore, optimism regarding delivery of a zika vaccine should be tempered by realism regarding a delivery date. the author reports no conflicts of interest in this work. zika virus. i. isolations and serological specificity zika virus in the americas -yet another arbovirus threat pan american health organization. zika virus infection. washington dc: paho/who concern over zika virus grips the world who director-general summarizes the outcome of the emergency committee regarding clusters of microcephaly and guillain-barré syndrome zika virus, what cdc is doing? centers for disease control and prevention full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika without symptoms in returning travellers: what are the implications zika virus -symptoms, diagnosis & treatment zika virus and birth defects--reviewing the evidence for causality rapid risk assessment. zika virus epidemic: potential association with microcephaly and guillain-barré syndrome zika virus and pregnancy: what obstetric health care providers need to know zika: panama has 'first microcephaly case outside brazil zika virus, french polynesia, south pacific cdc broadens zika virus travel alert for pregnant women zika virus: rumours and theories fuel 'information war the global spread of zika virus: is public and media concern justified in regions currently unaffected? probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus zika virus: your questions answered possible association between zika virus infection and microcephaly -brazil detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study novel vaccine strategies against emerging viruses a systems biology approach for diagnostic and vaccine antigen discovery in tropical infectious diseases temporal and spatial analysis of the - ebola virus outbreak in west africa mosquitoes and their control local transmission of zika virus infection is possible in australia but should be contained by current vector control measures introducing new vaccines in developing countries a review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available the guardian bbc news here's why we don't have a vaccine for zika (and other mosquito-borne viruses) pregnancy in the time of zika: addressing barriers for developing vaccines and other measures for pregnant women dengue vaccine aces trailblazing trial. protection against virus raises hopes of vaccine development for dengue and even zika key: cord- - ua dzjk authors: olawoyin, omomayowa; kribs, christopher title: coinfection, altered vector infectivity, and antibody-dependent enhancement: the dengue–zika interplay date: - - journal: bull math biol doi: . /s - - - sha: doc_id: cord_uid: ua dzjk although dengue and zika cocirculation has increased within the past years, very little is known about its epidemiological consequences. to investigate the effect of dengue and zika cocirculation on the spread of both pathogens, we create a deterministic dengue and zika coinfection model, the first to incorporate altered infectivity of mosquitoes (due to coinfection). the model also addresses increased infectivity due to antibody-dependent enhancement (ade) within the human population. central to our analysis is the derivation and interpretation of the basic reproductive number and invasion reproductive number of both pathogens. in addition, we investigate how model parameters impact the persistence of each disease. our results identify threshold conditions under which one disease facilitates the spread of the other and show that ade has a greater impact on disease persistence than altered vector infectivity. this work highlights the importance of ade and illustrates that while the endemic presence of dengue facilitates the spread of zika, it is possible for high zika prevalence to prevent the establishment of dengue. countries are endemic for the disease (kumar et al. ) . dengue is transmitted to humans mainly through the bite of infected female aedes aegypti mosquitoes and is caused by one or more viral serotypes of the flaviviridae family [denv- through are known to infect humans; identification of a fifth, sylvatic serotype termed denv- has been claimed (mustafa et al. ) ]. although this illness is typically self-limiting, with infection by one serotype resulting in lifelong immunity to that specific serotype, severe forms of the disease can cause dengue hemorrhagic fever and dengue shock syndrome (kumar et al. ) . currently, no treatment exists for dengue and only one controversial vaccine (dengvaxia ® ) has been licenced. as a result, mosquito control strategies remain the primary method of preventing dengue transmission. closely related to dengue is zika, another disease of international concern. zika was first discovered in uganda in and has since spread across the globe, with outbreaks in yap island ( ), french polynesia ( ) , and more recently the americas ( ) (gao et al. ). the zika virus (zikv) is of the same family as the dengue serotypes and is also transmitted to humans primarily by a. aegypti mosquitoes (although it can also be sexually and vertically transmitted within the human population). while some clinical symptoms of zika, such as acute fever, nausea, rash, joint pain, and myalgia, are similar to dengue, zika is unique in that it can cause serious complications in the form of guillain-barré syndrome and congenital zika syndrome (gao et al. ) . due to having a shared vector, cocirculation of dengue and zika is common in many geographical regions and increases the likelihood of dengue-zika coinfections within human and mosquito populations. to date, clinical studies have reported human coinfections in countries such as colombia, new caledonia, nicaragua, and haiti (carrillo-hernández et al. ; dupont-rouzeyrol et al. ; lovine et al. ; waggoner et al. ) . however, because of the rapid introduction of zika into countries that are endemic with dengue, similarities in symptoms between the two diseases, underreporting, and the lack of proper serotesting in developing countries, it is believed that the prevalence of coinfections is higher than currently perceived (rückert et al. ) . in a. aegypti mosquitoes, infection with multiple arboviruses has been shown to affect viral dissemination, transmission, and replication (abrao and da fonseca ; magalhaes et al. ; rückert et al. ) . researchers in chaves et al. ( ) reveal that for dengue and zika specifically, coinfection can impact mosquito infectivity. the results of chaves et al. ( ) indicate that while the number of dengue virus cdna copies in coinfected mosquitoes is higher than in monoinfected mosquitoes (up to times higher), zika cdna copies are lower in coinfected mosquitoes than in their monoinfected counterparts ( - times lower). this suggests that coinfection may cause mosquitoes to be more likely to transmit dengue and less likely to transmit zika. within humans, dengue and zika can display complex viral interactions in the form of antibody-dependent enhancement (ade). ade occurs when antibodies from a previous infection bind to a pathogen in a subsequent infection and, instead of neutralizing the pathogen, increase viral uptake and replication (whitehead et al. ) . many in vitro studies (e.g., charles and christofferson ; dejnirattisai et al. ; durbin ; paul et al. ) have shown that dengue antibodies cross-react with the zikv, increasing zika infection of cells and production of viral progeny by over -fold. the reciprocal effect of zikv antibodies increasing dengue virus titers has also been reported (kawiecki and christofferson ; stettler et al. ). although these cross-reactive ade effects have not been confirmed in vivo in humans by field data, they have been observed in vivo in mice (bardina et al. ) and macaques (george et al. ) . thus, given the well-known ade across denv serotypes and the consistent in vitro results, the potential is clear for immunity to one of the two viruses to enhance transmission of the other virus within the human population. while many mathematical models have been developed to understand the dynamics of zika and dengue individually (e.g., andraud et al. ; braselton and bakach ; wiratsudakul et al. ) , only a few have considered both viruses simultaneously. the first two studies to do so also included chikungunya, an arbovirus transmitted by the infamous a. aegypti (isea and lonngren ; okuneye et al. ), but largely excluded the possibility of coinfection. isea and lonngren ( ) focused on analyzing the stability of a nontrivial equilibrium in a system which considers only single transmission of the three viruses. they also introduce a second model that incorporates coinfections within the human population, but its analysis was limited to finding a nontrivial equilibrium. both models exclude sexual transmission of zika between humans, coinfection within the mosquito population, and altered infectivity of humans (due to possible ade) or mosquitoes (due to coinfection). meanwhile, the model in okuneye et al. ( ) investigates the impact of a dengue-chikungunya-zika superinfection hierarchy within humans, where (based on relative incidence data) infection with dengue completely replaces infection with chikungunya or zika, and infection with chikungunya replaces zika. furthermore, the authors include sexual transmission of zika, assume that dengue vaccination can reduce zika susceptibility, and consider the possibility of ade of dengue over zika (but not ade of zika over dengue) by altering the susceptibility of hosts and vectors to zika, rather than altering zika infectivity of these populations. okuneye et al. ( ) found that under this superinfection hierarchy, dengue-induced ade and dengue vaccination had only minor impacts on zika transmission. another mathematical study considered dengue and zika coinfection in order to examine the impact of dengue vaccination on a zika outbreak (tang et al. ) . the model captures vaccination indirectly through initial conditions. every combination of susceptible and infectious populations is given a different infection rate, so in principle the model could accommodate both ade in humans and altered infectivity of coinfected vectors, but qualitative analysis was limited to computation of the basic reproductive number (the number of secondary infections that a single infected individual can make in a completely susceptible population), and numerical analysis set all the infection rates equal to each other (without units) except the rate at which coinfected mosquitoes infect naive humans (with either virus) and the rate at which zikamonoinfected mosquitoes infect dengue-recovered humans. comparison of two graphs verified that when these latter two rates were increased by a factor of . , zika incidence increased. the study's main conclusion was that a high mosquito birth rate together with dengue vaccination could increase zika incidence and cause a zika outbreak to occur earlier and have a higher peak than observed with low mosquito birth rates. more recently, wang and zhao ( ) published a similar study, with dengue and zika spreading in a population where the dengue vaccine is available. they assumed perfect vaccine efficacy and found a monotone increase in zika's basic reproductive number with increases in vaccination. however, the authors also assumed that mosquitoes cannot be coinfected with zika and dengue, thus precluding any possibility of considering altered infectivity for coinfected vectors. we therefore propose a study which simultaneously explores the three facets of what we call the dengue-zika interplay: coinfection of humans and vectors, altered vector infectivity, and ade of dengue and zika. in this article, we develop the first zika and dengue transmission model that includes coinfection (in humans and mosquitoes), altered vector infectivity, and ade for both viruses (i.e., viral enhancement of zika given dengue antibodies and enhancement of dengue given zika antibodies). the goal of the present study is to better understand the epidemiological consequences of the dengue-zika interplay. in particular, through a deterministic mathematical model that utilizes a system of nonlinear ordinary differential equations, we seek to answer the following research questions: . how does the endemic presence of dengue affect zika's ability to spread in a region? . how does invasion of zika affect the endemic presence of dengue? with zika rapidly spreading across the globe to regions endemic with dengue, examining the complex interactions between the two pathogens is vital to clarifying the public health impact of the cocirculation of both diseases and potentially informing future vaccine development and control strategies. the current study is placed in the context of dengue and zika cocirculation in el salvador. the total human population (given in table ) represents the calculated atrisk population for zika in el salvador during the / outbreak, as described in shutt et al. ( ) . since zika and dengue are spread by the same vector and are in similar geographic regions, we assume that this number also represents the population at risk for dengue during that time. a visual representation of the deterministic dengue and zika coinfection model that we consider is shown in fig. , with the state variables and parameters described in tables and , respectively. in this model, we make the following assumptions: . human and mosquito populations remain constant. . only one dengue serotype is cocirculating in the study region [an assumption also made in isea and lonngren ( ) and okuneye et al. ( ) ]. . there is no disease-induced death in the human population as the case-fatality rate for each disease is low (moraes et al. ; paho ) . . ade occurs after a person has recovered from a primary infection and is subsequently infected with the other virus. ade produces an increased viral load. . all humans are equally susceptible to infection by a given virus (and the same for mosquitoes), but humans who have recovered from a primary infection and then become infected with the other virus exhibit altered (increased) infectivity due to ade. . any dengue/zika coinfection in humans and mosquitoes is sequential (i.e., requires two infection events) rather than simultaneous. humans are born fully susceptible to dengue and zika at a rate of μn h , where μ is the natural birth/death rate for humans and n h is the total human population. susceptible individuals can become infected with dengue from either a dengue-infected (i dv ) or coinfected female mosquito (i cv ). the mosquito-to-human dengue infection rate is given by β hd . this rate is modified by a factor of ν d to indicate the altered infectivity of coinfected mosquitoes. once infected with dengue, humans can recover or become coinfected with zika (by a zika-infected (i zv ) or coinfected female mosquito (i cv ), or by sexual transmission from a zika-infected (i z ) or coinfected (i c ) human) and transition into the r d or i c class, respectively. in a similar manner, fully susceptible humans become infected with zika from a mosquito in the i zv or i cv compartment, olawoyin and kribs ( ) or by a human in the i z or i c compartment. the mosquito-to-human zika infection rate is given by β hz and is modified by a factor of ν z for coinfected mosquitoes. once infected with zika, humans either recover (with the zika recovery rate given by γ z ) or become coinfected with dengue through a mosquito in i dv or i cv . when a coinfected human recovers from dengue, he or she becomes dengueimmune and enters the j z class. meanwhile, singly infected humans who recover from dengue join the r d compartment where they are susceptible to further infection with the zikv. for individuals in the j z class, the rate of zikv infection is modified by a factor of k z which represents the relative zika infectivity given the effect of ade. furthermore, coinfected humans who recover from zika transition into the j d class and are immune to zikv infection. once in j d , an individual's dengue infectivity is modified by a factor of k d due to the higher viral load induced by ade. on the other hand, individuals who recover from a single infection with zika enter r z and are susceptible to further infection with dengue. lastly, individuals immune to dengue or zika (i.e., those in j d or j z ) that undergo subsequent infection with the secondary virus recover and become immune to both diseases (r c ). in the vector population, mosquitoes are born into the susceptible class (s v ) at a rate of μ v n v , where μ v is the natural birth/death rate for mosquitoes and n v is the total mosquito population. susceptible mosquitoes are infected with dengue after feeding on a dengue-infected or coinfected human. in this case, the human-to-mosquito dengue infection rate is given by β vd . likewise, susceptible mosquitoes can become infected with zika after feeding on a zika-infected or coinfected human, with a human-tomosquito zika infection rate of β vz . these singly infected mosquitoes can be coinfected with both pathogens after feeding on a human infected with the second virus. the system of nonlinear differential equations corresponding to the dengue and zika coinfection model described above is given by ( ). ( ) a majority of the baseline parameter values used for this model were obtained from previously published literary sources as indicated in table . to obtain the zika and dengue transmission modification factors, we observed the difference in viral cdna copies between monoinfected and coinfected mosquitoes described in chaves et al. ( ) . based on this study, the number of dengue virus cdna copies in coinfected mosquitoes was . - times higher than in monoinfected mosquitoes, while zikv cdna copies were - times lower in coinfected mosquitoes than monoinfected mosquitoes. assuming that these ranges correspond to the disease transmission capability of coinfected vectors, we use values within the ranges for ν d and ν z . since the only studies on the effect of ade on zika and dengue have been conducted at the cellular level (i.e., comparing viral titers and replication), the direct effect of ade on dengue or zika transmission is unknown. however, due to the large rise (up to two orders of magnitude) in viral load caused by ade, we assume that ade increases the likelihood of disease transmission and take k d and k z to be greater than one. under the assumption that human infection with the zikv is completely independent of denv immunity (i.e., k z = ) and that the ability of vectors to transmit zika is independent of their coinfection with dengue (i.e., ν z = ), system ( ) simplifies tȯ and i zv = i zv + i cv . since the total human and vector populations, n h and n v , respectively, are constant, ( ) can be rewritten aṡ a similar reduction of ( ) can be obtained when k d = and ν d = . in this case, the model simplifies to ( ), with the exception that there is no sexual transmission term, and the z's in the subscripts of ( ) are replaced with d's. to find equilibrium values of ( ), we set all of the differential equations to zero. some of the equilibria are detailed in table and describe scenarios when no disease is present within the population (disease-free equilibrium), or when only one disease is present (dengue-only or zika-only equilibrium). although we were unable to find an analytic expression for a copersistence equilibrium, numerical explorations suggest the existence of a stable copersistence equilibrium when both the dengue and zika invasion reproductive numbers (irns) exceed . these irns are detailed in sect. . existence criteria for the unique single pathogen equilibria are described in lemma and theorem . , where a, b, and c are as described in sect. . in addition, the proof of lemma is in "appendix." theorem for ( ), a unique zika-only equilibrium exists iff table , all of the nonzero points of the zika-only equilibrium are expressed in terms of i * z , where i * z is the solution to the quadratic equation a i * z + bi * z + c = . in this equation, using the quadratic formula, we have . since a > , when c > , ) and one negative by this last inequality, we have (γ z +μ)(β vz β hz n v +(β s μ v +β vz μ)n h ) > β s β vz μn h . finally, multiplying by n h and subtracting β s β vz μn h on both sides of the inequality, we obtain b > . this implies that for c > , i * z < . thus, the only biologically feasible solution for i * z occurs iff c < and is given by to derive the brn of the zikv and denv coinfection model, we use the nextgeneration operator method proposed by van den driessche and watmough ( ). evaluating the f and v matrices obtained from this method at the disease-free equilibrium, we have the brn is the spectral radius of f v − and is given by the fractions β vd μ v and β hd μ+γ d in r d are the product of the denv infection rates and the average time a human or vector remains infected with dengue ( μ v days for vectors and μ+γ d days for humans). the second term under the radical in r z is similar to r d and represents vector transmission of zika. however, r z also includes sexual transmission of zika within the human population in the form β s μ+γ z , where β s is the sexual transmission rate and μ+γ z is the average duration of infection in humans. the next-generation operator method ensures the local asymptotic stability of the disease-free state when r = max{r d , r z } < . invasion reproductive numbers (crawford and kribs ; martcheva ; porco and blower ) for autonomous systems can be derived by extending the next-generation matrix method (van den driessche and watmough ) to consider any resident (non-invading) infections as non-infectious classes (mitchell and kribs ) . this method establishes conditions for the local asymptotic stability (las) of the boundary equilibria: the dengue-endemic equilibrium is las when dengue can spread alone but zika cannot invade it (r d > ,r z < ), and the zika-endemic equilibrium is las when zikv can spread alone but dengue cannot invade it (r z > ,r d < ). as noted in sect. , numerical explorations indicate that when both irns exceed , a copersistence equilibrium is las. the dengue irn, which describes the ability of dengue to spread in a population endemic with zika, is calculated with i d , i c , j d , i dv , and i cv categorized as the infectious classes. evaluating the f and v matrices at the zika-only endemic equilibrium, we obtain dengue's irn is the spectral radius of f v − and is given byr the zika irn describes the ability of zika to spread in a population endemic with dengue and is computed in a similar manner as the dengue irn. however, in this case i z , i c , j z , i zv , and i cv are categorized as the infectious classes. evaluating f and v at the dengue-only endemic equilibrium, we obtain zika's irn is the spectral radius of f v − and is given bỹ notice that when k z = ν z = ,r z = r z . furthermore, in a similar manner as k vd and k hd , the k vz and k hz expressions describe the relative zika infectivity of vectors and humans, respectively. it is worth noting that the four parameters which describe the dengue-zika interplay do not appear in either virus's brn but do appear in the two irns, specifically human ade (k d , k z ) through the terms k hd and k hz , and altered infectivity for coinfected vectors (ν d , ν z ) through the terms k vd and k vz . in order to visualize how various aspects of the dengue-zika interplay impact the persistence of each disease, we plot irn threshold curves forr d =r z = on the r d versus r z axis. as seen in figs. and , this results in four distinct regions of possible model outcomes (extinction of both diseases, e , persistence of only dengue, e d , persistence of only zika, e z , and copersistence of both pathogens, e dz ). in these figures, the curve above the r z = line isr d = and the curve to the right of the r d = line isr z = . figure illustrates that ade (i.e., k d , k z > ) causes zika and dengue to benefit from the presence of each other. in particular, due to ade, the presence of dengue makes it possible for zika to persist in regions where it would not have been able to persist on its own (i.e., in regions where r z < ). similarly, this figure shows that the reciprocal effect of zika presence on dengue is also true with ade. in fact, as the likelihood of disease transmission by recovered individuals increases across various orders of magnitude, the e dz region widens and makes it easier for dengue and zika to copersist. to disentangle the effects of altered infectivity of hosts from that of vectors, we let k d = k z = (while keeping ν d and ν z at their baseline values) and obtain fig. . since ther d =r z = curves appear to be straight lines, it seems, at first glance, that altered infectivity of vectors does not affect persistence of zika and dengue. however, after figure) curves. this graph shows the relatively minimal effect of altered vector infectivity on transmission of the viruses enlarging the irn threshold curves, it is clear that zika slightly facilitates the spread of dengue (as seen in the e dz region where dengue is able to invade even though r d < ) while dengue hinders the spread of zika (as evidenced by the narrow e d region where zika is not able to establish itself even though r z > ). this minor impact of ν d and ν z on the persistence of each pathogen can be attributed to the fact that coinfected mosquitoes have a higher likelihood of transmitting dengue and a lower likelihood of transmitting zika than monoinfected mosquitoes. a more detailed exploration of how the altered infectivity parameters impact dengue and zika dynamics is conducted by plotting ther d = r d andr z = r z curves on the ν versus k axis. as shown in fig. , these curves divide the plane into two distinct regions, one where the irn is greater than the brn and the other where the irn is less than the brn. if zika-recovered individuals are more than . times as likely as their zika-naive counterparts to transmit dengue, the dengue irn will always be greater than its brn. on the other hand, any level of ade (i.e., any k z > ) causes the zika irn to be greater than its brn. although cocirculation of zika and dengue is common due to the pathogens' shared vector, the impact of cocirculation on the presence of each pathogen has not received great mathematical modeling attention. in this study, we develop and analyze the first the results of this work differ from those of the previous mathematical modeling studies to consider dengue and zika cocirculation. specifically, our derivation of explicit expressions for the irns of the two viruses makes clear the role of each of the two factors (ade in humans and coinfection in mosquitoes) which we assume to affect the infectivities of both viruses. while the complexity of the model in isea and lonngren ( ) prevents the authors from deriving any reproductive numbers, tang et al. ( ) compute their model's brn only, in which neither infectivity-altering factor appears, since the brn is defined in terms of the disease-free state. the r d and r z expressions in our brn are structurally similar to those in okuneye et al. ( ) and wang and zhao ( ) . however, unlike ours, the brn expressions derived in these latter two papers include both vaccination and ade parameters, because those studies assume that ade alters susceptibility, rather than infectivity, including of vaccinated individuals. for them, ade alters the viruses' brns to the extent that vaccinated individuals are present in the disease-free state. since ade increases the viral load during a second infection, until field studies confirm the nature of its effects during transmission, the question of whether the increased viral load increases susceptibility or subsequent infectivity remains open. we consider an increased viral load throughout the course of an infection to affect primarily infectivity. our calculation of the irns for both diseases therefore permits a more systematic and interpretable study of how ade and coinfected vectors create a complex and asymmetric interplay between dengue and zika viruses by changing their infectivities. the expressions for weighted-average infectivities in each irn show how key parameter values impact them. through fig. , we see that altered infectivity of hosts has a greater impact on the irns than the altered infectivity of vectors. in addition, the k d and k z parameters can be used to determine when the presence of one disease makes it easier or harder for the other to spread (i.e., when each pathogen's irn will be greater than or less than its brn). regardless of the level of ade, we find that zika will always spread more easily in dengue-endemic regions than it would on its own. however, this is not the case with dengue. for dengue, the effect of ade has to be high enough (i.e., k z > . ) in order for the presence of zika to facilitate the establishment of dengue. this allows for interesting scenarios where zika and dengue will have opposite effects on each other. for example, when k z > and < k d < . , high dengue propagates the spread of zika (i.e.,r z > r z ), but high zika prevalence impedes the invasion of dengue (i.e.,r d < r d ). in addition to fig. , figs. and provide valuable insight on how the zika and dengue viruses affect each other on the population level. with or without altered infectivity of humans, the presence of zika makes it possible for dengue to persist in a population in which it would not be able to persist by itself. however, our results show that ade (i.e., k z > ) is essential for zika to benefit from the presence of dengue. without ade, it is possible for zika's brn to be greater than , but zika not be able to successfully invade a population because of the presence of dengue. both of these results are due to the baseline ν d but ν z values that allow coinfected mosquitoes to be better at transmitting dengue than zika. furthermore, as altered host infectivity parameters increase, the region of copersitence of both viruses widens, showing a mutualistic relationship between zika and dengue due to ade. from this research, it is clear that the impact of ade on the infectivity of hosts plays a crucial role in dengue-zika dynamics. however, there are no experimental studies that address the epidemiological consequences of ade, specifically how it affects dengue infectivity of zika-immune individuals or zika infectivity of dengueimmune persons. currently, the studies that address ade do so on the cellular level, describing its impact on viral titers. we argue that due to the effect of the k d and k z parameters on irn and brn comparisons and the persistence of zika and dengue, there is a need for studies that focus on estimating these values. using experimentally validated k d and k z estimates would allow us to draw more concrete conclusions on the population-wide impact of zika and dengue, which can potentially inform vaccine development efforts. in future, we plan to extend our study of this dengue-zika interplay model to consider vaccination and include more than one dengue serotype. explicitly incorporating potential zika and dengue vaccines will give a clear picture of the possible impact of vaccinations and whether or not the use of one vaccine can indirectly exacerbate the burden of the other pathogen. in addition, since multiple dengue serotypes typically cocirculate within particular regions (and have been shown to exhibit ade with each other), it would be beneficial to examine how the presence of more than one dengue serotype with zika affects the long-term persistence of the various pathogens. infection of mosquito cells (c / ) by dengue- virus interferes with subsequent infection by yellow fever virus dynamic epidemiological models for dengue transmission: a systematic review of structural approaches enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity a survey of mathematical models of dengue fever cocirculation and simultaneous co-infection of dengue, chikungunya, and zika viruses in patients with febrile syndrome at the colombian-venezuelan border 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infection and transmission by aedes aegypti mosquitoes estimating the reproductive number, total outbreak size, and reporting rates for zika epidemics in south and central america specificity, cross-reactivity and function of antibodies elicited by zika virus infection reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission viremia and clinical presentation in nicaraguan patients infected with zika virus, chikungunya virus, and dengue virus dynamics analysis of a zika-dengue co-infection model with dengue vaccine and antibody-dependent enhancement prospects for a dengue virus vaccine dynamics of zika virus outbreaks: an overview of mathematical modeling approaches world health organization ( ) global strategy for dengue prevention and control - . who library cataloguing-in-publication data assessing the effects of temperature on the population of aedes aegypti, the vector of dengue publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors acknowledge the national science foundation as this material is based upon work supported by the national science foundation graduate research fellowship program under grant no. . any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the national science foundation. it is important to note that when k d = ν d = ,r d = r d . epidemiologically, k vd is the average relative dengue infectivity of mosquitoes in a setting where zika is resident and k hd is the average relative dengue infectivity of humans within that setting. while the proportion of susceptible mosquitoes that get zika before dying () and the proportion of zika infectious mosquitoes (n v ) at the time that dengue arrives have relative dengue infectivity of ν d , the relative dengue infectivity of the proportion of susceptible mosquitoes that die before contracting zika (in the k hd expression, the proportion of susceptible humans who get zika before dying but recover from zika prior to getting dengue (, the proportion of humans infected with zika when dengue arrives who recover before getting dengue ( thus, a unique dengue-only equilibrium exists iff r d > . key: cord- - thdg up authors: payne, kelly; kenny, peter; scovell, jason m.; khodamoradi, kajal; ramasamy, ranjith title: twenty-first century viral pandemics: a literature review of sexual transmission and fertility implications in men date: - - journal: sex med rev doi: . /j.sxmr. . . sha: doc_id: cord_uid: thdg up introduction: the st century has seen a series of viral pandemics that have collectively infected millions of individuals. to understand factors that may contribute to viral spread and address long-term health sequelae for survivors, it is important to review evidence regarding viral presence in semen, sexual transmission potential, and possible effects on fertility. aim: to review the current literature regarding the sexual transmissibility of recent viral pandemics and their effects on semen parameters and fertility. we review evidence for the following viruses: ebola, zika, west nile, pandemic influenza, severe acute respiratory syndrome (sars), and sars-corona virus- (sars-cov- ). methods: a literature search was conducted to identify relevant studies. titles and abstracts were reviewed for relevance. references from identified articles were searched and included, if appropriate. main outcome measures: the main outcome measure of this study was reviewing of peer-reviewed literature. results: both the ebola virus and zika virus are present in semen, but only the zika virus shows consistent evidence of sexual transmission. current evidence does not support the presence of the west nile virus, pandemic influenza, sars, and sars-cov- in semen. the zika virus appears to alter semen parameters in a way that diminishes fertility, but the effect is likely time limited. the west nile virus and sars have been associated with orchitis in a small number of case reports. viruses that cause febrile illness, such as pandemic influenza, sars, and sars-cov- , are associated with decreased sperm count and motility and abnormal morphology. sars and sars-cov- may interact with angiotensin-converting enzyme receptors present in the testes, which could impact spermatogenesis. conclusions: we have reported the presence in semen, sexual transmission potential, and fertility side effects of recent viral pandemics. overall, semen studies and fertility effects are highly understudied in viral pandemics, and rigorous study on these topics should be undertaken as novel pandemics emerge. payne k, kenny p, scovell jm, et al. twenty-first century viral pandemics: a literature review of sexual transmission and fertility implications for men. sex med rev ;xx:xxx–xxx. the st century has seen a series of viral pandemics that have affected the health of millions of individuals and touched every corner of the globe. as one potential means of controlling viral spread, it is necessary to understand what viruses are present in semen, how long they remain detectable, and what their potential is for sexual transmission from male to female partners. in addition, with so many survivors of viral pandemics alive today, knowledge is needed about how these viruses affect the male genital tract and what implications they may have for future fertility. despite the importance of these questions, to our knowledge, these topics have not yet been the subject of systematic review. here, we detail several important foundational principles that affect viral infection in the testes and comment on the differences in immune response seen between men and women. then, we present the state of current research regarding presence in semen, sexual transmission, and fertility effects for the zika virus (zikv), ebola virus (ebov), west nile virus (wnv), pandemic influenza, severe acute respiratory syndrome (sars), and sars-coronavirus- (sars-cov- ) ( table ) . a literature search of pubmed, embase, cochrane, ovid, and clinicaltrials.gov was conducted by independent authors, k.p. and p.k. the literature search was limited to english publications or publications translated into english, and databases were searched from dates of their inception to june . articles with only abstracts available were included. to review the immune status of the testes, searches were conducted using the following keywords: testes, immune privilege, and blood-testes barrier. to review the difference in immune response between men and women, searches were conducted with the following keywords: gender difference in immune response and sex difference in immune response. searches were also conducted for each of the recent viral pandemics with the following key words separated by "or": genitalia, testis, orchitis, prostate, reproductive, semen, sperm, hormones, endocrine, luteinizing hormone, folliclestimulating hormone, gonadotropins, testosterone, fertility, fecundity, sexual transmission, erectile function, erectile dysfunction, sexual function, sexual dysfunction, and libido. case studies, case series, reviews, and meta-analyses were included. both human, animal, and basic science data were included. for each viral pandemic, articles were selected if they met any of the following inclusion criteria: discussion of semen parameters, identification of virus location within the genital tract, discussion of sexual transmission, or discussion of fertility related processes including spermatogenesis, libido, and sexual function. article titles and abstracts were reviewed by k.p. and p.k. to identify relevant studies. full-text articles were then reviewed to determine if they met the stated selection criteria, yielding total articles ( figure ). in any consideration of viral infection of testicular tissue and seminal fluid, it is important to review the function of the bloodtestis barrier (btb). the btb provides protection from autoimmune cell destruction, making the testes an immuneprivileged site. evolving understanding of the btb indicates that it comprises both a physical component and an immune regulatory component. the physical component of the btb comprises a layer of sertoli cells connected by tight junctions, separating undifferentiated spermatogonia from differentiating germ cells in the form of spermatocytes, spermatids, and spermatozoa. local immunosuppression is carried out by constitutive expression of anti-inflammatory factors by testicular cells, most prominently transforming growth factor beta. , testosterone plays an additional role in establishing immune privilege by regulating the numbers of testicular macrophages and lymphocytes and downregulating their expression of proinflammatory transcription factors, cytokines, and adhesion molecules. an in vitro study that treated macrophages with testosterone demonstrated decreased expression of toll-like receptor and reduced sensitivity of toll-like receptor to lipopolysaccharide, a key antigenic molecule for identifying foreign pathogens. furthermore, a study of rats pretreated with estrogen to suppress testosterone production showed a much more rapid rejection of an intratesticular allograft than untreated controls, underscoring testosterone's immune-modulating effects. despite its immune-privileged nature, the testicular environment is still capable of mounting an effective inflammatory response. in fact, the testes are of equal susceptibility to infection as any other tissue type. given the suppression of adaptive immune mechanisms, it is simply more reliant on the innate immune mechanisms of macrophages, natural killer cells, and antimicrobial secretory products such as interferons and defensins. , , nonetheless, there is evidence that the testes can act as a reservoir for pathogens that are no longer detectable systemically. jenabian et al have found that in the testicles of men with hiv, hiv viral rna has remained detectable in the testes even when levels were undetectable in the blood secondary to antiviral therapy (although this is not known to cause increased transmission risk). this becomes especially important in the study of viruses such as the ebola and zika, where asymptomatic individuals may still be capable of sexual transmission of the virus. sex-based differences in immune response present an additional consideration for viruses that affect the reproductive tracts. increasing evidence suggests that the intensity, severity, and mortality of viral disease differs between genders. literature examining this link has generally observed that women mount a stronger immune response to antigens, infections, and vaccines than do men. this effect is most widely observed in the cytokine response to antigens. on inoculation with viruses or direct stimulation of immune toll-like receptors , , , and , men exhibit lower levels of proinflammatory interferon alfa and higher amounts of anti-inflammatory interleukin . helper t cells in men also demonstrate lower cluster of differentiation (cd ), cd , and cd :cd ratio than those in women. the x chromosome contains many important genes for immune function, including tlr , cd ligand (cd l), and forkhead box p (foxp ). notably, it also contains the most micrornas of all chromosomes, which regulate protein-coding genes and modulate cellular activities. the y chromosome, by contrast, uniquely contains none. and, although x inactivation equalizes gene expression for most of the x chromosome, genes within non-recombining regions may be expressed more in women. in addition, other immune-regulatory genes present on both the x and y chromosomes have unequal expression levels, and tissue distribution between the sexes is perhaps due to sex hormones. , the first randomized controlled trial to separate the effect of sex hormones and chromosomes was conducted by palaszynski et al in , using mice in which the sex-determining region y (sry) gene was moved from the y chromosome to an autosome. this allowed separation of sex chromosome complement (xx or xy) from gonadal type (ovaries or testes). the authors reported that among ovariectomized (-sry) mice, those with xy had greater immune response than those with xx, suggesting that the male complement was immune stimulatory. this effect was not detected when comparing intact males (þsry) with xx and xy complements, suggesting that testosterone tempers the effect of the xy complement. supporting this, when testosterone was added to the ovariectomized female (-sry) xy mice, their elevated immune response was suppressed. in , robinson et al similarly found that the increased immune response of female mice to influenza a was eliminated by removal of the gonads. although they did not find any immune response to testosterone or dehydroepiandrosterone in male mice, administration of high-dose estradiol attenuated the induction of tumor necrosis factor alpha and c-c motif chemokine ligand by times and resulted in increased rates of survival compared with no or low-dose estradiol. building on this, administration of b-estradiol to female mice resulted in less secretion of tumor necrosis factor alpha and interferon gamma as well as decreased neutrophil recruitment, improving disease tolerance. taken as a whole, current evidence suggests women exhibit stronger immune response to viral infections than men. this response typically manifests as better long-term outcomes and to subsequent reexposure but also as more severe disease acutely. the size of the effect can be substantial; in the h n pandemic, infected women suffered more than -fold risk of death than men. , further exploration of the mechanisms behind the sexbased differences may allow for modulation of the immune response with hormone treatments. the zikv was first recognized in in a sentinel monkey from zika forest, uganda. the first suspected human case followed in , but the virus did not demonstrate mosquitoborne transmission until . in , the zikv emerged in brazil during preparation for its upcoming international sporting events, creating a large epidemic through the americas. it entered the continental united states in with cases in florida and texas, peaking in . no new cases have been reported since . however, similarities to other flaviviruses such as dengue fever have made the virus difficult to recognize clinically, with several epidemics first noted after rises in associated conditions such as congenital microcephaly and guillain-barre syndrome. the zikv in semen the zikv was first suspected to be in semen following case reports of travel-associated transmission to sexual partners in non-endemic areas. since then, multiple studies have confirmed the presence of zikv rna in semen by reverse transcription polymerase chain reaction (rt-pcr). e a prospective study by mead et al found that semen was positive with zikv in % of men, including % of those tested within days of symptom onset. infectious zikv was detected in % of rna-positive semen samples collected within days of symptom onset, but % of samples obtained after days of disease onset were infectious. evidence from joguet et al although the reported persistence of the zikv varies widely from days to months after the onset of symptoms, it is widely agreed that viral rna persists longer in semen than in other bodily fluids. this reflects a potential reservoir in the male genital tract, perhaps because of the immune privilege of the testes. supporting this, a systemic analysis by counotte et al reported a median duration of zikv rna in semen of days with a maximum of days, vs days in saliva and days in the female genital tract. the persistence of zikv rna in men with vasectomies further indicates that the virus may also replicate in distal genital tract tissues, such as the bulbourethral glands, prostate, and seminal vesicles. several factors are noted to influence persistence of the viral rna in semen. in the analysis from counotte et al, increased persistence was associated with increased age, absence of joint pain, conjunctivitis, and less frequent ejaculation. conversely, men who ejaculated more than times a week cleared viral rna more than days earlier, and for men who reported vasectomies, although the rate of viral rna detection was similar, the viral load was decreased from . to . log rna copies per milliliter. the first suspected case of sexual transmission of the zikv occurred in , when a male patient returned home to the united states from senegal. he developed symptoms of arthralgia, rash, and hematospermia shortly after returning, followed by his wife days later. serologic results confirmed zika infection, although only convalescent phase samples were positive in the wife, and semen samples were not collected. although it is possible that this transmission occurred via other bodily fluids such as saliva, no other family members were affected. after the subsequent zika outbreaks, in a systematic review moreira et al compiled demonstrated cases of sexual transmission from men to women, women to men, and men to men. among the cases examined, sexual transmission was confirmed with positive serum antibodies, zika rna in semen, and semen cultures. in addition, they demonstrated that zikv rna was detected in semen as late as days and infectious virus in semen up to days after symptom onset. notably, transmissions were from entirely asymptomatic patients, who represent the majority of zika cases. further complicating the control of zika transmission, a prospective cohort study by paz-bailey et al demonstrated that the virus exhibits intermittent shedding, making it difficult to determine when an individual has cleared the virus. the number of days between symptom onset and positive samples was up to in serum, in urine, and in semen. taken together, these data represent potential challenges in preventing sexual transmission of the virus, which is of particular concern in nonendemic areas such as the continental united states where mosquito-borne transmission is rare. although mathematical modeling suggests that sexual transmission alone is not likely to drive or sustain a zikv outbreak, serial semen screenings may be effective in preventing the zika-associated congenital microcephaly and guillain-barre syndrome in patients who are or wish to become pregnant. several case reports have described genitourinary symptoms in male zika cases, although their impact on the male reproductive system is as of yet unclear. in a cohort study, paz-bailey et al noted evidence of hematospermia ( . %), painful ejaculation ( . %), and penile discharge ( . %) in patients with the zikv, suggestive of local inflammation and tissue damage. although fever cannot be excluded as a cause, mouse models suggest direct tissue damage may be involved. e examining this effect, huits et al microscopically analyzed semen samples from patients with symptoms of the zikv in cohort studies, demonstrating macrohematospermia and microhematospermia in of patients tested and transient oligospermia in of patients. building on these results, joguet et al monitored the reproductive hormones as well as sperm count, motility, viability, and morphology of patients with the zikv on days , , , , , , and after symptom onset. they detected that total and motile sperm counts were about % lower on day compared with day but recovered by day . the long-term effect on sperm morphology is less clear. in the same study, morphology characteristics recovered to normal by day . results also reflected an early increase of follicle-stimulating hormone and inhibin b, which recovered over time. by contrast, in a cross-sectional study after the epidemic in brazil, avelino-silva et al examined serum hormones and semen samples in patients year after symptoms. in all patients, serum hormones were normal, and semen rt-pcr rna was negative, but impaired motility was seen in all samples tested and low count was noted in of patients. given the recency of the zika outbreaks, the long-term effects of the virus on fertility characteristics should continue to be monitored. history of the ebov ebola virus disease (evd) first emerged in with simultaneous outbreaks in zaire (in a village near the ebola river) and in south sudan, where it is thought to have originated in african fruit bats. recurrent outbreaks took place in in cote d'ivoire and in in kikwit, zaire. most recently, e saw a west african outbreak of unprecedented scale in sierra leone, liberia, and guinea with greater than , cases and , deaths. the large number of evd survivors means that understanding its persistence in semen, its potential for sexual transmission, and its fertility implications is a matter of considerable importance for the control of viral spread. across multiple studies, it has been shown that the crosssectional percentage of ebov survivors of the e west african epidemic with a positive rt-pcr for ebov rna in the semen ranges from . to . %. e with more living ebov survivors from this epidemic than ever before, this number represents a considerable pool of individuals. while disease modeling has shown that % of individuals will clear the virus from semen at days and % will clear it at days, viral ebov rna has been detected in the semen for as long as days after initial infection. , it has also been cultured in the semen up to days after initial infection. as expected, the percentage of individuals with a positive semen rt-pcr for ebov rna has been demonstrated to decrease in a predictable manner over time. deen et al showed in a cohort of male adult survivors of evd in sierra leone that at e months, % of individuals had positive rt-pcr results in semen; at e months, % had positive results; and at e months, % had positive results. these results were echoed by keita et al in a study including evd survivors in guinea where . % of individuals had positive rt-pcr in semen at months, . % at months, . % at months, . % at months, . % at months, and . % at months. interestingly, limited data indicate that older men may be more likely to have a longer length of time in which semen is positive for ebov rna. soka et al showed that men older than years of age were more likely to have a positive semen test than men younger than years of age. animal models have been used to further characterize the presence of filoviruses in the gonadal tissue and have shown viral infiltration of the testicular interstitium and the seminiferous tubules. in situ hybridization (ish) and immunohistochemistry performed on non-human primates (nhps) infected with the marburg virus localized the virus to the interstitium between the seminiferous tubules. in nhps infected with ebov, perry et al similarly showed viral replication in the interstitial tissues of the reproductive tract, including the seminal vesicles, epididymis, prostate, and testis. perry et al also identified the marburg virus in the seminiferous tubules of nhps. indeed, the virus was found to be most prominently localized to sertoli cells, leading to breakdown of the btb and inflammatory cell invasion into the testicular environment. another study in nhps found ebov rna to be localized to the tubular lumen of the epididymis of of twelve rhesus monkey sampled but did not find any ebov localized to the testis. in the lumen of the epididymis, macrophages were found to be the ebov reservoir. overall, it appears that viral persistence may be established in the interstitial tissues of the reproductive tract, where it is transmitted by tissue macrophages across the btb and into the seminal fluid. although the ebov in the semen is clearly demonstrated, it remains far less certain what potential the virus has for sexual transmission. since the first known outbreak of a filovirus (the marburg virus), there have been documented events of suspected human-to-human filovirus sexual transmission. the first case of suspected ebov transmission occurred during the kikwit outbreak, wherein in a -year-old woman was shown to have weakly positive igm antibodies to igm days after exposure from her convalescent partner. the individual was later negative for igg antibodies on a serum sample, making it difficult to confirm whether she was in fact asymptomatically infected. in the west african outbreak, the first confirmed sexual transmission occurred in march . a man transmitted the virus to his sexual partner days after onset of evd, which was confirmed by whole viral genome analysis of his semen compared with an acute blood sample of his female partner where the samples were found to differ by only nucleotide. in october , a suspected sexual transmission event occurred in conakry, guinea. a man presenting with evd was found to have an ebov genome that did not match the country's current strain of transmission but instead differed by only nucleotide submissions from a sample of his brother-in-law's blood collected during prior acute infection. the man's sister was positive for ebov igm at the time of his infection; however, the transmission could not be confirmed as her husband's semen was negative for the ebov at the time a sample was collected. it was thought that the transmission from the male survivor occurred at days after disease onset. the longest recorded convalescent period before sexual transmission occurred days after disease in n'zerekore, guinea in march . the acute blood sample and semen sample from the male survivor differed by mutations from a female sexual partner with acute evd and subsequent cases from the disease cluster. in total, the sexual transmission resulted in a cluster of cases with deaths. sexual transmission from the ebov, while possible, remains a rare event. the world health organization in their guide to clinical care for survivors of evd recommends abstinence or barrier protection during sexual activity for months after the onset of evd. the ebov and fertility given the extremely high mortality associated with evd and the limited number of laboratories with the capability to study such a pathogenic virus, there is much that remains unanswered about the potential reproductive sequelae of evd. in rhesus monkeys, it has been shown that although marburg infection had a deleterious effect on sertoli cells, the overall reproductive function of the sertoli cells was unaffected. spermatogenesis was unaffected, and tissue morphology was normal. further studies are needed to determine if semen parameters or reproductive capacity are altered in male survivors of evd. sexual health complaints are commonly reported among evd survivors. a health clinic for the management of evd survivors in sierra leone reported findings from a group of patients from to . of men, . % reported erectile dysfunction and . % reported loss of sexual desire. an evd survivors' clinic in liberia reported on evd survivors from to . in this cohort, which included men, % reported erectile dysfunction, whereas % reported decreased libido. the causal mechanism for these complaints remains to be determined. although physiologic conditions can play a role, it is also likely that psychosocial factors including residual stress, trauma, stigma, and grief contribute as well. the wnv was first recognized in north america in new york city during the summer of . the virus was previously known to cause small human outbreaks in africa and the middle east but has now established itself as a major worldwide seasonal pandemic. between and , there have been , documented cases of the wnv in the united states, with particularly high-intensity seasonal outbreaks in and . the most concerning sequelae of the wnv is neuroinvasive disease, which occurs in less than % of infected individuals but carries a % fatality rate. there is very little evidence to suggest that wnv is present in semen. while wnv infection in blood, cerebrospinal fluid, and urine has been well studied, to date, there is only study that describes the evaluation of the wnv in semen. gorchakov et al the wnv has been further studied in a cultured line of human sertoli cells. when the wnv was introduced to these cells, it was noted that the sertoli cells supported a robust wnv infection with titers comparable with that of the zikv, a pathogen known to have high infectivity in semen. in a small case series presenting postmortem autopsy results for individuals deceased from neuroinvasive wnv, the wnv was identified by immunofluorescence in the testes and prostate in of men examined. this individual was years of age and on chronic immunosuppression secondary to a kidney transplant, raising the possibility that immunosuppression could contribute to wider systemic dissemination of the disease. owing to the limited nature of currently available evidence, further studies are needed to assess larger sample size populations before any definitive conclusions can be drawn about the presence of the wnv in semen. nevertheless, the studies presented suggest that the wnv in semen is a possibility that bears further investigation. analysis of semen during the period of acute illness is also needed to assess semen infectivity at that time. given the poorly established evidence that the wnv is even present in semen, it follows that the possibility of sexual transmission is even less well characterized. there is only a single case report of male-to-female possible sexual transmission of wnv when a previously healthy middle-aged woman developed viral meningoencephalitis weeks after unprotected intercourse. her husband had serologically confirmed wnv. although no mosquito bite was reported, the women lived in a mosquito endemic area. interestingly, the food and drug administration recommends that all donor semen for assistive reproductive technology be tested for the wnv using a nucleic acid test in any donation made between june and october each year, the season when the virus is most active. further work is needed to determine if this type of testing should be made more routinely available to men with confirmed wnv desiring to father a pregnancy. the only available report of a possible compromise in fertility from the wnv comes from a case report of an autopsy on a year-old man deceased from the wnv. in this case, thickening of the tubular basement membrane was observed with frequent foci of tubular necrosis and absence of spermatogenesis. immunohistochemical staining did not identify wnv antigen in testicular tissue; however, transmission electron microscopy (tem) of formalin-fixed testicular tissue did reveal enveloped particles fitting the size, structure, and location of flavivirus particles, leading the authors to speculate that this was likely wnv causing orchitis. no studies could be identified reporting on semen parameters during or after acute wnv infection, and no studies could be identified detailing possible changes in sexual function. more evidence is required to substantiate a link between the wnv and reduced fertility. the influenza virus is among the most common infectious illnesses worldwide. the virus came into prominence among humans and pigs in the pandemic, shortly after mutations of avian strains enabled transferal to humans and pigs. amplified by the movement and proximity of world war i soldiers, the virus soon infected million people and killed million, more than the military and civilian deaths of world war i combined. since then, type a influenza has produced pandemic outbreaks in , , and most recently in . these events are triggered by reassortment in avian or swine reservoirs of genes for key hemagglutinin (h) and neuraminidase (n) surface glycoproteins, by which the virus is subtyped. the h n and h n strains of type a influenza currently circulate among humans, which along with influenza b cause seasonal flu (who ). notably, in the h n pandemic, young people were particularly at risk, whereas of older than the age of years had antibodies, likely from a historical infection or vaccination with a similar strain. the presence of influenza in semen has not been reported. however, the virus demonstrates extrapulmonary symptoms in many major organ systems, a large number of which have since been found contain to functional influenza receptors. , no studies to date have explored if those receptors are present in the human genital tract, but such tropism has been noted in turkeys. , given the variability of viral tropism among animals, further work is need to explore whether such tropism exists among humans or is reflected by the presence of viral particles and rna in semen. there is a similar dearth of information on sexual transmission of influenza, possibly because of other, more likely routes of infection. evidence suggests that while infection by direct contact is possible, it is not likely to be a significant mechanism compared with transmission via aerosol and respiratory droplets. these airborne routes are both particularly infectious within close contact and should deter high-risk populations from sex during the duration of viral shedding. as a pyrogenic virus, influenza affects sperm quality. macleod noted that medical students who presented with febrile illness had decreased sperm count, motility, and morphology parameters. morphology and motility recovered at weeks, as did sperm count at weeks. buch and havlovec reported a case of a semen donor who suffered a -hour febrile viral illness, who then had decreased sperm count and reduced egg penetration ability and weeks later. other studies have found similar effects from non-febrile heating such as saunas or laptops. , a case study by sergerie et al first detailed the timeline of genital tract effects following febrile influenza. after days of high fever due to the virus ( e c), a -year-old man had decreased total sperm count at days , , and before normalizing at day . motility percentile was similarly decreased but recovered sooner at day . the authors measured the dna fragmentation index and found it to increase from % before the fever to %, %, % and % at , , , and days, respectively. sperm dna fragmentation by terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay increased from % before fever to % at day , but this was not statistically significant (p < . ). evenson and jost found that underlying dna changes included decreased free sh groups and alteration of nuclear protein composition, causing latent effects on sperm chromatin structure and stability. murine models suggest that the viral particulates are also damaging. sharma et al first intraperitoneally inoculated mice with influenza a and recorded a significant increase in numerical and structural alteration of meiotic chromosomes, particularly aneuploidy. subsequent mouse studies noted similar dna damage was induced by both inactivated and purified influenza indicating a possible direct cytotoxicity by the viral particles. therefore, while evidence argues against a local inflammatory response, , the transient sperm changes observed are likely due to a combination of systemic fever and direct dna damage resulting in apoptosis and a transient decrease in fertility. history sars is respiratory disease caused by a novel coronavirus that first appeared in the guangdong province of southern china in . the virus rapidly became an epidemic and spread globally to countries and , people before being contained in . subsequent research uncovered a natural reservoir within horseshoe bats and civets acting as an intermediate host in local meat markets. although no cases have been reported since , the presence of this animal reservoir, the high reported case-fatality rate ( %), and a single reappearance of the virus have driven continued monitoring of the virus near its site of origin. sars in semen investigation into the effect of novel coronaviruses on fertility began after the discovery that angiotensinconverting enzyme (ace- ), which is expressed in the testis, acts as a functional receptor for the sars virus. , zhao et al first reported detection of the virus within testicular epithelial cells and leydig cells through combined ish and tem. however, no other research teams have been able to mirror these results. e ding et al examined all tissues that express ace- in autopsies of patients with sars and were surprised to find that the testes were uniformly negative for the sars rna and signature n protein despite high levels of ace- expression in the testes. similarly, gu et al noted focal testicular atrophy in all autopsies with confirmed sars but found no evidence of sars in parenchymal tissue by ish and tem. therefore, although semen has not been directly examined for sars, indirect evidence suggests that it is unlikely to be a reservoir. similar to other respiratory viruses, sars spreads most readily through respiratory droplets, fomites, and aerosol. to date, no sexual transmissions of sars have been reported. given that most studies do not find sars in genital tissues, any transmission of sars after intercourse is more likely due to direct contact and close proximity rather than through genital secretions. a mechanism for genital injury from sars was furnished in by xu et al. they analyzed the pathologic changes in testicular autopsy specimens from patients with sars compared with those of controls. all patients with sars demonstrated testicular atrophy grossly. the sars samples exhibited extensive germ cell destruction with increased apoptosis on tunel assay. histologically, basement membrane thickening, peritubular fibrosis, leukocytes, and vascular congestion indicative of local inflammation were noted. ish that produced positive readings in the lungs of the patients with sars was negative in the testes. however, extensive deposits of igg were detected within the seminiferous epithelium, interstitium, sertoli cells, and some germ cells of patients with sars compared with controls. this potentially represents an autoimmune orchitis secondary to the immune response to sars. further research is needed to explore this possibility and the persistence of orchitis. in addition, data are needed to ascertain whether the germ cell destruction in sars-associated orchitis is reflected in semen characteristics. history sars-cov- is a novel coronavirus known to cause the disease "coronavirus disease- " or "covid- ". it emerged in late in wuhan, hubei province, china, and likely has its origin in bat populations. on march , , the world health organization declared covid- to be a worldwide pandemic. it has been estimated to cause severe disease in % of cases. older individuals are particularly susceptible to severe illness, with % of deaths occurring in individuals aged years and older. given the recent and novel nature of the covid- outbreak, data remain limited. thus far, there are mixed findings regarding the presence of sars-cov- in semen. pan et al have reported a case series of adult chinese men diagnosed with covid- who showed no sars-cov- in semen at a median follow-up of days. unpublished data from chinese patients in recovery from covid- showed no sars-cov- in rt-pcr of semen. testicular biopsy from deceased individual in this study likewise was negative for sars-cov- viral rna. in addition, paoli et al showed negative rt-pcr in both urine and semen samples of an individual who tested positive for sars-cov- by nasopharyngeal swab. in contrast, li et al reported that of ( . %) patients in a cohort of patients in shangqiu, china, had virus detected in their semen by rt-pcr. of them, were in the acute stage of infection, and were recovering from the virus. in providing analysis and commentary on these findings, paoli et al nevertheless recommended that the small caseload and recency of infection in patients should give readers caution before drawing sharp, fatalistic conclusions. further studies will be essential to confirm these findings. although sars-cov- is not known to be present in semen, precluding the ability for sexual transmission, the intimate nature of physical sexual contact still dictates caution to prevent viral spread. given the presence of sars-cov- in respiratory droplets, saliva, mucus, and fecal matter, as well as current recommendations to maintain feet of distance between individuals to prevent the spread of infection, physical sexual intimacy presents a high-risk scenario for viral transmission, particularly for non-monogamous partners who do not live with one another. , in the chinese case series reported by pan et al, individuals ( % of the cohort) described scrotal discomfort around the time of onset of viral illness, suggesting sars-cov- may induce a viral orchitis. studies on semen quality from covid- survivors have not yet been reported. chen et al have proposed a theoretical link between covid- and reduced fertility based on the virus' strong interaction with angiotensin-converting enzyme (ace ). molecular modeling conducted by the authors showed the virus have a unique structure that allows it to penetrate deep into the hydrophobic pocket of ace . ace is highly expressed in the renal tubular cells, the intestines, leydig cells, and the seminiferous ducts in the testes, raising the possibility that the virus may have a deleterious testicular effect. by contrast, pan et al demonstrate with single-cell transcriptome data that ace rna is sparsely expressed in the testis and argue that this is an unlikely medium of viral entry into target host cells. even so, it is still unknown exactly what function ace serves in the testis. corona et al analyzed these studies and recommended that those of reproductive age consider andrological consultation and evaluation before safely pursuing reproduction. larger studies will be imperative to understand currently conflicting evidence and to deduce what potential fertility sars-cov- may have. in this article, we have reviewed the presence in semen, possibility of sexual transmission, and fertility implications of each of the major recent viral pandemics: zika, ebola, west nile, pandemic influenza, sars, and sars-cov- . the zikv has been reported in semen up to days after disease onset but appears to be present for a median time of days for most individuals. sexual transmission of the zikv has been repeatedly documented, even among entirely asymptomatic individuals, and is of particular concern given its ability to microcephaly in a developing fetus. short-term fertility may be negatively affected by the zikv based on several reports of reduced sperm count, altered morphology, and impaired mobility in semen samples. fortunately, this effect appears time limited. the ebov has also been detected in semen, with studies indicating its presence for an average of days, with a maximum reported duration of days. reports of sexual transmission remain rare, with only suspected cases documented in the history of the virus. semen parameters do not appear altered, although a proportion of survivors decreased sexual function and diminished libido. the wnv has only been reported in semen or to be transmitted sexually in isolated case reports. likewise, only a single autopsy report suggests the possibility of wnv-induced orchitis. influenza, a respiratory pathogen, has not been found in semen or been shown to be sexually transmissible. research demonstrating its impact on fertility has focused on the effects of febrile illness on semen parameters, showing that febrile episodes are linked to transiently decreased sperm count, motility, and morphology. the sars virus has not been shown to be present in semen, but several small case series of autopsy reports have found testicular damage and atrophy in individuals deceased from sars, which may be linked to secondary autoimmune orchitis. sars-cov- , the most recent viral pandemic included in this review, is expected to behave similar to sars virus, but further data are required to validate these assumptions. current evidence from small case series shows that it is not present in semen and thus is unlikely to be sexually transmitted. for both sars and sars-cov- , there is speculation that the viruses' interaction with ace , which is present in the leydig cells and the seminiferous ducts of the testes, could have implications for spermatogenesis. further studies are needed to explore this possibility. we have reported the presence in semen, sexual transmission potential, and fertility side effects of recent viral pandemics. overall, semen studies and fertility effects are highly understudied in viral pandemics, and rigorous study on these topics should be undertaken as novel pandemics emerge. specifically, further semen studies and fertility studies are needed to understand the transmission potential 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a focal point of zika virus infection date: - - journal: j biomed sci doi: . /s - - - sha: doc_id: cord_uid: zr e lh zika virus (zikv) belongs to the flavivirus genus of the flaviviridae family. it is an arbovirus that can cause congenital abnormalities and is sexually transmissible. a series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital zikv syndrome and its underlying pathophysiological mechanisms. endoplasmic reticulum (er) and er-related proteins are essential in zikv genome replication. this review highlights the subcellular localization of zikv to the er and zikv modulation on the architecture of the er. this review also discusses zikv interaction with er proteins such as signal peptidase complex subunit (spcs ), er membrane complex (emc) subunits, and er translocon for viral replication. furthermore, the review covers several important resulting effects of zikv infection to the er and cellular processes including er stress, reticulophagy, and paraptosis-like death. pharmacological targeting of zikv-affected er-resident proteins and er-associated components demonstrate promising signs of combating zikv infection and rescuing host organisms from severe neurologic sequelae. zika virus (zikv) is a mosquito-borne virus that belongs to the flaviviridae family together with other notable flaviviruses such as dengue virus (denv), west nile virus (wnv), japanese encephalitis virus (jev), and yellow fever virus (yfv). zikv was first isolated from a febrile rhesus monkey in april and was subsequently isolated from aedes africanus mosquitoes months later in zika forest of uganda [ ] {dick, # }. despite its discovery more than a half-century ago, zikv received little attention due to sporadic cases of human infection with mild and self-limiting symptoms [ ] . zikv was put under scrutiny following its first outbreak in the yap island of micronesia in . during this outbreak, suspected cases of zikv infection were reported, and at least % of these patients were either serologically or molecularly confirmed for zikv infection [ ] . the same study estimated that island residents ( %) were infected, of which were symptomatic [ ] . zikv-infected patients typically present mild clinical symptoms such as fever, maculopapular rash, conjunctivitis, and arthralgia [ ] . nevertheless, the second zikv outbreak in french polynesia in provided the first compelling relationship between zikv infection and a neurological complication, where a woman was diagnosed with guillain-barré syndrome (gbs), an autoimmune disease typically affecting motor neuron functions, a week following the onset of zikv-like symptoms [ ] . this epidemic also recorded a -fold increase of gbs incidence, where gbs-diagnosed patients ( %) were serologically positive for zikv [ , ] . the sudden spike of microcephaly cases among newborns in brazil following the - zikv outbreak triggered the brazilian ministry of health and the world health organization to declare zikv as a national and international public health emergency [ , ] . the causative link between zikv and congenital neurological anomalies was established based on several evidence including the detection of zikv rna within the amniotic fluid acquired from zikv-infected mothers with confirmed fetal microcephaly case [ , ] . subsequent clinical and pre-clinical findings further supported the causal relationship between zikv infection and microcephaly in newborns [ ] [ ] [ ] . zikv is believed to infect cells through receptormediated endocytosis. these putative receptors include cluster of differentiation (cd ), tyrosine-protein kinase receptor tyro , and axl, where overexpression of these receptors in zikv-impervious hek t cells rendered the cells susceptible to zikv infection [ ] . in particular, the role of axl in zikv infection has been extensively investigated due to its crucial role in dengue virus infection [ ] . during zikv infection, axl mediates zikv entry indirectly whereby the phosphatidylserine extension on zikv lipid membrane binds to growth arrest-specific (gas ), one of the ligands for axl that serves as a bridge for zikv and axl interaction, resulting in clathrin-mediated virus internalization [ , ] . the acidic microenvironment within the endosome promotes fusion between the virus envelope proteins and the endosomal membrane resulting in the release of zikv genome into the host cell cytosolic space [ , ] . the zikv genome is a positive-sense, single-stranded rna ((+)ssrna) of approximately , bases in length [ ] . zikv genome contains a single open reading frame that encodes three structural and seven non-structural (ns) proteins. these structural proteins consist of capsid (c), pre-membrane (prm), and envelope (env) proteins, which are predominantly involved in viral pathogenesis and virion structure. the seven non-structural proteins, ns , ns a, ns b, ns , ns a, ns b, and ns proteins, largely contribute towards the purposes of viral pathogenesis, replication, and immune evasion [ ] . zikv utilizes host translational machinery to produce a single polyprotein that is further cleaved by viral ns b-ns serine protease and host cell protease into functional viral proteins [ ] . these viral proteins are then distributed to cellular compartments for various functions [ ] . zikv proteins localize to distinct subcellular compartments zikv proteins are primarily distributed within and in close proximity to several endomembrane compartments including the endoplasmic reticulum, golgi apparatus, endosomes, lysosomes, autophagosomes, and nucleus [ ] . molecular cloning and expression of individual zikv proteins reveal distinct and specific organellar localization. zikv capsid localizes to several compartments including the nucleus and the golgi [ ] . in addition to capsid, ns b and ns a also localize to the golgi. three zikv proteins namely env, prm, and ns a are distributed to the er as indicated by their co-localization with calreticulin expression. ns , which contains nuclear localization signals, forms punctate distribution in the nucleus [ ] . however, these data should be interpreted with caution as the subcellular localization of these cloned viral proteins may differ in zikv-infected cells. for example, individual expression of zikv ns localizes to the mitochondria, but ns is instead localized at the er when co-expressed with ns b [ ] . similarly, although ns protein is primarily detected in the nucleus of zikv-infected cells [ ] , zikv ns -rna polymerase has been shown to interact with zikv ns -helicase during viral rna replication in the er [ ] . inhibiting this interaction reduces ns helicase activity. subcellular localization of viral proteins is affected by viral proteins interaction, which is crucial for productive infection. importantly, understanding the mechanisms of viral protein transport and viral protein-protein interaction may provide novel therapeutic targets. the subcellular distribution of certain zikv proteins was corroborated in a separate study where interatomic analyses using proximity-dependent biotin-identification (bioid) labeling and flag-based immunoprecipitation (ip) coupled with mass spectrometry (ms) uncover indepth molecular interactions between zikv proteins with various host organelles and proteins. in general, zikv proteins mainly interact with host proteins that are involved in protein processing, vesicle trafficking, rna processing, and lipid metabolism [ ] . zikv capsid interacts with multiple nucleolar proteins, whereas ns protein targets and disrupts the cajal bodies in the nucleus [ ] . this observation is consistent with the aforementioned experimental study that showed nuclear localization of zikv capsid and ns protein [ ] . the study also identified numerous interactions between viral proteins (prm, env, ns a, ns b, ns a, and ns b) with various er proteins, which highlights the importance of the er in zikv life cycle [ ] . zikv has been reported to be dependent on several er proteins including er-associated signal peptidase complex (spc) proteins, er translocon, and er membrane complex (emc) proteins. spc, especially spc subunit (spcs ), is crucial for zikv pathogenesis as knocking out spcs in t cells significantly reduced zikv infection and drastically impaired the production of infectious zikv particles [ ] . a study using pooled crispr/cas cell survival enrichment assay identified zikv strongly depends on er membrane complex (emc) during early-stage zikv replication [ ] . emc is a highly conserved oligomeric complex residing on the er membrane and is crucial for transmembrane protein folding and lipid trafficking [ ] . the dependency on six emc subunits (emc -emc ) was verified with sirna assays, where depletion of these proteins significantly impaired the replication of several zikv strains [ ] . another study reported that zikv ns b physically interact with emc subunit and depletion of this subunit markedly reduced the level of zikv ns a and ns b protein [ ] , indicating that emc proteins are required for viral protein biogenesis. further investigation using denv ns b identified two marginally hydrophobic domains at the n-terminal of ns b to be crucial for ns b dependence on emc [ ] . since zikv ns b shares moderate sequence identity, high sequence similarity, and similar topology with other flaviviruses [ ] , it is plausible that the two weak hydrophobic domains of zikv ns b are the specific determinants for zikv dependency on emc proteins. in addition to its direct interaction with viral proteins, emc proteins also associate with er sec translocon and oligosaccharyltransferase (ost) complex proteins, both of which are also important for zikv infection [ , ] . sec is a major component of the er translocon that facilitates the entry of nascent polypeptides into the er lumen for protein processing. zikv dependency on sec translocon was validated in a separate study wherein myolactone treatment, a sec α inhibitor, dramatically reduced zikv expression [ ] . zikv replication was restored in cells expressing mutant sec α that conferred resistance against myolactone inhibition [ ] . zikv dependency on ost complex, an integral part of the translocon consisting of eight er-transmembrane protein subunits that catalyzes co-translational n-glycosylation, is based on emc , emc , emc and emc interaction with ost complex subunits namely stt a/b, rpn / , and ddost [ ] . additionally, zikv proteins including ns b have been reported to directly interact with these ost subunits [ ] . zikv dependency on ost complex is corroborated by marceau et al. that reported a significant abrogation of zikv rna replication in stt a knockout cells [ ] . importantly, treatment with ngi- , a small molecule ost complex inhibitor, significantly reduced zikv rna replication with an ec value of . μm [ ] . the inhibitory activity of this non-toxic molecule (cc = . μm) is independent of n-linked glycosylation activity of the ost complex as zikv replication was drastically impaired in hap cells containing wild-type or catalyticinactive stt a [ ] . the specific mechanism of zikv dependency on stt a is unclear, but immunoprecipitation and electron microscopy studies showed physical interactions between denv proteins and stt a/b, forming viral rna replication complexes that reside within the er in close proximity to denv -vesicle packets [ ] . since zikv proteins also directly interact with stt a and zikv infection induces the formation of vesicle packets [ , ] , it is plausible that zikv depends on ost complex in a similar manner. in short, zikv proteins interact with various host proteins and exploit their functions to accommodate the progression of the viral life cycle. er and er-related proteins are especially important in the virus genome replication, which may be useful in developing antiviral therapies. the following sections of this review will discuss the structural and molecular changes in the er following zikv infection. throughout the course of infection, viruses induce various modifications on the host cells' metabolisms and their cytoarchitecture to support the progression of virus life cycle. one of the common targets is the er [ ] . among the important functions of the er include protein processing, synthesis of essential biosynthetic compounds (primarily lipids and proteins), metabolism of steroids and carbohydrates, and storage of calcium ions (ca + ) for intracellular signaling [ ] . structurally, the er is made of a continuous network of membraneenclosed flattened sacs and tubules. the dynamic and fluid nature of these structures enable this organelle to accomplish tubule extension, membrane curvature and shape alteration in response to stress-induced or factordemanding conditions such as cell division and differentiation [ ] . likewise, zikv infection is also capable of exploiting the organelle's plasticity and remodels the er structure for their replicative benefit. recent transmission electron microscopy analyses on zikv-infected cells frequently observed significant expansion of the er [ , , ] . this structural modification typically consisted of proliferating er lamellae and dilated er lumen, which cumulatively increases the overall er size and volume. enlargement of the er is believed to occur in response to virus-induced er stress, due to a supply-demand imbalance that will be elaborated in the next section. zikv infection also forms distinct aggregates of intricate er tubular network that resembles sponge-like matrix called the convoluted membranes (cm) (fig. ) [ , [ ] [ ] [ ] . cm aggregates are frequently seen bordering zikv virions and other zikv-induced structures, with occasional virions also found within the cm itself [ , ] . the function of the cm in zikv infection is unclear; nevertheless, evidence from other flaviviruses studies suggest that the cm possesses roles closely associated to protein synthesis and processing [ ] . interestingly, cm aggregates are not observed in zikv-infected neural progenitor cells, suggesting cell-type-specific factors are required to develop the cm [ ] . no discernible difference in the cm structure is observed between the african and asian zikv strains [ , ] . next, vesicle packets (vps) are also commonly observed in zikv-infected cells ( fig. ) [ , [ ] [ ] [ ] . vps refers to an aggregate of closely packed single-membrane vesicles that are encapsulated within an er cisterna. individually, these vesicles measure between and nm in diameter with a narrow channel of approximately nm that connects the vesicle lumen and the cytosol [ , ] . the radial dimension of these vesicles varies according to the host cell type, thereby hinting possible involvement of cell-type-specific factors in its formation [ ] . modest radial difference was also noted between zikv lineages [ ] ; however, more morphologically pronounced difference between the zikv lineages is observed in the vesicle's shape whereby african strain tend to form ovoid vesicles, whereas the asian strain's vesicles are generally more spherical [ ] . nevertheless, these differences, especially the latter, remain contentious due to limited experimental measurements and warrants additional investigations across various cell types to support these observations. despite these differences, the vesicles' pore maintain a comparable diameter of nm regardless of the virus strain and the host cell type, suggesting that a conserved cellular or viral machinery mediates pore formation [ ] . zikv exploits the er dynamic characteristic and remodels the er structure to generate virus-induced structures, vesicles (ve), vesicle packet (vp), convoluted membrane (cm) and zippered er (zer), for the benefit of virus replication. blue and green box depicts schematic representation of host and viral factors involved in vesicle and virus particle (vi) formation, respectively. zikv ns a utilizes the host reticulon . a (rtn . a) to facilitate membrane curvature during vesicle invagination into the er lumen (blue box). viral genome replication takes place within this vesicle. neosynthesized viral rna genome is released into the cytosol and could undergo either translation, virus particle assembly, or another round of genome replication. virus particle assembly takes place in apposed er leaflet. separate zikv ns a independently recruits viral genome rna, ns b-ns , and unprocessed c-prm-env complexes, and subsequently congregates at the virus particle assembly site through ns a oligomerization (green box). once assembled, ns b-ns proteolytical activity cleaves the recruited c-prm-env complex to generate individual capsid, prm and env protein. cleaved capsid protein interacts with capsid-dense lipid droplets and viral rna to generate nucleocapsid core followed by env and prm proteins encapsulation of the nucleocapsid core invagination of zikv-induced vesicles into the er lumen is mediated through the host reticulon . a (rtn . a) that facilitates er membrane curvature (fig. , blue box) [ ] . silencing of rtn . a impairs ns a protein stability and results in significant reduction of virusinduced vesicles and virus replication [ ] . typically, these vesicles contain the virus' replication complex and therefore carries out the virus rna synthesis or viral replication [ , ] . following viral replication, neosynthesized viral genome rna is released into the cytosol through the vesicle's narrow channel, where it would consequently either undergo another round of genome replication, translation or virion assembly [ , ] . in the latter case, zikv virion assembles and buds into the er lumen directly apposed the narrow pore of the replication vesicle (fig. , green box) [ ] . although detailed mechanistic insights of virion assembly remains uncertain, recent study identified that zikv ns a independently recruits viral (+)-ssrna, ns b-ns , and unprocessed c-prm-env complexes to the virus particle assembly site, possibly through ns a oligomerization [ ] . the same study postulates that virus particle assembly proceeds through ns b-ns protease complex cleaving the recruited c-prm-env complex. cleaved capsid protein form nucleocapsid core with the viral rna and capsid-dense lipid droplet; and subsequently, it is encapsulated by prm and env proteins, which leads to eventual virion budding into the er lumen [ ] . ultrastructural analyses within the dilated er lumen consistently identified virus particles arranged in a two-dimensional paracrystalline array [ , ] . this array is located in close proximity to the replication vesicles and is therefore largely comprised of fully assembled virions and viral-like particles (enveloped virions without viral genome) [ ] . moreover, zikv-infected huh cells also exhibited a unique structural alteration of collapsed er cisterna or more commonly known as zippered er (zer) (fig. ) that reminisce the observation reported in avian infectious bronchitis virus (ibv) infection, a (+)-ssrna virus from the coronaviridae family [ , ] . this finding presents the first identification of such structure among flavivirus-infected cells and has only been observed in zikv-infected huh cells, suggesting that cell-typespecific factors are possibly required to generate this structure [ ] . the role of zer in zikv infection is unclear; however, zer are frequently found adjacent to vps suggestive of functions associated with vesicles formation, which is consistent with observations made in ibvinfected cells [ , ] . nonetheless, this unique finding warrants further research to elucidate the mechanistic details and biological relevance of the zer. in addition to the direct modulation of the er structure, zikv also induces the formation of viroplasm-like structures, which is incidentally the first reported observation of such structure in flavivirus infection [ ] . contrary to other zikv-induced structures, viroplasms are cytoplasmic inclusion of viral replication components and various relevant host factors associated with virus replication that are formed through reorganization of cell membrane or cytoskeletal elements [ ] . these structures are significantly larger than vps and can measure up to nm in diameter [ ] . viroplasms are primarily located in the perinuclear region and in close proximity to the er, mitochondria, and microtubules to facilitate viral genome replication [ ] . the spatial segregation of virus-induced enclosed structures (viroplasms and vesicles) is postulated to create a subcellular microenvironment concentrated with factors required for virus genome replication, and simultaneously provides physical barriers against rna nucleases and the host immune responses [ , ] . the latter, in particular, is consistent with an investigation on wnv that virusinduced structures confer protection, albeit partially, against the host immune mechanism [ ] . in summary, zikv and several zikv proteins localize to the er and eventually leads to the remodeling of the reticular architecture and exploitation of the organelle's unique characteristics. these modifications create an ideal environment for viral genome replication, but simultaneously introduce additional burden and stress on the host cell leading to the activation of several cellular responses, which will be discussed in the following sections. one of the fundamental roles of the er is the folding of secreted and membrane proteins, which depends on a multitude of factors including supply of atps, stable ca + concentration, and a balanced redox environment [ ] . disruption of this specialized environment within the er, whether through glucose deprivation or viral infection, may lead to overwhelming accumulation of misfolded and unfolded proteins-a condition described as er stress [ ] . under normal conditions, glucose regulated protein (grp ), one of the er molecular chaperones, binds to er stress sensor transmembrane proteins: protein kinase rna-activated (pkr)-like er resident kinase (perk), activating transcription factor (atf ), and inositolrequiring enzyme (ire ). in the presence of misfolded or unfolded protein, grp will bind to exposed hydrophobic residues of these proteins to induce proper protein folding [ , ] . accordingly, during er stress, the accumulation of misfolded or unfolded proteins saturates the free pool of grp and titrates the bound grp away from the stress sensors leading to activation of respective sensor's molecular pathways [ ] . these molecular initiatives and their corresponding downstream effects are collectively known as the unfolded protein response (upr). this evolutionarily conserved countermeasure induces pro-survival responses including global arrest of protein synthesis, upregulation of protein degradation factors, and enhanced protein folding capability [ ] . however, under severe er stress conditions, upr will instead trigger apoptosis. following zikv infection, the accumulation of misfolded virus polyproteins in the er lumen overwhelms the er protein-folding capacity leading to er stress and triggers the activation of the upr (fig. ) [ ] . additional evidence of er stress and upr activation are demonstrated in the elevated expression of grp and other chaperones such as calnexin, calreticulin, and protein disulfide isomerase (pdi) in zikv-infected neural cells in vitro and in vivo [ , , ] . increased expression of these er stress markers was accompanied by impaired indirect neurogenesis and microcephalic phenotype in mice [ ] . this finding is consistent with a previous report wherein the induction of upr in cerebral apical progenitor cells tipped neuronal differentiation towards direct neurogenesis at the expense of indirect neurogenesis, leading to depleted intermediate progenitors, reduced overall cortical neuron output, and diminished cerebral volume, which ultimately caused microcephaly in vivo [ ] . in addition to elevating the expression of er chaperone proteins, zikv has been shown to induce upr by activating the stress sensors as summarized below (fig. ) . atf is a type ii er transmembrane protein that contains a transcription activation domain (tad) on its cytosolic-facing n-terminal and two independent golgilocalization signals (gls) on its luminal-facing cterminal [ ] . following the dissociation of grp that unmasks the gls, atf translocate to the golgi apparatus and undergoes sequential proteolytic processing by fig. zikv-induced er stress initiates host cell unfolded protein response (upr). zikv infection induces er stress due to the increased amount of unfolded/misfolded viral (red strand) and host cell (grey strand) protein aggregates in the er lumen. the accumulation of these partially processed proteins leads to the overwhelming demand of correct protein folding. to facilitate protein folding, grp (orange) dissociates from the stress sensors (ire , atf , and perk) and binds to the misfolded/unfolded proteins. expression of other chaperones is also elevated to address the overwhelming protein folding demand during zikv infection. binding of misfolded protein to perk (green) activates the sensor and triggers phosphorylation of eif that in turn stimulates ) global translational block except for selective mrna involved in upr, and ) formation of stress granules. however, zikv bypasses global translation block and inhibits stress granules formation, but the mechanistic details of these viral interference are still unclear. activation of atf (yellow) promotes the protein proteolytical processing in the golgi apparatus into atf n, atf n nuclear translocation, and expression of upr target genes including xbp . activated ire (purple) splices xbp transcript which produces an active transcription factor (xbp s) that stimulates expansion of the er volume, and expression of chop and erad factors such as edem- . alternatively, ire can trigger ask -p mapk pathway that enhances chop apoptotic activities and promotes other apoptotic-related activities under severe er stress condition. blue pointed arrows denote activation, blue blunt-end arrows denote inhibition, and red pointed arrows denote increased expression/activity site- and site- proteases, liberating tad-containing atf n-terminal (atf n) [ ] . subsequently, atf n enters the nucleus and binds to er stress response elements in the promoter region of upr-target genes, including x-box binding protein (xbp ) transcription factor, a downstream effector of ire stress sensor pathway, and grp to autoregulate the upr [ ] [ ] [ ] . zikv infection has been shown to activate the upr via atf , where atf proteolysis and atf n nuclear translocation were induced in vitro and in vivo [ ] . ire is a single-pass transmembrane protein. the protein's er luminal domain serves as a stress sensor, whereas the cytosolic domain is sub-divided into kinase and ribonuclease (rnase) domains. under er stress, grp releases from ire luminal domain and binds onto the misfolded/unfolded proteins. this dissociative event frees the luminal domain and in turn, permits binding of the misfolded/unfolded protein onto the liberated domain [ , ] . consequently, ire oligomerizes at its luminal domain, which brings the kinase domains in close proximity leading to auto-phosphorylation of the kinase domains [ ] . the phosphorylation event at the activation loop is postulated to induce conformational changes of the rnase domain that permit a more efficient binding of rna substrates [ ] . activated ire cleaves off nucleotides from xbp transcripts, resulting in a translational frameshift to produce a potent transcriptional activator (xbp s) that elevates the expression of upr-target genes to promote protein folding, processing, and secretion [ , ] . xbp s also modulates the expression of factors involved in er-associated protein degradation (erad), a cellular process of eliminating aberrant proteins through retro-translocation and proteasomal degradation [ ] . alternatively, ire protein can also mediate er stressinduced apoptosis following chronic stress damage by triggering activation of apoptotic signaling kinase (ask ), which consequently activates p mitogen-activated protein kinase (p mapk) and jun n-terminal kinase (jnk) leading to apoptosis [ ] . jnk mediates apoptosis primarily by inhibiting the anti-apoptotic b-cell lymphoma (bcl- ) and activating the pro-apoptotic bim protein [ ] , whereas p mapk promotes apoptosis by phosphorylating ccaat-enhancer-binding protein (c/ebp) homologous protein (chop) at serine residues and to enhance its transcriptional activity and induce apoptosis [ , ] . chop is expressed downstream of all three er stress sensors, indicating overlapping upr pathways during severe er stress [ ] . chop forms heterodimers with other c/ebp family transcription factors; therefore, the basic domain in chop renders the transcription factors incapable of binding to their respective dna binding sites, which reduces the expression of their target proteins including bcl- [ ] . chop also contains a tad and thus, can bind to a unique sequence to induce the expression of target genes including bim [ , ] . zikv infection has been shown to promote xbp splicing and xbp s translocation into the nucleus, leading to elevated expression of xbp downstream effectors such as er degradation-enhancing α-mannosidase-like (edem- ) and chop, indicating the activation of the ire arm of the upr [ ] . edem- is an enzyme involved in the erad process that recognizes and facilitates retrotranslocation of misfolded proteins to the cytosol for subsequent proteasomal degradation and thus, alleviating stress damage [ ] . instances of aggravated er stress were also demonstrated in zikv-infected cells, which lead to elevated expression of chop protein and initiation of erstress-induced apoptosis [ ] . pharmacological intervention using ire inhibitor, μ c, prevented microcephaly and impaired zikv replication in mouse fetal brains [ ] . modulation of ire -ask pathway during zikv infection is still unclear. previous study reported that ectopic expression of xbp promotes expansion of the er, a visual sign often manifested in response to er stress [ ] . this morphological change is hypothesized to reduce the local concentration of misfolded/unfolded proteins and accommodate the overwhelming demand for protein folding-a significant portion of which is neosynthesized viral polyproteins [ , ] . consistent with increased expression of xbp , ultrastructural characterization of zikv-infected cells reported significant er enlargement extending across the cytoplasm and is related to zikv-induced er stress [ , , , ] . similar to ire , release of grp proteins enable misfolded/unfolded proteins binding onto exposed perk luminal domain leading to oligomerization and autophosphorylation of perk protein to activate its downstream pathway [ , ] . perk phosphorylation, in turn, induces eif α phosphorylation (p-eif α) that mediates three interlinked upr mechanisms: expansion of protein folding capacity, suppression of nascent protein production and induction of apoptosis in the event of severe stress [ ] . phosphorylation of α subunit in the eif complex hinders the assembly of preinitiation complex (pic) by blocking the activity of eif b guanine exchange factor, leading to global suppression of mrna translation [ ] . however, selected mrnas, typically transcripts for upr machinery, can bypass this translational block [ ] . interestingly, viruses have evolved various mechanisms to overcome this translational block, but the mechanism of this process in zikv infection is unclear. recent analyses of zikv infection in vitro and in vivo reported a substantial increase of eif α phosphorylation and elevated expression of several downstream perk effectors such as atf , atf , chac , and chop [ , ] . importantly, intracerebroventricular administration of pharmacological perk inhibitor in zikv-infected mice restored appropriate neurogenesis balance and rescued infected mouse embryos from microcephalic phenotype. however, unlike ire inhibitor, perk inhibitor did not affect zikv replication [ ] . perk inhibitor also prevented microcephaly in placental zikv inoculation model, which mimics natural zikv vertical transmission [ ] . in summary, zikv localizes to the er for viral genome replication. these additional transcriptional and translational processes impart significant burden on the er leading to er stress and upr induction. during the early stage of upr, upr effectors elicit adaptive responses to mitigate er stress. these responses include the upregulation of chaperones and protein processing enzymes to promote and rectify protein folding, induction of erad to degrade misfolded/unfolded proteins, global translational arrest, and activation of autophagy and/or reticulophagy. beside upr, er stress also concomitantly induces several other cellular processes. specifically, stress granules assembly and er autophagy impart fundamental importance in regulating translational arrest and er homeostasis, respectively. zikv have been reported to subvert these processes to allow the progression of viral replication. er stress signal can be transmitted to other cellular components to rescue cell survival. a clear example to illustrate stress signal transmission is the generation of stress granules (sgs) in the cytoplasm to stall initiation of translation [ ] . sgs assembly are generally triggered through two distinct categories of mechanisms: ) eif α-independent and ) eif α-dependent mechanisms [ ] . in particular, the latter category, or more specifically eif α-phosphorylation, is mediated by pkr, perk, general control non-derepressible- (gcn ), and heme-regulated inhibitor kinase (hri) in response to diverse cellular stress signals [ ] . sgs are ribonucleoprotein (rnp) structures primarily composed of non-translating mrnas, stalled translation initiation complexes, and rna-binding proteins [ ] . the formation and components of sgs are reviewed in details here [ ] . sgs assembly during global translational arrest negatively impact viral genome translation as the sgs reduce the accessibility of translational machinery complexes [ ] . following the relief of translation suppression, the sgs are disassembled via several mechanisms, one of which involves eif α dephosphorylation by growth arrest and dna-damage-inducible (gadd ) protein [ ] . this allows the pic to resume protein translation at the surface of the sgs, resulting in sgs shrinkage and disappearance [ ] . viruses have adopted several mechanisms to repress the assembly of stress granules and utilize sg protein components for viral polyprotein synthesis instead [ ] . for example, herpes simplex virus genome encodes γ . protein that functionally mimics the activity of gadd and directs the dephosphorylation of eif α [ ] . alternatively, coronaviruses repress the assembly of stress granules by asserting an inhibitory effect on pkr activation and upregulating the expression of gadd [ ] . likewise, zikv also suppresses the formation of stress granules in favor of virus replication by upregulating the expression of gadd [ ] . consistent with this finding, pharmacological inhibition of gadd -mediated eif α dephosphorylation rescued sgs assembly and decreased zikv particles production [ ] . additionally, another study reported that zikv proteins, namely capsid, ns , ns b-ns , and ns a proteins, suppressed sgs assembly; however, this modulation was observed in an eif α-independent manner [ ] . zikv capsid inhibited sgs assembly by forming stable complexes with sg core proteins, ras gtpase-activating proteinbinding protein (g bp ) and caprin- , but not with t-cell-restricted intracellular antigen related (tiar) protein [ ] . zikv utilized these sg core proteins for viral protein and virion production. beside regulating eif α phosphorylation and hijacking key sg proteins, rna viruses were reportedly capable of interfering sgs assembly through cleaving and redistributing sgs nucleating factors [ ] . it was previously reported that zikv ns b-ns protease could cleave host antiviral factors to impair intrinsic host defense; intriguingly, zikv did not mediate the cleavage of sg factors even though sgs assembly was affected by zikv ns and ns b-ns protease [ ] [ ] [ ] [ ] [ ] . nonetheless, zikv infection was found to facilitate the redistribution of tiar to viral replication sites, which correlates to viral replication output [ , , ] . hence, it is unclear how zikv ns , ns b-ns , and ns a proteins block sgs formation and whether these viral proteins affect eif α phosphorylation. interestingly, a recent study also identified a key sg-interacting protein, human antigen r, exhibits substantial anti-zikv effect potentially by destabilizing the zikv rna [ ] . as previously discussed in section , the induction of er stress initiates several adaptive countermeasures such as upregulation of upr pro-survival factors and er enlargement to repair stress-induced damages and restore normal cellular functions. interestingly, initial studies in mammalian and yeast cells revealed that a fraction of cells also contain autophagosome-like structures following er stress induction [ , ] . these autophagic vacuoles expel selectively excised er membrane containing aberrant protein aggregates [ ] . this process, known as reticulophagy, is an additional mechanism that occurs in parallel with upr to regulate er volume and homeostasis [ ] . autophagy, including reticulophagy, is also a host innate defense mechanism as these autophagosomes have been shown to incorporate viral proteins for degradation [ ] . reticulophagy, also known as er-phagy, is mediated by er-phagy receptors such as family with sequence similarity member b (fam b) and reticulon- (rtn ) proteins that reside on the er membrane. these autophagy receptors sequester er fragments via its lc -interacting-region (lir) domain interaction with autophagosomal-presenting microtubuleassociated protein light chain (map lc ) [ ] . similar to other er-shaping proteins, fam b also possesses a reticulon homology domain (rhd), a motif that promotes membrane curvature [ ] . in zikv-infected cells, depletion of fam b protein renders notable er expansion and significant upregulation of zikv replication activity [ ] . this is due to the proteolytic activity of zikv ns b-ns that cleaves exposed rhd located on fam b protein, which impedes the oligomerization of fam b proteins and consequently, prevents er membrane excision and reticulophagy from taking place [ ] . this efficiently blocks host cell innate defense mechanism from degrading zikv proteins. to summarize, zikv virus bypasses the upr by inhibiting stress granules assembly and reticulophagy to ensure continuous viral protein translation and virion production while simultaneously protecting the virus from host cell defense mechanisms. prolonged er stress exacerbates the stress condition and leads to the activation of another arm of the upr: cell death. countless studies showed that zikv infection causes cell death through several cell death mechanisms such as paraptosis and apoptosis [ , ] apoptotic events in zikv-infected cells occur through both the intrinsic and extrinsic pathways based on the activation of pathway-specific caspase- and caspase- , respectively [ , ] . interestingly, zikv env protein is sufficiently capable to induce pro-apoptotic expression profile that represents intrinsic apoptosis including elevated expression of tumor protein p , which correspondingly mediates the expression and activity of its downstream targets by suppressing anti-apoptotic bcl- and facilitating expression of pro-apoptotic bcl- associated x (bax) [ ] . this modulation consequently reduces mitochondrial membrane permeability; thus, aiding the release of cytochrome c, formation of apoptosome, and initiation of caspase cascade, which eventually leads to cell death [ ] . additionally, increased level of several apoptotic markers, such as tumor necrosis factor alpha and receptor-interacting protein , in zikv-positive microcephalic neural specimens are suggestive of extrinsic apoptotic activation [ ] . as previously mentioned, er stress elevated the expression of chop to initiate apoptosis in zikvinfected cells [ , ] . prolonged er stress could also trigger non-apoptotic cell death as demonstrated by an investigation using time-lapse and electron microscopy that revealed the formation of extensive erderived vacuoles within the cytoplasm of zikvinfected cells, which eventually resulted in paraptosislike death [ ] . this observation is consistent with earlier report that showed cytoplasmic vacuolization in zikv-infected human skin biopsy specimens [ ] . vacuole formation was inhibited with class i pi k/akt inhibitor treatment. more importantly, pan-caspase inhibitor, zvad-fmk, only slightly rescued cell survival but did not affect vacuolization [ ] , implying that certain zikv strains, namely hd , pf and nc , induce cell death primarily through paraptosislike death. the role of pi k/akt pathway in zikvinduced cell death was verified by a recent study that reported treatment with ar- , a celecoxib derivative kinase inhibitor, significantly inhibited the replication of zikv in a mice and improved mice survival mainly through akt down-regulation [ ] . zikv, like other viruses, is an intracellular parasite that largely depends on the host biosynthetic, energetic and structural resources for successful viral replication and propagation. in particular, exploitation of these resources is manifested through the manipulation of the endoplasmic reticulum architecture and the processes that take place within or in proximity to the er as summarized in fig. . briefly, zikv infection leads to structural changes of the er such as er enlargement due to the accumulation of misfolded/unfolded zikv proteins (fig. d ) and the formation of convoluted membranes, vesicle packets, zippered er, and viroplasm-like structures for viral rna replication (fig. e) . the accumulation of misfolded/unfolded proteins induce er stress and triggers the upr (fig. f) . in parallel, er stress also initiate several response mechanisms such as stress granules assembly to impede global protein translation (fig. g ) and reticulophagy to remove damaged er (fig. h ). however, zikv are able to bypass these processes through various strategies, which eventually lead to paraptosis-like cell death (fig. i) . although the number of zikv cases have subsided, zikv remains a significant threat due to the sporadic and unpredictable nature of its outbreak. in addition, zikv shares the same vectors with another widespread flavivirus, denv [ ] , which is projected to increase exponentially in the future [ ] . thus, the potential re-occurrence of outbreaks, coupled with devastating neurological complications, warrants for extensive research to completely understand the virus pathophysiology for antiviral drug development. this is effectively demonstrated by several studies included in this review that employ multiple omics technologies to identify and target er-associated zikv dependency factors and er stress sensors. this strategy could pave the way in developing anti-zikv drugs. other cell receptors such as dc-sign can also serve as zikv entry receptor. b) acidic endosomal microenvironment facilitates endosomal fusion thereby, releasing zikv rna genome that is immediately bound onto cytosolic ribosomes. c) translation of the viral polyprotein (orange) likely initiates in the cytosol and continues with cotranslational insertion into the er membrane via sec translocon complex (blue). post-translational processed zikv proteins induce er structural alterations such as d) enlargement of the er, and e) formation of convoluted membrane (cm), vesicle packets (vps), zippered er (zer) and viroplasms. simultaneously, accumulation of misfolded and unfolded zikv proteins in the er lumen results in f) the induction of er stress and thus, initiates the host unfolded protein response (upr) and other intrinsic defense mechanisms including reticulophagy and stress granule formation. zikv proteins g) impede formation of stress granules and h) inhibit reticulophagy to sustain viral replication. zikv infection also induces i) er-derived cytoplasmic vacuolization, a visual characteristic of paraptosis. blue pointed arrows denote activation, blue blunt-end arrows denote inhibition. ve, virus-induced vesicle; vi, virus particle zika virus. i. isolations and serological specificity zika virus zika virus outbreak on yap island, federated states of micronesia zika virus infection complicated by guillain-barre syndrome -case report guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a 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via downregulation of the pi k/akt pathway and protects zika virus infected a mice: a host-targeting treatment strategy an overview of mosquito vectors of zika virus the current and future global distribution and population at risk of dengue publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions mimr and ask contributed to the topic conception and writing of the manuscript together. mimr prepared the figures. mimr, ask, nnr and ry edited and revised the manuscript. all authors read and approved the final manuscript. this work was supported by the grant [frgs fp - a] from the ministry of higher education, malaysia.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.received: september accepted: january key: cord- -iex lr authors: niu, xuefeng; zhao, lingzhai; qu, linbing; yao, zhipeng; zhang, fan; yan, qihong; zhang, shengnan; liang, renshan; chen, peihai; luo, jia; xu, wei; lv, huibin; liu, xinglong; lei, hui; yi, changhua; li, pingchao; wang, qian; wang, yang; yu, lei; zhang, xiaoyan; bryan, l. aubrey; davidson, edgar; doranz, j. benjamin; feng, liqiang; pan, weiqi; zhang, fuchun; chen, ling title: convalescent patient-derived monoclonal antibodies targeting different epitopes of e protein confer protection against zika virus in a neonatal mouse model date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: iex lr the zika virus (zikv) outbreak and its link to microcephaly triggered a public health concern. to examine antibody response in a patient infected with zikv, we used single-cell pcr to clone heavy and light chain-paired monoclonal antibodies (mabs) that bind to zikv envelope (e) proteins isolated from memory b cells of a zikv-infected patient. three mabs ( b , c , and a ) that showed the most potent and broad neutralization activities against the african, asian, and american strains were selected for further analysis. mab b showed an ic value of . ng/ml against the circulating american strain gz . epitope mapping revealed that mabs b and c targeted residue k of the lateral ridge (lr) epitope of the ediii domain, but b has a broader lr epitope footprint and recognizes residues t , g , e , and n as well. mab a recognized residues d , k , and k of the edii domain. interestingly, although the patient was seronegative for denv infection, mab c , originating from the vh - and vk - germline pair, neutralized both zikv and denv . administration of the mabs b , c , and a protected neonatal scid mice infected with a lethal dose of zikv. this study provides potential therapeutic antibody candidates and insights into the antibody response after zikv infection. zika virus (zikv) is a member of the flaviviridae family which includes dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), west nile virus (wnv), and tick-borne encephalitis virus (tbev) [ , ] . zikv is mainly transmitted by aedes mosquitoes but can also spread through sexual contact, blood transfusions, or via mother-to-child transmission during pregnancy [ , ] . zikv was first discovered in africa in [ ] and was confined within the equatorial zone of africa and asia until the outbreak in yap island, which was then transmitted to french polynesia and other southern pacific islands in [ , ] . it is believed that the adaptation and infectivity of zikv in mosquito-vectors contributed to the spread of the virus from asia to the americas [ ] . the zikv outbreak and associated increase in microcephaly cases in brazil increased global awareness [ ] ; to date, more than countries have reported zikv infections [ ] . it is known that zikv can cross the placental barrier, leading to fetal microcephaly, and can cause neurological complications in adults, such as guillain-barré syndrome [ ] [ ] [ ] . currently, there are no approved drugs or vaccines to mitigate the risk of zikv infection. the zikv surface is formed by copies of each envelope (e) glycoprotein and associated membrane (m) protein [ , ] . e proteins are arranged as dimers, with three parallel dimers connected to form a raft, and with rafts covering the viral surface [ ] . the e protein mediates viral entry into host cells and membrane fusion and is the major target for neutralizing antibodies and vaccine immunogens [ ] . the flavivirus e ectodomain consists of three distinct domains, edi, a -stranded beta-barrel that acts as a bridge between edii and ediii [ ] ; edii, a finger-like structure that is responsible for the dimerization of soluble e protein monomers and viral fusion [ ] ; and ediii, an immunoglobulin-like segment that is involved in host cell receptor recognition and viral fusion [ , ] . in recent years, a number of neutralizing antibodies (nabs) have been isolated from individuals infected with zikv [ ] [ ] [ ] [ ] [ ] . these nabs mainly recognize edii, ediii, and tertiary or quaternary epitopes that constitute e ectodomains. although ediii-targeted antibodies represent a relatively small population of e protein-binding antibodies, their presence is associated with serum neutralizing activity against zikv [ , ] . among these nabs, ediii-targeted antibodies and edii/e-dimer epitope (ede)-targeted antibodies showed the most potent neutralization activities. in this study, we cloned and characterized e-targeted monoclonal antibodies (mabs) from a chinese patient who returned to china from a visit to venezuela. selected mabs were evaluated for their neutralizing activities in vitro and in vivo via a zikv-infected neonatal severe combined immunodeficiency (scid) mouse model. the patient was a -year-old male who returned from venezuela in february . he was hospitalized in guangzhou th people's hospital (guangzhou, china). zikv rna was detected in serum, saliva, and urine samples by rt-pcr. the patient manifested relatively mild symptoms including fever, rash, sore throat, and fatigue, and recovered and was discharged approximately weeks after the onset of symptoms with no detectable zikv. the patient tested serologically negative for denv - infection using an ns based elisa kit (euroimmun, lubeck, germany), indicating that the patient had no previous exposure to denv - before infection with zikv [ , ] . single b cell sorting, rt-pcr, sequencing, and cloning freshly isolated peripheral blood mononuclear cells (pbmcs) were stained with a cocktail of antibodies including anti-human cd -fitc (invitrogen, carlsbad, ca), igg-apc-h /cd -pacific blue/cd -percp-cy . (bd biosciences, franklin lakes, nj), and anti-his-pe (miltenyi biotec, bergisch gladbach, germany). for antigen-specific memory b cells, we used zikv e protein (cat. no. -v b ; sino biological inc., beijing, china) as a probe. after washing, cd − cd + cd + igg + his + memory b cells were sorted using a multi-laser ariaii sorter. individual b cells were sorted into -well pcr plates containing µl lysis buffer per cell. the lysis buffer contained . µl rnasin inhibitor (promega, madison, wi), µl x first-strand buffer, . µl . m dtt, and . µl igepal (sigma-aldrich, st. louis, mo). pcr plates with sorted cells were frozen on dry ice and then stored at − °c or subjected to reverse transcription. rt-pcr and cloning into expression vectors was performed as previously described [ ] . briefly, µl random hexamers ( ng/µl; promega), µl dntps (each at mm), and . µl superscript iii (invitrogen) were added to each well, followed by incubation at °c for h. the igg heavy and light chain variable regions were amplified independently by nested pcr [ ] . first round pcr was performed using µl cdna directly following reverse transcription, with hotstart taq plus dna polymerase (qiagen, hilden, germany) and the primer mix. the pcr programme was initiated by min incubation at °c, followed by cycles of °c for s, °c (first round) or °c (second round) for s, and °c for min, followed by °c for min before cooling to °c. using % agarose gels, the pcr products were evaluated, excised from the gel (approximately bp for the heavy chain and bp for kappa and lambda chains), and sent for sanger sequencing after purification. human antibody sequences were analysed using imgt/v-quest (http://www.imgt.org/) [ ] . fulllength igg was expressed by co-transfecting hek- t cells with equal amounts of paired heavy and light chain plasmids based on the backbone of the pci-neo vector [ ] . culture media were harvested four days after transfection and purified using protein a agarose (ge healthcare, chicago, il). zikv particles were successfully isolated from the infected patient's blood plasma or urine. the virus was passaged once in suckling mouse brains and cultured in vero cells to prepare stocks, which were stored at − °c before use. denv (hawaii strain), denv (new guinea-c strain), denv (h strain), and denv (h strain) were prepared in vero cells. recombinant zikv e and ediii protein (cat. no. -v b and -v h, respectively) were purchased from sino biological inc. the e protein was derived from zikv strain sph (ku ), isolated from brazil in . denv ediii protein (cat. no. -v b), denv ediii protein (cat. no. -v y ), and denv e protein (cat. no. -v b ) were also purchased from sino biological inc. nunc maxisorp plates (thermo fisher scientific, waltham, ma) were coated with zikv e or e domain iii protein ( µg/ml) and incubated at °c overnight. after blocking for h, the plates were washed six times with phosphate-buffered saline (pbs) and transfected with antibody supernatants at a : dilution with blocking buffer or a serial dilution of purified mabs. secondary antibody (goat anti-human igg; ab ; abcam, cambridge, uk) was applied at a : dilution in blocking solution and then the plates were incubated at room temperature for h, and tmb substrate. absorbance values were determined at nm using a biotek plate reader (biotek instruments, winooski, vt). neutralization activity of purified antibodies was measured using a flow cytometry-based neutralization assay with vero cells as previously reported [ , ] with minor modifications. briefly, × cells were plated in a -well plate h before the experiment. purified human mabs were serially diluted in dmem (gibco; thermo fisher scientific) supplemented with % feta bovine serum (fbs; gibco) and incubated with zikv ( × pfu) for h at °c under % co . the cells were then incubated with µl of the mixture for h at °c under % co , after which ml/well mem medium containing % fbs was added and incubated for another h. zikv-infected and uninfected cells were used as positive and negative control, respectively. cells were then trypsinized, fixed, and permeabilised with fixation and permeabilization solution (bd biosciences) on ice for min, followed by staining with g antibody ( µg/ml) diluted with x perm/wash buffer and staining with anti-mouse igg fitc ( : in x perm/wash buffer) on ice for min. after washing, the percentage of e-positive cells was measured using bd facscanto ii (bd biosciences). the antibody dilution that neutralized % of the viruses (half-maximal neutralizing inhibitory concentration; ic ) was calculated by nonlinear, dose-response regression analysis with graphpad prism version . (graphpad software inc., la jolla, ca). epitope mapping was performed by shotgun mutagenesis as previously described [ ] . zikv prm-e protein expression constructs (based on zikv strain sph ) were subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library [ , ] . each residue within prm-e was changed to alanine, with alanine codons mutated to serine. in total, zikv prm-e mutants were generated ( % coverage)which were confirmed by sequencingand arrayed into -well plates. each zikv prm-e mutant was transfected into hek- t cells and incubated for h. cells were then fixed in % (v/v) paraformaldehyde (electron microscopy sciences, hatfield, pa) and permeabilised with . % (w/v) saponin (sigma-aldrich) in pbs plus calcium and magnesium (pbs++). for mapping, cells were sequentially incubated with . µg/ml mabs and . µg/ml alexafluor -conjugated secondary antibody (jackson immunoresearch laboratories, west grove, pa) diluted in pbs++, % normal goat serum (sigma-aldrich), and . % saponin. cells were washed three times with pbs++/ . % saponin, twice with pbs, and then mean cellular fluorescence was recorded using a high-throughput flow cytometer (htfc; intellicyt, albuquerque, nm). antibody reactivity against each prm-e mutant relative to the wildtype (wt) protein was calculated by subtracting the signal from mock-transfected controls and normalizing to the signal from wt prm-e-transfected controls. binding competition between mabs b , c , and a and five other ediii-targeted antibodies was determined using a real-time, label-free, bio-layer interferometry assay on an octet red biosensor (fortebio, fremont, ca) as previously described [ ] . the experiment was performed at °c in pbs buffer with shaking at , rpm. ni-nta biosensors (forte-bio) were first loaded with μg/ml his-tagged-e protein for s and then associated with the first mab ( b , f , d , c , a , b , d , or f ) for s. an irrelevant mab, d , was used as negative control and pbs was used as blank solution. the biosensors were then dipped into the second mab and incubated in the presence of the first mab. the capacity of additional binding was monitored by measuring further shifts for s. all mabs were evaluated at concentration of nm, except for b ( nm), d ( nm), and f ( nm), for saturation measurement. the ni-nta biosensors were regenerated with mm glycine-hcl (ph . ; ge healthcare) and re-charged with mm nicl . the response of mab binding to the e protein was compared and the data were processed using biaevaluation software (biacore, uppsala, sweden). the use of pregnant scid and suckling mice in this study was carried out in strict compliance with the association for the assessment and accreditation of laboratory animal care. the experimental protocol was approved by the guangzhou institute of biomedicine and health (gibh) institutional animal care and use committee. scid beige mice were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). one-day-old suckling mice (n = - ) were intraperitoneally inoculated with zikv at . × pfu. zikv mabs were administered as a single dose h after virus infection. mouse brain, spleen, and serum samples were then collected days after virus infection for rna extraction ( ; qiagen). zikv rna detection was determined by one-step qrt-pcr ( ; qiagen) on a cfx real-time pcr system (bio-rad laboratories, hercules, ca) using published primers and conditions. briefly, µg rna together with a mixture of . µl sybr green, . µl rt-mix, and . µl each of forward and reverse primers were placed in -well pcr plates in µl reaction volumes. amplification was performed at °c for min, °c for min, followed by cycles of °c for s, °c for s, and °c for s. viral load was expressed on a log scale as viral rna copies per µg after comparison with a standard curve. three replicates were conducted for each sample. flow cytometric data were analysed using flowjo version . (tree star inc., ashland, or). the ec (half-maximal effective binding concentration) and ic values obtained from the elisa assay of zikv e/ediii binding activity to mabs and the neutralization assay, respectively, were calculated using a dose-response inhibition model and sigmoidal curves were generated using graphpad prism version . (graphpad software inc.). statistical significance of the difference in viral loads between groups was determined using one-way anova. p values < . were considered statistically significant. to clone e-targeted mabs from a zikv-infected patient, we used recombinant zikv e protein as a probe to isolate e-specific memory b cells from a chinese patient that had returned from venezuela and was identified as infected with zikv during border entry. thirty-one mabs that bind to e protein were successfully cloned days after the onset of symptoms. we first used a facs-based neutralization assay to screen neutralizing activities in the cultured media of hek- cells transfected with expression plasmids for each heavy and light chain of the mabs. eight mabs with the best neutralizing activities and binding to e protein were selected for further analysis. the binding activities of these eight mabs to zikv e, ediii, and denvi ediii proteins were assessed ( figure and table ). mabs b , c , d , and b bound both zikv e and ediii, while mabs a , a , e , and a bound e protein but not ediii ( figure (a) ). mabs b and c showed strong binding activities to zikv e and ediii protein in the nanomolar range. we then measured the neutralization activities of purified mabs against the zikv gz strain. zikv gz has the same genomic sequence as the current circulating zikv strain in south america and was the same as the zikv strain isolated from the patient [ ] . three mabs, b , c , and a , showed the best neutralization activities with ic values of . , . , and ng/ml, respectively; thus, we further analysed these mabs for epitope identification and in an animal model (figure (b) ). we also tested the neutralizing activities of these three mabs against two other zikv isolates, mr (the first zikv isolated from the zika forest in uganda) [ ] and prvabc (a zikv isolated from puerto rico). all three antibodies cross-neutralized mr and prvabc (table ) . intriguingly, one of the mabs, c , showed binding to denv ediii protein but not to other denv ediii proteins. the ed of c with denv ediii was . ng/ml, which was comparable to zikv ediii binding ( . ng/ml). we then measured the neutralization activity of these three representative mabs against denv and found that mabs b and a showed no denvi ediii cross-reactivity and failed to neutralize denvi virus at µg/ml, whereas c showed potent neutralizing activity at an ic of . µg/ml (table ) . neutralizing mabs recognized either the ediii or edii lateral ridge (lr) ediii has been recognized as a major target site for nabs that bind to the same or different epitope region. we used a bio-layer interferometry (bli) competition assay to probe whether our e-targeted mabs bind to the same epitope region. a his-tagged e protein, captured to a ni-nta biosensor, was first saturated with one mab, after which competitive binding of a second mab was assessed. the binding competition between mabs b , c , and a and five other zikv e-targeted mabs was measured (figure (a) ). mabs c , d , d , f , and f but not a could block the binding of mab b to e protein, indicating that b and these five mabs share the same or overlapping epitopes on zikv ediii. interestingly, mab c binding to e protein was blocked by mab b and no other etargeted mab, indicating that b may bind to a broader epitope footprint on ediii, whereas c binds to a smaller epitope region that overlaps with the binding region of b . we next mapped the detailed epitope residues for mabs b , c , and a using a shotgun alaninescanning mutagenesis library of zikv prm and e protein variants [ , ] . mab b recognized an epitope region that includes residues t , g , e , n , and k along the lr region of ediii. notably, mab c only recognized k on the lr region as a key residue (figure (b and c) ). amino acid sequence alignment between e proteins of zikv and denv revealed that residue k on zikv e protein corresponds to k on denv , which is not present on the e proteins of denv , denv , or denv ( figure (d) ). mab a is an edii-targeted antibody that recognized residues d , k , and k on the edii. we confirmed that an individual mutation of these key residues reduced binding activity by more than % when compared with that of wt prm-e protein (figure (b) ). analysis of the immunoglobulin heavy chains of b , c , and a revealed that they were derived from germlines hv - * , hv - * , and hv - - * , respectively (figure (a) ). the shm rates of these heavy chains compared with their predicted germline sequences were relatively low, at . % for b h, . % for c h, and . % for a h, which is lower than that of antibodies isolated from annual trivalent inactivated influenza vaccine (tiv) donors [ ] and chronic human immunodeficiency virus (hiv)- patients (> %) [ , ] . compared with their germline sequences, the shms of a h were in the cdr , cdr , and cdr regions, whereas shms of c h and b were found sporadically in the cdr , fr , cdr , fr , and cdr regions ( table ). we paired the heavy chain predicted germline (hgl) of c h ( c hgl) with the light chain (kappa) of c ( c k) and similarly paired a hgl with a k and expressed these heavy chain germline-reverted antibodies in hek- cells. these germline-reverted antibodies showed reduced binding activities to e protein at . µg/ml for a gl, which is about times lower than that of mature a ( . µg/ml), and . µg/ml for c gl, which almost completely lost binding ability compared with that of mature c ( . µg/ml; table ). therefore, shms are necessary for strong binding to e protein, but only a low level of shms is needed to improve binding. to evaluate the protective effects of e-targeted mabs, we developed a zikv lethal infection mouse model. intraperitoneal infection of -day-old scid neonates with . × pfu zikv gz caused % lethality within days; neonatal mice that received no treatment showed disease symptoms, including ruffled fur, trembling and shaking, and body weight loss ( figure (a) ). neonatal mice treated with mab b at either , µg, or µg h after infection survived the lethal infection (figure (b) ). titration of viral load from brains and spleens collected days after infection revealed a dose-dependent decrease in viral rna in mice treated with mab b , whereas mice that received no treatment had much higher viral rna levels in the brain and spleen (figure (c and d) ). treatment with µg mab b resulted in the best protection, with no detectable viral rna in the brain and spleen. we also demonstrated in subsequent experiments that zikvinfected neonatal scid mice survived and did not exhibit weight loss after treatment with µg of either mab c or a (figure (e and f) ). in a separate experiment, an unrelated mab, g , which is specific for h n influenza virus, showed no protective effects on zikv-infected neonatal scid mice (data not shown). therefore, both ediii-targeted and edii-targeted mabs can effectively protect neonatal scid mice from lethal zikv infections. the emergence of zikv in south america has raised a global health concern due to the link between zikv infection and microcephaly in infants and guillain-barré syndrome in adults. the search for and development of vaccines and therapeutics to prevent and control zikv infection are thus necessary. for example, nabs have been found effective in combating emerging viruses, such as the middle east respiratory syndrome coronavirus (mers-cov), ebola virus, and influenza virus [ , [ ] [ ] [ ] . in the present study, we cloned zikv e proteinbinding mabs from the memory b cells of a zikvinfected chinese patient and selected three mabs for further study. we characterized the zikv e protein epitopes recognized by these mabs and analysed the shm pattern. two of the most potent mabs, b and c , are ediii-targeted and showed neutralizing activities against three different zikv strains, including the south american circulating strain (gz ), african strain (mr ), and american strain (prvabc ). mab b recognized several residues in the lr region of ediii, and an individual mutation in these residues led to a > % decrease in binding activity compared with that of wt prm-e protein. a previous study also demonstrated that an e k mutation alone abolished the neutralizing activity of the lr-targeted mab zka [ ] . meanwhile, the heavy and light chains of mab c were derived from the vh - and vk - germlines, respectively. it has been reported that zikv mabs with vh - /vk - paired antibodies are present in five of six people that were sequentially infected with denv and zikv; thus, these mabs were determined as recurrent antibodies that recognize both zikv and denv viruses [ ] . our findings, along with observations by other studies [ ] , suggested that vh - may be preferentially enlisted in response to zikv and denv infections. it is interesting to note that both mab b and c recognized k in the lr region of zikv ediii. mabs reported by others, such as z and z , also recognized k on zikv ediii or k on denv ediii [ ] . although mab c neutralized the african zikv strain mr , another reported mab, zikv- , that also recognized k of the zikv e protein and neutralized the h/pf/ strain, failed to neutralize the mr strain [ ] . this difference may because the current zikv strains gz and prvabc possess e in the ediii region, whereas african strain mr has d in the ediii region. therefore, k in lr region of ediii appears to be a key target residue for ediiitargeted antibodies to exert neutralizing activities. mabs that recognized k showed potent in vitro neutralizing activity, supporting the notion that residue k is a hotspot for effective neutralizing activity of ediii-targeted mabs [ , , , , ] . it is important to note that most reported e-targeted neutralizing mabs are ediii-targeted, whereas only a few neutralizing mabs are edii-targeted. mabs showing broad neutralization activities against flaviviruses have been reported to recognize the edii targeting either the fusion loop epitope (fle) or ede regions [ , ] . antibodies targeting the fle are conserved among flaviviruses and can bind to zikv e protein. it has been reported that fle-targeted antibodies show poor neutralizing activities but have strong infection enhancement in vitro [ ] . meanwhile, antibodies targeting the ede show potent neutralizing activities against zikv and can protect against zikv infection in mouse and rhesus macaque models [ , ] . notably, mab a in our study showed potent neutralizing activities against all three tested zikv strains and is likely an edii-targeted mab. it also recognized residues d , k , and k and is similar to the reported ede-targeted antibody zikv- , which recognizes d , k , and q [ ] . both mabs recognize the same two residues in the edii region. structural analysis indicated that mab zikv- is cross-linked with e monomers within dimers and neighbouring dimers in the zikv particle [ , ] . therefore, mab a may not exhibit the same interaction with the virus particle as zikv- . recently, another ede-targeted mab, zikv- , was reported to neutralize multiple zikv strains and recognize residues d , m , r , and k in the edii region [ ] ; both mab a and zikv- recognize residues d and k . notably, mab a , zikv- , and zikv- were all isolated from memory b cells of zikv table . cdr , fr , cdr , and fr amino acid sequence of b h, c h, and a h with their corresponding vh germline sequences and predicted cdr sequences. convalescent patients. it is possible that residues d , k , and k are hotspots for ede-targeted neutralizing antibodies. future structural biology analysis is required to confirm if mab a is indeed an edetargeted antibody. antigen-specific b cells undergo a process termed shm to increase antigen affinity. interestingly, the neutralizing mabs b , c , and a had relatively low shm rates in their vh genes, which is much lower than the antibodies isolated from annual tiv vaccine donors [ ] and chronic hiv- patients [ , ] . it is possible that when a person is exposed to zikv infection, e-targeted antibodies that require low shm rates to bind and neutralize zikv were generated. even with relatively low shm rates, these limited mutations appeared essential for conferring binding activities. when the mabs c and a heavy chains were reverted to their predicted germline sequence and paired with their respective mature c and a light chains, binding activity to zikv e protein was found almost completely impaired and times lower than that of mature a , respectively. therefore, (d) viral load in the spleen (n = - ). total rna was extracted from the homogenates of the brain and spleen. zikv genomic rna was evaluated using one-step qpcr. viral loads are expressed as the genome copy number per microgram tissue. (e) body weight changes in zikv-infected mice treated with mab a or c at µg/mouse. (f) survival curve of zikv-infected mice treated with mab a or c . data are representative of two independent experiments and presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significance (one-way anova). a few mutations are sufficient but necessary to confer neutralizing activities against zikv infection. in addition to evaluating the neutralizing activities of representative mabs in inhibiting zikv infection in cultured cells, we also tested the protective efficacy of these mabs in a neonatal scid mouse model. zikv infection in wt mice does not result in disease, whereas suckling wt mice are susceptible to infection [ , ] . mice lacking interferon signalling, such as a (type i ifnar ko) [ ] , interferon regulatory factor (irf) / / triple ko [ ] , and ag (type and type ifn ko) [ ] , are susceptible to zikv infection with detectable zikv in the brain, spinal cord, and testes; these mice died within - days post-infection. in this study, we developed a scid beige suckling mouse model for evaluating the protective ability of mabs against zikv infection. scid beige mice are deficient in t, b, and nk cells and are thus more suitable for evaluating the net effect of anti-zikv mabs. zikv infection of scid beige suckling mice led to neurological symptoms and high viral loads in the brain and spleen, which was eventually lethal. all three mabs tested (ediii-targeted mabs b and c and edii-targeted mab a ) showed protective effects in zikv-infected scid mice. therefore, nabs against zikv cloned from convalescent patients have the potential to be further developed for treating zikv infection. there is an opinion that e-targeted mabs may mediate antibody-dependent enhancement (ade) of virus infection. we found that mab b enhanced zikv infection in k cells at a very low concentration of ng/ml. however, we believe that ade is not a critical concern because the concentration of mabs used for treatment are much higher. nevertheless, engineering of the mab fc fragment to minimize fc gamma receptor-mediated infection would be beneficial in improving the practical usage of these neutralizing mabs. overall, our study findings provided insights into the antibody response after zikv infection and demonstrated the potential of mabs in zikv treatment. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus: a previously slow pandemic spreads rapidly through the americas effect of acute zika virus infection on sperm and virus clearance in body fluids: a prospective observational study the emergence of zika virus and its new clinical syndromes isolations and serological specificity zika virus outside africa evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes assessing the global threat from zika virus zika virus: recent advances towards the development of vaccines and therapeutics guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study possible association between zika virus infection and microcephaly -brazil zika virus associated with microcephaly the . 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müller, janis a.; dietzel, erik; harms, mirja; krüger, franziska; heid, christian; sowislok, andrea; riber, camilla frich; kupke, alexandra; lippold, sina; von einem, jens; beer, judith; knöll, bernd; becker, stephan; schmidt-chanasit, jonas; otto, markus; vapalahti, olli; zelikin, alexander n.; bitan, gal; schrader, thomas; münch, jan title: the molecular tweezer clr inhibits ebola and zika virus infection date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: evk g ebola (ebov) and zika viruses (zikv) are responsible for recent global health threats. as no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer clr may inhibit ebov and zikv infection. this small molecule has previously been shown to inactivate hiv- and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. we found that clr indeed blocked infection of ebov and zikv in a dose-dependent manner. the tweezer inhibited infection of epidemic zikv strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. our findings show that clr is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent. zika virus (zikv) was first isolated in from a febrile rhesus macaque in the zika forest of uganda (dick et al., ) . since then, sporadic zikv infections occurred in africa and asia (haddow et al., ; hayes, ) . until , zikv infection usually has been associated with mild symptoms and thus, the virus had not been considered a threatening pathogen. however, since the recent outbreaks it is evident that zikv causes drastic birth defects, most prominently microcephaly (mlakar et al., ; rasmussen et al., ) , and is associated with neurological disorders, such as guillain-barré syndrome (cao-lormeau et al., ; krauer et al., ) . since , countries and territories reported ongoing zikv transmission (http://www. who.int/emergencies/zika-virus/en/). in brazil alone, . million persons had been infected (shuaib et al., ) , prompting the who to declare zikv a public health emergency of international concern. currently, there are no specific treatments or vaccines against zikv making development of effective preventive measures an urgent public health need (barrows et al., ; paixão et al., ) . zikv is transmitted mainly via mosquito bites but cases of sexual transmission also https have been reported (d'ortenzio et al., ; moreira et al., ; petersen et al., ) . like other members of the flaviviridae family (e.g. dengue virus), zika virions are surrounded by a host-membranederived lipid bilayer containing envelope glycoproteins responsible for cell entry (sirohi et al., ) . ebola virus (ebov) was discovered in near the ebola river in the former zaire. since then, outbreaks sporadically occurred in africa, with the most severe in , reaching epidemic proportions and a death toll of more than , people (centers for disease control and prevention (cdc), ) . ebov belongs to the family of filoviridae, forms enveloped filamentous infectious particles, and causes hemorrhagic fever, a rare and deadly disease with a high fatality rate. in addition to being a global health concern, the virus also is considered a potential biological threat agent. ebov likely is transmitted from infected bats to humans where it may spread through personal contact, contaminated objects, or sexual intercourse (mate et al., ; pandey et al., ) . noteworthy, even after recovery, ebov is found in some body fluids, including semen, up to several months (deen et al., ) . as for now, no licensed vaccine or medicine is available to prevent or manage future ebov outbreaks (sharma and ketki jangid, ) . molecular tweezers are novel drug candidates for the treatment of amyloidosis and related conditions. a lead compound, clr , has been used in many in vitro and in vivo studies to date schrader et al., ) . binding of clr to lysine residues in polypeptides disrupts noncovalent molecular interactions that are important for the abnormal self-assembly that leads to the formation of toxic oligomers and amyloid aggregates (schrader et al., ; sinha et al., ) . in fact, clr prevents assembly and promotes disaggregation of amyloid fibrils that are associated with neurodegenerative diseases ferreira et al., ; prabhudesai et al., ; richter et al., ) . therapeutic effects of clr have been demonstrated in animal models of parkinson's disease (lulla, ; prabhudesai et al., ; richter et al., ) , alzheimer's disease malik et al., ) , familial amyloidotic polyneuropathy (fap) (ferreira et al., ) , and desmin-related cardiomyopathy and was found to be safe in mice at doses -times higher than those showing therapeutic effects . we have recently established that clr also disrupted the formation of amyloid fibrils in semen (lump et al., ) , which enhance hiv- infection (münch et al., ; usmani et al., ) . surprisingly, clr also inhibited hiv- infection through a direct virusinactivating mechanism: the tweezer preferentially bound to raft-rich regions in the viral membrane resulting in the disruption of the lipid bilayer and loss of infectivity. in line with the membrane targeting activity, clr is inactive against non-enveloped viruses such as human adenovirus but destroyed other enveloped viruses, including hepatitis-c virus, human cytomegalovirus, and herpes simplex virus, demonstrating that clr represents a broadly active antiviral compound with prospects for microbicide or drug development (lump et al., ) . thus, we evaluated here whether clr might also block infection of re-emerging enveloped viruses. we show that clr indeed inhibits infection of replication-competent ebov and zikv as well as marburg, rabies, and sars corona virus (sars-cov) pseudoparticles. clr lost antiviral activity in the presence of serum, but remained active in the presence of semen, urine, saliva or cerebrospinal fluid. thus, this broadly active compound represents a promising lead for further development as a microbicide to protect from the sexual acquisition of zikv or ebov, or as a topically applied drug for the therapy of viral infections of the skin, mucosa or the respiratory tract. clr and clr were synthesized as described previously (fokkens et al., ; talbiersky et al., ) and . mm stock solutions were prepared in pbs. zikv envelope protein had been ordered from fitzgerald industries international, acton, usa. vero e cells were used for propagation and infection with zikv as described . tcid values (tissue culture infectious dose ; tcid /ml) were determined according to reed and muench, multiplied with a factor of . to obtain pfu/ml (plaque forming units) and, by normalizing to the number of cells, used to calculate the respective moi (multiplicity of infection). sw (human epithelial colon carcinoma) cells were kindly provided by ninel azoitei (center for internal medicine i, university of ulm, ulm, germany). hff (human foreskin fibroblasts), human glioblastoma (a ) or neuroglioma (h ) cells were kindly provided by jens von einem and karin danzer (ulm university medical center, ulm, germany). hepatic huh- cells were kindly provided by s. pöhlmann (göttingen, germany). the reporter cell line tzm-bl was obtained through the nih arrrp. virus stocks of r -tropic hiv- nl - th were generated by transient transfection of t cells as described (münch et al., ) . methods describing the effect of clr on pseudotyped lentiviral particles ( . .), ebola virus infection ( . .), the detection of zikv infection by a colorimetric mtt assay ( . .) or by cell-based zikv immunodetection assay ( . .), flow cytometry ( . .) and confocal microscopy ( . .) as well as the rna release assay ( . .) and the antiviral activity of clr in body fluids ( . ) can be found in the supplement. we have shown previously that clr inhibits hiv- infection by disrupting the viral membrane (lump et al., ) , suggesting that the antiviral activity of the tweezer is independent of the presence of the viral glycoproteins. to test this hypothesis, we generated luciferaseencoding retroviral particles harboring glycoproteins derived from ebov, the closely related filovirus marburg virus, rabies virus (rhabdoviridae) or sars-cov (coronaviridae). these pseudoparticles were exposed to clr and then added to huh- cells. clr , which lacks hydrophobic side arms and has no anti-hiv- activity (lump et al., ) , served as a negative control. infection rates were determined three days later by quantifying luciferase activity and showed that clr , in contrast to clr , efficiently blocked infection by all tested pseudoparticles (fig. a) . the half-maximal inhibitory concentrations (ic ) of clr against the four pseudoparticles were similar and ranged between . μm for rabies and . μm for marburg virus (fig. a) . these data corroborate our previous findings that clr targets the viral membrane rather than a viral glycoprotein (lump et al., ) . we tested next whether clr blocked infection by the replication-competent bsl pathogen ebov. vero e cells were inoculated with gfp-encoding ebov (ebihara et al., ; hoenen et al., ) that had been exposed to clr or clr . after days, the number of virus-induced plaques revealed that clr , but not clr , reduced the infectious titer in a dose-dependent manner with an ic of . μm (fig. b) . next, we determined the effect of clr on zikv infection. vero e cells were inoculated with the african zikv mr strain that was isolated in from a sentinel rhesus macaque (dick et al., ) in the absence or presence of clr or clr and viral replication was monitored by light microscopy. in the absence of clr , zikv caused a profound cytopathic effect (cpe) as evidenced by large plaques caused by detachment of cells. clr had no effect on this phenotype ( fig. a) . however, exposure to μm clr prevented formation of the virus-induced cpe entirely. to quantitatively assess the antiviral activity, we used a colorimetric mtt assay measuring the virally induced cpe (müller et al., ) . in the absence of compounds or in the presence of clr , zikv resulted in more than % dead (detached) cells. in contrast, clr suppressed cpe formation with an ic of . μm (fig. b ). the mtt assay allows an indirect measurement of zikv infection as the tetrazolium dye is metabolized only in living, non-infected cells. to evaluate the effect of clr on zikv more directly, infected cells were stained for the viral e protein and analyzed by confocal microscopy (schandock et al., ) . upon treatment with or μm of clr , e protein-specific fluorescence could not be detected, whereas clr had no effect, as expected ( fig. c ). lack of e protein expression in cells infected with clr -treated virus was confirmed by flow cytometry (fig. d, supplementary fig. s a ). of note, clr was not cytotoxic at concentrations active against zikv ( supplementary fig. s b ), and did not cause alterations in cell morphology (compare confocal images and dot plots of uninfected cells vs. μm clr exposed cells in fig. c and d). thus, clr prevents zikv infection of vero e cells. we hypothesized that if clr inhibited zikv infection by a similar mechanism to other enveloped viruses, its activity would be directed against the viral membrane (lump et al., ) . indeed, when clr was added to cells, rather than to the virus, and washed out prior to infection, no antiviral effect was observed in a cell-based zikv immunodetection assay (fig. a) . we performed next a time-course analysis in which zikv was exposed to μm clr for to s prior to infection. already after exposure for only s, clr reduced infection by ∼ %. infection declined further with increasing incubation time and was nearly at the level of uninfected cells after s incubation (fig. b) . in agreement with a direct activity against the , rhabdoviridae (rabies virus) or the coronaviridae family (sars-cov), were exposed to indicated concentrations of clr or clr and then used to infect huh- cells. after three days, infection rates were determined by quantifying firefly luciferase activity and subtracting background activity derived from uninfected cells. values represent % reporter gene activities ± sd derived from triplicate infections, normalized to values obtained for cells infected in the absence of tweezers. (b) analysis of replication competent ebov was performed using confluent vero e cells in -well plates. rgebov-egfp was preincubated with clr or clr and mixtures were added to the cells. after days, samples were analyzed by counting the number of plaques per well and calculating the corresponding plaque forming units per milliliter (pfu/ml) for each inhibitor and dilution. displayed values represent the mean of three independent experiments ± sd. ic values were calculated by graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). light microscopy images of vero e cells infected with zikv mr in the absence or presence of μm clr or clr . images were taken days post infection (dpi). (b) zikv mr was incubated with . - μm clr or clr before these mixtures were used to infect vero e cells. after days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . values represent mean ± sd of percentages derived from triplicate infections. ic was determined using graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (*** denotes p < . ) (c) confocal microscopy images of vero e cells infected with clr -or clr -treated zikv at day post infection. cells were stained for zikv e protein (green) and nuclei (hoechst, blue) and imaged using confocal microscopy. scale bar: μm. (d) flow cytometry of infected vero e cells. virus was pretreated with pbs, clr or clr and added to cells. h later, cells were fixed, permeabilized, and stained with an anti e protein antibody, and quantified using an alexa coupled secondary antibody. percentages indicate the fraction of protein e positive cells. ic, isotype control. a.e. röcker et al. antiviral research ( ) - virion, the ic of clr increased from . μm to . μm when a fold higher multiplicity of infection was applied (supplementary fig. s ). clr was shown to destroy hiv- membrane integrity through interaction with raft-rich regions in the retroviral envelope (lump et al., ) . in contrast to hiv- , the zikv particle is relatively densely packed with glycoproteins (sirohi et al., ) , which might hamper the interaction of the tweezer with the zikv membrane. to determine whether clr might inhibit zikv infection through direct interaction with glycoproteins, increasing amounts of recombinant viral e protein were titrated to clr , and then these solutions were assayed for anti-zikv activity. as shown in fig. c , even e-antigen concentrations of μg/ml did not affect the antiviral activity of clr , suggesting that the tweezer did not reduce zikv infectivity through binding to the viral e protein. interestingly, we also observed that elevated e-antigen concentrations of and μg/ml reduced infection in the absence of clr (fig. c) , which is likely due to the competition between the virion-associated e protein and the recombinant e protein for cellular receptors. to test whether clr interaction with the viral particle results in loss of membrane integrity, as was shown in the case of hiv- , (lump et al., ) , we measured viral rna genome release. sucrose cushion purified zikv was incubated with clr , clr , pbs or triton x- , as a positive control, for min at °c and the amount of total rna in the solution was determined fluorometrically. as expected, no viral for virus treatment, zikv was incubated with clr for min at room temperature before the mixtures were added to vero e cells. for cell treatment, clr was added directly to the cells; after h, the medium was replaced and the cells were infected with zikv mr . dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; *** denotes p < . ) (b) zikv was incubated for the indicated times with pbs or μm clr before the mixture was added to vero e cells. dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells infected with clr -treated zikv to cells infected with zikv that had been incubated with pbs for the same time period (*** denotes p < . ). (c) indicated concentrations of zikv e protein were titrated to μm clr or pbs before zikv was added. mixtures were used to inoculate vero e cells. dpi, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . the values shown are mean ± sd from triplicate infections. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells treated with different concentrations of zikv e protein and clr to cells that had been treated with the same concentrations of e protein and pbs (*** denotes p < . ). (d) zikv mr was incubated with pbs, μm triton x- , . - μm clr or μm clr for min at °c. samples were inactivated by uv light of a laminar flow for min. then, μl of the samples were used to determine rna concentrations using the quantifluor ® rna system and a quantus fluorometer (promega). background values obtained from control samples using cell supernatant of uninfected cells were subtracted from the respective signals. rna levels of virus stock incubated with pbs were subtracted from these values. data points represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare samples treated with different clr concentrations, clr or triton x- to pbs-treated virus (*** denotes p < . ). a.e. röcker et al. antiviral research ( ) - rna was detectable when zikv was incubated with buffer or clr (fig. d ). in contrast, incubation with triton x- resulted in readily detectable viral rna suggesting that the detergent lysed the zikv particle. similarly, elevated amounts of rna were detected upon treatment of zikv with clr , demonstrating the physical destruction of the viral particle by the tweezer. of note, clr itself did not act as detergent but targets the viral membrane (lump et al., ) . next, we analyzed whether clr was active against epidemic zikv strains. the fb-gwuh- isolate was derived in from the fetus of a pregnant finnish tourist returning from south america (driggers et al., ) , and the prvabc- isolate represents the current american epidemic strain, isolated in from a puerto rican patient (lanciotti et al., ) . both zikv strains were exposed to clr upon infection of vero e cells. cell-based immunodetection assay and flow cytometry experiments demonstrated that the tweezer suppressed infection of both strains with ic values of . μm for fb-gwuh- ( fig. a and supplementary fig. s ) , and . μm against prvabc- (fig. b) , respectively. as zikv can be transmitted via sexual intercourse, we studied whether zikv infects cells derived from the anogenital region and if so, whether infection can be blocked by clr . zikv effectively infected cell lines derived from cervix (fig. a, supplementary fig. s ) , colon (fig. b, supplementary fig. s ) , and primary foreskin cells (fig. c, supplementary fig. s ). viral infectivity was suppressed by clr but not clr , with ic values in the μm range ( fig. and supplementary fig. s ). because zikv is neurotropic (cao-lormeau et al., ; mlakar et al., ) and clr has been shown previously to penetrate through the blood-brain barrier (bbb) when administered systemically richter et al., ) , we also tested whether clr blocked zikv infection of brain-derived cells. both a glioblastoma and h neuroglioma cells were susceptible to zikv infection and clr entirely abrogated infection at μm (fig. a) . finally, we confirmed these findings obtained in human cell lines using primary murine cerebellar neurons. zikv infected neuronal bystander cells, and this was blocked by clr (fig. b) . there was no apparent toxicity of clr in the primary neurons at this concentration. . . the antiviral effect of clr is abrogated by serum but not urine, saliva, semen, or csf its broad antiviral activity makes clr an interesting lead compound for antiviral prevention or treatment. for a systemic application, clr must retain antiviral activity in blood. to test whether this was the case, clr was diluted in human serum, and then exposed to zikv. as shown in fig. a , serum concentrations of . % and higher abrogated the anti-zikv activity of the tweezer. similarly, serum also abrogated the anti-hiv- activity of clr ( supplementary fig. s ), precluding its use as a systemically applied antiviral drug. in contrast, clr remained active and inhibited zikv infection in the presence of up to % of urine (fig. b) , saliva (fig. c ), cerebrospinal fluid (fig. e ) and % human semen (fig. d) . thus, clr inactivates zikv in the presence of body fluids associated with virus transmission, and could be administered locally to halt virus replication, or applied topically as a microbicide to prevent e.g. sexual virus transmission. thereafter, vero e cells were infected and days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sd (n = ). ic values were determined with graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (** denotes p < . ; *** denotes p < . ). zikv was incubated for min at °c with . - μm clr or clr . next, mixtures were used to inoculate hela (a), sw (b), or hff (c) cells. days later, infection rates were determined via quantification of the viral e protein using a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - . discussion our results establish clr as a broad-spectrum antiviral agent, which not only inhibits infection of established viral pathogens (lump et al., ) , but also of emerging viruses, such as ebov and zikv. we characterized here mainly the effect of clr on zikv, as there is currently no specific antiviral therapy nor a preventive vaccine available. clr blocked zikv infection of primary brain-derived murine cells (fig. b) as well as human cell lines derived from the anogenital region (fig. , supplementary fig. s ) and the brain (fig. a) with ic values between and μm. these values are in the same range as ic values obtained against ebov ( μm, fig. b ) and hiv- ( - μm) (lump et al., ) . several lines of evidence demonstrate that the antiviral activity of clr is directed against the zikv particle itself. first, treatment of cells with clr prior to infection had no antiviral effect (fig. a) . second, the ic of clr increased with increasing viral concentrations ( supplementary fig. s ) . third, the integrity of the zikv particle was lost upon clr treatment, as shown by the release of viral rna from clr -treated virions (fig. d) . these findings were in agreement with our previous observations that clr selectively disrupted viral membranes (lump et al., ) . interestingly, the selectivity stems from clr 's preferential interaction with membranes containing high levels of sphingomyelin and cholesterol, two lipids that are enriched in membranes of enveloped viruses, such as hiv- or ebov (bavari et al., ; brügger et al., ; chazal and gerlier, ; lorizate et al., ) . the selective interaction of clr with lipids that are enriched in the viral but not the cellular membrane may also explain its minimal effects on cell viability ferreira et al., ; lopes et al., ; prabhudesai et al., ; sinha et al., ) (fig. c , d, supplementary fig. s b ). clr is slightly cytotoxic at concentrations of ∼ mm, corresponding to selectivity indices of - , which is in a reasonable range for drug development (buschmann and mannhold, ; food and drug administration, ) . . clr inhibits infection of human and murine brain cells. (a) human glioblastoma (a ) or neuroglioma (h ) cells were infected with zikv in the presence or absence of . - μm clr . two days later, cells were stained for zikv e protein (green) and nuclei (with hoechst; blue) and imaged by confocal microscopy. scale bar: μm. (b) primary murine cerebellum cultures were infected with zikv mr that had been incubated with . - μm clr for min at °c. dpi, cultures were fixed, permeabilized and stained for the neuronal protein βiii tubulin (red), zikv e protein (green) and nuclei (with hoechst; blue). scale bar: μm. clr lost antiviral activity in the presence of serum, precluding application as a systemic antiviral drug. this finding was unexpected because clr has been found previously to be stable in mouse and human plasma, with a half-life of ∼ . h in mice after sc or iv injection . moreover, clr showed therapeutic effects in animal models of alzheimer's and parkinson's diseases malik et al., ; prabhudesai et al., ; richter et al., ) . these data suggest that the loss of antiviral activity in the presence of serum likely was due to binding of clr to serum proteins. the concentrations of clr required to prevent amyloid formation in vitro and in vivo are in the sub-micromolar range, which is - orders of magnitude lower than those needed to block viral infection. presumably, the vast majority of clr was bound to serum proteins and hence unavailable to act on the virus, whereas the minority of the tweezer remains free at concentrations allowing to exert anti-amyloid activity. this interpretation also is supported by the observation that clr retained anti-zikv activity in semen, urine, saliva, and csf. the total protein concentrations in these body fluids are ∼ - orders of magnitude lower than in serum (hu et al., ; vibhakar et al., ) . thus, a larger percentage of clr molecules likely are free in these biofluids and hence antivirally active. clr has been described previously as a novel bi-functional microbicide counteracting sexual hiv- transmission both through directly targeting virus infectivity and by inhibiting the infection-promoting activity of amyloids in semen (lump et al., ) . we show here that the tweezer also inhibits pathogenic zikv infection of cells derived from the anogenital region in the presence of semen. interestingly, both zikv and ebov are present in semen of infected individuals (atkinson et al., ; deen et al., ; moreira et al., ) and can be transmitted by sexual intercourse, similar to hiv- (d'ortenzio et al., ; mate et al., ; moreira et al., ) . thus, application of clr as a microbicide in the vaginal or rectal tracts might protect from acquiring different viral pathogens. interestingly, clr has been shown to penetrate the bbb richter et al., ) , where it may also block replication of neurotropic viruses such as zika or rabies (cao-lormeau et al., ; ludlow et al., ; mlakar et al., ) . however, it needs to be considered that clr may only block cell-free virus infection but not cell-to-cell viral spread, which may limit its utility as therapeutic agent. therefore, the effect of clr on cell-tocell viral transmission needs to be explored. another means of application of clr is topical, e.g., by directly administering clr on virus-infected body surfaces, such as the skin or mucous membranes, to treat, e.g., hsv-induced herpes labialis or genitalis. topical medications may also be inhalational, such as medications against flu or respiratory syncytial virus (rsv), or applied to the surface of tissues other than the . after min of incubation, the mixtures were used to infect vero e cells. days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ), except for csf and serum: mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of clr but in presence of the same respective body fluid concentration (* denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - skin, such as eye drops applied to the conjunctiva, for example, in cmvinduced retinitis. to address the challenge of emerging and re-emerging viral pathogens, it is imperative to develop broad-spectrum classes of antiviral agents as the conventional one-bug-one-drug paradigm is insufficient (zhu et al., ) . current direct-acting antiviral drugs are highly successful but have a narrow spectrum of coverage and are only available against a very limited number of viral pathogens. moreover, drug development is slow and expensive, and it typically takes more than a decade to get market approval. currently, no specific antiviral treatment exists against most, if not all, emerging and re-emerging viruses. broad-spectrum antivirals may offer certain advantages as they reduce time and cost of drug development, allow off-label use, and could be applied even before a viral threat is diagnosed (bekerman and einav, ; zhu et al., ) . clr is broadly active against enveloped viruses as it disrupts lipid bilayers containing elevated levels of sphingomyelin and cholesterol (lump et al., ) , which are typically enriched in viral membranes (bavari et al., ; brügger et al., ; chan et al., ; chazal and gerlier, ; lorizate et al., ) . we have shown that clr inactivates major viral pathogens such as hiv- , hsv- , cmv, and hcv, as well as zikv and ebov. it is highly likely that clr also blocks other enveloped viruses, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) corona viruses, influenza or rsv. most notably, resistance development against clr is very unlikely to occur, as this would require changes in the lipid composition of the cellular and the viral membrane, which is difficult to envisage. in conclusion, clr represents a promising prototype of a broad-spectrum antiviral agent. all data were analyzed using graphpad prism version . for windows, graphpad software, la jolla california usa (www.graphpad. com). the significance level was calculated using one-way one-way analysis of variance (anova) (non-parametric, grouped), followed by bonferroni's multiple comparison test. p values of < . were considered significant (*, p < . **, p < . ***, p < . ). detection of zika virus in semen safety and pharmacological characterization of the molecular tweezer clr -a broad-spectrum inhibitor of amyloid proteins' toxicity protection of primary neurons and mouse brain from alzheimer's pathology by molecular tweezers a screen of fda-approved drugs for inhibitors of zika virus infection lipid raft microdomains combating emerging viral threats the hiv lipidome: a raft with an unusual composition drug selectivity: an evolving concept in medicinal chemistry guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study ebola outbreak in west africa -case counts retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides virus entry, assembly, budding, and membrane rafts. microbiol ebola rna persistence in semen of ebola virus disease survivors -final report zika virus (i). isolations and serological specificity evidence of sexual transmission of zika virus zika virus infection with prolonged maternal viremia and fetal brain abnormalities in vitro and in vivo characterization of recombinant ebola viruses expressing enhanced green fluorescent protein molecular tweezers targeting transthyretin amyloidosis a molecular tweezer for lysine and arginine guidance for industry; 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xu, yi; su, ling; zhu, xun; chen, minxia; zhu, weijin; xia, huimin; huang, xi; gong, sitang title: epidemiologic investigation of a family cluster of imported zikv cases in guangdong, china: probable human-to-human transmission date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: lgkqwm u zika virus (zikv) is an emerging mosquito-borne flavivirus that can potentially threaten south china. a chinese family of four returning from venezuela to china was found to be positive for zikv when the youngest son's fever was first detected at an airport immigration inspection. they were isolated temporarily in a local hospital in enping city, guangdong province, where their clinical data were recorded and urine and saliva were collected to isolate zikv and to obtain viral sequences. all of them except the mother presented mild symptoms of rash and fever. envelope gene sequences from the father, daughter and son were completely identical. phylogenetic analysis demonstrated that this strain is similar to several imported strains reported in recent months, which are all clustered into a group isolated from zika outbreaks in brazil. together with the climatic features in venezuela, new york and guangdong in february, it can be concluded that our subjects are imported cases from venezuela. with the same viral sequence being shared between family members, neither direct human-to-human nor vector transmission can be ruled out in this study, but the former seems more likely. although our subjects had mild illness, epidemiologists and public health officials should be aware of the risk of further expansion of zikv transmission by local competent vectors. although zika virus (zikv) was first identified early in in uganda, africa, outbreaks in french polynesia in significantly accelerated the spread of this virus to other parts of the world. zikv is a reemerging mosquito-borne flavivirus circulating in a wide range of regions including africa, south america, and asia. zikv infection can cause serious damage to the central nervous system, such as infant microcephaly and guillain-barré syndrome. [ ] [ ] [ ] the virus has proven to be neurotropic in animals, and a recent experiment in vitro also showed that it can infect human neural progenitor cells derived from induced pluripotent stem cells. another study showed that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are also permissive to the most recent zikv isolates. an animal model of zikv infection has been established in ag mice by foot pad injection. by far, aedes aegypti is considered the principal transmission vector of zikv, although aedes albopictus, which caused several outbreaks of dengue fever in guangdong province of south china in the last two decades, may play a role in the spread of this virus because a. albopictus may be a competent vector. there are over chinese in venezuela, which is one of the regions most heavily affected by zikv infection in south america. with frequent people shuttling between south america and guangdong, there is a potential risk of spreading zikv to south china, where a. albopictus are active in densely populated communities. in this study, a family of four flying from venezuela to guangzhou of guangdong province was found to be zikv positive in their peripheral blood. to gain a better understanding of transmission among communities, the phylogenetic relationship between the isolates from this family and others from diverse regions of the world was analyzed. because this virus may be transmitted directly by body fluids, [ ] [ ] [ ] it was also necessary to explore this possibility in this family. four hospitalized individuals from a family (father, mother, daughter and son) were diagnosed with zikv infection at enping people's hospital. this family had lived in venezuela for more than two months before february ; then they flew to new york on that day and stayed there for~ days. finally, they flew from new york to guangzhou, china on february ( figure ). these four infected individuals were first confirmed by real-time reversetranscription polymerase chain reaction (rt-pcr) in baiyun international airport of guangzhou, where the youngest one (the son) had developed fever, and the family was then isolated by the local department of public health. the infected individuals then lived in a shared ward (without other patients) in the infectious disease department of enping people's hospital. the room had been screened against mosquitoes, and each bed was also covered with a bed net to prevent spreading by local competent vectors. at the time of hospitalization, the subjects' clinical history and results of a general physical examination, blood tests and routine urine tests were documented. the youngest one (a -year-old boy) was the first case whose manifestation was fever and maculopapules. saliva, urine and peripheral blood were collected from the patients during the onset and recovery period and were stored at − °c. all samples were tested for zikv rna by real-time pcr, and some urine was utilized to isolate virus. informed consent was obtained from all patients before sample collection. the study protocols were reviewed and approved by the scientific and ethical committee of guangzhou women and children's medical center. before rna extraction, urine samples were concentrated with amicon ultra- . centrifugal filter units with ultracel- membrane (millipore, shanghai, china). the qiaamp viral rna mini kit (qiagen, hilden, germany) was used to extract viral rna from urine and saliva according to the manufacturer's instructions. rna was eluted in μl of ave buffer and stored at − °c until use. samples positive for zikv were selected to amplify genes encoding envelope protein (e) and nonstructural protein (ns ). complementary dna (cdna) was synthesized from viral rna by using the revertaid first strand cdna synthesis kit (thermo fisher, salt lake city, usa) according to the manufacturer's instructions. four pairs of primers were designed to generate overlapping dna fragments covering the e and ns gene regions by using the software oligo . (http://www.oligo.net/. supplementary table s ). polymerase chain reaction (pcr) was performed with ex taq hs dna polymerase (takara, dalian, china) under conditions of initial heating of °c for min, followed by cycles of °c for s, °c for s and °c for . min. the sequences of the pcr products were identified by using the sanger sequencing method. before phylogenetic analysis, the e and ns coding sequences of four individuals in our study were aligned with other reference sequences using clustal . software (http://www.clustal.org/clustal /). the reference sequences selected were those with highly similar blast (megablast, http://blast.ncbi.nlm.nih.gov/blast.cgi) scores. phylogenetic trees were drawn using the maximum likelihood method in the tamura-nei model with gamma-distributed evolutionary rates in mega . (www.megasoftware.net). an initial tree was made automatically with the nearest-neighbor-interchange method. the gaps/missing data treatment was set as complete deletions. bootstrap analyses with replications were utilized to determine confidence values for groupings within the phylogenetic trees. other parameters were set to default style. the clinical characteristics of four individuals with zikv infection are summarized and compared in table . in this family, whose members were living together for a long period before and after the first onset, the -year-old son was the first patient, and he had both fever and rash . before returning to china, they had no symptoms of any infectious disease, but the son and the daughter both had a history of mosquito bites in venezuela according to the father's memory, although the specific date of biting was not clear. the -year-old daughter was the second one who had symptoms, which included fever and rash, with the rash occurring on the second day after her brother had fever and rash. the -year-old father was the latest patient; the only symptom he had was a rash, and the rash was distributed uniformly on his neck and back ( figure ). his neck rash first occurred on the third day after his son showed fever and rash. the -year-old mother did not have any symptoms during the whole period of observation. a time axis based on rash indicates the sequence, the time interval and the date of positive detection for zikv ( figure ). the four family members had been isolated since february , and they were released on march . the clinical laboratory showed results that the overall symptoms of the four subjects were mild. the daughter and the son were subjected to laboratory blood tests twice and thrice, respectively (table ) . unlike most other viral infectious diseases, low white blood cell counts were not a common feature of our subjects. detection of zikv in saliva and urine from four hospitalized patients. real-time pcr based on taqman probes was used to detect zikv rna. among the four tested urine samples collected on february , patient (father), patient (daughter) and patient (son) were found to be positive for zikv. only patient was positive for zikv in a saliva sample (detected on the night of february , when the samples were collected). the urine and saliva samples from patient (mother) were all negative for zikv detection. nucleic acid sequencing, alignment and phylogenetic analysis pcr products were successfully obtained only from patient , patient and patient . after sequencing and fragment assembly, the e gene sequences of the three patients were found to be exactly the same. the e gene sequence of patient is identical to z (genbank: ku . , submitted by wu et al, guangdong provincial center for disease control and prevention), the viral sequence from the same patient. we found that all our sequences were similar to those of strains circulating in south america, especially brazil ( figure ). to further investigate the origin of the zikv isolated from our subjects, phylogenetic trees were constructed using the maxim likelihood method. zikv infection is becoming a global public health concern since microcephaly cases were linked to zikv infection during pregnancy. so far, the data suggesting that microcephaly cases in brazil might be linked with zikv infection are only epidemiological. , growing evidence has indicated that zikv can damage the fetus, causing intrauterine growth restriction. , a recent report that zikv can be detected in amniotic fluid of fetuses with microcephaly substantiated this point. with the establishment of a prevention and control system for emerging infectious diseases in china after the outbreak of severe acute respiratory syndrome in , imported cases from affected areas with fever will be strictly examined for specific pathogen infections. travelers with fever would usually be intercepted for further quarantine in an international airport when they have a history of being bitten by a mosquito in an area affected by zikv. in our study, when the youngest son had a fever, the whole family needed to be examined. because the four subjects in this family showed zikv positivity in their peripheral blood, isolation of the whole family was obligatory. south american countries closest to the equator have been deeply plagued by zikv infection in recent years. for example, zikv infection has been frequently reported in counties such as brazil, suriname, colombia and venezuela. neighboring caribbean countries including guatemala, haiti, puerto rico and dominica have also been affected. throughout the whole year, these areas have a warm and humid climate suitable for the survival of mosquitoes. the temperature in february in north america, especially northern regions such as new york, is below °c; thus, the family members in our study were unlikely to be bitten by mosquitoes during their -day stay in new york. thus, the zikv isolates carried by this family could only have come from venezuela (figure ). rash and low-grade fever seem to be the main symptoms for most zikv-infected individuals. rash was reported to be the most frequent symptom (presented in . % cases), followed by fever and arthralgia. in general, symptoms of zikv infection (mild flu-like symptoms) are milder than those of dengue virus (denv) infection; the latter often include high-grade fever with myalgia, headache, arthralgia and nausea. leukopenia (o /mm ) has been detected iñ % of dengue fever patients. in our study, one adult and two children had the symptoms of fever, rash and conjunctival congestion, and no one complained of any pain. unexpectedly, the three patients with fever all had normal leukocyte counts ( /mm ). whether the milder symptoms of zikv infection (milder than denv infection) mean a higher leukocyte count is unclear because of the lack of data obtained in past reports on zikv outbreaks. hence, if the mother without any symptoms in our study is considered only a carrier of zikv (not a patient), this proportion of patients in our study is almost consistent with previous reports, in which rashes were presented by almost all patients. information about pruritus, the second most common clinical symptom in the confirmed cases in previous study, was not obtained from the family while inquiring them regarding their case history. perhaps this symptom is transient and unnoticeable at the time of disease onset. rt-pcr based on taqman probes may be the most convenient tool to detect zikv infection in suspected patients , because enzyme-linked immunosorbent assays (elisas) for igm antibody against zikv would cross-react with other flaviviruses, , and the results must be validated by a plaque reduction neutralization test, a labor-intensive and costly method. moreover, compared with blood drawing, urine and saliva collection are more acceptable options for suspected individuals, and the latter have an advantage for viral detection and isolation because virus can be detected at higher titers and for a longer period in urine than in serum. a previous study figure phylogenetic tree based on e gene sequences of zika virus isolates. e gene sequences from the father, daughter and son (genbank: ku ) in our study were used for alignment with other reference sequences. all isolates are indicated with related information (genbank number, country, year and host, some with sample type). phylogenetic trees were drawn using the maximum likelihood method by the tamura-nei model with gamma-distributed evolutionary rates in mega . . an initial tree was made automatically with the nearest-neighbor-interchange (nni) method. gaps/missing data treatment was set as complete deletion. bootstrap analyses with replications were utilized to determine confidence values for groupings within the phylogenetic trees. other parameters were set to default style. first imported familial zikv cases in china y yin et al showed that a patient had prolonged shedding of viral rna in saliva and urine for up to days after symptom onset. nevertheless, positive taqman rt-pcr detection is not usually a guarantee of successful sequencing or isolation of target viruses. urine and saliva usually need to be concentrated before rna extraction or viral inoculation, which was performed in our study. like other flaviviruses, the structure of the single polyprotein encoded by zikv genomic rna is ′-c-prm-e-ns -ns a-ns bns -ns a-ns b-ns - ′, in which the e (envelope) protein is the main antigen recognized by the host immune system, and ns is the principal component of the replication complex of the virus. in previous research, e gene sequences of zikv isolates were usually utilized to construct phylogenetic trees based on experience from molecular study of dengue virus. our phylogenetic tree constructed based on the e protein coding gene is topologically similar to that based on the complete zikv genomic sequence (figure ) , meaning that the phylogenetic tree based on the e gene is strong enough to distinguish all clusters of zikv isolates from all over the world. because complete genome sequencing of an rna virus is a time-consuming procedure, constructing a phylogenetic tree using e protein gene sequences is a simpler method for molecular epidemiologic study, especially in an outbreak. in our phylogenetic tree constructed from e protein gene sequences, zikv can be divided into two lineages, african and asian. , although there is a very short evolutionary distance between our strains and the venezuela imported strain ku . -ve ganxian, the diversity of the strains is apparent, and the latter is nearer to kj . -h/pf/ french polynesian strains. , currently, all cases reported in china seem to be imported from south america and pacific islands, which also indicates there was a great diversity of strains circulating during early in venezuela. our strains are more similar to the strains circulating in south america in late , where there were more strains isolated from patients. a phylogenetic tree based on ns gene sequences cannot efficiently distinguish different subgroups of zikv from various sources of isolates (supplementary figure s ) because the ns gene sequence is more conserved than that of e or even ns , which is a gene sequence used more frequently to build phylogenetic trees. currently, the e gene sequence is the most commonly used sequence in molecular epidemiologic studies of flaviviruses because it has the greatest genetic diversity (supplementary figure s ) . it is worth noting that strains of the african lineage were almost solely isolated from aedes mosquitoes circulating in the african continent according to the phylogenetic tree, probably because zikv did not draw much attention before massive microcephaly cases were reported in brazil. zikv is spread primarily by a. aegypti, a dominant vector for several mosquito-borne viruses in the western hemisphere. zikv was detected in a. albopictus from gabon, a country in central africa. no direct evidence has demonstrated that this virus could be spread by a. albopictus, a local vector for dengue virus in south china, yet the threat is quite real. the fact that zikv can propagate in the c / cell line derived from a. albopictus suggests that this mosquito may be a competent vector and have the potential for spreading the virus, although there have not been any reported isolates from mosquitoes in south china. a previous experiment conducted in laboratory indicated that zikv could be detected in the midgut and salivary glands of a. albopictus ten days after oral infection. outbreaks of denv have proved that south china may be a permissible environment for transmission of emerging viral diseases such as zikv, and imported cases may be sources of subsequent autochthonous cases. , a key difficulty in preventing the spread of infection is that a high proportion of infected individuals have no symptoms. similar to the mother in our study, asymptomatic individuals with viremia may be a source of infection when they are exposed to competent vectors. if the son's fever had not been found at the airport entrance, our subjects would not have been suspected of being infected with zikv, and thus their viremia would not have been confirmed. it is difficult to convince zikv carriers to be examined even if they are returning from an affected region. furthermore, in addition to the mosquito-borne route, other complex modes of transmission such as body fluid transmission will make controlling zikv infection even more difficult than controlling dengue fever. the priorities for control include strengthening the education of travelers from infected areas to non-epidemic regions, identifying virus carriers, preventing local transmission caused by imported cases, and an effective vector management program. in our study, four subjects in one family had lived and traveled together for the past few months, so they had similar opportunities for exposure to mosquito bites. however, because they are a family, the probability of human-to-human transmission by close contact between family members should not be ignored. if their zikv infections were all caused by mosquito-biting, from a time axis showing when symptoms occurred, the incubation periods of three of the patients were all more than five days (figure ), which is consistent with previous reports ( - -day incubation period according to the world health organization website). denv transmission by non-blood routes is almost impossible, so vector transmission is nearly the only way. thus, as for clustered cases in one family, if the vector transmission is considered, zikv can be comparable to denv because they have the same transmission dynamics via vectors. until now, there have been no reports of denv case numbers in one family being more than two, especially when they share the same virus sequence. therefore, because vector transmission of denv between family members is less likely, so is that of zikv. because the zikv isolated from three members is identical and there was a to day interval between initial symptom occurrence, human-to-human transmission may also be a plausible explanation. moreover, viral isolation from urine and saliva samples (collected on february ) of three patients was performed successfully using inoculation of suckling mice (data not shown), indicating that during onset, there was viral shedding in these three patients' body fluids, which was infectious to their family members. regarding direct transmission, sexual contact may be a route, for a relative case had been reported previously but with no direct evidence. because an in vitro experiment demonstrated that human skin cells are permissive to zikv, it might be possible that zikv can be transmitted by other bodily fluids such as saliva and urine. rt-pcr positivity for zikv rna together with successful viral isolation from the three patients' urine 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the americas first report of autochthonous transmission of zika virus in brazil we thank dr de wu (guangdong provincial center for disease control and prevention, china) for helpful discussions on zika virus isolation. this work was supported by a grant ( y - ) from the science and technology program of guangzhou, china. key: cord- -wnf fozk authors: chan, m.y.; smith, m.a. title: infections in pregnancy date: - - journal: comprehensive toxicology doi: . /b - - - - . - sha: doc_id: cord_uid: wnf fozk infections during pregnancy may affect a developing fetus. if left untreated, these infections can lead to the death of the mother, fetus, or neonate and other adverse sequelae. there are many factors that impact infection during pregnancy, such as the immune system changes during pregnancy, hormonal flux, stress, and the microbiome. we review some of the outcomes of infection during pregnancy, such as preterm birth, chorioamnionitis, meningitis, hydrocephaly, developmental delays, microcephaly, and sepsis. transmission routes are discussed regarding how a pregnant woman may pass her infection to her fetus. this is followed by examples of infection during pregnancy: bacterial, viral, parasitic, and fungal infections. there are many known organisms that are capable of producing similar congenital defects during pregnancy; however, whether these infections share common mechanisms of action is yet to be determined. to protect the health of pregnant women and their offspring, additional research is needed to understand how these intrauterine infections adversely affect pregnancies and/or neonates in order to develop prevention strategies and treatments. . intrauterine infection, comprehensive toxicology, nd edn, vol. , pp. - , isbn - - - - , . /b - - - - . - . preterm birth babies born before completed weeks of gestation. sepsis a life-threatening condition where a whole body inflammatory response is activated due to an infection. sequelae a pathological condition resulting from a prior disease or injury. stillbirth a baby born dead after completed weeks of pregnancy. torch an acronym for toxoplasmosis, other, rubella, cytomegalovirus (cmv), and herpes infections that are some of the most common infections associated with congenital anomalies. vertical transmission when an infection is transmitted directly from the mother to an embryo, fetus, or baby during pregnancy or childbirth. in the s, serious birth defects in infants born to women infected with rubella during their first trimester provided the first evidence that adverse pregnancy outcomes could result from maternal infection. it has become well recognized that infections during pregnancy, in addition to affecting the mother, may affect or be passed to the developing fetus. infections are caused by bacteria, viruses, or parasites that have invaded body tissues or have released toxins disrupting normal functions and eliciting an inflammatory response to help combat the microorganism. infections, or the inflammatory responses to an infection, can result in adverse effects including preterm delivery, birth defects, developmental delays, or stillbirths. the main focus of this article is to explore the variety of pathogens that can lead to adverse effects to the fetus or neonate. there are various factors that can impact infection and susceptibility during pregnancy, some of these will be examined in detail, focusing on new data and information. databases searched for this information included pubmed, google scholar, and web of science focusing on publications from to . since the publication of the previous comprehensive toxicology nd edition, new sections included in this article discuss the transmission of intrauterine infections; the factors impacting infection; examples of classic and new pathogens capable of causing intrauterine infections; and an updated table of additional selected pathogens detailing the exposures, symptoms, and outcomes of infection. bacteria, viruses, and other organisms are able to be passed from mother to child, a phenomenon known as transmission. the specific symptoms of transmitted infections depend on the individual pathogen and the stage of pregnancy at the time of infection. transmission occurs in the womb during pregnancy or during birth. pathogens can enter the uterus and infect the fetus through several routesdthe most common routes are vertical transmission, such as hematogenous route where pathogens gain entry to the fetus by passing through the placenta, and ascending transmission, such as pathogens traveling up the reproductive tract of the pregnant woman. other less common routes are passage from the abdominal cavity and through the fallopian tubes, invasive medical procedures, or the birthing process (richardson et al., ) . the main routes of intrauterine infection during pregnancy are vertical transmissions. a vertically transmitted infection is caused by a pathogen transmitted directly from the mother to an embryo, fetus, or baby during pregnancy or childbirth; this type of transmission is also known as mother-to-child transmission. vertical transmission during the perinatal period can be subcategorized as a perinatal infection. vertical transmission generally refers to transplacental transmission. the pathogen reaches the placenta through the mother's blood (hematogenous transmission). these pathogens must have the ability to overcome the placental barriersdsyncytiotrophoblast interface, decidual-trophoblast interface, and physical obstacles (robbins and bakardjiev, ) . pathogens may overcome these barriers through a damaged syncytiotrophoblast interface; invasion of the uterine-trophoblast interface directly or cell-to-cell transport (e.g., listeria monocytogenes); or immune cells may allow for barrier crossing (transport of human immunodeficiency virus mediated by leukocytes) (robbins and bakardjiev, ; lagaye et al., ) . when the placenta is infected, this may allow for the pathogen to invade the fetus. ascending infections occur when infectious pathogens residing in the external genitalia of the mother access the amniotic sac. upon infection, the amniotic sac may become compromised and rupture. once ruptured, the pathogens may inoculate the amniotic fluid, spread as a biofilm over the exposed amnion, and invade choriodecidual tissue planes (kim et al., ; redline, ) . the fetus can become infected by aspirating the microorganism to the lungs, ingesting the pathogens, or by the pathogen penetrating the ear canal. see section on chorioamnionitis for details of post amniotic fluid infection. susceptibility of infection in women differs due to natural variation; however, a number of other factors also influence this susceptibility. for intrauterine infections, there are some common factors that impact susceptibility to infection. epidemiologic studies have shown that pregnant women have an increased incidence of contracting a variety of infections like influenza, varicella, measles, severe acute respiratory syndrome, tuberculosis, listeriosis, pneumocystis, toxoplasmosis, and malaria (sappenfield et al., ; riley et al., ; jamieson et al., ) . the severity of infection has been suggested to vary at different stages of pregnancy, likely from the unique immunological alterations that occur during different stages of pregnancy (faucette et al., ) . to accommodate the genetic differences between the mother and the fetus and to prevent allogenic rejection of the fetus, the maternal immune system shifts toward a t-helper bias (jamieson et al., ) . maternal hormone levels change during pregnancy, and interplay between the sex hormones and immune system may alter the efficiency of the maternal immune system. these changes are thought to contribute to an increase in susceptibility toward certain infectious diseases which may change the severity of illness the mother experiences and fetal mortality rates. pregnant women may be considered immunosuppressed, though not in the traditional sense of the word. a fetus derives half of its genetic material from its father; thus, the fetus is essentially a foreign tissue in the maternal uterine environment. the fetus' susceptibility to rejection from its mother's immune system has been compared to an organ transplant (jamieson et al., ) . evidence suggests that the maternal immune system tolerates fetal antigens by suppressing cell-mediated immunity while retaining normal humoral immunity and response (jamieson et al., ) . though only occurring locally at the maternal-fetal interface, this suppression prevents the rejection of the fetal tissue and has been suggested that it may affect maternal response to infection (jamieson et al., ; erlebacher, ) . the exact immunological tolerance is not completely understood. during pregnancy, there is a systematic shift from t-helper (th ) to t-helper (th ) cell-mediated responses in the mother. in a th -dominated response, the th -type t lymphocytes and proinflammatory cytokines amplify cell-mediated immunity allowing for recognition of the body's own cells that engulf the pathogen and express pathogen-related antigens on their surfaces (jamieson et al., ; corr et al., ) . when shifting to the th cell-mediated response, the th -stimulating cytokines dominate and suppress the th t cell responses locally allowing for adequate humoral immune response while the cell-mediated immunity is compromised (thaxton and sharma, ) . hormonal changes during pregnancy promote this shift from th to th . hormones can contribute to a modified immune response by altering the functions of immune cells and can have effects on the outcome of infection during pregnancy (robinson and klein, ) . during pregnancy, steroid sex hormone levels change drastically and are considerably higher than at any other time during a woman's life, particularly pregnancy-associated hormones including estradiol, estriol, progesterone, corticosteroids, and prolactin (robinson and klein, ) . the interplay between sex hormones and the immune system is complex and often contradictorydestradiol can enhance innate immunity in cellmediated and humoral adaptive immune responses, yet progesterone can suppress the maternal immune response and alter helper t cell responses (kourtis et al., ; robinson and klein, ) . research is needed to fully understand the changes in hormones and the immune system during pregnancy and the interplay between the two systems. pregnancy is a stressful change in a woman's body. additional stressors beyond that of a pregnancy impact not only the health of the mother but also the development of the fetus. stress can either enhance or suppress the immune system and thereby change the mother's susceptibility and severity to certain infections. mechanisms linking maternal stress to infant development are complex. stressors can enhance or suppress immune function, which can change susceptibility to infections. the duration of stress impacts the health of the individual. suppressed immunity can occur by decreasing immune cell number and function or increasing active immunosuppressive mechanisms. maternal stress may be mediated through biological and behavioral mechanisms like the neuroendocrine pathway, which can result in the activation of the maternal-placental-fetal endocrine systems or an immune/inflammatory pathway, where maternal stress may modify the characteristics of systemic and local immunity by increasing susceptibility of the intrauterine and fetal environments to the immune inflammatory processes (wadhwa et al., ) . it is likely that the placenta plays a vital part in mechanisms linking maternal stress and infant development. traditionally, intrauterine infections were thought to have originated from pathogens in the vaginal tract that ascend into the intrauterine environment. the placenta was considered sterile during gestation and the presence of any bacteria in clinical cultures was a diagnostic for intrauterine infection (hillier et al., ) . however, the presence of placental or membrane bacteria has been observed in the absence of histological infection and recognition that the presence of bacteria does not always have the expected corresponding clinical outcomes when observed (stout et al., ; leviton et al., ; han et al., ; buhimschi et al., ; steel et al., ; mysorekar and cao, ) . of all the cells in the human body, only % are human somatic and germ cells; the remaining % are made of microflora (savage, ) . the microflora found in and on humans are composed of diverse niche communities of microbial species varying by site or organ. as a whole, all these communities make up the microbiome. with the initiatives set by the national institutes of health with the human microbiome project in , the microbiome has been found to be increasingly integral with the healthy functions of a human (turnbaugh et al., ; peterson et al., ) . human-microflora interactions occur across body sites and have roles in metabolism, immunity, development, and behavior of the host (o'hara and shanahan, ; cabreiro and gems, ) . the communities consist of a balance of commensal bacteria and beneficial bacteria that help humans with normal functions. when the balance is upset, it can result in disease and unfavorable alterations; under certain conditions, an outgrowth of potential pathogenic bacteria, a decrease in the number of beneficial bacteria, or an imbalance of commensal microflora may contribute to the pathogenesis of disorders which may affect the susceptibility rate for infectionsdincluding intrauterine infections (round and mazmanian, ; hill and artis, ; honda and littman, ; o'hara and shanahan, ) . infection with a pathogen leads to a change in the normal microbiome which can have an effect on the intrauterine system (hacquard and schadt, ) . for instance, a decrease in the diversity in the placental microbiome has been correlated with lower birth weight in human infants (zheng et al., ) . many of the diverse groups of oral cavity bacteria have been found in the intrauterine environment and associated with adverse pregnancy outcomes suggesting that the oral cavity may act as a reservoir for infection (mysorekar and cao, ; fardini et al., ; han, ) . these bacteria are capable of hematogenous transmission, similar to the transient bacteremia possible during periodontal infections (fardini et al., ; han, ; offenbacher et al., ) . for example, maternal oral infections, like acute gingival infection and chronic periodontal infections, are multifactorial disorders where microbial dental biofilms are considered the primary agent initiating inflammation (chambrone et al., ; offenbacher et al., ; xiong et al., ) . although inflammation is typically restricted to local periodontal tissues, the pathogens can stimulate bacteremia and translocate to distant tissues (champagne et al., ; xiong et al., ) . microorganisms have the ability to transfer from one location to another, as noted by studies on periodontal disease and its impact on pregnancy. as we continue to discover the complex roles and interactions between the human body and the microbiome that inhabits the body, it is likely that correlations between the health of the intrauterine microbiome and the susceptibility to intrauterine infection will be discovered. once infected, there are a multitude of adverse pregnancy outcomesdpreterm births, chorioamnionitis, infections of the central nervous system (cns), and sepsisdwhich are detailed in the following section (richardson et al., ) . premature births, or preterm births, are defined as births that occur prior to completed weeks of gestation, and they are associated with %- % of pregnancies (liu et al., ) . premature births are the leading cause of neonatal morbidity and mortality in the developed and developing world (goldenberg et al., ) . infants who are born prematurely have a higher risk of serious disability or mortality than their full-term counterparts, with those infants born before the th week of pregnancy being especially at risk of prenatal mortality and morbidity. potential problems for premature babies are an increased risk of short-term complications due to immaturity of organ systems, neurodevelopmental disorders, intellectual disabilities, vision/hearing impairments, or even death (cdc, e; romero et al., ) . intrauterine infection is a frequent and important mechanism that may contribute to nearly % of preterm births (goldenberg et al., ; agrawal and hirsch, ; romero et al., ) . however, this number may be higher because many infections are likely to be subclinical and the pathogenesis is not detected due to the lack of sensitivity of conventional culture techniques (racicot et al., ) . outcomes of infection in a pregnant host are dependent upon various factors including gestational stage of fetus, previous exposure and immunity of the mother, variable immunity among individuals, the placenta's ability to protect the fetus from infection, and the developing immune system in the fetus (richardson et al., ) . there are different pathways by which preterm birth from bacterial infection are known to occur. two major events that are required for bacteria to induce preterm labor are ( ) bacterial colonization in gestational tissues or the fetus and/or ( ) bacterial counts reaching a threshold that elicits maternal immune inflammatory response that will induce preterm labor (romero et al., ; richardson et al., ) . preterm labor is more frequent when a fetal inflammatory response is triggered as diagnosed by an increase in il- and il- and a decrease in il- (romero et al., ) . compared to term infants, preterm births have a higher incidence rate of temperature instability, respiratory distress, apnoea, hypocalcaemia, seizures, jaundice, kernicterus, feeding difficulties, periventricular leukomalacia, and hospitalizations (saigal and doyle, ) . effects of preterm birth can manifest later in childhood (huddy et al., ) . studies on low birth weight have shown that most of these infants showed sequelae like cognitive defects, academic underachievement, grade failures, and the need for increased remedial assistance during mid-childhood and adolescence continuing during life (saigal and doyle, ) . there is still much about the pathogenesis and mechanisms of preterm birth that is not understood and animal models, such as sheep, have/are being utilized to gain knowledge that could lead to methods and treatments that can improve neonatal outcomes (racicot et al., ; goldenberg et al., ) . the condition in which the membranes that surround the fetusdthe chorion and amnion, and the amniotic fluiddare infected by bacteria is commonly referred to as "chorioamnionitis." chorioamnionitis complications are associated with significant and/or long-term adverse outcomes for the mother (postpartum infections and sepsis) and/or the infant (stillbirth, premature birth, neonatal sepsis, chronic lung disease, and brain injury) (tita and andrews, ) . it has been estimated that one of every three preterm neonates is born to a mother with intraamniotic infection, identified with either cultivation or molecular microbiologic techniques (berger et al., ) . it is important to also note that microbial colonization of chorioamniotic membranes does not always result in a fetal or maternal inflammatory response (romero et al., ) . chorioamnionitis is typically associated with bacteria that have low virulence and enter the uterus by ascending from the lower reproductive tract; it is rare that transmission is spread hematogenously (one notable exception: listeria monocytogenes) (richardson et al., ; tita and andrews, ) . the presence of organisms in the amniotic cavity elicits an intense inflammatory response (racicot et al., ) . the inflammatory response may intensify and progress into fetal inflammatory response syndrome which predisposes preterm neonates to shortand long-term consequences (goldenberg et al., ) . a maternal fever is an important criterion for the diagnosis of chorioamnionitis. the presence of maternal fever of greater than c ( . f) and at least two of the following criteria: maternal leukocytosis (greater than , cells/mm ), maternal tachycardia (greater than beats/min), fetal tachycardia (greater than beats/min), uterine tenderness, and/or foul odor of the amniotic fluid. these thresholds are associated with higher rates of neonatal and maternal morbidity (polin and committee on fetus and newborn, ) . additional details of the mechanisms of chorioamnionitis can be found in richardson, pollak et al. ( ) and in tita and andrews ( ). adverse effects to the cns is a common result of intrauterine infections in neonates. transversal of the blood brain barrier (bbb) must occur for a pathogen to access the cns (polin and harris, ) . the bbb secretes cerebrospinal fluid (csf) while physically separating the brain from the intravascular department. the purpose of the bbb is to control the flow of molecules and ions that enter and leave the brain in order to maintain a neutral environment (kim, ; richardson et al., ) . the extent of effect depends on the maturity of the developing brain, timing of infection, or amount of pathogens (kim, ) . there are an estimated six cases of bacterial meningitis per , people worldwide (ninds/nih, b); however, there is little known about bacterial meningitis in the fetus (ninds/nih, b). generally, pregnancy has not been associated with an increased risk of bacterial meningitis with the exception if the cause is due to streptococcus pneumoniae, listeria monocytogenes, or cryptococcus neoformans (baldwin and roos, ; adriani et al., ) . clinical effects of meningitis are sepsis and increased intracranial pressure; chronic changes can occur such as hydrocephalus, encephalomalacia, or subdural effusion (kim, ) . neonatal meningitis is the most common and serious bacterial infection during the neonatal period, with the most common pathogens being enterobacteria, e.coli, and group b streptococcus (kim, ) . hydrocephaly is a condition where excess csf accumulates in the brain: it is also known as hydrocephalus or "water on the brain" (ninds/nih, a). the excessive accumulations cause an abnormal expansion of the brain's ventricles producing pressure on the brain (ninds/nih, a). due to the malleability of an infant's skull, the added pressure on the brain may result in an enlarged head size for the infant as the skull widens to relieve pressure (richardson et al., ) . causes of hydrocephalus are still not well understood; possible causes in the fetus are infections, genetic defects, developmental disorders, meningitis, premature birth complications, or traumatic head injury (richardson et al., ; ninds/nih, a). microcephaly is a medical condition present at birth or developed within the first few years of life wherein the circumference of the head is smaller than normal because the brain has either not developed properly or growth has stopped (ninds/nih, c). it is most often caused by genetic abnormalities during the early months of fetal development, but it may be induced if the mother is infected or affected by pathogens, drugs or alcohol, or certain toxic chemicals during pregnancy (ninds/nih, c). recently, pregnant women infected with the zika virus had a high incidence of babies born with microcephaly. see sections examples of infection during pregnancy, viral infections, and subsection zika virus, for examples and a discussion. the result of having microcephaly varies. babies born with microcephaly will have smaller than normal heads that fail to grow through infancy. microcephaly can be an isolated condition or occur in combination with other major birth defects (cdc, b) . depending on the severity of microcephaly, problems can range from mild to severe and are often lifelong, such as impaired cognitive development, developmental delay, facial distortions, dwarfism or short stature, hyperactivity, seizures, difficulties with coordination and balance, feeding problems, hearing loss, vision problems, and other brain or neurological abnormalities (ninds/ nih, c; cdc, b). severe microcephaly can also be life-threatening (cdc, b) . yet some will develop normally and their head will grow bigger especially if they are otherwise growing and developing normally (ninds/nih, c). infections during pregnancy may result in developmental delays for the infant. pathogens that are able to interact with the cns have capabilities of causing morbidities in fetuses and premature infants. some of these impairments may not manifest at birth or in the neonatal period but later in life. many neonates are able to survive major insults, like infection, without any evidence of impairment due to the plasticity of the developing brain and improvements in medical care; yet, in others, these insults can cause varying degrees of long-term neurodevelopmental impairment such as cerebral palsy, with half of those continuing to develop cognitive and behavioral deficits (mwaniki et al., ; stoll et al., ) . the exact mechanisms whereby pathogens lead to developmental delays are not yet known. molecular events such as the release of inflammatory cytokines by brain cells during infection may have a significant role in the brain injury (dammann and leviton, ) . for instance, ascending intrauterine infections have been suggested to significantly increase the risk of fetal brain damage due to the initiation of fetal inflammatory response syndrome (berger and soder, ; dammann and leviton, ; de vries, ). yet, whether there are exposure-specific or pathogen-specific patterns by which cytokines act in response to inflammation during infection is unknown. sepsis is a systematic syndrome that occurs in response to infection of the blood and stems from an additional medical condition, like chorioamnionitis or a cns infection (nigms/nih, ) . during sepsis, intravenous coagulation may occur, blocking blood flow to vital organs and creating hypoxic and ischemic conditions. mortality in cases of sepsis is due to the failure of multiple organsdtypically, a single organ fails leading to the dysfunction of other organ systems. there is early and late onset of neonatal sepsis. early onset is typically a consequence of fetal infection in utero or during parturition within the first week of life. sepsis can begin in utero when the fetus inhales or swallows infected amniotic fluid; during parturition, sepsis is capable of developing within the hours or days after birth when colonized skin or mucosal surfaces are compromised (polin and committee on fetus and newborn, ) . maternal treatment with intrapartum intravenous antimicrobial agents is the only intervention proven to reduce the incidence of early-onset neonatal sepsis (cdc, b; polin and committee on fetus and newborn, ). late-onset sepsis can result from colonization during birth via vertical transmission of a colonized mother's anorectal and vaginal areas; case reports have suggested possible horizontal transmission in the postnatal period through breast milk (berardi et al., ; polin and committee on fetus and newborn, ) . depending on the pathogen and the neonatologists' discretion, a full course of antimicrobials may be used to treat the neonate (rubin et al., ) . vancomycin has been a frequent choice of empiric antimicrobial treatment of neonates with suspected late-onset sepsis (rubin et al., ) . chorioamnionitis is a major risk factor for neonatal sepsis and increased risk of early-onset sepsis (polin and committee on fetus and newborn, ; wolfs et al., ) . additionally, pathogens associated with neonatal meningitis have the ability to induce neonatal sepsis. as mentioned previously, neonates that survive a cns infection may experience developmental delays and impairment. a study showed that the degree of impairment was more likely to be severe in septic preterm neonates than in nonseptic neonates (mwaniki et al., ) . infections that are acquired in utero or during birth are a significant cause of fetal and neonatal mortality which can contribute to early and later childhood morbidity. organisms shown to cause these congenital conditions are included in the torch complex. the torch acronym represents toxoplasma gondii, other infections, rubella, cytomegalovirus, and herpes viruses as a concept emphasizing that these infections have been shown to cause stillbirths, perinatal morbidity, and %- % of all congenital anomalies (neu et al., ; stegmann and carey, ) . these conditions are far from the only kinds that can cause congenital conditions. specific examples of types of pathogens that affect pregnancy and the developing fetus are listed in table . to explore some of the mechanisms unique to pathogens (bacterial, viral, parasitic, and fungal), a few examples are elaborated in this section. listeriosis is predominantly a foodborne disease associated with the consumption of contaminated foodsdprimarily in dairy products. populations most susceptible to l. monocytogenes infection are people with a compromised immune system, older people, pregnant women, and neonates. these populations account for at least % of reported infections (cdc, a) . pregnant women are about times more susceptible to l. monocytogenes infection than healthy, nonpregnant adults; about one in seven ( %) cases of listeriosis occur during pregnancy (cdc, a; c) . infection during pregnancy can cause spontaneous preterm labor, fetal loss, and illness or death in newborn infants (cdc, c; richardson et al., ) . symptoms of infection may include gastrointestinal symptoms, influenza-like illness with fever, headache, myalgia, and/or backache, but % of the infection cases are asymptomatic (mylonakis et al., ; jackson et al., ) . animal studies on rhesus monkeys (smith et al., ) , guinea pigs (williams, ) , and gerbils (roulo et al., ) have shown a relationship between increasing bacterial load and adverse pregnancy outcomes. a fetus becomes infected when l. monocytogenes crosses the placenta, which has been suggested to be a reservoir for reinfection (bakardjiev et al., ; de luca et al., ) . neonatal infection occurs in % of the cases of maternal infection; % of the cases may make a full recovery, but in . % of cases, there may be long-term sequelae (mylonakis et al., ) . infection may lead to severe health conditions such as septicemia, pneumonia, or meningitis and in about % of cases, the occurrence of preterm delivery with birth weights lower than normal or stillbirth may occur (mylonakis et al., ) . septicemia is the main symptom that neonates display with a %- % mortality rate. other symptoms include respiratory problems, pneumonia, and the formation of minute abscesses in bodily organs (farber and peterkin, ) . a neonate can develop late-onset listeriosis through natural birth if the mother's vaginal tract has been colonized by l. monocytogenes (cdc, c) . bacteremia can be caused by ascending infection, transplacental passage of the organism, or by inhalation of amniotic fluid (becroft et al., ; de luca et al., ) . second-and third-trimester infections can result in premature delivery followed by neonatal illness or preterm delivery of a stillborn (farber and peterkin, ) . for additional discussion of mechanisms, see richardson, pollak et al. ( ) . streptococcus are classified by their hemolytic properties with two species known to affect pregnancydalpha-hemolytic streptococci (group a strep) and beta-hemolytic streptococci (group b strep). we will be detailing examples from group a strep and group b strep in the following section because of their potential impact on pregnancy. a group a streptococci, streptococcus pneumoniae or pneumococcus, is a significant human bacterium. it is part of the normal microbiome in the upper respired tract, but opportunistic, if conditions allow. it is one of the most common causes of severe pneumonia and can cause pneumococcal meningitis, which is the most common form of meningitis and the most serious (ninds/nih, b; cdc, d) . coexisting disease in the mother increases the risk of contracting pneumonia in pregnancy. a pregnancy complicated with pneumonia can have adverse fetal effects; % of women experienced preterm labor and of the preterm births, % had respiratory failure (munn et al., ) . pneumonia in pregnancy can result in inflammation and edema of alveoli, restricting the number available for oxygen transport. fetal oxygen delivery decreases when the maternal oxygen saturation falls to < % (goodnight, ) . birth weights were significantly lower at delivery during pregnancies complicated by pneumonia (goodnight, ) . invasive group b streptococci (group b strep) or streptococcus agalactiae is the leading infectious cause of maternal chorioamnionitis, puerperal endometritis, and early-onset neonatal sepsis (phares et al., ; cdc, b) . it causes invasive disease primarily in infants, pregnant/postpartum women, and older adults, with the highest incidence among young infants (phares et al., ) . there are two main types of group b strep diseasedearly-onset disease and late-onset disease. early-onset disease occurs during the first week of life and late-onset disease occurs from the first week through the first three months of life. during early-onset disease, group b strep commonly causes sepsis, pneumonia, and, sometimes, meningitis (cdc, ) . for late-onset disease, the same illnesses are found as in the early onset, but meningitis is more common during this type of disease (cdc, ) . the primary risk factor for neonatal infection transmission is colonization of the mother's genital tract through direct exposure during birth, or in fetuses through ascension into the amniotic fluid then into the placenta. group b strep can also cause miscarriages, stillbirths, and preterm deliveries (cdc, ) . the exact mechanisms are generally unknown (cdc, ). campylobacter are found ubiquitously in the environment and considered normal intestinal flora in most mammals and birds. the consumption of raw or undercooked poultry or crosscontamination of other foods is the most common source of campylobacteriosis in industrialized nations, responsible for more than % of all sporadic cases in humans (dasti, ; allos, ) . more than % of all human campylobacter infections are caused by campylobacter jejuni (c. jejuni) and campylobacter coli, with increasing rates of infection reported during the summer months. infants are one of the two most infected populations, the second group are patients between and years (dasti, ) . c. jejuni, campylobacter fetus (c. fetus), and other strains have caused stillbirths/abortions in pregnant women (smith, ) . c. jejuni and c. fetus are two strains known to cause illness in pregnant/postpartum women and their fetuses/newborn infants. though most cases have no complications, enteritis caused by c. jejuni infection can result in stillbirth or premature labor (allos, ) . transmission of the pathogen to offspring can occur either transplacentally or through vaginal delivery (smith, ; richardson et al., ) . additional details into the mechanisms of campylobacter intrauterine infection and the impacts on the fetusdsuch as stillbirthdmay be found in the second edition of comprehensive toxicology, volume , intrauterine infections (richardson et al., ) . rubella was the first virus recognized to result in birth defects. there are a number of viruses that can adversely affect the developing fetus (table ) . viruses act through different mechanisms of action; there are some common features of viral infections that adversely affect pregnancy. transmission occurs vertically from the mother to the fetus. if the placenta is not infected, the virus does not appear to spread to the fetus. various factors influence the likelihood of the virus infecting the fetus such as infection state of the mother and gestational age of the fetus. outcomes of viral infections are generally spontaneous abortion, stillbirth, prematurity, and organ pathogenesis. in the following section are provided examples of viruses, additional examples may be found in table . cytomegalovirus (cmv) is a herpes virus that infects people of all ages. once infected, the virus stays within a person's body for life. in the united states, it is estimated that %- % of adults are infected with cmv by the age of (cdc, a). most healthy children and adults infected with cmv exhibit no symptoms; those that do exhibit symptoms tend to be like other illnessesdfever, sore throat, fatigue, and swollen glands. cmv can cause serious disease in pregnant women and their babies, people who care for infants and children, and the immunocompromised. it is the most common viral infection in the developing fetus; approximately in children is born with congenital cmv infection. transmission of this virus is generally through direct contact with body fluids. cmv can be transmitted from a pregnant woman to her fetus during pregnancy by the virus crossing the placenta and infecting the fetus' blood. congenital cmv infection in developed countries occurs between . % and . % of all live births (peckham, ; alford et al., ; bonalumi et al., ) . most infants born with congenital cmv infection ( %) appear healthy at birth and % of the healthy appearing infants never develop symptoms or disabilities. health problems or disabilities may appear or more years after birth or never (cdc, a). fetal damage from cmv infection can include growth retardation and cns abnormalities (kimberlin et al., ) . additional details regarding the mechanisms of cmv may be found in the second edition of comprehensive toxicology, volume , intrauterine infections (richardson et al., ) . influenza viruses are a group of rna viruses and are the leading cause of serious wintertime respiratory morbidity worldwide. studies about the effects of influenza-related illness during pregnancy have shown an impact on the health of pregnant women. specific changes in a woman's immune system, heart, and lungs during pregnancy increase the susceptibility to severe illness, hospitalizations, and possibly death. there is an increased chance for serious problems for unborn babies, including premature labor and delivery (mcneil et al., ) . there are three distinct genera of influenza viruses: a, b, and c. for this example, we will be looking at the outcomes specific to the subtype of influenza adthe h n influenza a virus. women hospitalized with h n infection were estimated to have a three times increased risk of delivering preterm (yates et al., ) . emergency cesarean deliveries were frequently reported as urgent or emergent in one study regarding the h n influenza (haberg et al., ) . the unusually high cesarean section delivery is a likely effect of deteriorating maternal condition that impacts the overall fetal status (mendez-figueroa et al., ) . infants of ill mothers can test positive for h n influenza, but it is an unusual occurrence. this raises the possibility of vertical transmission, but additional studies are needed on the impacts of maternal infection with influenza on neonatal outcomes. long-term follow-up studies on infants born to women with influenza infection should be performed to fully understand the impacts on overall outcomes when evaluating influenza in pregnancy. vaccines are available and recommended to pregnant women; see centers for disease control and prevention ( ) for recommendations (cdc, b). hepatitis refers to the inflammation of the liver. though inflammation can be caused by various means, most commonly it is due to a virus. there are five main types of hepatitis virusesda, b, c, d, and e. hepatitis a and e are generally caused by the intake of contaminated food or water; hepatitis b, c, and d typically occur as parenteral contact with infected bodily fluids. although hepatitis e has a high vertical transmission rate ( %), there is still limited data on complications (ornoy and tenenbaum, ; kumar et al., ) . for this example, we concentrate on hepatitis b due to the effects it can cause on the pregnancy, to the fetus, and potential teratogenic effects of drugs used to prevent infection. maternal hepatitis b virus (hbv) infection has not been shown to increase major congenital defects in offspring. reports of a higher incidence of low birth weight and prematurity have been noted during acute infection compared to the general population (jonas, ). the higher incidence may be attributed to the changes in the maternal immune system contributing to a suppressed immune response against hbv. a high frequency of hbv perinatal transmission occurs in up to % of infants born to women positive for hbv infection. hbv is able to cross the placental barrier but presumed to be the cause of a minority of infections for those mothers not immunized (jonas, ) . transmission of the hbv can occur prenatally when the virus enters the placenta through maternal blood and crosses the placental barrier gaining access to the fetus or during birth when the newborn is exposed to infected cervical secretions. increased likelihood of intrauterine transmission has been associated with the level of hbv dna in the mother. prevention of hbv transmission can be performed by identifying the hepatitis b surface antigen in pregnant women and providing hepatitis b immune globulin and hepatitis b vaccine to the infants within h of birth (cdc, f). the virus was first identified in africa in and was named after the zika forest in uganda, where it was first isolated (dick, ) . zika virus is a single-stranded rna virus spread to people when they are bitten by an infected aedes species mosquito (arbovirus). it is related to other flaviviruses including dengue and chikungunya (triunfol, ) . transmission is typically through exposure to infected blood but transmission can result from vertical transmission from a pregnant woman to her fetus, sexual transmission, laboratory exposure, and potentially through blood and organ transplant (musso et al., ; foy et al., ; cdc, a) . many zika infections are asymptomatic; common symptoms of zika are fever, rash, joint pain, conjunctivitis, muscle pain, and headache which may appear - days after infection, last several days to a week, and are generally mild (cdc, g; schuler-faccini and rasmussen, ) . severe disease is uncommon; fatalities are rare (cdc, g); however, guillain-barre syndrome has been reported in patients following suspected zika virus infection (oehler et al., ) . the virus usually remains in the blood of an infected person for a week and can persist longer in other secretions such as semen. once a person has been infected, they are likely to be protected from future infections schuler-faccini and rasmussen, ) . the centers for disease prevention and control (cdc) acknowledges the link between zika virus infection in pregnant women and subsequent birth defectsdspontaneous abortion and fetal demise schuler-faccini and rasmussen, ) . a pregnant woman already infected with zika can pass the virus to her fetus during pregnancy or around the time of birth . brain defects linked with zika in the fetus include decreased total brain tissue with resulting microcephaly, calcium deposits in the brain indicating brain damage, excess fluid in the brain cavities and surrounding the brain, absent or poorly formed brain structures, and abnormal eye development cdc, b; schuler-faccini and rasmussen, ) . zika can be sexually transmitted and can be passed from an infected partner before their symptoms start, while they exhibit symptoms, and after their symptoms end (who, a; schuler-faccini and rasmussen, ) . the zika virus can remain in semen ( - weeks) longer than in any other body fluids, including vaginal fluids, urine, and blood (foy et al., ; higgs, ; oster, ; musso et al., ) . brazil's ministry of health made an announcement as of december for ".women in the northeast of the country not to get pregnant for the foreseeable future" (npr, ) . in jan. , the cdc issued a travel notice alert level regarding the zika virus in south america, central america, mexico, the caribbean, and puerto rico. the world health organization has declared a public health emergency of international concern on feb. , (who, b . since , countries and territories reported evidence of vector-borne zika virus transmission (who, a). as of summer , the united states reported locally acquired mosquito-borne cases all from the state of florida (cdc, a). parasitic infections affect pregnant women worldwide and directly or indirectly lead to a variety of adverse fetal effects. these effects include intrauterine retardation of growth, congenital malformations, and fetal loss. depending on when the infection takes place and the stage of pregnancy the fetus is in, severe fetal consequences may occur during development or be asymptomatic until later in the pregnancy. malaria is a protozoan disease transmitted by female mosquitoes of the genus anopheles. the parasites multiply in the host's liver and subsequently infect the red blood cells causing symptoms of fever, headache, and vomiting; if not treated, the infection can become life-threatening by disrupting the blood supply to vital organs. in , there were an estimated million malaria cases and , malaria deaths; young children, pregnant women, and those nonimmune travelers are the most at risk when infected (who, ) . in areas with high transmission rates, more than % of malaria deaths occur in the age group of children under the age of five (who, b). pregnant women have increased susceptibility to p. falciparum malaria. malaria during pregnancy increases the risk of maternal and fetal anemia, stillbirth, spontaneous abortion, low birth weight, and neonatal death (who, a). the effects of low birth weight have an odds ratio of two to seven times higher effects when it is the woman's first pregnancy compared to women who have had multiple pregnancies prior (desai et al., ; white et al., ) . in malaria-endemic countries, pregnant women are at increased risk of developing severe p. falciparum malaria with a very high mortality rate, approximately % (white et al., ) . maternal hiv infection also predisposes pregnant women to malaria, congenital malaria, and exacerbates reductions in birth weight (steketee et al., ) . the risk of infant death is high if maternal malaria occurs late in pregnancy (bardaji et al., ; white et al., ) . congenital malaria occurs in roughly % of neonates but clears spontaneously in % of cases (falade et al., ) . the condition is typically defined as the presence of asexual forms of malaria parasites in the peripheral blood within the first days of life but frequently reported with the presence of the parasites in cord blood (menendez and mayor, ) . p. falciparum contributes to %- % of low birth weight deliveries (white et al., ) . with increased malaria prevention (early diagnosis and treatment) and control measures, the malaria mortality rates have fallen globally among all age groups. there is, however, a concern of p. falciparum resistance in some countries to artemisinin, one of the core compounds in world health organization recommended treatments for uncomplicated malaria (who, b). toxoplasmosis is caused by the protozoan parasite toxoplasma gondii (t. gondii) that infects most species of warm-blooded animals. it has a complex life cycle. sexual reproduction can only occur in the digestive tract of felidae hosts (cats). asexual reproduction can occur in any warm-blooded vertebrate. humans can be infected by eating undercooked meat of animals harboring tissue cysts, consuming contaminated food or drinking water, contact with contaminated environmental samples, blood transfusion or organ transplant, or by vertical transmission. in humans, those infected are generally asymptomatic; when symptoms occur, it is usually mild and flu-like lasting for weeks to months and until the cycle returns to a dormant state (cdc, b). the parasites form tissue cysts and may remain throughout the life of the host (cdc, b). if a woman has contracted toxoplasmosis before becoming pregnant, the fetus will be protected due to the mother's developed immunity (woudwyk et al., ) . so maternal-fetal transmission only occurs when a pregnant woman is newly infected with toxoplasma. global estimates of maternal-fetal transmission are approximately %- % of newly infected pregnant women (carlier et al., ) . although transmission rates during the first trimester of pregnancy are low (below %), when it does occur the damage to the fetus is the most severe at this time (carlier et al., ) . the second and third trimesters' transmission rates increase to %- % and %- %, respectively; the highest proportions of transmission occur in the last few weeks before birth (carlier et al., ) . transmission may result in a miscarriage, stillbirth, or a child with congenital toxoplasmosis. infants infected before birth may be asymptomatic at first but may develop later in life with potential vision impairment/loss, mental disability, and seizures (cdc, b) . treatment is available for pregnant women, newborns, and infants involving sulphonamides or spiramycin, but due to the parasites residing within cells in a less active phase, it is difficult for medication to completely eliminate the cysts (richardson et al., ; cdc, b) . fungi are ubiquitous, found outdoors, on many inside surfaces, and on human skin. most fungi are harmless, but some types can be harmful to health. anyone can be affected by fungal diseases (cdc, c). serious fungal infections are uncommon during pregnancy; in some cases, infection may occur with higher frequency during pregnancy, potentially increasing maternal mortality and cause fetal loss or prematurity (ostrosky-zeichner and rex, ). an extreme outcome of a fungal infection is the spread of the fungus through the blood to the spinal cord leading to fungal meningitis. candida is a genus of yeasts and is the most common cause of infections worldwide (manolakaki et al., ) . there are over species. many of the species are harmless commensals or endosymbionts under normal conditions; when conditions are sufficient, they can invade and cause disease. candida albicans (c. albicans) and candida parapsilosis (c. parapsilosis) are the two most likely species to cause candidiasis and candidemia. up to % of pregnant women may have vaginal colonization with candida spp. (goldenberg et al., ; digiulio, ) . candida spp. have been isolated from amniotic fluid of spontaneous preterm birth mothers (maneenil et al., ) . cases studied have shown intraamniotic infection can cause fetal death or fetal candidiasis with impaired cns outcomes in humans (marelli et al., ; benjamin et al., ; darmstadt et al., ; siriratsivawong et al., ) . in neonatal candidiasis, c. albicans and c. parapsilosis are the most commonly isolated species and cause the majority of fungal infections in very low birth weight infants. though c. albicans is not specifically associated with the risk of preterm delivery, there has been some evidence that suggests the elimination of the yeast reduces the risk of a late miscarriage and preterm birth (maneenil et al., ) . c. albicans is usually vertically transmitted, whereas c. parapsilosis is mostly transmitted by contact other than parentchild (waggoner-fountain et al., ; reef et al., ; chapman and faix, ; abi-said et al., ) . pathogenesis likely involves ascension of the infection from maternal candida infection then leading to amniotic fluid infection and chorioamnionitis/funisitis (siriratsivawong et al., ) . approximately %- % of nosocomial blood infections in neonates are fungal infections; . % of these infections are caused by candida spp. (blaszkowska and goralska, ) . the mortality rate is the second highest ranging from % to % (blaszkowska and goralska, ) . at risk neonates can develop candidiasis, a dermatitis caused by candida spp. the dermatitis usually resolves on its own in neonates born full-term; however, the condition may advance to candidemia in very low birth weight infants possibly leading to death. some of the risk factors for dermatitis are vaginal birth, extreme prematurity, low birth weight, hyperglycemia, and steroid administration. congenital cutaneous candidiasis can present in a variety of symptomsddiffuse skin eruption in the absence of systemic illness to severe systemic disease leading to stillbirth or early neonatal death (darmstadt et al., ) . cutaneous eruptions occur in % of cases and are typically detected within h of birth (darmstadt et al., ) . diagnosis of congenital candidiasis may be delayed due to the similar appearance to more common rashes in newborns which increases risk for adverse sequelae (siriratsivawong et al., ) . adhesion is a major virulence factor for this fungus. candida spp. has the capability of forming biofilm which allows it to adhere to skin, mucus layers, and surfaces of tubes/catheters that may be used on neonates during hospitalization. there is evidence that candida spp. has the ability to form biofilms on intrauterine devices (iud). due to iud application directly affecting the vaginal ecosystem, there may be a need for the consideration of candida spp. internal exposure for those women planning to get pregnant after removing the iud (caliskan et al., ) . since the s' outbreak of rubella, it has become well recognized that infections during pregnancy can affect the mother and fetus. for most women, pregnancy is a normal and healthy state. but there are factors that may make pregnant women more susceptible to infection. changes in the immune system and the altering effects of hormones affect the immune system's susceptibility and responses to infection. stress is common during pregnancy, but high levels may increase the risk of certain problems with delivering a healthy neonate by influencing the already altered immune functions. the diversity in the human microbiome has been correlated with the health of humans; as we learn more about the interconnecting relations between the microbiome and our health, promoting specific diversity may be able to protect the mother and fetus during pregnancy in the future. infection has long been established as the leading cause of preterm births which has its own risks such as the infection of the cns or low birth weight, which can allow the neonate to be more susceptible to infections postpartum. chorioamnionitis indicates infection of the intrauterine environment and is a major risk factor for neonatal sepsis, which can cause those neonates to commonly present with multiple insults or lead to a stillbirth. stillbirths are an end to a pregnancy when the conditions in the mother are unable to keep the fetus alive. intrauterine infections are a diverse group of organisms that can cause a range of adverse outcomes in pregnancy. the torch complex is a reminder of organisms that can produce congenital defects when a woman is infected during pregnancy. the mechanisms of some infections are currently unknown and additional research is needed to understand how these pathogens cause adverse outcomes in pregnancy. those that are understood may be preventable; others, like the zika virus, are emerging and are only now being studied as outbreaks occur. with the ever globalizing world, the risk of being exposed or contracting these infections rises as organisms can travel across continents in mere hours. environmental factors such as changes in climate may allow for some organisms to move into more populated areas, exposing more individuals. the understanding of intrauterine infections is vital to ensure the health of all future mothers and their offspring. many factors influence the susceptibility of becoming infected. contracting an intrauterine infection has health impacts on the mother and fetus. the fetus's development is a major concern after being exposed to an intrauterine infection but predicting the outcome for any individual is difficult with our current knowledge. there are many known organisms that are capable of producing congenital defects during pregnancy. however, there are new emerging organisms that have yet to be studied. additional research to understand the mechanisms and risks associated with these organisms is critical for the health of all potential mothers and their offspring. see also: . . maternally mediated developmental toxicity. . . epigenetics and the developmental origins of health and disease. the epidemiology of hematogenous candidiasis caused by different candida species bacterial meningitis in pregnancy: report of six cases and review of the literature intrauterine infection and preterm labor congenital and perinatal cytomegalovirus infections listeriosis: a resurgent foodborne infection campylobacter jejuni infections: update on emerging issues and trends listeria monocytogenes traffics from maternal organs to the placenta and back impact of malaria at the end of pregnancy on infant mortality and morbidity severe coxsackie virus b infection in preterm newborns treated with pleconaril epidemic listeriosis in the newborn neonatal candidiasis among extremely low birth weight infants: risk 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regional, and national causes of child mortality: an updated systematic analysis for with time trends since fluconazole treatment of intrauterine candida albicans infection in fetal sheep candida infection and colonization among trauma patients fetal candida infection associated with an intrauterine contraceptive device effect of respiratory hospitalization during pregnancy on infant outcomes neonatal characteristics and outcomes of pregnancies complicated by influenza infection during the pandemic congenital malaria: the least known consequence of malaria in pregnancy perinatal echovirus infectiondrisk of transmission during a community outbreak pandemic influenza a (h n ) in pregnancy: a systematic review of the literature pneumonia as a complication of pregnancy potential sexual transmission of zika virus long-term neurodevelopmental outcomes after intrauterine and neonatal insults: a systematic review listeriosis during pregnancy: a case series and review of cases microbiome in 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biobehavioural perspective. paediatric and perinatal epidemiology vertical and horizontal transmission of unique candida species to premature newborns a double-blind, randomized, placebo-controlled trial of acyclovir in late pregnancy for the reduction of herpes simplex virus shedding and cesarean delivery world malaria report . geneva: who. who ( a) malaria | high-risk groups world malaria report . geneva: who. who ( a) who | zika virus and complications ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations dose response, infectivity and stillbirths in pregnant guinea pigs inoculated with listeria monocytogenes inflammation-induced immune suppression of the fetus: a potential link between chorioamnionitis and postnatal early onset sepsis study of the uterine local immune response in a murine model of embryonic death due to tritrichomonas foetus periodontal disease and adverse pregnancy outcomes: a systematic review influenza a/h n v in pregnancy: an investigation of the characteristics and management of affected women and the relationship to pregnancy outcomes for mother and infant the placental microbiome varies in association with low birth weight in full-term neonates key: cord- -aq s x authors: andersen, petter i.; ianevski, aleksandr; lysvand, hilde; vitkauskiene, astra; oksenych, valentyn; bjørås, magnar; telling, kaidi; lutsar, irja; dumpis, uga; irie, yasuhiko; tenson, tanel; kantele, anu; kainov, denis e. title: discovery and development of safe-in-man broad-spectrum antiviral agents date: - - journal: international journal of infectious diseases doi: . /j.ijid. . . sha: doc_id: cord_uid: aq s x abstract viral diseases are one of the leading causes of morbidity and mortality in the world. virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. however, broad-spectrum antiviral agents (bsaas, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of the general population from emerging and re-emerging viral diseases, reinforcing the arsenal of available antiviral options. here, we review discovery and development of bsaas and summarize the information on safe-in-man agents in a freely accessible database (https://drugvirus.info/). future and ongoing pre-clinical and clinical studies will increase the number of bsaas, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections. viruses are one of the major causes of morbidity and mortality in the world (dalys and collaborators, ; disease et al., ; howard and fletcher, ; who, ) . antiviral drugs and vaccines are used to fight viral infections in human (de clercq and li, ; marston et al., ) . previously, there has been a focus on "one drug, one virus" dogma, which relied on targeting virusspecific factors. a counterpoint to this is the "one drug, multiple viruses" paradigm, which came with the discovery of broadspectrum antiviral agents (bsaas), small-molecules that inhibit a wide range of human viruses (bekerman and einav, ; de clercq and montgomery, ; debing et al., ; ianevski et al., ; rada and dragun, ; sidwell et al., ) . this paradigm was based on the observation that different viruses utilize similar pathways and host factors to replicate inside a cell (bosl et al., ) . although the concept of bsaas has been around for almost years, the field received a new impetus with recent outbreaks of ebola, zika, dengue, influenza and other viral infections, the discovery of novel host-directed agents, as well as development of drug repositioning methodology. drug repurposing, also called repositioning, redirecting, reprofiling, is a strategy for generating additional value from an existing drug by targeting disease other than that for which it was originally intended (nishimura and hara, ; pushpakom et al., ) . this has significant advantages over new drug discovery since chemical synthesis steps, manufacturing processes, reliable safety, and pharmacokinetic properties in pre-clinical (animal model) and early clinical developmental phases (phase , i and iia) are already available (figure ) . therefore, repositioning of launched or even failed drugs to viral diseases provides unique translational opportunities, including a substantially higher probability of success to market as compared with developing new virus-specific drugs and vaccines, and a significantly reduced cost and timeline to clinical availability pizzorno et al., ; zheng et al., ) . here, we detail the steps of bsaa repurposing, from discovery of novel antiviral activities in cell culture to post-market studies. moreover, we summarized currently available information on bsaas in freely available database, focusing on those antivirals, which have been already tested in human as antivirals, antibacterials, antiprotozoals, anthelmintics, etc. finally, we discuss future perspectives of using safe-in-man bsaas for treatment of emerging and re-emerging viral infections as well as viral and bacterial co-infections. discovery of novel bsaa activities in immortalized cell cultures and co-cultures the discovery of novel activities of bsaas starts with exposing cells to the candidate antiviral agent at different concentrations and infecting the cells with a virus or mock. immortalized cancerous cell cultures and co-cultures, which express appropriate viral receptors, are most commonly used in this first step. the halfmaximal cytotoxic concentrations (cc ) for a compound are calculated based on their dose-response curves obtained on mockinfected cells. the half-maximal effective concentrations (ec ) are calculated based on the analysis of curves obtained on infected cells. statistical analyses can help to determine if the differences between cc and ec are significant, given the inherent variability of the experiment (meneghini and hamasaki, ) . a relative effectiveness of a drug is defined as selectivity index (si = cc /ec ). cell viability assays and cell death assays are commonly used to assess the cytotoxicity and efficacy of bsaas ( figure a ). cell viability assays include mtt, mts, resazurin or similar assays, mitochondrial membrane potential-dependent dyes-based assays, esterase cleaved dye-based assays, atp-adp assays, and assays that measure glycolytic flux and oxygen consumption. other cell death assays include ldh enzyme leakage assays, membrane impermeable dye-based assays, and apoptosis assays, such as annexin v, tunel, and caspase assays (shen et al., ) . for example, the cell titer glo (ctg) assay quantifies atp, an indicator of metabolically active living cells, whereas cell tox green assay uses fluorescent asymmetric cyanine dye that stains the dna of dead cells (bosl et al., ; bulanova et al., ; ianevski et al., ; muller et al., ) . viral strains or cell lines expressing reporter proteins are also used to assess the efficacy of bsaas in infected cells. for example, tzm-bl cells expressing firefly luciferase under control of hiv- ltr promoter allowed quantitation of bsaa action on hiv- infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay (sarzotti-kelsoe et al., ; xing et al., ) . rfp-expressing rvfv, nanoluc-expressing chikv and rrv, as well as gfp-expressing fluav, hcv and hmpv also allowed identification of novel activities of several bsaas (andersen et al., b; bosl et al., ; de graaf et al., ; habjan et al., ; ianevski et al., ; jupille et al., ; kittel et al., ; lee et al., ; utt et al., ) . in addition, qpcr/rt-qpcr, rna/dna sequencing, rna/dna hybridization, immuno-and plaque assays as well as crispr-cas systems could be used for detection of inhibitory effects of bsaas (boonham et al., ; fischer et al., ; konig et al., ; laamiri et al., ; landry, ; perez et al., ; sashital, ; zhou et al., ) . interestingly, crispr-cas , sirna and shrna approaches were used for identification of bsaa targets (deans et al., ; puschnik et al., ) . novel anti-hsv- and anti-ev activities of emetine were discovered recently using ctg/plaque assays in human nonmalignant rpe cells. moreover, novel antiviral activities of the drug were identified using rfp-expressing rvfv, and gfp-expressing hmpv or fluav strains in rpe cells (andersen et al., a) . given that emetine also inhibits zikv, ebov, rabv, cmv, hcov-oc and hiv- infections (chaves valadao et al., ; macgibeny et al., ; mukhopadhyay et al., ; shen et al., ; yang et al., ) , and that it is an fda-approved anti-protozoal drug, it may represent a promising safe-in-man bsaa candidate. immortalized cell cultures/co-cultures and reporter viral strains represent excellent model systems for the discovery of novel activities of safe-in-man bsaas. however, these genetically modified systems have certain limitations (attenuated or incomplete virus replication cycle, accumulation of mutations during repeated cell and virus passaging, defective innate immune responses and viral counter-responses, etc.) (carter and shieh, ) . thereby, novel antiviral activities of bsaas should be further validated in primary human cells using different viral strains (including wild-type viruses), different viral loads, different times of compound addition, different endpoint measurements and compound concentration range. primary cell cultures give more accurate images of drug responses (alves et al., ; denisova et al., ; koban et al., ; postnikova et al., ) . they have a low population doubling level and therefore more closely recapitulate the physiological conditions observed in vivo. primary cells are cells isolated directly from tissues or blood using enzymatic or mechanical methods. the cells are characterized by their high degrees of specialization, are often fully differentiated and thus require defined culture conditions (serum-free media) in order to preserve their original phenotype. peripheral blood mononuclear (pbmc), placental, amniotic and fetal primary cultures as well as vaginal/cervical epithelial and male germ cells have been used intensively to validate bsaa activity (barrows et al., ; denisova et al., ; fink et al., ; rausch et al., ; robinson et al., ) . although primary cell cultures are relevant systems for validation of bsaas, there are technical difficulties limiting their use, such as ethical issues, purity of population of primary cells, and limited shelf life of the cells. in addition, age, race, sex and other genetic and epigenetic factors of donor cells should be considered to determine common biological effect across a significant number of donors thereby avoiding minor variants (lee et al., ; zhang et al., ) . the obstacles associated with use of human primary cell cultures can be bypassed using human embryonic stem cells (escs) and human induced pluripotent stem cells (ipscs). escs are isolated from surplus human embryos, whereas ipscs are obtained by reprogramming somatic cells. these cells proliferate extensively and retain multi-lineage activity, which allows them to generate virtually any cell type of the body. the escs-and ipsc-derived cells have been used successfully to investigate the efficacy of several bsaas against hbv, zikv, chikv and hsv- infections (table s ) (ferreira et al., ; iwasawa et al., ; lanko et al., ; simonin et al., ; xia et al., ; zhou et al., ) . ipscs, escs and primary tissue cells can be used to generate complex cultures termed organoids. organoids are miniature and simplified version of organs. establishing human airway, gut, skin, cerebral, liver, kidney, breast, retina and brain organoids allowed researchers to study toxicity and efficacy of several safe-in-man bsaas against coronaviruses, influenza, enteroviruses, rotaviruses and flaviviruses sacramento et al., ; watanabe et al., ; xu et al., ; yin et al., ; yin et al., ; yin et al., ; zhou et al., ) . however, ipscs, escs and ipscs/escsderived organoids have the same disadvantages as human primary cells (genetic differences, line-to-line and organoid batch-to-batch variability). on the other hand, these models allow researchers to predict the behavior of viruses in vivo and, therefore, to reduce animal use and in cases where animal models are unavailable to initiate clinical trials. for example, novel anti-zikv activities of enoxacin, amodiaquine and niclosamide were discovered recently using human neural progenitor cells, human pluripotent stem cell-derived cortical neural progenitor cells, and human induced neural stem cells, respectively (cairns et al., ; xu et al., ; zhou et al., ) . enoxacin is an oral broad-spectrum fluoroquinolone antibiotic, which also possesses anti-hcv and anti-hiv- activities in immortalized cell cultures (kashiwase et al., ; young et al., ) . amodiaquine is an anti-malaria drug, which also possesses antiviral activities against denv, hcv, rrv, sinv, wnv, efv, ebov, lasv, rabv, vzv, and hsv- in immortalized cell cultures (boonyasuppayakorn et al., ; hulseberg et al., ; mazzon et al., ) . niclosamide is an orally bioavailable anthelmintic drug which inhibits the broadest range of viruses in vitro and, in if the drug is repositioned from another disease (i.e. its safety profile in man is available) it could bypass the pk and safety studies in man. some cases, in vivo (cairns et al., ; fang et al., ; huang et al., ; hulseberg et al., ; jurgeit et al., ; kao et al., ; mazzon et al., ; stachulski et al., ; wang et al., ; wu et al., ) . these safe-in-man bsaas represent promising drug candidates. in vitro and ex vivo models do not fully reflect the complexity and physiology of living organisms. therefore, several in vivo models have been developed to test novel antiviral activities of bsaas. these include immunocompetent and genetically or chemically immunocompromised mice, guinea pigs, hamsters, ferrets, pigs, macaques and other animals ( figure b ) (alves et al., ; haese et al., ; louz et al., ; morrison and diamond, ; taylor, ; thangavel and bouvier, ) . pk/pd studies determine drug absorption, dosage and half-life of bsaas. toxicological studies determine if the drugs have any adverse effects on the tissues and organs of the animals and define the dosage of adverse effects (alabaster and in vivo pharmacology training group, ; parasuraman, ; rizk et al., ) . studying the efficacy of bsaas is generally done by treating the animal with the drug or vehicle and infecting it with a virus of interest. endpoints are usually body weight/ mortality (depending on the virus), histopathology, virus titers in organs, presence of clinical signs and development of immunity (oh and hurt, ; smee and barnard, ) . although animal models can give the initial characterization of bsaa, it is important to keep in mind that they differ significantly from humans, with respect to symptoms, disease manifestation, susceptibility, immune responses, pathogenesis, and pharmacokinetics (barré-sinoussi and montagutelli, ; shanks et al., ) . often animals require higher concentration of an experimental antiviral as compared to effective in vitro concentrations. moreover, it is relatively difficult to achieve micromolar ec in vivo. for example, aminoglycoside antibiotics, kasugamycin and neomycin were successfully tested against zikv, fluav, and hsv- infections in mice (gopinath et al., ) . polyether antibiotic salinomycin also showed anti-fluav effect in mice (jang et al., ) . in addition, investigational anticancer agent, flavopiridol, was effective against fluav in mice . these findings support further development of these and other bsaas. clinical trials are the most critical and time-consuming step of a drug candidates' journey to being approved ( figure c ). however, safe-in-man bsaas make this journey relatively short, because they have been already at phase , i and, sometime, at iia of clinical trials as antibacterial, antiprotozoal, anticancer, etc. agent; i.e. they have been administered at sub-therapeutic doses to healthy volunteers to ensure the drugs are not harmful to the participants. thus, safe-in-man bsaas enter phase ii and iii, which assess the efficacy, effectiveness, safety and side effects of the drugs in clinic. it is important, however, to differentiate acute and chronic viral infections when repurposing bsaas, given that drug concentrations and duration of the treatment could be different, and therefore, drug safety issues should be considered. for phase ii, patients with the viral disease in question are invited to join the study, where they are administered the bsaas at the ideal therapeutic doses. phase iii is the longest of the phases, and includes multiple levels of securities to the studies, such as the use of placebos and double-blinded studies, to ensure the data is as unbiased as possible. upon completing phase iii, depending on its performance and efficacy, bsaas may end either being approved or dropped. the u.s food and drug administration (fda) estimates that only - % bsaa candidates that enter phase iii are approved for use in the public (u.s food and drug administration, ). after approval and marketing of the drug, phase iv may be initiated to follow up on the use of the drug in public, to surveil for rare effects (u.s food and drug administration, ; umscheid et al., ) . forty-eight safe-in-man bsaas undergo clinical studies as antivirals. there are currently compounds in phase i, agents in phase ii and compounds in phase iii clinical trials. for example, nitazoxanide, remdesivir and brincidofovir are under clinical investigations against different viral infections (nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct ). twenty-one bsaas were approved by fda, ema or other agencies. these bsaas altogether target viruses. for example, favipiravir, also known as t- , was approved against fluav in japan; cidofovir is an injectable antiviral medication used as a treatment for cmv retinitis in people with aids; ribavirin, also known as tribavirin, is used for treatment of rsv and hcv infections; pleconaril is used against viruses in the picornaviridae family, including enterovirus and rhinovirus; and valacyclovir is used against cmv, ebv, hbv, hsv- , hsv- and vzv infections. twenty bsaas are undergoing surveillance studies (phase iv). azithromycin, chloroquine, cyclosporine, ezetimibe, mycophenolic acid, nitazoxanide and rapamycin progressed to phase iv studies without approvals from national or international authorities (nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct ). we have developed a database for safe-in-man bsaas, which is available at https://drugvirus.info/ (figure ) . the drug annotations were obtained from pubchem, drugbank, drugcentral, pubmed and clinicaltrials.gov databases (table s ) ursu et al., ; wishart et al., ) . the information on virus families was exported from virus pathogen database and analysis resource (table s ) (pickett et al., ) . the database summarizes activities and developmental status of bsaas. we decided to set no limits for ec , si and statistical significance because the studies were not harmonized (different cell lines, assays, end-point measurements, time of compound addition, etc.). the database allows interactive exploration of virus-bsaa interactions. it also includes information on bsa targets. a feedback form is available on the website. the database will be updated upon request or as soon as a new safe-in-man bsaa emerges or novel activity for an existing bsaa is reported. altogether, the database contains approved, investigational and experimental safe-in-man bsaas, which inhibit human viruses, belonging to viral families. the bsaas inhibit viral or host factors and block viral replication, reduce the viral burden to a level at which host immune responses can deal with it or facilitate apoptosis of infected cells (table s ). analysis of bsaa targets and structures (figure ) revealed that the most abundant are nucleotide and nucleoside analogues which inhibit viral rna and dna polymerases. imatinib, erlotinib, gefitinib, and dasatinib, which inhibit tyrosine kinases, are the most abundant hostdirected bsaas. most of the host targets (except bcl-xl protein) are essential for viral replication but redundant for the cell, which is critical for reducing putative toxicities associated with blocking cellular pathways. the limited diversity of the targets and scaffolds could slow down the development of bsaa concept. emerging bsaas, such as , -dimethoxyindan- -one, saliphenylhalamide, and gs- (denisova et al., ; kuivanen et al., ; muller et al., ; muller et al., ; patil et al., ; sheahan et al., ) , whose safety profiles in humans are not yet available, are not included in the database. however, they could serve as valuable antivirals in the future, pending the results of further pre-clinical and clinical investigations. sars-cov- is a novel strain of coronaviruses which is associated with a cluster of cases of pneumonia in china (zhou et al., ) . coronaviruses (cov) are a broad family of virusesthat includes sars-cov, mers-cov, hcov- e, hcov-oc , hcov-nl and hcov-hku strains. hcov- e, hcov-oc , hcov-nl and hcov-hku strains are usually associated with mild, self-limiting upper respiratory tract infections, such as the common cold. by contrast, people infected with mers-cov, sasrs-cov or sars-cov- could develop severe respiratory illness, and many of the infected have died. no vaccines and drugs are available for prevention, prophylaxis and treatment of coronavirus infections in humans (eurosurveillance editorial, ) . however, safe-in-man bsaas could be effective against sars-cov- and other coronaviruses ( figure ). for example, teicoplanin, oritavancin, dalbavancin, monensin and emetine could be repurposed for treatment of covid- . teicoplanin, ritavancin, dalbavancin and monensin are approved antibiotics, whereas emetine is an anti-protozoal drug. these drugs have been shown to inhibit several corona-as well as some other viral infections . moreover, chloroquine and remdesivir were shown to effectively inhibit sars-cov- infection in vitro. in addition, clinical investigations into the effectiveness of lopinavir, ritonavir, remdesivir, hydroxychloroquine and arbidol against covid- have started recently (nct ; nct ; nct ; nct , nct ). bsaas could be combined with other antiviral agents to obtain synergistic or additive effects against certain viruses zheng et al., ) . several combination therapies which include bsaas (such as abacavir/dolutegravir/lamivudine (triumeq), darunavir/cobicistat/emtricitabine/tenofovir (symtuza), lopinavir/ritonavir (kaletra), ledipasvir/sofosbuvir and sofosbuvir/velpatasvir) became a standard for the treatment of hiv or hcv infections. several synergistic drug combinations, such as obatoclax/saliphenylhalamide and gemcitabine/pimodivir, could enter clinical studies and become effective treatment of zikv and fluav infections (fu et al., ; kuivanen et al., ) . by contrast to individual drugs, combinations of - bsaas could be used to target an even broader range of viruses (foucquier and guedj, ; zheng et al., ) . such combinations could serve as front line therapeutics against poorly characterized emerging viruses or re-emerging drug-resistant viral strains. for example, a cocktail of nitazoxanide, favipiravir, and niclosamide could be developed for the treatment of viruses belonging to families. fifty bsaas possess not only antiviral but also antibacterial activity (figure ; table s ) (schor and einav, ) . moreover, of the agents are approved as antibiotics ( withdrawn). these agents with dual activity could be used for treatment of viral and bacterial co-infections or for the protection of patients from the secondary infections. for example, azithromycin could be used against fluav and chlamydophila pneumoniae, haemophilus influenzae, mycoplasma pneumoniae or streptococcus pneumoniae infections (nct ) (mandell et al., ) . in addition, bsaas showed activity against a wide range of other medically important human pathogens, including fungi, protozoa and parasites (table s ) (montoya and krysan, ) , pointing out that some pathogens utilize common mechanisms to infect hosts. moreover, structure-activity relationship analysis of bsaas suggests that some agents, such as doxycycline, artesunate, omeprazole, nitazoxanide, suramin, azithromycin, minocycline and chloroquine, could have novel antibacterial, antiprotozoal, antifungal or anthelmintic activities (figure ). if confirmed, this could lead to development of broad-spectrum anti-infective drugs. bsaas could also serve as treatment of other co-morbidities, therefore, simplifying the therapy and lowering its cost (table s ). for example, the concomitant actions of ezetimibe and statins could be beneficial for treatment of both hypertension and several viral infections in patients with these co-morbidities (nct , nct , nct , nct , nct , nct ). here we reviewed a process of bsaa development and summarized information on safe-in-man agents in freely available database. we hope that further pre-clinical and clinical studies on bsaas will be harmonized, and data collection will be standardized. furthermore, the follow-up studies as well as the results of on-going, finalized or terminated clinical trials will be made publicly available to allow prioritization and translation of emerging and existing bsaas into clinical practice. this would allow bsaas to play a pivotal role in the battle against emerging and re-emerging viral diseases. discovery of novel as well as repositioning existing safe-in-man bsaas may shorten time and resources needed for development of virus-specific drugs and vaccines. in the future, bsaas will have a global impact by decreasing morbidity and mortality from viral and other diseases, maximizing the number of healthy life years, improving the quality of life of infected patients and decreasing the costs of patient care. the authors declare no conflict of interest. this study was supported by the european regional development fund, the mobilitas pluss project mobtt (to d.k.). approval was not required. we thank katarzyna kolasa for illustrations. this manuscript has been released as a pre-print (doi: https:// . /pre-prints . .v ). we thank the european regional development fund, the mobilitas pluss project mobtt . figure . safe-in-man broad-spectrum antiviral agents and coronaviruses they inhibit. a snapshot is taken from https://drugvirus.info/ website. different shadings indicate different development status of bsaas. grey shading indicates that the antiviral activity has not been either studied or reported. the fall and rise of in vivo pharmacology research models and tools for the identification of antivirals and therapeutics against zika virus infection novel antiviral activities of obatoclax, 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. /fimmu. . sha: doc_id: cord_uid: wfakzb w in the last decades, a number of infectious viruses have emerged from wildlife or re-emerged, generating serious threats to the global health and to the economy worldwide. ebola and marburg hemorrhagic fevers, lassa fever, dengue fever, yellow fever, west nile fever, zika, and chikungunya vector-borne diseases, swine flu, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and the recent coronavirus disease (covid- ) are examples of zoonoses that have spread throughout the globe with such a significant impact on public health that the scientific community has been called for a rapid intervention in preventing and treating emerging infections. vaccination is probably the most effective tool in helping the immune system to activate protective responses against pathogens, reducing morbidity and mortality, as proven by historical records. under health emergency conditions, new and alternative approaches in vaccine design and development are imperative for a rapid and massive vaccination coverage, to manage a disease outbreak and curtail the epidemic spread. this review gives an update on the current vaccination strategies for some of the emerging/re-emerging viruses, and discusses challenges and hurdles to overcome for developing efficacious vaccines against future pathogens. since the start of this century, a certain number of new or neglected pathogens have emerged from wildlife reservoirs and spilt over into human populations, causing severe diseases ( - ). factors such as urbanization, globalization, travels, international commerce, aging, and climate changes have contributed to favor emergence, spread, and transmission of pathogens. contacts among humans and potential zoonotic reservoirs are increasing, the number of travelers and their movements is growing, the aged population are more susceptible to infections, and the geographic distribution of pathogens within a previous endemic zone is changing ( , ) . during the last decades, the global community faced several outbreaks of emerging and reemerging infectious diseases, with high threats to the health security, biodefense, and economy worldwide ( , ) . the occurrence of significant disease outbreaks-such as sars (severe acute respiratory syndrome) originating in china in ( ) , the h n swine flu pandemic from mexico ( ) , mers (middle east respiratory syndrome) that occurred in saudi arabia in ( ) , the west african outbreak of ebola virus (ebov) in late ( ) , the zika virus (zikv) outbreak originating in brazil in ( ) , the health emergence in nigeria caused by lassa virus ( ) , and the ongoing coronavirus disease (covid- ) pandemic ( ) -has renewed interests in developing strategies to faster prevent, treat, and/or control emerging and re-emerging viruses with high epidemic potential. usually, there is little or no knowledge about identity, epidemiology, and pathogenesis of a new infectious agent appearing for a first time in a certain geographic area (as in case of novel coronaviruses or new influenza variants), as well as the potential to spread out from the zoonotic reservoir, making hard to predict if, where, and when a disease outbreak will occur. the world health organization (who) and the national institutes for allergy and infectious diseases (niaid) published a list of pathogens to be prioritized for research and development, given their epidemic potential. this non-exhaustive list comprises viruses, bacteria, protozoa, and fungi, causing diseases for which efficient countermeasures do not actually exist, or require new therapeutics ( , ) . as proven by historical records, vaccination has played a pivotal role in reducing morbidity and mortality from devastating infectious diseases, successfully leading to disease eradication (i.e., smallpox), and generally decreasing infectious disease burdens. even in presence of therapeutic options, vaccines are the valuable means to prevent infections and overall represent the much wanted achievement. however, even with worldwide efforts, getting a vaccine to the public takes time, and side effects, dosing issues, and manufacturing problems can all cause delays. thus, we have to use this time with great concern. generally speaking, in case of newly emergent diseases, conventional strategies might raise some issues. the unpredictable identity of largely unknown emerging pathogens, the lack of appropriate experimental animal models, and the time and costs for faster developing, producing, licensing, and globally distributing effective vaccine candidates are some of the major challenges to overcome in case of pandemic threats. hence, new and/or alternative approaches in vaccine design and development are required to rapidly face outbreak situations ( ) . this review will discuss the current vaccination strategies for some of the emerging and re-emerging viruses, as well as the approaches that might be suitable in face of global pandemic threats. emerging and re-emerging pathogens represent a constant epidemic threat to humanity not only for the public health consequences but also for the economic, social, and political effects they may globally provoke. therefore, a major public awareness and preparedness would be fundamental in fighting emerging infectious diseases. the terms "emerging and reemerging infectious diseases" mainly refer to two major categories of infectious diseases: newly emergent infections, caused by novel pathogens; and re-emerging infectious diseases, caused by microbes reappearing after previous control, and/or eradication ( ). almost % of emerging infectious diseases are zoonoses, with the great majority of them originating in wildlife, and the number is constantly increasing. climate changes have been related to the emergence of vector-borne diseases in severe environmental conditions, but this is a most debated issue, as well as the contribution of agricultural practices ( ) . in addition, the chances of infectious disease spreading could also include livestock/wildlife animal markets and consumption of those. a study where australia was used as a model of urbanization has proposed a relation among increasing pandemic threats and urbanization: it ascribes the increased threat of pandemic to the high number of major city residents, the exponential intensification of international air traffic, and the commuter mobility network ( ) . basically, an epidemic is an event that occurs when there is an increase, often sudden, in the frequency of a disease above what is normally expected in that population, in that area; while pandemic (from: παν = all, and δεµoσ = people) refers to an epidemic that spreads over several countries or continents at the same time, usually affecting a large number of people ( ). in the last decades, a certain number of viruses came to light for the first time or reappeared, giving rise to significant epidemics and pandemics (figure and table ) . epidemic outbreaks of viral diseases were mostly caused by flaviviruses generally transmitted by vectors, including west nile virus (wnv) ( , ) , zikv ( , ) , yellow fever virus (yfv) ( , ) , and dengue virus (denv) ( , ) . vector-borne diseases, including chikungunya fever caused by chikungunya alphavirus (chikv) ( , ) , are extremely difficult to eradicate because viruses are maintained in nature by propagation among vectors and hosts, without human-human contact. moreover, dry and hot climate conditions seem to foster mosquitoes to bite humans than animals, increasing the risk of spreading diseases with a devastating impact ( ) . today, most areas of the world are endemic for at least one flavivirus, with denv being the most prevalent, and approximately - million people are infected each year. among viral hemorrhagic fevers, lassa fever (lf) is a rodent-borne acute disease caused by lassa virus (lasv) ( ) , endemic in many west african countries, including nigeria that experienced a high mortality rate in the outbreak ( ) . ebola virus disease (evd) and marburg virus disease (mvd) are caused by members of the filoviridae family, ebov ( ), and marburg virus (marv) ( ), respectively. the - ebola outbreak in west africa was the largest since the virus was first discovered in , with a case fatality rate for zaire ebolavirus of % ( ) , while the largest recorded mvd outbreak occurred in angola in ( ) . concerning pandemics, flu pandemics were reported three times during the twentieth century; genome analysis of pandemic influenza viruses dated (h n ), (h n ), and (h n ) demonstrated that all viral strains fully or partially originated from non-human reservoirs, and that the ultimate origin of ha (hemagglutinin) genes are from avian influenza viruses ( ) , with the strain likely being the ancestor of the subsequent epidemic variants. hence, the influenza pandemic has been called the mother of all pandemics ( nhps, non-human primates. frontiers in immunology | www.frontiersin.org during the first months, after the first reported case ( ) . the age of deceased people was below years in almost % of cases, a peculiarity compared with the seasonal influenza epidemic. the mortality rates observed in and flu were of . % and - % due to h n and h n , respectively, and ranging from . and . % in ( ) . the h n pandemic has been commonly referred to as swine flu for the swine origin of the virus, first isolated in mexico and united states in april . the viral genome sequencing indicated that the virus contains a combination of genes never reported in swine or humans before. it has been demonstrated that the swine has become a reservoir of h viruses with the potential to cause future pandemics ( ) . a (h n )pdm virus monovalent vaccine was produced in late ( ) , but the virus has not been eradicated and it continues to circulate as a seasonal variant, causing hospitalization, and death ( ) . in november , a first case of sars was reported in guangdong (china), and after months, the coronavirus (cov) causing the disease, named sars-cov, spread in countries, giving rise to lower respiratory tract infections, with a poor outcome in % of cases ( , ) . middle east respiratory syndrome-cov, the causative agent of mers, was isolated in . the coronavirus has caused isolated mers outbreaks thereafter, becoming endemic in arabian peninsula, with a case fatality rate of . % ( ) ( ) ( ) . on march , , who has declared the coronavirus disease (covid- ) outbreak a global pandemic. the disease is caused by a novel coronavirus, known as sars-cov- , that shares almost % of the genome with that of sars-cov ( , ) . actually more than . million (as of august , ) of people are infected, with an overall case fatality rate of . - . % ( ) . in case of global public health emergencies, governmental and private organizations, vaccine developers, and regulatory authorities should all massively collaborate in selecting and funding the most suitable vaccine platform and strategy to quickly act and curtail disease outbreaks. at the outset of a disease outbreak, gaps in knowledge of identity, pathogenesis, epidemiology of the new emerging pathogen, time required to study the immune responses correlating with the outcome of the viral infection, and the lack of appropriate preclinical models susceptible to infection for testing a vaccine candidate pose several barriers and impediments to expedite vaccine design and development, and thus to ensure global vaccination coverage in time. in the fight against newly emergent viruses, vaccine design might benefit from a range of platform technologies, including nucleic acid vaccines, viral-vector vaccines, and recombinant protein-based vaccines (likely to be administered with adjuvants) ( , ) . compared with conventional vaccines, such as live attenuated and inactivated vaccines, molecular-based platforms might offer a more versatile tool against new emergent viruses, allowing a more fast, low-cost, and scalable vaccine manufacturing. essentially, these platforms rely on the use of a system to deliver and present a new antigen (or a synthetic gene) to rapidly target an emergent pathogen. theoretically, once a platform has previously met safety and efficacy requirements to be moved and advanced into the market, a candidate vaccine against a new virus might profit from the same system, production, and purification protocols, only replacing the disease target antigen (or inserted gene), thus streamlining the vaccine discovery. in inactivated vaccine, the virus is rendered uninfectious using chemicals, such as formaldehyde or heat. this technology, conceived in the nineteenth century, is used for few vaccines still in use (i.e., inactivated polio, whole cell pertussis, and hepatitis a) ( ) . live attenuated vaccines are obtained by passing the virus through animal or human cells until it picks up mutations that make it unable to cause the disease (i.e., measles, mumps, chickenpox, etc.); the attenuated smallpox was used for the massive vaccination campaign that successfully eradicated the infection ( ) , and currently, attenuated influenza viruses are used as vaccines against the seasonal influenza ( ). the advantages of live attenuated vaccines are the intrinsic adjuvant properties, the ability to infect cells (figure ) , and to activate the innate immune response. interestingly, a safe sars-cov- inactivated vaccine (picovacc) has been recently described as being able to induce specific neutralizing antibodies (nabs) in experimental animal models ( ) , and a phase iii clinical trial (nct ) will soon assess efficacy and safety of this candidate in health care professionals ( table ) . nucleic acid vaccines include either mrna or plasmid dna (pdna) vaccines (figure ) . two types of mrna vaccines were developed: conventional non-replicating mrna vaccines and self-amplifying vaccines (or viral replicons). the in vitro enzymatic transcription (ivt) of a dna template plasmid, containing the promoter sequence for the dna-dependent rna polymerase, provides a mature mrna molecule, with the open reading frame that encodes the target antigen, the and flanking untranslated regions (utrs), the cap, and the terminal poly(a) tail. self-amplifying rna (sam) vaccines are commonly based on alphavirus genomes, where genes coding for the structural proteins are replaced with that encoding the target antigens, while the rna replication machinery sequences are conserved, allowing intracellular antigen-encoding rna amplification and higher antigen expression levels than the conventional mrna vaccines ( , ) . once the mrna vaccine is delivered to the host cells and reaches the cytoplasm, it is translated in vivo by the host cellular machinery, providing the corresponding posttranslationally modified antigen (figure ) , thus mimicking the in vivo natural infection. mrna vaccines activate the innate immune system, triggering host immune sensing receptors, and successively promoting adaptive immune responses ( ) . several figure | platforms for vaccine manufacturing: a graphical overview. nucleic acid, viral-vector, protein-based, live attenuated and inactivated vaccines are schematically illustrated. nucleic acid vaccines: conventional non-replicating mrna vaccine, containing the target gene sequence, can be encapsulated into a delivery system to aid its cellular uptake. once released from endosome into the cytosol, it is translated by the host cellular machinery into the target antigen. a pdna carrying a gene target reaches the nucleus to achieve transcription and translation into the cytosol. pdna can be internalized by somatic cells (i.e., myocytes) and then the secreted antigen can be taken up by apcs or naïve b cell, priming immune responses. viral vectored vaccines: defective viral vector, carrying a transgene cassette, can be employed as a system to deliver a transgene and allow the expression of the heterologous antigen within the infected cell. a recombinant replicating viral vector retains the ability to replicate and produce progeny virus particles that can then infect cells, leading to transgene expression and ag processing and presentation. protein-based vaccines: recombinant subunit vaccine or a vlp can be taken up by apcs for mhc presentation and b-cell recognition through bcr. virus vaccines: compared with an inactivated virus, a live attenuated virus retains the ability to replicate and infect cells, mimicking the natural infection. apcs, antigen-presenting cells; mhc, major histocompatibility complex; ag, antigen; pdna, plasmid dna; ep, electroporation; bcr, b-cell receptor; and vlp, virus-like particle. technological innovations have allowed to overcome some of the concerns associated with instability, half-life, inefficient in vivo delivery, and high innate immunogenicity of mrna platform ( ) . mrna vaccines do not produce infectious particles and potentially do not integrate into the host genome, reducing safety issues, and no anti-vector immunity is elicited. they can be quickly produced (likely within the time required to get genomic information from the new emergent virus), saving time and cutting costs. thus, the mrna platform offers a promising attractive alternative to conventional vaccines, should a disease outbreak occur. no rna vaccine has been yet licensed for humans, but encouraging results from preclinical and human clinical trials have shown that mrna vaccines are able to induce safe and long-lasting immunity against different infectious viral diseases, including zika ( ), influenza ( - ), ebola ( ), dengue ( ) , and other viral diseases ( ) . a sars-cov- mrnabased vaccine entered clinical phases just months after the identification of the viral genome sequence (nct ), and a phase iii study (nct ) will assess its effectiveness to prevent covid- ( ) ( ) ( ) . a clinical study (nct ) is currently evaluating a similar vaccine in healthy adults ( table ) . the dna-based strategy, like the mrna-based technology, offers a valuable platform to design and deliver any target of choice, due to safety profile, stability, ease of gene manipulation, and large-scale vaccine manufacturing, in short ( , ), on cellular uptake and in vivo long-term gene expression, potentially providing advantages over mrna vaccines in terms of protein coding capacity, and amount and extent of antigen production. unlike mrna, pdna needs to cross both plasma and nuclear membranes to enter into a cell target, reach the nucleus, and achieve transcription (figure ) . advances in pdna delivery devices (i.e., use of gene gun; in vivo electroporation, ep), and delivery systems (i.e., encapsulation in lnps; adsorption to polymers), have greatly enhanced molecular stability, delivery efficiency, uptake, and antigen expression. in addition, the use of optimized pdna formulations and encoding molecular adjuvants, to be administered in prime-boost strategies or simultaneously with other vaccine platforms, has generally improved the low protective immunestimulatory profile of pdna ( ) . however, some potential safety concerns should be considered, including long-term persistence upon administration, which could eventually lead to genomic integration events, antibodies against bacteria-derived plasmids that could potentially trigger autoimmune diseases, and unwanted side effects due to encoded and co-delivered molecular adjuvants ( , ) . even though no dna vaccine has been yet licensed for use in humans (four for veterinary use), this platform has shown great promise for several emerging viral diseases, including ebola and marburg ( ), mers ( ), west nile ( ), dengue ( ), chikungunya ( ) , and other viral diseases ( ) , and more recently for covid- ( ) . currently, dna-based vaccine candidates, encoding the s protein from sars-cov- , have moved into clinical phase i/ii development ( , , ) (table ). recombinant viral vector-based platform employs either live replicating often attenuated or non-replicating viruses as vector vaccines (figure ). viral vector vaccines represent the biotechnological evolution of live attenuated and inactivated vaccines: a viral backbone devoid of the replication machinery to be used as a shuttle to express in vivo the chosen target antigen. several viral backbones have been exploited to generate viral-vector vaccines. targeted deletion of replication genes represents the non-empirical way of virus attenuation, allowing the generation of a wide array of viral vectors, engineered by insertion of a transgene cassette. the modified virus ankara (mva) is an attenuated form of the vaccinia virus (vacv), derived from more than passages in chick embryo fibroblasts, a method that empirically modifies the viral genome, without affecting the immunogenicity ( ) . it is able to infect multiple cell types but cannot replicate inside the infected cells, ruling out the safety concerns related to the use of live vaccines. one of the drawbacks in the use of a viral vector vaccine is that multiple immunizations lead to the host response against the structural viral proteins, limiting the efficacy of vaccination, as demonstrated in a study based on cellular immune response. to overcome this limitation, the heterologous prime-boost regimen has been introduced in several clinical trials, where two different viral vectors or a pdna prime-viral vector boost were tested ( ) . risks of integration into the host genome do potentially exist, as some viral vectors enter to the nucleus of cells to achieve transcription and replication. a major restrain in the production of viral vector vaccines is the time-consuming manufacturing; several attempts to accelerate vaccine production are in development, like selecting cell lines with higher yield or choosing the best promoter for transgene expression to reduce vaccine doses ( ) . among the available viral vectors, the adenoviruses are the most used in priming the immune response, being able to induce humoral and cellular responses ( ) . a pre-existing anti-vector immune response jeopardized the vaccine response in adenoviral-based clinical trials ( ) . to avoid pre-existing immunity, adenoviral vectors of non-human origin or rare serotypes have been used as vaccine platform. the use of chimpanzee adenoviral vectors proved to be safe and effective in clinical trials conducted against ebola ( ) and respiratory syncytial virus (rsv) ( ) . vesicular stomatitis virus (vsv), a single-stranded negative sense rna virus that naturally infects livestock, represents an attractive safe alternative over other viral vectors due to low risk of pre-existing immunity, lack of dna molecules during replication, and ability of vsv-based vaccines to induce effective humoral responses ( ) . for humans, two viral-vector vaccines are available: imojev, a japanese encephalitic virus (jev) vaccine; and dengvaxia, a dengue vaccine (both from sanofi pasteur). both are produced using the chimeric yfv as vector: two of the yfv genes have been replaced by genes encoding the pre-membrane (prm) and the envelope (e) protein of jev or denv, and the chimeric viruses are propagated in cell culture ( , ) . conversely, several viral vector vaccines have been licensed for veterinary use because of the less stringent regulatory requirements ( ) . to face covid- , an adenovirus type vector expressing sars-cov- s protein (ad -ncov) has been advanced into phase ii trial (nct ), while a phase iii study (isrctn ) is currently investigating the chimpanzee adenoviral vector (chadox ncov- ), expressing the same protein ( , , ) ( table ) . recombinant protein-based vaccines consist of immunogenic proteins from the target pathogen. once identified, recombinant proteins can be produced on a large scale, in bioreactors, using heterologous expression systems, like bacteria, yeast, plants, insect, or mammalian cell lines, depending on the posttranscriptional pattern of modification required ( ) . vaccines based on recombinant proteins represent a safe platform because they do not contain pathogen-derived genetic information, and the manufacturing does not require manipulation of live pathogens. they might represent a platform of choice when a fast response to an epidemic is on demand, as the vaccine production can start once the genome of the new virus has been sequenced, even before the virus isolation. protein-based vaccines can be obtained producing recombinant virus subunits (suvs) that can be administered in combination with adjuvants to improve the host immune response against the recombinant viral antigens ( ) . recombinant proteins derived from viral capsid can selfassemble into virus-like particles (vlps), high ordered and repetitive structures devoid of the viral genome. vlps display antigenic epitopes in their original conformation in high copy number, they retain the size and geometrical organization of the original virus (mainly icosahedral or rod shape), preserving the viral immunogenicity due to the ability to crosslink b cell receptor on b cell surface ( ) , and to be taken up by antigenpresenting cells (apcs) ( , ) (figure ) . several strategies have been proposed to improve dendritic cell (dc) uptake, by expressing targeting molecules such as antibodies directed against endocytic receptors, and to augment immunogenicity, through simultaneous delivery of maturation stimuli, like tlr agonists ( , ). when not able to self-assemble into a vlp, the selected antigen can be expressed as chimeric protein: several vlp platforms are available for the display of heterologous antigens on the viral coat proteins. recombinant vlps from plant virus, like tobacco mosaic virus ( ) , or alpha mosaic virus ( ) , are easily produced, competing for speed and cost of production with vlp platform based on mammalian viruses ( ) . the most used vlp platform is the hbcag-vlp, the core antigen from hepatitis b virus (hbv) ( ) . it is also possible to chemically attach the heterologous antigen to a preformed vlp by using conjugation methods ( ) . although this strategy could increase the manufacturing costs, it might be suitable when the expression of recombinant antigens affects the vlp assembly. to table ) . it is worth mentioning that kim and colleagues designed and developed a sars-cov- subunit vaccine within weeks of the identification of sars-cov- s protein n-terminal domain s sequence. delivery of recombinant subunit vaccines by microneedle array resulted in potent antibody response in mice ( ) , and vaccination with a sars-cov- spike s -fc fusion protein induced antibody responses in small animal models and nabs in monkeys ( ) . in table are listed the vaccine candidates that currently moved into clinical trials for preventing the viral infectious diseases discussed in the following section. west nile virus includes five lineages; among them, lineage was classified as the most virulent, while lineage is considered more attenuated. however, during a serious outbreak in hungary in , the sequencing of lineage showed some genetic mutations that demonstrated the increased virulence of this strain and its explosion throughout the central europe ( , ) , causing renewed interest in the development of a vaccine against wnv. years after the epidemic that hit the united states, no wnv vaccine has been yet released for human use, while four vaccine formulations are on the market for veterinary use, three based on the whole inactivated virus (wn innovator, vetera wnv, and prestige wnv), and one on recombinant vaccine expressing wnv prm/e into a canarypox backbone (recombitek equine wnv) ( , ) . these vaccines completely protect horses from viral infection but require subsequent administrations and several booster doses overtime. for the development of a vaccine for humans, many different platforms were used in preclinical studies, and many of them entered into phase i/ii trials, including hydrogen peroxideinactivated whole virus (hydrovax) vaccine (nct ) ( ), a recombinant truncated form of wnv e protein ( ), recombinant chimeric live attenuated viral vectors, employing yfv ( ), or mva ( ) delivering wnv prm/e proteins (nct ), pdna vaccines encoding prm/e (nct ) ( , ) . all the envelope-based vaccines induced nabs against both wnv lineages and , but some candidates are unable to generate long-lasting antibody responses, requiring multiple administrations ( , , ) . thus, further improvements are needed for the development of next-generation vaccines ( ) . recently, a wnv replicationdeficient vaccine candidate with a deletion of the non-structural protein ns has been shown to protect mice from a highly lethal viral challenge, after a single dose, without adverse effects ( ) . during the outbreak in brazil, an abnormal microcephaly number and other birth defects in newborns were reported ( ) . for this reason, vaccination of pregnant and of reproductiveage women became an urgency. shan and colleagues developed a candidate vaccine, using a live attenuated viral strain containing a deletion in the region of the virus genome. this vaccine induced strong and protective antibody response, after a single injection in mice and macaques, and reduced viral rna in placental and fetal tissues in infected mice ( ) . the immunized mice also developed a robust t-cell response ( ) . although promising, this attenuated virus-based formulation does not meet the safety standards required to be used to vaccinate pregnant women, whose prophylaxis requires a vaccine that fulfill higher safety standards. a number of different replication-deficient viral vectors have been recently developed and are currently under evaluation. immunization of mice with a vaccine based on mva delivering the zikv prm and the e structural proteins (mva-zikv) elicited nabs and potent zikv-specific cd + t-cell responses, mainly with an effector memory phenotype ( ) . a rhesus adenovirus serotype vector (rhad ), expressing zikv prm and e proteins, induced high titer of zikv-specific antibodies after the first prime, offering complete protection against subcutaneous zikv challenge, in mice ( ) , and rhesus monkeys ( ) . these adenoviral-based vaccines induced antibodies that were also maternally transmitted ( ) . in addition, abbink et al. using the rhesus macaque model demonstrated that a complete anti-zikv immunity can only be achieved through vaccination with a combination of different vaccine platforms ( ) . zikv vaccine candidates currently in phase i clinical trials include inactivated and live attenuated vaccines, mrna and pdna vaccines, and recombinant viral-vectored vaccines, mainly targeting the prm and e proteins ( ) . a dna-based vaccine encoding the prm signal sequence from jev and zikv e proteins moved into phase ii (nct ), showing immunogenicity and safety in humans ( ) . a protective and efficacious vaccine against yfv is currently available. to date, the main type of yf vaccine produced on a large scale is based on the live attenuated d virus vaccine. this vaccine is obtained after numerous passages of asibi virus strain in mouse and chicken embryo that generate a strain with accumulated mutations in the envelope protein. these mutations affect the virus binding to the host receptor, reducing its neurotropism and vicerotropism, and mosquito transmissibility ( ) . because the vaccine is produced in chicken embryo, there are issues related to manufacturing costs and vaccine availability. the interruption of vaccination coverage against yf in endemic countries has caused major outbreaks in africa and south america in and , which exhausted the d vaccine stockpiles leading to the use of an emergency "fractional dose" campaign in the democratic republic of congo ( ) . thus, the fluctuating demand for doses during outbreaks makes the accessibility to the vaccine still a problem to be solved. the need for a vaccine against denv has become an urgency only in recent decades. dengue fever is caused by four distinct virus serotypes, denv - , able to circulate simultaneously in endemic areas, making extremely difficult the development of a broad protective vaccine. recently, the food and drug administration approved the first dengue vaccine by sanofi-pasteur, named cyd-tdv or dengvaxia ( , ), a tetravalent live attenuated virus vaccine on yfv backbone, whose release has generated controversy due to evidence that the administration can increase the risk of a more severe form of the illness in people with a pre-existing immunity toward other denv strains ( , ) . for this reason, the use of dengvaxia is strictly limited, depending on age (between and ) and serostatus of recipients to vaccinate (exclusively individuals who had a previous denv infection), generating concerns about its costbenefit balance. studies for the development of a safer vaccine are still ongoing, and candidate vaccines include a tetravalent dengue purified inactivated virus vaccine, currently in phase i/ii clinical trial (nct ), and two live attenuated tetravalent chimeric tdv (denvax), and tvd / (tetravax-dv) vaccines, currently in phase iii clinical trials (nct ; nct ) ( ). no vaccine is actually available to prevent chikv infection. among the candidates in ongoing studies, two of them achieved and completed phase i or ii trials: vla and mv-chik vaccines. vla candidate (by valneva) is a live chikv (la réunion isolate lr opy ) attenuated by a partial deletion of the gene encoding the non-structural replicase complex protein. this vaccine induced immunity lasting over months after a single shot immunization (nct ). mv-chik vaccine is a live attenuated measles-vectored chikv vaccine that induced chikv-specific nabs and shown to be well tolerated by all the participants (nct ) ( ) . recently, moderna therapeutics tested a vaccine based on engineered mrna encoding chikv structural polyproteins (mrna- ) in a phase i clinical trial. as shown in preclinical studies, this formulation induced strong immune responses after one single injection, totally protecting mice from developing the disease ( ) . several vaccine platforms have been tested in preclinical animal models and shown to be able to protect animals from marv infection and to induce both humoral and cellular immune responses. these include vlps ( ) , dna vaccines ( ), recombinant adenoviral vectors ( ) , and rvsv ( , ) . many works have emphasized the use of a multivalent vaccine formulation to achieve protection against different filoviruses. vaccination with a single dose of a trivalent formulation based on rvsv expressing glycoproteins from ebov, sudan ebolavirus (sudv), and the angola strain of marv elicited antibodies specific for the three glycoproteins in non-human primates (nhps) and a balanced t-cell response sufficient to protect against the viral challenges ( ) . similarly, vlps delivering a trimeric hybrid glycoprotein from marv, ebov, and sudv fully protected vaccinated animals from marv challenge, inducing specific nabs ( ) . using an enhanced dna-based platform encoding the envelope glycoprotein from marv and ebov, shedlock and colleagues showed that a polyvalent-filoviral vaccine candidate, delivered by in vivo ep, elicited in preclinical models robust nabs and cytotoxic t cells, completely protecting animals from the viral challenge, after a single dose administration ( ) . actually, a multivalent phase i study (nct ) is evaluating safety and immunogenicity of two heterologous and two homologous prime-boost regimens using a mva multi-filo and ad zaire ebola (ad .zebov) vaccines ( ) in healthy volunteers, with the aim to analyze the protective response to different filoviruses. coronaviruses are a group of single-stranded rna viruses that have been present in humans for at least - years and all originated in bats ( , ) . earlier than , six coronaviruses had been known to cause diseases in humans: hcov- e, hcov- , hcov-nl , hcov-hkn , sars coronavirus (sars-cov), and mers coronavirus (mers-cov) ( ) . in late and early , a novel coronavirus was discovered to be the cause of a rapidly spreading outbreak of respiratory disease, including potentially fatal pneumonia, in wuhan, china. the virus, provisionally designated -ncov and later given the official name sars-cov- , owing to its similarity to sars-cov (then named sars-cov- ), was isolated and the viral genome sequenced. sars-cov- was characterized as a beta-coronavirus ( ) . the disease caused by the virus was officially named coronavirus disease (covid- ) by who. coronaviruses are capable of adapting quickly to new hosts through the processes of genetic recombination and mutation in vivo. point mutations alone are not sufficient to create a new virus. however, this may occur when the same host is simultaneously infected with two coronavirus strains, enabling recombination of genomic fragments of hundreds or thousands of base pairs long and thus making a new virus ( , ) . this susceptibility enabled the emergence, in approximately two decades, of three new human coronavirus species with epidemic potential: sars-cov- , mers-cov, and sars-cov- . coronaviruses enter cells via binding to a host receptor followed by membrane fusion. the angiotensin-converting enzyme (ace ) was identified as the cell receptor for sars-cov ( ) , and recently also for the new sars-cov- ( ), while mers-cov binds the dipeptidyl peptidase (dpp ) receptor, also known as cd ( ) . the s protein is used for virus-cell receptor interaction during viral entry ( ) . transmission of the virus during the viremic stage of disease is primarily via respiratory secretions (droplets) or direct contact. sars-cov- is extremely contagious, with an estimated basic reproduction number (r ) of . - . ( ) . in contrast, the r for both sars-cov- and mers-cov is less than ( ). it soon became apparent that infected individuals might be capable of transmitting the virus during the prodromal period ( ) . social distancing strategies (quarantine and community containment) represent the only efficacious means of controlling coronavirus spread in the absence of effective drugs or vaccine against the pathogens. of importance, for preventing the spread of the disease caused by contact with patients or contaminated fomites, hygiene measures are also mandatory, such as washing hands with soap and water or with alcohol-based preparations. indeed, coronaviruses are able to survive on various surfaces for few days but can be inactivated by disinfection ( ) . finally, because it has been demonstrated that the overlap between human and animal ecosystems have given to coronaviruses the opportunity to cross the species barrier, to prevent future zoonotic diseases, a coordination with veterinary experts as well as stricter laws governing the trade of wild animals would be necessary. humans are extremely exposed to these pathogens because these viruses had not previously circulated in the human population, as testified by the absence of antibodies against coronavirus in healthy people. in addition, the innate immune response has demonstrated to be insufficient in controlling coronavirus infection because decreases in viral load are coincident with the specific antibody response ( , ) . in this context, vaccines represent a much expected resource. a hopeful premise is represented by the successful containment of coronavirus epidemics in farm animals by vaccines, based on either killed or attenuated virus ( ) , and concerning sars-cov- by the finding that specific antibodies are detectable in % of patients with covid- , - days after symptom onset ( ) , and that the magnitude of antibody titers positively correlated with viral neutralization potency ( ) . after the sars outbreak, several vaccines were formulated based on various strategies, as recombinant s protein-based vaccines, attenuated and whole inactivated vaccines, as well as vectored vaccines. pre-clinical data showed animal protection from challenge with sars-cov- . however, sterilizing immunity was not always achieved ( ) . in few cases, the use of live virus as a vaccine resulted in complication including lung damage, eosinophil infiltration, and liver damage in animal models. moreover, a study of vaccination with inactivated sars-cov- in nhps reported enhancement of disease caused by specific epitopes on the s protein [reviewed in ( ) ]. another issue is related to the length of a protective immune response. both humoral and cellular responses have been found important for lasting protection. in long-term studies of recovered sars patients, antibody responses waned after approximately years, while t-cell responses persisted, suggesting that the latter is required for long-lasting immunity. concerning mers-cov, the vaccines proposed target the s protein ( ) ( ) ( ) , including mucosal vaccine for intranasal administration ( ) . however, cases of enhanced lung diseases were also reported in preclinical models of vaccination in mice ( ) . new mers-cov vaccines in development also include live attenuated, protein subunit, and dna vaccines ( , ) . recently, a small animal model that replicates mers-cov transmission has been developed ( ) and will help the preclinical studies. following the alarming data and casualties provoked by covid- , a strong effort by the research community is going on at the moment, and who has been informed of dozens of vaccines in preparation using different platforms, as mentioned in section "vaccine platforms." some of these candidate vaccines are already in phase i/ii clinical trials, while others have been advanced to phase iii studies ( , , ) (table ). however, it is possible that a sars-cov- vaccine will not be available for another - months. recently, a rhesus macaque model that recapitulates sars-cov- infection has been developed to study immunopathogenesis and test vaccine candidates ( , ) . therapy based on passive administration of anti-coronavirus antibodies, isolated from patient sera, also represents a much wanted option for the treatment of coronavirus diseases ( ) , and a global effort is pursued in this direction to treat patients before the achievement of a validated vaccine. in addition, researchers are trying to produce in laboratory specific and protective anti-coronavirus antibodies. in the case of sars outbreak, a monoclonal antibody (mab) with neutralizing activity, being able to block receptor association, was identified and described ( ) . moreover, neutralizing mabs have also been produced to fight mers-cov infection. in a collaborative study by us and chinese researchers, mabs targeting the receptor (cd /dpp ) binding domain of mers-cov spike glycoprotein were reported ( ) . japanese researchers have also investigated anti-cd mab for mers-cov and have identified the humanized mab ys as a promising candidate ( ) . finally, in the case of sars-cov- outbreak, dutch researchers claimed the identification of a human mab named d able to block sars-cov- infection ( ). recently, a mab able to cross-neutralize sars-cov- has been identified from memory b cells of a sars-cov-infected individual. the antibody, named s , engages the s receptor-binding domain, recognizing a highly conserved protein/glycan epitope distinct from the receptor-binding motif ( ) . more recently, other potent neutralizing antibodies were isolated by different research institutions ( ) ( ) ( ) . amidst the gamut of high-affinity antibodies with the potential to neutralize human pathogenic viruses, single-domain antibodies, referred to as nanobodies or nbs ( kda), and nanobody-based human heavy chain antibodies ( kda) derived from camelids might be harnessed as useful therapeutics for the ongoing covid- pandemic ( ) . camelid heavy-chainonly antibodies (hcabs) are composed of two heavy chains with a single variable domain (vhh) as the target-binding module. recombinant vhhs, devoid of the effector domains, act as single-domain antibodies and harbor advantageous features over conventional antibodies (higher thermal and chemical stability, higher solubility, smaller size, lower susceptibility to steric hindrances, ease of manufacturing, and simple structure) to have been recently proposed as prospective therapeutic candidates against various infectious pathogens ( ). vhhs isolated from a llama subcutaneously immunized with perfusionstabilized sars-cov- and mers-cov s proteins have been recently characterized and shown to be able to neutralize s pseudotyped viruses in vitro, interfering with the host cell receptor binding ( ) . interestingly, sars-cov- s-directed vhh cross-reacted with sars-cov- receptor binding domain (rbd) and neutralized sars-cov- s pseudoviruses in vitro as a bivalent human igg fc-fusion format, underscoring the potential of vhhs to treat coronavirus diseases ( ) . because of the global spread of diseases caused by flaviviruses, understanding the cross-reactivity of anti-viral immunity among these viruses is of crucial importance for predicting the evolution of viral disease outbreaks. recently, the analysis of pbmcs isolated from individuals infected by denv or vaccinated with denv tv or yf d vaccines, and pulsed with a pool of antigens from autologous and heterologous flaviviruses, indicated that both cd and cd t-cell responses were specific, with little or no cross-reactivity, despite the high level of homology ( ) . individuals preexposed to denv infection developed t-cell responses against non-structural zika proteins rather than structural envelope protein, suggesting that previous flaviviral infections biased the t-cell response toward more cross-reactive non-structural epitopes ( ) . studies enrolling mothers who gave birth to microcephalic babies after zikv infection, showed serological evidence of a pre-existing anti-dengue response, suggesting that vaccination against denv does not protect against zikv microcephaly ( ) . however, cross-reactive antibodies between zikv and denv have been described, mainly targeting the structural dimeric envelope protein ( , ) . the antigenic sequences are both linear and quaternary, with nabs mainly recognizing the latter. the high-conserved e protein fusion loop induces cross-reactive but weak nabs that can be a marker of worst outcome during subsequent flaviviral infections ( ) . a research concerning zikv-specific b-cell responses in three denv-experienced donors showed that months after the infection, the pool of antibodies comprised both poorly nabs derived from pre-existing denv-induced memory b cells, associated with an enhanced zikv infection in vitro, and potent zikv-specific antibodies originated de novo ( , ) . the possibility that wnv-specific antibodies may drive the infection by other flaviviruses is still controversial, even if cross-reactivity was demonstrated. plasma samples from convalescent human wnv patients were shown to enhance zikv infections by antibody-dependent enhancement (ade) phenomenon ( ) ; conversely, mice previously infected with zikv and challenged with wnv showed enhanced protection toward the second infection ( ) . the immunological flavivirus cross-reactivity, the ade phenomenon (discussed below), genetic mutations that increase the virulence, potential pre-existing immunity concerns, combined with the necessity to increase cost-effectiveness of marketable products are among the issues that have limited the development of successful vaccines until now. the use of t-cell inducing vaccines or proteins with mutations into conserved envelope fusion-loop epitopes might be useful to overcome the cross-reactivity hurdle ( ) . known as ade of viral infection, ade is a phenomenon occurring when antibodies facilitate virus entry into the host cells, driving viral replication and increasing infectivity, with subsequent severe outcomes. among the several stumbling blocks in realizing a safe vaccine, ade is a phenomenon largely underestimated, but that can produce severe adverse effects, rendering vaccinated individuals more predisposed to develop harsh symptoms after infection ( ) . the first report of ade dates ( ) . the molecular mechanisms disclosed the involvement of fcγr ( ) and complement receptors ( ) . when an antiviral antibody (induced by vaccination or viral infection) with no neutralizing or sub-neutralizing activity is produced, it can act like a bridge between the virus and the fcγr expressed on the surface of immune cells, leading to viral uptake (figure ) , as demonstrated for denv, zikv, wnv, influenza, sars-cov, mers-cov, and ebov ( ) . the role of complement receptor has been demonstrated in ebov response: two antibodies directed against epitopes in close proximity bind the c q, forming an immune complex able to enhance the virus entry into a target cell ( ) , whereas in an animal model of mers-cov, c a and c protein level increase was observed after passive immunization ( ) . the first licensed vaccine against denv (cyd-tdv-dengvaxia) caused hospitalizations in two large multicenter phase iii trials; after result revision, it has been estimated that in seronegative individuals, it can produce adverse effects ( ) , and who recommendations are to vaccinate only seropositive individuals in endemic areas of age older than years. using a mathematical model of denv transmission to formulate hypothesis on vaccine trial results, it was speculated that "seronegative recipients gain transient protective cross-reactive immunity akin to that observed for natural infection, " increasing the risk of severe disease after infection, while vaccination of seropositive subjects results in boosting the immune response, producing a protection comparable with the one obtained in individuals who has had two natural infections ( ) . the most severe adverse effect after vaccination was registered when a formalin-inactivated vaccine against rsv produced an increase of severe illness in vaccinated infant (hospitalization: % rsv vaccinated vs. % vaccinated against parainfluenza) and two deaths ( ) . afterward, a role for the th response was hypothesized in generating the rsv-mediated ade ( ) , and it was demonstrated that the formalin-inactivated virus produced ade in monkeys ( ) , suggesting that the carbonyl groups on formaldehyde-inactivated rsv were responsible for the th response in mice ( ) . moreover, the observation that formalin inactivation produced an alteration of antigens, leading to the production of non-nabs, whose avidity did not mature, and the activation of complement were also reported for a measles vaccine ( ) . the low-avidity non-nabs are produced in absence of tlr activation (and affinity maturation), and they figure | antibody-dependent enhancement on dengue infection. antibodies generated from a previous denv infection can recognize but do not neutralize another denv serotype and can lead to antibody-dependent enhancement (ade) of entry of the latter virus into host cells. the pre-existing non-(or sub-) neutralizing antibodies bind denv through the fab domains and mediate viral entry into fcγr-expressing cells. on engagement by the fc domains, the virus-antibody immune complex is internalized by the activating fcγriia within the endosome. co-ligation of fcγriia and lilrb (leukocyte immunoglobulin-like receptor-b ) to opsonized denv drives the inhibitory signal cascade via immunoreceptor tyrosine-based inhibition motif (itim) pathway, abrogating the expression of isgs (interferon stimulated genes). ligation of fcγriia to immune complex also increases th cytokine production and reduces ifnγ, inhibiting the jak/stat signaling pathway, overall resulting in the suppression of the antiviral response and increase of viral replication. nabs, neutralizing antibodies; and itam, immunoreceptor tyrosine-based activation motif. trigger complement activation ( ) , enhancing viral infection. to induce potent nabs, the tlr activation has been obtained using a th -polarizing adjuvant ( ) , in association with the candidate vaccine exposing the epitopes of interest. antibody-dependent enhancement has been reported also in many studies focusing on the development of sars and mers vaccines, demonstrating that vaccination with the whole s glycoprotein can increase the susceptibility to viral infection with a mechanism not linked to the virus receptor expression on the host cells ( ) , and especially when antibodies are induced with low titer ( ) . while for many flaviviruses the mechanism of ade has been explained through evidences that antibodies developed during a primary infection can enhance entry of a heterologous virus via fc-receptor during a secondary infection, for mers-cov and sars-cov, it has also been proposed that nabs that strongly bind the rbd region of the s surface protein can induce conformational changes that enhance the virus entry via canonical viral-receptor-dependent pathways, mimicking viral receptor binding ( , ) , and antibodies targeting a specific region of the s protein enhanced the viral infection in a sars model of nhps ( ) . the high sequence homology and the similarity in structure shared among sars-cov, mers-cov, and sars-cov s glycoproteins raises reasonable concerns about the development of covid- vaccines based on the s protein. in potential pandemic settings, the clinical development of vaccines is the main aim. however, apart from technical reasons, the vaccine production might be delayed also for economic considerations and safety issues. other strategies may be based on self-disseminating vaccines and induction of trained immunity. to control zoonosis, the formulation of self-disseminating vaccines acts at the level of animal, insect, or environmental reservoir, to directly interfere within the animal-to-human transmission ( ) . they are essentially based on replicating viral vectors engineered to express the disease antigen and to target a certain animal population ( ) . global vaccination of animals could be achieved to effectively contain an emerging pathogen within the wildlife reservoir, avoiding its global spread. feasibility concerns, costs, and safety issues should be considered when using this strategy to control reservoirs linked to the emergence of high-risk pathogens. in addition, which animal pathogen will cause a human disease is generally unpredictable. it is interesting to underline that a vaccination of great apes with an engineered specific cmv-based vector has been proposed as a strategy to potentially interrupt (or at least decrease) the zoonotic transmission of ebola virus to humans, being able to protect animals from the lethal viral challenge ( , ) . trained immunity-based vaccines (tibv) might be formulated to stimulate broader anti-infectious responses compared with conventional vaccines for their capacity to increase innate immunity and enhance adaptive responses ( ) . this strategy exploits the ability of innate immune cells (monocytes, macrophages, nk cells) to undergo extensive metabolic and epigenetic reprogramming, following certain vaccinations or infections, and to become primed for a quite long period of time to respond more potently to autologous or heterologous re-infection, mounting the so-called "innate immune memory." triggering of pattern recognition receptors (prrs) by microbial effector stimuli results in increased production of pro-inflammatory cytokines and/or reactive oxygen species, and in enhanced immune responses, regardless the primary stimulation ( ) . many infectious stimuli are considered potent activators of trained innate immunity, including β-glucan and chitin (components of fungal cell wall), lps (a component of the cell wall of gram-negative bacteria), and the bacille calmette-guérin (bcg) vaccine ( ) . thus, tibv should contain pathogen-associated molecular patterns (pamps) to target prrs and subsequently induce trained immune cells. bcg vaccine, vacv, and live attenuated influenza vaccines, together with immunostimulants, could be ascribed to this category of vaccines ( ) . it is worth mentioning that a whole-cell killed bacterial vaccine might have played a role in preventing pneumonia and mortality during the influenza pandemic ( ) . recently, a work by berg and colleagues showed that bcg vaccination is associated with the flattening of the curve in the spread of covid- , suggesting that bcv vaccine might serve as a protective factor against the disease ( ). however, it should also be noted that an enhanced immune response mediated by reprogrammed immune cells could contribute to the development or maintenance of inflammatory, neuroinflammatory, and chronic metabolic disorders ( ) . the phenomenon of "trained immunity" occurring in the brain is known as microglial priming. exposure of primed microglial cells to a second stimuli can cause an augmented inflammatory response, leading to neuroinflammation and production of neurotoxic molecules. the hyperglycemia condition that characterizes type diabetes could long term affect the cellular metabolism of monocytes and macrophages, leading to increased cytokine production and subsequent diabetes complications, including atherosclerosis. an augmented activation of innate cells may also result in the induction and maintenance of chronic inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or sarcoidosis ( ) . the covid- pandemic experience, combined with the previous viral disease outbreaks, should give blueprints for rapidly responding to the emergence of high-risk pathogens in the future. it is a common belief that vaccines would be the only means of providing long-term immunity and preventing viral diseases. despite the great progress made in vaccine research, we are still unable to produce successful vaccines in a timely manner. human trials take a long time and given a huge list of vaccine candidates, it is hard to choose the most promising one. while the who proposed a solidarity vaccine trial to test all the candidates in rolling trial until they fail, to increase the chances of succeeding, some vaccine stakeholders are considering extreme alternatives for emergency use: intentionally infect young healthy volunteers at low risk in controlled "human challenge trials" to define which vaccine will work ( ) . although these approaches are already used for studying influenza ( ) and dengue diseases ( ) , it is hard to ethically accept this option without a validated therapy. vaccines go through regulatory pathways before the final approval and licensure. in epidemic or pandemic settings, we need to carefully develop a vaccine, as quickly as possible, that adequately proved to be safe and effective ( ) . scientists need to fill the gaps in understanding the epidemiology of novel viruses, to identify potential zoonotic reservoirs or spill-over hosts, and the way of transmission of pathogens. once the pathogen is identified, preclinical models need to be developed to study virus-host interactions and early test vaccine candidates, defining the immune correlates of protection. pathogen-specific epitopes need to be identified to guide structure-based vaccines that will elicit protective antibodies, minimizing the induction of non-or weakly nabs that would promote ade of viral infection ( ) . moreover, data sharing and collaboration among academia, government, and companies will be essential to coordinate a strategic approach in face of next pandemic threats ( ) . all authors equally contributed to this work and read and approved the final manuscript. this work was supported by prin "nanotechvax tackling biological barriers to antigen delivery by nanotechnological vaccines" prot. zeccm, and consiglio nazionale delle ricerche (cnr), italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." mt was supported by postdoctoral fellowship from cnr, italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." references . nii-trebi ni. emerging and neglected infectious diseases: insights, advances, and challenges infectious disease threats in the twenty-first century: strengthening the global response the epidemic of -novel-coronavirus ( -ncov) pneumonia and insights for emerging infectious diseases in the future major factors affecting the emergence and re-emergence of infectious diseases climate change and multiple emerging infectious diseases infectious disease and economics: the case for considering multi-sectoral impacts emerging infectious diseases: a proactive approach 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journal: transfusion doi: . /trf. sha: doc_id: cord_uid: t x gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks , , , and following index donations from donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by months. urine and saliva detection decreased significantly after weeks and was undetectable by months. of donors who were enrolled in the acute pre-seroconversion stage of infection % ( / ) developed multiple zikv related symptoms week post index donation, compared to only % ( / ) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* , yuelong guo , ritchard g. cable , joseph e. kiss , michael paul busch , grier page , stacy endres-dighe , steve kleinman , simone glynn , alan mast and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) . american red cross, rti international, american red cross blood services, blood systems inc., blood systems research institute, university of british columbia, nih/ nhlbi, blood research institute, nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over , donors were enrolled from us blood centers for ferritin testing. the study population was enriched for racial minorities [ african-american (aa), asian (as), hispanic (hisp)] and for "super donors" ( , who had completed donations in two years without low hemoglobin deferral). the minority donors and the remaining non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml). results/findings: across all subjects, % had ais and % had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed % % decreased risk for ais compared to nhw, while hisp donors had % higher risk. daily use of exogenous iron reduced risk for lf and ais by to %, respectively, while the estimated benefit from less-than-daily use was lower ( to % protection). regular use of antacids was associated with a % or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by % - %, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean ( sd) *p < . compared to batf / , ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to % of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d and d of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration ( % on d vs % on d of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized ( %) and macrophage-depleted ( %) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than % at h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at h posttransfusion. at h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* , amanda l richards and krystalyn e hudson . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod otii mice are predisposed to have autoreactive cd t cells. study design/method: four cohorts of hodxotii f mice ( - mice/ cohort) were bled monthly for months to assess for autoab production. peripheral rbcs were stained with anti-complement (c ) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd and ter to assess for the presence of rbc progenitors. statistical analysis between hod otii autoab vs. hod otii autoabvs. hod -otii was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd t cells were not deleted in the thymus of hod otii mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod otii . however, as they aged, - % of hod otii were positive for rbc autoab by months. thereafter, $ % of the autoab mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in of the cohorts, - % of autoab mice were female. hod otii autoab mice also had enlarged spleens compared to hod otii autoaband hod -otii mice ( . g vs. . g and . g, resp., p< . ). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd hi ter inter ) were observed in the spleens of hod otii autoab mice but not in hod otii autoaband hod -otii ( . % vs. . % and . % resp., p< . ). moreover, autoab and c deposition were found ( . - % and - %, resp.) on ter rbcs in all of the hod otii autoab mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b -a a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards , christopher a tormey and krystalyn e hudson* . background/case studies: red blood cell (rbc) alloimmunization occurs in up to % of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c bl/ (b ) mice were treated with pbs, or anti-ly g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at - hours post-transfusion. anti-hod alloantibody generation was assessed days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n ), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n ). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls ( / experiments, p< . ). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l ), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p< . ); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b -a b cxcr pd and ccr expressions characterize responders to rbc immunization benoît vingert* , , , marie tamagne , , , sadaf pakdaman , , , anoosha habibi , , , philippe bierling , , , , , rachid djoudi and france pirenne , , , . efs ile de france, laboratory of excellence gr-ex, imrb u -eq , ap-hp, universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd t cells have a th profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd t cells with a cxcr pd phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr pd hi profile, with a differentiated expression of ccr . ccr is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr and cxcr can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr pd lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr pd cells were compared in groups of transfused sickle cell patients : alloimmunized (n ) and non-alloimmunized patients (n ). the analysis was also performed in non-transfused healthy controls (n ). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr pd hi subpopulation expression was identical between transfused groups and controls. ccr and cxcr expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr expression was very strong independently of the expression of pd . in the aim to determine the help of the circulating cxcr pd cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr pd subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr pd cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr on circulating cxcr pd cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr pd profile and the ccr expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* , ashley bennett , kathryn girard-pierce , connie arthur , amanda mener , patty zerra , christopher a tormey , jeanne hendrickson and sean stowell . emory university, yale-new haven hospital, yale university, emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod kel). to examine the role of cd t cells, pic/kel primed b recipients were cd t cell depleted prior to transfusion. in addition, b recipients were adoptively transferred with cd t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < . ); pic/kel primed recipients transfused with (hod kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < . ) and transfer of cd t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < . ). conclusion: these results demonstrate that cd t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c bl/ ) mice or oti mice, whose cd t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after , , , , and hours and on days - . results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after hours and peaking at hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is - % versus > % in wt recipients at hours (p< . ), whereas transfusion of wt platelets into either oti or wt recipients is approximately % at hours after transfusion. adoptive transfer of oti cd t cells into wt mice recapitulates the effect, with significant mova platelet clearance at hours compared to wt platelet clearance (p< . ). conclusion: this work extends the ability of cd t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c bl/ mouse blood and treated with uv r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv r treated wbc-rich prp, or uv r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c bl/ donor blood. a second transfusion of untreated wbc-rich c bl/ prp was given weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p< . ) or necrotic (p< . ) wbcs, but not those given uv r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c bl/ wbcs were reduced in recipients of either uv r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so ) of venous blood is generally assumed to be around - % as measured from a central venous line. however, a recent investigation of so levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so distribution (mean . % . % [yoshida et al. ; blood transfusion , ] . the present study was undertaken to determine the distribution of so in lr-rcc produced at a medium-size blood center using a novel non-invasive so probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so levels. study design/method: the so from units of lr-rcc were examined on five consecutive days representing % of the collected units during the period at a regional blood center where all the units were processed at room temperature within hours of blood collection. so was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a u ; pendar technologies, cambridge ma). in addition to so , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n ) from human volunteers were stored in as- under normoxic, hyperoxic, or hypoxic conditions for up to days (so ranging from < to > %) prior to uhplc-ms metabolomics analyses in presence of c, n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so carried out non-invasively at a blood center yielded a similar wide distribution as previous study from units of lr-rcc procured and sampled invasively within hours after blood collection [yoshida ibid]. the shape of the so distribution appeared near normal with the mean of . % . %, median . %, range < % to > % and inter-quartile range (iqr) of . %- . %. male donors showed higher so compared to female donors (p< . ). no correlations were observed between so levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so levels ameliorate the energy and oxidative metabolic lesion. lower so levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so levels was observed from lr-rcc manufactured at a blood center using -hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a -fold increase in the absolute lymphocyte count (ko . . x /l vs. wt . . x /l, p . ), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko . . fl vs. wt . . fl, p . ). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd foxp regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd foxp tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd foxp tregs derived exosomes and their functions involved in cd tregs mediated immune-modulation were seldom reported. study design/method: cd t cells were freshly purified from pbmcs, cultured with anti-cd /cd antibody packaged beads and il- , and then polarized with tgf-b and rapamycin into cd foxp treg cells. the harvest cells were co-cultured with cd /cd beads stimulating cd cd effector cells in the transwell plate. the supernatant derived from cd tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd , cd , tsg and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir- , let- b, let- d were measured by qpcr. the precipitated exosomes were further purified by cd immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd treg cells could suppress the proliferation of effector cells with a small decline (p> . ), which means some non-contact factors involved in the cd treg mediated immune modulation. a total number of . . / cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle - nm in diameter ( . . nm by nta). cd and cd were expressed on these background/case studies: regulatory t cells (tregs), containing cd and cd subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd foxp regulatory t cells (ntregs) in inflammation conditions (including instability of foxp , conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd regulatory t cells in inflammation and transfusion. study design/method: human cd tregs were induced with tgf-b and rapamycin from cd t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd tregs when encountering with inflammation were test by foxp expression, th and th cells conversion in inflammations conditions (il tgf-b il il and il tgf-b il b il ) on day , day and day . in vivo, cd tregs were transfused into cia mice and then their survival in mice and foxp express were evaluated to reveal the stability of cd tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd tregs when induced factor tgf-b and rapamycin were removed by testing the foxp expression on day , day and day . results/finding: ex vivo induced human cd treg were foxp ( . . %) and did not secret il a (both in supernatant and % of cells). foxp express in cd tregs were maintained after induced factor tgf-b and rapamycin were removed on day , day and day . in vitro, foxp , il and ifn-c expression has no significant difference when compared with controlled tregs on day , day and day and did not secret il a when encounter with inflammation conditions (il tgf-b il il and il tgf-b il b il ). in vivo, cd treg cells were transfused into cia mice on the peak of disease onset ( days after the first collagen immunization, has inflammation condition in vivo) to test cd tregs survival. cd tregs were found in cia mice foot ( . . %), blood ( . . %) and spleen cells ( . . %) hours after transfusion and their % of foxp were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd tregs are stable in inflammation and transfusion and can maintain foxp expression when induced factor were removed, these make cd treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd /cd /cd expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p s k and its downstreanm proteins, especially the protein s , which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s related protein translation inhibition. xiaoyun fu* , , mikayla anderson and james c zimring , . bloodworksnw research institute, university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day (one day past their expiration). bioactive lipids including common fatty acids, oxylipins, and lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in stored rbc units. for example, arachidonic acid (aa) ranges from . - . mm, linoleic acid (la) ( . - mm), dihomo-c-linolenic acid (dgla) ( . - . mm), eicosapentaenoic acid (epa) ( . - . mm), docosahexaenoic acid (dha) ( . - . mm), and alpha-linolenic acid (ala) ( . - . mm). ten oxylipins including hetes, hodes, and dihomes, and lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of analytes quantified, showed a significant difference in concentration among different blood types by one-way anova testing (fdr< . ). the ab rh blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh ). the fold increase of o rh-/o rh among pufas ranges from . to . , suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna- was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna- targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps ( nm) surrounded by a thick fluorescent silica shell ( nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r rnaprobe . and r dnaprobe . ) with mirna- concentration, down to a -nm limit of detection. hybridization assays in % human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in % human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. . . a . . . . a . . . . a . . total atp,lm/ghb . . a . . . . a . . . . a . . extracellular lactate,mm a extracellular glucose,mm a a a extracellular na ,mm a extracellular k ,mm a a a a p< . , paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n ) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by % ( / , ) compared to the previous year without pathogen inactivation ( / , , p . , chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased ( / , vs / , ). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count ( . x ) than unaffected controls ( . x ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above . x . in vitro quality of single dose amotosalen/uva treated platelets in % plasma/ % pas- after days of storage crystal stanley , marguerite kelher , nero evero , melissa vongoetz , betsy donnelly and anna erickson* . belle bonfils memorial blood center, university of colorado, cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas- to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct ) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas- and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in % plasma/ % pas- , collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, . . platelets in ml, were collected on the trima apheresis platform in % plasma/ % pas- . a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n , were ml (t) and ml (c) with doses of . . (t) and . . (c). all pc were stored under the same conditions and evaluated on day and day for physical/metabolic characteristics. results/finding: on days and all t and c pc had ph c ! . . the dose recovery for t was % %. on day , t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table ) . conclusion: trima pc in % plasma/ % pas- treated with the inter-cept blood system for platelets using the sv set and stored for days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan , , yang yu , li-ping sun , shu-fang wang , rui wang , lei-ying zhang and deqing wang* . the department of blood transfusion, the pla general hospital, the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d , d and d . we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in % final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. levels of k and lactic acid (la) were tested using automatic biochemical analyzer. k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. treated hips-cms with d ssrbcs, d k and cell culture media for h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: d ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d ssrbcs stop beating, but beating patterns restored at h. hips-cms treated with d ssrbcs stop beating, and beating patterns did not restored at h. levels of k and la in ssrbcs changed most obviously. only d k solution made hips-cms stop beating and can restore in h; d k, d k and la solution did not influence the beating pattern in at the end of the treatment for h, hips-cms treated with d ssrbcs show obvious shrinkage. at the end of the treatments for h, cells treated with d k and d ssrbcs both show obvious shrinkage, the shrinkage in d ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. gene expression array results show a total of genes were differentially expressed in d ssrbcs group compared with naive group. there was no consistent separation within the d k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. further study should be applied to signal pathways on ssrbcs induced cytotoxicity. large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as- as compared to sagm. the presence of citrate in as- seems to be necessary to prevent hemolysis of thawed cells. during storage in as- , atp and , -dpg levels rapidly decline. recently developed additive solutions like pag m and as- have shown to better maintain , -dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag c in which the mannitol of pag m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at - c in pag c. study design/method: leukoreduced rcc (n ) in pag c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at - c. on day , rccs were glycerolized using acp (haemonetics v r , braintree, ma) to a final concentration of % (w/v), frozen and stored for at least two weeks at - c. after thawing and deglycerolization using acp , rcc were resuspended in pag c. during storage at - c, stability (hemolysis), atp and , -dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n ) resuspended in or sagm (n ). results/finding: pre-freeze storage in pag c resulted in increased , -dpg levels at day as compared to storage in sagm, resp. . . mmol/g hb and . . mmol/g hb. hemolysis during post-thaw storage in pag c remained below . % for days and was comparable with storage in as- . in sagm, hemolysis remained below . % for days. during the first weeks of post-thaw storage in pag c, both atp and , -dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag c showed significantly higher atp and , -dpg levels compared to as- or sagm. while in sagm and as- , , -dpg levels were undetectable after days post-thaw storage, in pag c, , -dpg levels only decreased to . lmol/g hb after days of storage. conclusion: pre-freeze storage in pag c resulted in increased , -dpg levels. as compared to as- , post-thaw storage in pag c showed comparable hemolysis while atp and , -dpg levels were much better maintained. based on a maximum allowed hemolysis of . % and an atp content of > . mmol/g hb, thawed rcc can be stored at - c for days in pag c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold ( c, c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n - ) in % iso using a trima or in % int using an amicus and stored for days at rt and c. samples were tested on day (baseline, bl), , , and of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at % hct. labeled blood was perfused through microfluidic channels (fluxion) coated with ug/ml type- collagen at s - shear rate. images were acquired every sec for min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p< . . results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day of storage compared to bl (bl: . . %, rt: . . %; p< . ). c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day (p . ) and compared to c-int by day (p< . ). conclusion: our work suggests that c storage of plts collected with a trima ap system in iso for up to days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at c. these results are surprising since both c-int and c-iso have been shown to express similar levels of cd p, pac- , and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table . comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after -week storage between unirradiated and irradiated groups (n ) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone ( mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin ( mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th cells and increase in cd regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th ,th ,tfh and tfr cd subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* , sarah kloss , sara crew and sandra j nance . american red cross, american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from plasma and serum clinical specimens. group contained a single hpa alloantibody specificity with or without hla antibodies (n ). group included specimens with hla antibodies alone and group consisted of patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa- , hpa- , hpa- , hpa- , hpa- , gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is % concordance observed for hpa- a, hpa- b and hpa- b antibodies. the pak-plus assay had difficulty discriminating hpa- b from hpa- a antibody when hpa- a antibody was present ( false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa- a when compared to hpa- b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within % of the cut-off for pakplus and < . adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut overexpression on cell motility. results/findings: fut overexpression increases both ley expression and cell migration, while fut knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac . as tumor promoter, hdac becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n ) and stored at c for up to days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means sem, and paired student's t-tests were used to determine statistical significance (p< . ). results/finding: on day , p-selectin levels were significantly higher in pre than bl (p . ). mirasol treatment caused a significant increase in pac- expression compared to pre (pre: . . %, post: . . %; p . ), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post- samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after day storage at c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day c-stored pas plts followed by incubation ( minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* , arthur p. bode , anne s hale , michael stanton , mark johnson and g. michael fitzpatrick . cellphire, bodevet, inc, cellphire, background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to , , and . % of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control ( -day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to % of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of % and % of the tcpc produced a significant decrease in blood loss. the lcps at % and % tcpc were as effective in mitigating blood loss as -day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to % of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of % and % of the tcpc reduced blood loss. these results suggest a starting dose above . % tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as % tcpc had similar efficacy signals as % tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso c. the study on pcr-ssp technique for the genotyping of cd - del.ac mutation and the genetic polymorphism of cd - del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd (platelet glycoprotein iv, scarb ) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd deficiency in china. cd gene mutation is the main reason that leads to cd deficiency. cd - del.ac (frameshift at aa ) mutation is one of the cd mutations that causes cd deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in pediatric patients who received both doses of the mmr vaccine at and months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are ! % for all mmr components. results/finding: table shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged . years ( . to . years). thirteen patients ( %) were chronically transfused at the time of serology. twenty-three patients ( %) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper , , franklyn cladis , richard saladino , darrell triulzi , barbara a gaines and mark yazer* . university of pittsburgh, children's hospital of pittsburgh of upmc, institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients ! years old and ! kg with evidence of hemorrhagic shock were eligible to receive up to cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (< ) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately months, trauma patients received wb: group o and group a recipients, % male, median (iqr) age was ( . - ) and % blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of ( - ) and % mortality rate. the median (iqr) quantity of wb transfused to group o recipients was . ( . - . ) ml/kg versus . ( - ) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean standard deviation haptoglobin concentrations for non-group o recipients was . . mg/dl on day , . . mg/dl on day , and . . mg/dl on day ; the corresponding haptoglobin concentrations for group o recipients were . . mg/dl, . . mg/dl, and . . mg/dl, respectively (p> . for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n ) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n ). the mean standard deviation platelet volume administered was cc for whole blood recipients versus cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) with anti-human igg only, and a positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a minute c incubation, followed by automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer and the breast milk showed anti-d with a titer between and . the patient had a consistent plasma anti-d titer of . the patient's mother chose to stop breast feeding after weeks, and the patient's hemoglobin was improved at and weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that % of c scd patients from the west indies and west and central africa are partial c and at ( %) risk for alloimmunization to the c antigen through transfusion of c rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce( g, t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce( - )-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of patients with genotype/rh phenotype data available, ( . %) were c antigen positive serologically. the allele frequency of rhce*ce( g, t) was . . in total, ( . %) patients possessed rhce*ce( g, t) in the absence of conventional c gene in trans. of the c antigen positive patients, individuals ( . %) were predicted to be partial c based on four molecular profiles [rhce*ce( g, t)/rhce*ce: ; rhce*ce( g, t)/rh*ce: ; rhce*ce( g, t)/rh*ce( g): ; rhce*ce( g, t)/rh*ce( g, t): ]. in these partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after transfusion exposures ( c-antigen negative units; mean: , range: - ), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce( g, t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* , megan l nguyen , melanie c proctor , david e krysztof , gregory a foster , erin k sash , sandy s dickson , joua yang , jeffrey m linnen , kui gao , jaye p brodsky and susan l stramer . american red cross, grifols diagnostic solutions inc., grifols diagnostic solutions, inc, quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on / / (fl, ga, sc, ms, al). following revised guidance on / / , testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on / / . travel history questions were discontinued on / / . confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of / / , , , donations were tested including , ( %) in , mps. no reactive donations were identified by mp-nat. of the , , id-nat donations, were initial reactive (ir) of which ( %) confirmed positive (cp) by subsequent testing (cp rate of : , ; positive predictive value of %; specificity of . %). five ( %) cp donations were id-nat repeat reactive (rr); ( %) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and in fl, of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from to days prior to donation. two donors with a travel risk reported clinical symptoms; cp donors ( %) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than copies (c)/ml to about Ê c/ml. at the time of writing, the longest period of detection in rbcs was days vs. days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from ir and all rr donors, ranging from to c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of were prepared by diluting nat plasma : and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n ) were sorted into categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm /high vl; igm /low vl. results/finding: of , donations collected april -december , were reactive for zikv rna. igm-index donations had higher vls (mean . x vs . x iu/ml) and higher proportions of simulated mp-detectable results ( % vs %) than igm donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm donations increased (table ) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r / systems lisa lee pate* , phillip c williamson , michael paul busch , susan rossmann , scott jones , ann butcher , john duncan , jean stanley and susan a galel . roche molecular systems, inc., creative testing solutions, blood systems research institute, gulf coast regional blood center -sugar land, qualtex laboratories background/case studies: in february , the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march , and testing of puerto rico donations began on april , . as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august , the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april , -february , using the investigational cobasv r zika for use on the cobas v r / systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a : dilution to simulate mini-pool testing. results/findings: a total of , , blood donations were screened using cobasv r zika. of ir donations, were repeat reactive (rr), non-rr and had no repeat testing. of the rr donations, were positive by altnat; of these were igm positive. all altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the rr donors that were not igm positive on index, enrolled in follow-up and all seroconverted. of non-rr donations, were altnat negative and is pending supplemental testing. / donors were igm positive on index. donors were igm negative on index; / enrolled in follow-up; remained igm negative and was gm inconclusive. of donations without repeat testing results, met criteria for positive ( was altnat positive, igm negative and altnat negative, igm positive). donation is pending additional testing. altogether, / ir donations met the criteria for true positive on the index donation. / ( %) true positive donations were reactive when retested in a simulated minipool. / were igm positive. conclusion: . % of the , , donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* , marc germain , gilles delage , maria esther lopes and yves gr egoire . hemorio, hemaquebec, h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia ( ) , and in brazil ( / ), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since % of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january st , through november, th , , from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: days, with % of the values lower than days); % of infected donors with symptoms lasting days; . donation/donor/year for wb and . for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x ( -proportion of refused donors) x ( proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain : , , for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from donations). the residual volume of mp plasma, . - . ml, is routinely discarded. beginning in april each blood center saved $ mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april - ) mp were combined into pools of donations; thereafter mp were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and % confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity ( % limit of detection < copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first months of samples is complete for , mp, comprised of , donations collected from april to october , . a total of pools were positive, with detected between april-june . the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over . % of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over . % of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in , zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* , sze sze chua , mars stone , michael paul busch and ai leen ang . health sciences authority, blood services group, blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on august . the numbers rose rapidly to cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since october . zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using blinded frozen samples consisting of replicates of half log dilutions of the who international standard for zikv and replicates of negative controls prepared by bsri. probit analysis was performed to determine the % and % limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a member blinded zikv reference panel from the usa-fda. results/finding: a total of , donations were screened from october to march , with false positive case and zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of . x copies/ml. zika igm was negative in the index donation sample but present in the -day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be . copies/ml at % lod and . copies/ml at % lod. the procleix zikv assay detected rna in out of patient samples and provided . % agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of in , donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types , and express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to reactivity on initial gel testing. if genotyping demonstrated weak d types , or , the intent was to manage the patient as rhd-positive. if weak d types , or were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in to weeks. results/finding: rhdgenotyping was performed on patient samples over months. of these patient samples, ( %) were weak d types or . the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types and patients have not received transfusions at this institution since they were genotyped. four of obstetric weak d types and patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this month study period serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using fda approved anti-d reagents. when reactivity with all reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of months we tested rhd-negative blood donors. there were ( . %) partial-d, weak d ( . %), and ( . %) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs - _ deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these donors showed that rhd-negative recipients received rbcs from of these donors. five of these recipients underwent antibody screening after an average follow-up period of months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which % of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d " with reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october to march , we performed routine d typing (neo, immucor) on obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of using at least antibody. solid phase and manual testing used the series and series reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh ) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all samples. two of ( . %) were d with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs - c, rs ; ivs c, rs ; and ivs a, rs ). two ( . %) were d and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four ( . %) patients had rhd alleles with known potential to make anti-d (rhd*dol , rhd*dar . , and with weak d type . ). one had weak d type , which has uncertain susceptibility to alloimmunization and one was weak d type , which has not yet been associated with anti-d. interestingly, two ( . %) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type is a variant of the rhd protein that comprises an amino acid substitution located in the th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c. g>c which is the first nucleotide of the exon of the rhd gene and thus could be implicated in exon skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v (agilent) and the nextseq platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of patients previously characterized by beadchip technology. interestingly, out of carry the c. - c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last patients, one has been previously characterized as rhd weak type carrying the c. g>c (p.gly ala). independently, sanger sequencing on unrelated rhd weak type samples pinpoint to a linkage disequilibrium between c. g>c (exac, maf . ) and the c. - c>t (exac, maf . ). in silico analysis of both mutation located close to the splice acceptor site of the exon does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon , mutant rhd c. g>c, mutant rhd c. - c>t and double rhd mutants c. g>c plus c. - c>t, we showed no influence on skipping of exon due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type between ala (transmembrane helix ) and val (transmembrane helix ) hampering membrane insertion. conclusion: the c. - c>t variation is always associated in cis with the missense mutation c. g>c on the allele rhd weak type . the c. - c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type red blood cells is due to the substitution of alanine at amino acid position to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has transmembrane (tm) and tilted ureapore a-helices, a long extracellular connector segment, and cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p. . we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and - aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, ) . results/finding: seven snmvs located within amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all at the exofacial ends (p.a t, p.w r, p.v d) are jk-weak; the two jkneg exceptions p.g e and p.g e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v m, p.e k, p.v i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the jk-neg variants are within aa (p. -p. ) of jk a/b at p. . none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n s and p.s p are adjacent to p. f and p. l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc a gene, which encodes the urea transporter ut-b . the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in . in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc a gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within aa from tm a-helix end v i, a t, w r, w r, g e, g e, v d* cytoplasmic n-terminal v m, g s, e k, l p in membrane tm and urea-pore a-helices r w, r q, g d, i t, a v, l r, a t ‡, a a §, l f, n s, s p, t m / * second nucleotide variant in this allele is synonymous (p.p p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a mbp region in q . - . with an lod score of . . using deep sequencing, we identified a potential deleterious mutation in the znf gene, which deletes bp resulting in loss of an entire zing finger domain. the identical del -znf mutation is present in all affected individuals, and is absent from all controls tested (n> ). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf del . none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc a gene, is a urea transporter that has been associated with renal function, we found that people with the znf del in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf , prevalent in southern spain due to a founder mutation, leads to ut-b dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-( -ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis( -ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at days and year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the pools included group a, group o and group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than - c within hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day (pool), day , and year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day , day , and year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp . ppm; mehp . ppm; deht . ppm; and meht . ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than % of its initial value. plasma stored in deht bags had an average plasticizer content % lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at g to separate rbcs from platelet-rich plasma (prp). prp was diluted -fold in pipes-saline with . mm pge and centrifuged at g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of - % and , - , platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted : (spdp %) with plasma from a patient with type vw disease (t vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of s - for seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was % (spdp/ffp > . ). results/finding: six batches of spdp/ffp were evaluated using subjects. there was no statistical difference between the spdp/ffp pairs (p . ). the mean ratio of spdp/ffp was . with a % ci of . - . . comparing spdp vs. spdp %, there was no difference (median ratio . , range: . - . ) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was % greater than in samples reconstituted with ffp. the lower limit of the th % ci is a difference of %, which is less than the a priori determined margin of noninferiority of %. even with % dilution with t vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* , qiyong peter liu , grantham c. peltier , ryan c. carney , ashley s. taylor , colby s. mcintosh , james a. bynum and andrew p cap . u.s. army institute of surgical research, velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: ( ) ffp; ( ) ffp with mm glycine; ( ) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; ( ) spdp pretreated with glycine-hcl ( mm); and ( ) spdp pretreated with glycine-hcl:glycine ( mm: mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood ( % hct with platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < . ). fibrin polymerization density was slightly diminished in rspdp vs. ffp ( . vs. . o.d., p < . ), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < . ). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < . ) and an additional twofold in pretreated spdps vs. rspdp (p < . ). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < . ). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples ( . % surface coverage vs. . - . %, p < . ). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* , anita tuip-de boer , ruqayyah almizraq , jason p. acker , philip j. norris , jennifer a muszynski and nicole juffermans . academic medical center, university of alberta, canadian blood services, blood systems research institute, nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from donors (blood type a or b). supernatants were prepared after - (fresh) and - days of storage (stored) for measurement of thrombin generation and ev analysis. a type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to % stretch using a cellstretcher. control cells were not stretched. after hours, il- and il- production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il- and il- production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p< . ) . incubation of stretched cells with stored wbf products resulted in higher il- production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by different methods from individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after - days (fresh) and - days (expiry). monocytes were co-cultured in media plus % rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table ) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin- production was higher after exposure to fresh wbf ( % control, p . ) or wbd at expiry ( % control, p . ). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* , alessandro tocchio , anita howell , kaushik sridhar , jason p. acker and utkan demirci . stanford university, canadian blood services, centre for innovation, background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of - g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at , , , , and days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from volunteers with four different age and sex categories (male, - years; male, > years; female, - years; female, > years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young ( . g/ml) and older female donors ( . g/ml) (p < . ). moreover, rbcs from young males ( . g/ml) were significantly less dense compared to rbcs profiled from older female donors ( . g/ml) (p < . ). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il- (pg/ml) il- (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p< . ). substantial correlations were also found between orp and free hemoglobin (p< . ) and orp and free heme (p< . ). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections vs (p< . ). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than days compared to rbcs stored for days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of to days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to days storage duration -reference group), medium age (at least rbc of - days storage), and oldest (at least rbc greater than days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every days, and b) a finer partition using cut-points every days. results/finding: , patients receiving , rbcs were included in the analysis. exposure to rbcs stored for more than days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for days or less) after adjusting for several fixed and time-dependent potential confounders (hr . ; % ci: . , . ; p . ). exposure to blood stored for at most - days yielded a similar hazard ratio (hr . ; % ci: . , . ; p . ). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than days compared to exclusive exposure to rbcs stored days or less was not significant (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . similar findings were obtained with partitioning exposure data into day intervals where exposure to rbcs stored for - days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for - days (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . conclusion: individuals exposed to rbcs stored for more than days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* , cynthia walser , tatsuro yoshida , andrew dunham and pedro cabrales . university of california san diego, new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o saturation < %) stored rbcs, or anaerobic/hypercapnic (o saturation < % and pco (@ c) $ mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as- after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either ) conventional; ) anaerobic; or ) anaerobic/hypercapnic conditions. rats ( - g) were hemorrhaged to % of blood volume, held in hypovolemia for minutes, and resuscitated to recover blood pressure to % pre-hemorrhage with prbc stored for either or weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. ( %) neg patient showed short term response and ( %) patients showed progressive disease. at the neg group standard eval ( %) patient showed response and ( %) had progressive disease. ( %) neg patient had long term response compared to ( %) pos patients. at the pos short term eval ( %) patients showed response and ( %) patients had progressive disease. at the pos group standard eval, ( %) patients showed response and ( %) patients had progressive disease. overall, ( %) pos patients responded compared to ( %) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd neutralizing substance could play a role in treatment response. alternatively, reduced cd expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a -hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate % replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of . m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* , laurie sutor , , germ an leparc , marjorie doty and william crews . carter bloodcare, ut southwestern medical center, oneblood background/case studies: anti-cd drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with . m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and . m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual ( th edition). each of the plasma aliquots was further separated into aliquots and stored at - c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. ( ) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than with the untreated or dtt-treated cells during the study. conclusion: long term storage of . m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque- ), pooled, suspended in cryopreservation media ( % dmso; : ) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips ( c, % co , h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o ) rbcs sensitized with either anti-d (positive control), anti-scianna- (sc ) or anti-anwj or lipopolysaccharide stimulated for h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/ monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed . % viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il- b, il- , il- , mip-a (p < . ), mip-b and gro (p < . ) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc -and anwj-sensitised rbcs resulted in a pi of . % and . . % respectively vs anti-d sensitized rbcs (pi: . %). a weak ( ) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi> %). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in patients, involving antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all patients had - positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients and typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c by hea precise-type. ega-treated rbcs gave reactions with the same anti-c reagent. patient rbcs gave variable reactivity (vw- ) with bio-rad seraclone and ortho bioclone anti-c. patient rbcs gave reactivity with all anti-c reagents when incubated for the maximum incubation time allowed. patient rbcs were jk(a ) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients and tested s with bio-rad seraclone anti-s ( - ), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat rbcs but not all manufacturers include reagent limitations regarding testing of dat rbcs. we describe cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and cases with false positive tests with anti-s (n ) and anti-jk a (n ) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with - positive dat and supports testing to dissociate igg from rbcs strongly dat before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/ opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r : prob(ab|al-loexp), so that prob(ab) prob(ab|alloexp)*prob(alloexp) and r ; rewriting prob(alloexp) prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar months before ( / - / ) and months after ( / - / ) was selected; for state b, a similar months before ( / - / ) and months after ( / - / ) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (< months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a -and -fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from to (state a) and to , (state b), which annualized, represents a potential gain of , (state a) and , (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from - , there were donors identified who had changed their gender from their birth gender; female donors changed their gender to male and male donors changed their gender to female. there were ( %) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to . ml/kg or % of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the % limit. variable volume scales [vvs] can be programmed to vary unit volume (up to ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by ml at ebvs < . l in donors ! yo, but increase by - ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the mos. before a mo. phased implementation of the vvs, and the subsequent mos. multivariable analysis [mva] by -mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods & , continued during impl and post-impl periods & , returning to the baseline rate in post-impl periods & (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods & . the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of . ml during post-impl periods & from the temporally matched baseline & pre-impl period . conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* and yves gr egoire . hema-quebec, h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, . the vaccine is produced with live and attenuated yfv, which can circulate for at least weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received , blood donors candidates; from those, , were accepted as a blood donor, after medical interview. the deferral rate was . %. at the same period of the year , there were , prospective donors, and blood donations. the deferral rate was . %. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, , additional donors, compared to same dates. that represents a . % increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from . % in to . % in . conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of ! . x is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: , apheresis collections from centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of . x for single (s), . x for double (d), and . x for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or % plasma) assuming i) a minimum dose (allowing for production loss) of . x for s and . x for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as %) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc rbc plt/p plt plt/rbc/p plt/rbc plt plt/rbc plt/p #donations citrate exposure (mls) - study design/method: a randomized ( : ), placebo-controlled, single blind, subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into cohorts, receiving increasing doses, ranging from / , - / of the lowest effective dose found in the above rabbit model. cohorts and received the / th dose, but cohort received two / th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for hrs post infusion and followed for up to days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of aes: were treatment emergent (teae), of which were treatment-related ( thrombosomes and control). all teaes were mild or moderate in severity. in cohorts and , / thrombosomes subjects had treatment related adverse events. one cohort subject developed an upper respiratory infection and elevated wbcs within hours post infusion, which resolved by hours, and an elevated d-dimer at hours post infusion, which resolved by day . this subject also had an elevation of prothrombin fragment at baseline, which increased post transfusion and peaked at hours with resolution by day . one cohort subject developed non-specific t-wave changes at and hours following her nd infusion that resolved by day without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort subject developed an igg platelet autoantibody on days - , which was undetectable on days - ; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days - , and negative on days - . background/case studies: cryopreservation of platelets (plts) could extend the shelf life from - days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with % dmso and stored at c. after thawing, the unit was reconstituted in thawed ffp spiked with either lm puromycin (pm) or nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after , and hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x- containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by -fold during hour storage. immunoblot analyses of the plts showed a -and -fold increase in pm incorporation after and hours of storage, respectively. massspectrometry revealed unique proteins that were synthesized after hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac , rap and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac , rap and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in , the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r - cooler with units of ffp, units of rbcs, and unit of whole blood. three to ml of platelets were collected via syringe from each unit at min (before storage in cooler or refrigerator) and after . , , , , and hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding) were measured by coulter counter, channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p< . deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c -a h molecular sieving: beyond genotyping ghazala hashmi , reinhard klemm and michael seul* , . biomolecular analytics, immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi http://bit.ly/ ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, * * samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for sensitized sickle cell anemia ("sca") patients (tb in cas-tro , http://bit.ly/ oplxhr, excluding le and e(variant) and assuming request per patient), presenting with up to allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only = plate holding * candidate units from actual black donors, followed by profiling of samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for of requests ( . %), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining pooled samples produced additional assignments to a second set of requests, for a total of assignments from only wells. in another scenario, sieving of a full plate of * samples, produced $ assignments for two successive batches of requests from sca patients, a yield exceeding . x. sieving alone typically fills - % of requests of moderate complexity ( ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez , monica kalvelage , ghazala hashmi* and michael seul . biomolecular analytics, lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou http://bit. ly/ ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including at the rhce locus) that encode mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag " (e.g. e ,c ,e ,c ) to specific combinations of "ag " (e.g. c e k fya and c e jsa ) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the c samples, that were also v and vs and, among the e samples, that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select specific "ee" pools of which were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b ( pools), co a|b ( ) and others. conclusion: molecularsieving of a single -well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c , e and jsa . these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam (landsteiner-wiener) and ackr (duffy). for longer genes, such as abo of > kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of > kb each was used for all physically confirmed ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v . . was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least clades representing clusters of to alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors ) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons - , and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors . a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos (b galnt ) cells, and cell-surface expression of fors antigen was immunologically monitored with a monoclonal anti-fors antibody. results/findings: we found that met thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon or of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met thr/ ser or exon / deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt genes is reminiscent of common ancestral origin of alpha , -gal(nac) transferase genes. the finding that at can synthesize fors implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of , cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period / / - / / . abo genotyping targeting specific snps for groups a, a , b, o , and o and, if needed, gene sequencing was conducted in cases with indeterminate results, and in cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two ( . %) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in % the predicted abo phenotype was a rh neg (table a ). the predominant donor race was caucasian ( %). four cbu with abo discrepancy were also evaluated by genotyping (table b) . in of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r / systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: , fresh and , frozen edta plasma samples from american red cross donors, collected from february - , were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of , valid results, a total of donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a -year old male in indiana, a -year old male in california, and a -year old female in kentucky. all donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna ( iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as a, the california donation genotype b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was % ( % exact ci: . % to %). conclusion: based on the confirmed-positive donations of , tested, the hev prevalence was . % ( % exact ci: . % to . %) with a detection rate of : , ( % ci, : - : , ). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than % of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using % confidence intervals. this analysis contains data from / / - / / . results/findings: among , , donations reported ( . % from firsttime and . % from repeat donors), there were respectively , and cp results for hbv, hcv and hiv with corresponding rates of . , . and . per , (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of : , : and . : for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv . vs . ; hcv . vs . ; hiv . vs . ). in general, higher rates for all markers were seen among minority donors, those in the - -year age group (also - year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when -month periods were compared. conclusion: data from major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* , melanie c proctor , deanna self , monique portugal , adrian gurrola , laura tonnetti , sonia bakkour , cheryl lobo , michael paul busch , susan l stramer and jeffrey m linnen . grifols diagnostic solutions inc., american red cross, blood systems research institute, new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening , unlinked whole blood donations collected from august th to april th in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of . results/finding: the procleix babesia assay detected all four babesia species with a % lod ranging from . - . copies/ml. the preliminary % lod in parasites/ml ranged from . - . p/ml for b. microti (n ), from . - . p/ml for b. duncani (n ), and from . - . p/ml for b. divergens (n ). of the , donations screened, initial reactive and confirmed positive donations were identified for specificity of . % ( %ci: . - . %). of the confirmed positive specimens, were reactive by both ifa and pcr, by ifa only and by pcr only. all confirmed positive samples were reactive in lysate pools of . donors of reactive donations resided in ct ( ), nj ( ), nh ( ) and me ( ) for an overall incidence of : , , and : , in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* , whitney r steele , ed p notari , james haynes , roger y dodd and susan l stramer . american red cross, american red cross (retired) background/case studies: from - , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from - . study design/methods: prevalence was calculated in -year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of . , . and . days for hbv, hcv and hiv, respectively. linear regressions were calculated with p< . (*) as significant. results/findings: from / / - / / , there were more than million donations from , , donors ( . % female, % first-time (ft), . % caucasian). there were significant decreases in donation prevalence for hbv and hcv (p . and . ), but no significant decrease in hiv during the years (see table for f and r values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p . and . ). prevalent ft donors were significantly more likely to be male ( . % -hbv, . % -hcv, . % -hiv; p< . ). incidence for all agents declined (significant only for hbv; p . ). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last -year period ( in - vs. in - ) . hcv incident donors in - were more likely to be male ( . % vs . % in - , p< . ) and were younger ( . % vs. . % in - < years, p . ). overall, incident donors were more likely to be caucasian males (p< . ). rrs for all agents decreased over time with rrs in - of in , , ; in , , ; and in , , for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the -year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in , mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as- rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with lm amustaline, and incubated for hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, > . log , or > . log pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was > . log , or > . log pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts- inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts activity of < % and high inhibitor ( . - ). mean age of cohort . years (range - ). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean . x /l, range - x /l) and low a-ipc (mean . x /l, range . - . x /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab mg/m ( patients) and cyclophosphamide mg/m (one patient). tpe continued until platelet count reached x /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of . days [range - days]) when they achieved a three-fold increase in a-ipc from baseline (mean . x /l, range . - . x /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean . x /l, range - x /l) and a-ipc (mean . x /l, range - . x /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of . days (range - days) mean platelet count was . x /l (range x /l) and mean a-ipc . x /l (range . - . x /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of . days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* , michelle n stram , joan sevcik , alesia kaplan , and joseph e. kiss , . department of pathology, university of pittsburgh medical center, blood systems inc., university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within - hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january , to november , was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts- activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the ttp patients identified, were excluded due to missing temporal data for important variables. the majority ( %) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients ( %) had a prior history of ttp and % had severe adamts deficiency on admission. the median time from tpe request to initiation was . hours (interquartile range: . - . hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table ) . treatment was not started within an -hour window in patients; the median time to cv access was significantly longer in these patients ( . vs . hours, p< . ). two of these patients had a prior history of ttp and only four patients had severe adamts- deficiency. the majority (more than %) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table ) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus - hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi- a transfusion vol. supplement s hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, through january, , we performed cytapheresis (cy) treatments (txs) for pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. pts ( %) had cml-at and received leukapheresis (lp) txs; pts ( %) had et and received thrombocytapheresis (tc) txs. cml-at pts presented with median wbc x /l (range - x /l), of which % had blast percent > % or blast count > x /l. median age was years ( - years); % were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. % of cml-at pts had no sxs of lks; % pts had sxs of either cns or pulm lks ( sxs), and % pts had sxs of both cns and pulm lks ( sxs). et pts presented with median platelet (plt) count of: x /l ( - x /l)and % pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was years ( - years); % pts were male. results/finding: all pts received a course of cy tx with following objectives: ) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and ) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) < x /l for cml-at pts, and plt ct < x /l for symptomatic et pts and < x /l for asymptomatic et pts. cml-at pts received median of lp txs (mean . txs/pt; range - txs). et pts underwent median of tc txs (mean . txs/pt; - txs). outcomes were evaluated by percentage of pts who: ) reached wbc (or plt ct) tx goal, and ) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved > % reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, % pts improved, % pts stabilized; and % pts worsened. in et cohort, % improved, % stabilized, and % were unchanged. for cml-at pts, median final wbc ct x /l (range - x /l); % pts received ind chemo. for et pts, median final plt ct x /l ( - x /l); % pts had resolution of thrombotic a transfusion vol. supplement s symptoms. % of cml-at pts and % of et pts expired within - days after course of cy tx. of expired pts, pts had both blast crisis and sxs of cns/ pulm lks; pt had intracranial hemorrhage or cva; and pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median - txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used % albumin or % albumin- % normal saline ( / ) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered ( % albumin vs / ), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where % albumin was used versus those that used / . covariates included were fluid types, age and gender. odds ratios (or) and % confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < . . results/finding: during the study period, procedures were documented for subjects ( % female), age range - years, of which , ( . %) received / . the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with % albumin had a significantly lower risk of having either event than by using / , [p . , or (ci): . ( . , . )] , and also had a significantly lower risk of causing hypotension [p . , or (ci): . ( . , . )] in addition to a lower risk of causing citrate toxicity [p . , or (ci): . ( . , . )]. age had a significant effect on having a hypotensive event [p . , or (ci): . ( . , . )] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a year period and compared it to published literature. study design/method: we conducted a -year retrospective study of ta procedures performed and aes were classified according to criteria described in table . during the study period, ta were performed using cobe spectra (software versions . and . ) and since the spectra optia apheresis system (version . ). literature search was conducted for data published on aes associated with ta. four studies from us and non-us studies (canada, europe and japan) were analyzed. trend for ae rates from - was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was . % ( of , procedures) during year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher ( . %, p< . ) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho . , p . ) over the years and significant down trend of moderate and severe aes with a spearman rho of - . (p . ) and - . (p . ) respectively. there were no fatalities during the study period. majority of aes were grade i ( %) and grade ii ( %): / ( . %) procedures were not completed due to aes. comparison of aes [ . % ( / , )] to both european [ . % (n , , / , ) ] and other us studies [ . % (n , / , )] showed a statistically significant difference (p< . ). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table ) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether platelet donors with a donation activity of up to platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* , jordy jurgens , jacoline buchner-doeven , joris roelofs , philip spinella , jennifer a muszynski , carel goslings and nicole juffermans . academic medical center, washington university school of medicine, nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells ( days old) and platelets ( days old) by washing. plasma was filtered through a . um filter. rats ($ grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $ % of their estimated blood volume, which was calculated to be ml/kg. hemorrhage continued until a mean arterial pressure of mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of ml/kg of blood products in a : : ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april , which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and -day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january and july (n ). because of missing data on patient characteristics, patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january to march , n ) and after (april to july , n ) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of baseline variable, generating pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common ( . % vs . %, p< . ) and ventilation time was longer ( h vs h, p . ) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group ( ml vs ml, p . ; and ml vs ml, p< . , respectively). however, -day mortality was not statistically different between the groups ( . % vs . %, p . ). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april , www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over , joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy . length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of -factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: -factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in . marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p . ) and ptt (p . ) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p . ) and plasma (p . ) after off-label use was significantly greater than on-label use. cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were . times (p . ) more with cell saver or anh, and . (p . ) times more with cpb. post-pcc thromboses were identified in cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to days. the transfusion service medical director reviews the case and gives final approval. we observed only patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight ( ) patients were in-patients continuously until delivery. five patients were discharged prior to delivery- moved to another state, was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was days (range - ). six ( ) patients delivered within days of approval. after approval, the mean number of additional specimens per patient was . (range, - ). no patient required transfusion prior to delivery. five patients received transfusion of at least rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only patients delivering within days of approval for extended specimens, patients avoided collection of at least specimen each, and patients avoided at least collections each. since new antibodies are not detectable for at least days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* , , jan m. binnekade , benjamin nota , pieter r tuinman , kirsten van de groep , olaf l cremer , janneke horn , marcus j schultz , robin van bruggen and nicole p juffermans . academic medical center, sanquin research and landsteiner laboratory, vu university medical center, university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in tertiary icus in the netherlands comparing patients who developed ai during icu stay with control groups: non-anemic patients with sepsis, non-anemic patients without sepsis, and patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron ( . vs. . mmol/l, p< . ) and transferrin saturation ( vs. %, p< . ), and low ferritin ( vs. mg/l, p< . ). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately . x cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to %, whilst leaving % of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than % of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately % of the cd fraction post separation and freeze thaw (table ) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table ) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd cd ) and early projenitor cells expressing oct and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: . routine recovery of the wcf at levels higher than current methods, independent of volume. . higher percentage recoveries of all cell types tested than can be achieved with existing methods. . markedly higher post-thaw recovery of viable nucleated cells than any current methodology. . almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving > . x lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd cell target of . x /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf plerixafor (g pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< . considered significant. results/finding: no alc and alc collections occurred among the patients. fenwal amicus was used for % of the no alc and % of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was hodgkin's and non-hodgkin's lymphoma (no alc); hodgkin's and non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc . , lymph . x /l) than alc (wbc . , lymph . x /l). equivalent whole blood (corrected for ac) was processed for no alc ( . l) and alc ( . l). for alc group, extra collections beyond cd target were: days: %, day: %, days: %, days: %, and days: %. significantly more patients were mobilized with g pl in no alc group (n ) than alc group (n ) and collections in alc group had mobilization discontinued after cd cell target reached. there was no significant difference in g ( . x lymph) compared to g pl mobilized collections ( . x lymph); both were significantly higher than the collections where mobilization had been discontinued ( . x lymph). days to wbc engraftment ( . no alc vs . alc) and platelet engraftment ( . no alc vs . alc) were not significantly different. median number of collections for no alc ( ) and alc ( ) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the . x lymph/kg or even the . x lymph/kg targets. implementation of a lymph target increased patients obtaining . x lymph/kg from % to %. only % had < . x lymph/ kg. discontinuation of mobilization once cd cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* , and nicolas pineault , . canadian blood services, university ottawa, canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost hours at room temperature (rt) as long as units are cryopreserved by -hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n ) were split with one half processed immediately (baseline - hours) and the second after hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd cells and cd cell (n ). primary nsg mice were transplanted with a ucb cell dose that contained a total of , annexinv neg viable cd cells. the latter was done to avoid any bias towards one group or another. short term platelets ( vs. hplt/ml, p . ) and leucocytes ( . % vs. . % hcd , p< . ) engraftment at -weeks were significantly reduced in stored mice vs. baseline (n ), and similar results were observed long-term at -weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated months post-transplant. strikingly, the frequency of human cd bm cells was -fold greater in baseline vs. stored mice (p< . , n ). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured -weeks post-transplants were reduced by % in unit , and by % in unit . conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($ mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to %, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of sec). total lymphocyte recovery was % and monocyte concentration was reduced %. furthermore, in a two-pass process platelets were reduced by %. in a -fold parallel system we tested rbc separation from plasma and achieved % separation at ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from clinical protocols from - . an infusion reaction was defined as any symptom from the time of nk cell infusion up to hours afterwards. a severe reaction was defined as any symptom with grade or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r . . . two major endpoints of interest were: ) infusion reaction with any symptom and ) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of nk cell infusions. there were ( %) patients with an infusion reaction of any symptom and there were ( %) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . ). infusion rate (ml/min/kg) was also similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . respectively). incubation of nk cell product overnight in il- vs il- had similar reaction rates for those with any symptom ( % had reaction with il- , % had reaction with il- , p . ) and those with severe reaction ( % had severe reaction with il- , % had severe reaction with il- , p . ). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median . x ) versus those without (median . x , p . ). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan , maryanne c herzig* , barbara a christy , james a. bynum and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at - . mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at - , cells/ well and cultured in well plates for - h in their respective medias. on day , mscs were washed, resuspended in pbmc media and incubated with or without , freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, - lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by h, with > fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was . %. inter-assay variation of msc preps run under identical conditions was . %. inhibition of pbmc proliferation was graded from - % over the range msc concentrations therefore an ec of msc cell number resulting in % suppression of pbmc could be determined for each msc prep. this ec however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute % or more of the us blood supply. differences between donors - years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged - were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the / academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml) were estimated for , , and - yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of donors contributed donations. donors were evenly split by gender, % were ft donors, and % were - yo. ft and rpt - yo donors had on average lower ferritin values at enrollment (p<. ), and a greater percentage were iron-depleted than donors - yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged - have sharply higher risk for iron depletion than donors - yo. odds for lf were to times greater in the younger donors, and for ais were -to fold higher. preliminary statistical models indicate yo donors may have greater risk for lf than or yo by to percentage points, controlling for other factors (p . ). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in - yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on / / by a large blood collector. testing was performed on successful - y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < ng/ml in females (f) and < ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations ( months for f and months for m) and counseled to take - mg of elemental iron daily for days. for m and f, a ferritin < ng/ml indicated absent iron stores (ais) and < ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! ng/ml in f and ! ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior months. an appreciable number of donors with no rbc donations in the prior months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* , joan williams , michelle humphries , nancy haubert , ben reynolds , michael phillips , randall spizman , ralph r vassallo , hany kamel , sally caglioti , german leparc , and phillip c williamson . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. study design/methods: over , serum samples from donors aged , and years were analyzed for ferritin levels using the beckman coulter au instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among , adolescents (ages - ; . % female) who donated blood from to . study design/method: abnormal risk factor levels were defined as hba c ! . %, sbp/dbp ! / mm hg and tc ! mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, , ( . %) adolescents had at least one abnormal risk factor ( . % of males, . % of females). of these, , adolescents had isolated abnormal risk factors, and , adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as ! . g/dl for men and ! . g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from blood services worldwide and complete data were available for blood services. deferral percentages for low hb varied from . % to . % among male donors and . % to . % among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with % lower hb deferral rates in men ( % confidence interval [ci] % to %) and % lower rates in women ( %ci % to %). iron supplementation was associated to % lower hb deferral rates among women ( %ci % to %) but there was no evidence of such an effect among men (p . ). each one-week increase in minimum donation intervals resulted in % lower hb deferral rates among women ( %ci % to %) but not among men (p . ). at the % level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: . % of all candidates for wb donation were deferred in continental france in . deferral was significantly more frequent in women ( . %) than in men ( . %), due to anemia in . % of deferred women and . % of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from to weeks. analysis (table) identified main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the previous years. conclusion: the main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, days stored or days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, ng/kg). blood was sampled every hours up to hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from . e (iqr . e - . e ) /ml in the fresh product to . e (iqr . e - . e /ml; p< . ) in the stored product (p < . ), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: cases of taco and matched controls were enrolled from , transfused patients who received , blood components from may until july . taco incidence was case per patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation ( % vs. %; p < . ), experienced longer intensive care ( vs. days; p . ) and hospital length of stay following transfusion ( vs. days; p< . ), and had higher mortality ( % vs. %; p . ). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from to days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july and to routinely extend ap outdate to day since february . this study reports a month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july -january , ap underwent rt on day . day and units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day had a second rt performed. from february -january , ap underwent rt on day with routine outdate extension to days by performing a second rt on day and a third rt on day , as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type ) or repeat rt positive with negative confirmatory culture (type ). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july , , ap were entered into inventory. of these, , ( %) were transfused prior to rt testing. the remaining ( %) underwent rt on day or day . of these ( . %) were rt positive ( type fp, returned to inventory; type fp, discarded), leaving a total available inventory of units tested by rt. of these, ( % of original inventory) were transfused before the end of day and the remaining ( % of original inventory) reached a day outdate. a total of ( % of original inventory) were transfused on day or day . of these, underwent a second rt on day ( rt positives; fp type one and fp type ) and underwent a third rt on day (no positive results). a total of ( % of original inventory) outdated on day . of these, underwent a second rt on day (no positive results). conclusion: to date we have performed rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted % to only %. a total of ap have been tested twice by rt ( on day and ; on day and ) with ( . %) positive results, both of which were deemed fp by repeat testing or culture. a total of units have been tested times (day , day and day ) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with to million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in . contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between - , parasites/ml. each parasite concentration in wb was tested x . an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at c for up to days; platelets were stored at c (rt) under agitation for days and plasma was frozen at - c. aliquots for culture were removed weekly from rbcs, daily from platelets and after days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at c for detection of live parasites for up to weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at c, rbcs from all units spiked with , parasites/ml were positive for up to days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with parasites/ml were positive for up to days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with , and parasites/ml were positive up to days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at c for up to weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* , marion lanteri and larry corash . cerus corporation, scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom ( - ), french ( - , swiss ( - ), and belgium( - hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately . million dlvbc-screened were issued with a day outdate after release into inventory days after collection, and $ . million amotosalen/uva-treated pc were released into inventory on day or , with a to day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored > . million conventional, non-dlvbc-screened pc and recorded str and fatalities. concurrently, zero definite and possible str were reported with , amotosalen/uva-treated pc, significantly fewer than with conventional pc (table ) ( . str per million vs. . per million, p< . ). one definite, possible, undetermined/indeterminate non-fatal str and contaminated "near miss" pc were reported with . million dlvbc-screened pc between and , for a reduced falsenegative rate compared with the prior five years ( . str per million vs. . per million, p < . ). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october for all platelets received at our institution. at time of receipt at the blood bank (day post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at c for three days. results/finding: a total of / , ( . %) platelet products were successfully cultured ( / [ . %] and / [ . %] in october and march respectively). over the -month period, two true positive cultures were obtained (incidence of in platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us . per product tested. the cost per averted case was $us , . conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january and december . the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the -year study period, a total of transfusions reactions were reported, of which were bcptr ( . % of transfusion reactions). of the bcptr, ( %) were associated with apheresis platelets, ( %) with red blood cells, and ( %) with plasma. recipient diagnoses spanned hematologic/oncology (n ), renal (n ), cardiac (n ), autoimmune (n ), and obstetrics (n ). an organism was identified in both the blood product and recipient in ( %) cases; in ( %) cases an organism was grown in the blood product but not the recipient; and in ( %) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in of the cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever ( %), chills ( %), nausea and vomiting ( %), pain ( %) and dyspnea ( %). blood pressure (bp) decreased in %, increased in %; % of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie , jenna lebedev , linda kapp , xiaohong wang , meghan delaney , lay see er and james c zimring* . bloodworksnw research institute, bloodworks nw, university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg -igg ), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all known variants. study design/methods: the heavy and light chain variable regions of an anti-k monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg , igg , and igg had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table ). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg or for any of the monoclonal ahgs tested. monoclonal anti-igg had a blindspot for igg - , due to the shorter hinge region on igg - . no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* , yves dominique pastore and maryse st-louis . chu sainte-justine, hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since , our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of , scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september , ( %) patients had been transfused and had antibodies with known blood group antigen specificity: anti-c, anti-e ( ), anti-hrb, anti-fya, anti-jka, anti-jkb ( ), anti-s, anti-m, anti-sc , anti-leb ( ). seventeen patients ( . %) were either d or partial d. rhce results showed that patients expressed a normal c antigen and expressed partial c. as for e antigen, had a normal antigen, bore a partial antigen and were weakly expressed. fy(a b ) phenotype was found in ( %) patients. a total of genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos , emilia sippert , mayra dorigan de macedo , sheila fatima perecin menegati and lilian castilho* , . hemocentro unicamp, university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa- a, il b- t cytokine polymorphisms, rhag g>a and hla-drb * alleles may predict a good responder phenotype (sippert et al, transfusion ) and that rhag a and hla-drb* alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included non-alloimmunized patients with scd, homozygous for hbs, receiving a range of - rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa- g>a, il b- c>t) and the rhag g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among non-alloimmunized patients, were homozygous or compound heterozygous for rh variant alleles. from those, had rhag a and/or hla-drb* alleles and at least one cytokine polymorphism (tnfa- a or ilb - t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and were confirmed by sequencing. the third sample was found to be rhce*cevs. ,rhce*cebi on sequencing (predicted phenotype v ,vs ). the samples were typed as v (or ce s ) and vs (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[ g]in samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising % of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies ( % of patients) bound young and old rbcs with no apparent prejudice. band- is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band- is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band- to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band- aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band- tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing type i and type ii patients, we found that type i is characterized by percollv r fractions (similar to healthy storage-matched controls) but increased band- tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by - percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band- tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band- suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* , burak bahar , jeanne hendrickson , krystalyn e hudson and christopher a tormey . yale-new haven hospital, yale university, background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam abstract algorithm, e value x - , word size , gap costs: existence exten-sion ). search results were restricted to bacteria and fungi, with a selective threshold of > % identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b - . , r . & p . ); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of alloimmunized patients reviewed, were culture-positive. of these, % of the anti-c/c group ( of patients) and % of the anti-k group ( of patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from - . %. overall, . % ( of patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of > % sequence identity. while . % ( of ) patients reviewed had positive cultures for klebsiella species, . % of these ( of patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed tegs performed on patients undergoing cv surgeries at our institution from jan to dec , . no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at minutes (min) all within reference range), "hypocoagulable" (r> min, k> min, a< degrees, ma< mm) and "hypercoagulable" (r< min, k< min, a> degrees, ma> mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of tegs analyzed, patients ( %) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients ( % vs. %, p . ). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused ( % vs. %, p . ). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused ( % versus %, p . ). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from liver transplants, performed from to in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, olts were performed. a total of patients was submitted to cs. the median age was years (range - yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in , % of the patients. the average meld score was , , and it was slightly higher in the cs group ( , vs , , p< , ) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was ml and mean reinfused blood volume was ml. allogeneic blood transfusion was required in , % patients in the cs group, compared to , % patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group ( , units vs , units, p< , background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july -december was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp ( and products, respectively) however, obp wastage occurred more frequently in the month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations ( versus products). this is skewed by one month when products were wasted due to expiration of product on the floor. cooler-related issues ( ) and products dwelling too long out of a controlled environment ( ) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were . %, . %, and . %, respectively, with a total exsanguination protocol waste rate of . %. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p . ). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d ) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d- (table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* , edward smith , thomas brown , foeks jeremy , metcalf suzanne , james johnson , peter davis , karafa sw badjie and abba zubair . department of laboratory medicine and pathology, transfusion medicine, mayo clinic, department of anesthesia, mayo clinic background/case studies: our institution performs an average of solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused o( ) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the o( ) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in % of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a y.o. female patient should not have received o( ) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with admissions during the hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight ( kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o( ) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o( ) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately , surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from - %, whereas transfusion rates for vaginal and robotic pfd surgeries range from . - . % and . - . %, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately % of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may -may in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p< . . results/finding: we identified patients for analysis, of whom ( . %) had a preoperative t&s ordered. two ( . %) of these patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine ( . %) of the patients required a second abo/rh specimen per hospital protocol; ( . %) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table ) . no abo/rh discrepancies were identified. one patient received unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution ( . % vs. %, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n ) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of . g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs- i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), units were subjected to the volume reduction while recording the time needed to process all units. this was performed twice for a total of units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of % (range %- %). in units between and days (n ), the estimated mean residual k was . meq (range . to . ). in the two mock mtp trials, the time to complete the procedure was approximately minutes and we estimate an additional - minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal ( ) and caesarian ( ) births. uc collections were divided into segments to test conditions. segment explants were placed on . % gelatin-coated gridded tissue culture plates ( explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the remaining tissue segments were soaked in (ab/am) saline solution for hr and hrs at c, respectively. tissue segments were frozen in cryo bags with a proprietary % dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process or times the total blood volume (bv) of the patient, up to a maximum of liters (l) per procedure, to obtain peripheral blood cd stem cells. as a consequence, a patient often would need to spend hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd pre-count and cd yield, normalized per liter of blood processed, was derived utilizing the patient's cd pre-count, the patient's weight in kilograms (kg), and the target cd dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd stem cells. the initial equation was modified to add an additional % to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in patients, representing both allogeneic and autologous donors, the average blood volume processed was . l. the range was . l - . l. the target dose was achieved in all patients. our previous practice for these patients would have required, assuming a standard bv procedure, processing an average of up to l per patient, with a range of - l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd yield. the result was a high correlation between these two ratios (r . ), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r . , confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses ( - ) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june and march . patients (n ) evaluated were diagnosed with malignant lymphoma (n ), multiple myeloma (n ) and primary amyloidosis (n ) and were mobilized according to standard protocols. collection cd cellularity target was established ! x e /kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by > years old, previous fludarabine, lenalidomide, or bendamustine treatments or ! previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v . . results/finding: the media (range) general collection parameters were: cd (day ) . /ml ( . - . /ml), blood volume processed ml ( - ml) and . ( - . ) exchanged volemias. seventeen patients were considered bad mobilizers, needed plerixafor and had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p . ]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n ), fever (n ) and flu syndrome; all grade ]. two patients could not undergo hematopoietic stem cell transplantation due low cd cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd ) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with % fetal bovine serum. bmsc and amsc at passage - were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses ( . /ml, /ml and . /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par ) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within min and hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: seconds, versus low, medium and high doses of amsc ( , , and seconds), and bmsc ( , , . seconds), p< . ), clot formation time (cft, p< . ) and increased alpha angle (p< . ) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par . no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* , neil bagamasbad , reynold dilag , melissa nasser , nicole bauer , jennifer wheeler and mary berg . department of pathology, university of colorado -anschutz medical campus, department of medicine, division of hematology, university of colorado hospital, scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from collection procedures using the mnc protocol and collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd )-positive (cd ) throughput, cd collection efficiency (ce%), platelet loss a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included and allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd throughput was significantly higher in the cmnc group than the mnc group. the cd ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient # , originally typed as an a , had bone marrow donor and cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient # is now typing as type o. patient # was originally typed as a and received a bone marrow transplant from a type b donor. patient # is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient # and patient # indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with % fetal bovine serum under either normoxia ( % o ) or hypoxia ( . % o ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd /cd and cd were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc ( . /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by %, but depressed the growth of amsc by % at day in comparison to normoxia. both bmsc and amsc equally expressed cd and cd but not cd under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: (control), versus (bmsc), and (amsc) seconds) by natem. hypoxia also significantly shortened ct ( (bmsc), (amsc) seconds, p< . as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd cells. study design/method: cryopreserved cd cells from healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef -alpha-yfp lentivirus ( . % concentration) and media (x-vivo- , human serum albumin(hsa), ng/ml each of cytokines (scf, tpo and flt -l) over days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of % dmso, % pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd cd -cd ra -cd cd f cells) phenotyping and cfu assays were done following first thaw (pt ), post-transduction (ptxn) and second cryopreservation-thaw (pt ). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd %, cfus were similar before and after pt . hscs ranged from to cells/ cd cells in the pt -tr arm compared to a range of to / cd cells after pt . viability, % cd and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt (table) . conclusion: dec of mpb human cd cells decreases tnc recovery, but has minimal effects on cd cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd % in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of % hydroxyethyl starch, % human serum albumin and % dmso at final concentration. pbsc were cryopreserved by direct immersion on - c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of consecutive days of neutrophil count > . x /l and platelet count > x /l after days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd was below x cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd on the day of the collection versus collected cd per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd was calculated. final laboratory count of cd per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss software. results/findings: among patients collecting hpc for autologous transplantation, , % needed only one day of hpc harvesting, while , % needed two days and , % needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was - , %. after comparing predicted values with cd collected in the final product, we found a very strong correlation of . (p< . ) for patients and a strong correlation . for healthy donors (p< . ). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; ! % cd , cd , cd and % cd , cd , cd , cd , hla-dr) study design/method: umbilical cord tissue (n ) was washed, blood vessels removed, cut into . - mm pieces, and washed twice in saline. fresh tissue was immersed in . % saline for same day culture, while frozen tissue was cryopreserved for at least hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a cm tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures > % confluence. all cells were tested on an msc flow panel at passage just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of days (fresh . , frozen . ), and days (total) for the msc's to reach passage (fresh . , frozen . ). all cells were ready for flow analysis in approximately weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p . ), or their growth rates (p > . for all). flow cytometry showed average ! % for positive markers and % negative markers. there was no statistical difference between fresh and frozen flow result (p > . ). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of up to years ( to ) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with % concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a c water bath and . ml aliquots were diluted at a : proportion with % human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using -aad marker through flow cytometric analysis. results/finding: ucb storage period was . years (mean) and cell recovery was . % (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p . ). post-thaw cell viability of . % (mean) showed no statistically significant correlation with unit storage period (p . ). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln vapor in a dmso-based cryoprotectant for yrs. ( . . ; n ). units were rapidly thawed and rinsed in dpbs, then pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a x grid pattern in msc-supportive medium and incubated for days, after which the tissue was discarded and media exchanged. cells were isolated on the th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a % success rate. cells were positive for the msc markers cd , cd , and cd ( . . %, . . %, and . . %, respectively) and negative for the hematopoietic markers cd / ( . . %). passage and passage doubling times were . . days and . . days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical a transfusion vol. supplement s research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd yield prediction algorithm ines bojanic* , nelly besson , ivana vidovic and branka golubic cepulic . department of transfusion medicine and transplantation biology, university hospital centre zagreb, terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version ) in adult and pediatric lvl. a prediction algorithm for cd cell yield was also tested. study design/method: we evaluated retrospectively lvl performed in adult patients, and lvl in pediatric patients treated in uhc zagreb from march till september . mobilization regimen combined chemotherapy and filgrastim; poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio : ). in patients weighting kg (n ), a rbc prime was performed. cd , lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd cell count and cd cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd values to real cd yield. results are presented as median (iqr). results/finding: in both groups, cd , ly and mo ces were high. target cd dose was successfully reached in procedure in ( , %)adults and in ( . %) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in ( . %) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd cells and cd cells collected/ blood volume was observed in both groups (r . and . in adults and children respectively, p< . ) suggesting cd yield could be predicted based on precd cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd yield and observed cd yield (r . and . in adults and children respectively, p< . ) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of . ( . - . )l of blood in adult procedures, and . ( . - . )l in pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd , ly and mo ce were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* , aniko barta , arpad batai , zoltan csukly , zita farkas , laszlo gopcsa , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) times per case weekly at a dose of million cells/kg. clinical response was assessed days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all patients had received cycles of msctreatment ( dose per cycle). the median age was years old ( - ) with a male/female ratio of : . distribution of the original malignancies (n): acute myeloid leukemia: ; acute lymphoblastic leukemia: ; myelofibrosis: ; myelodysplastic syndrome: ; multiple myeloma: ; t-cell lymphoma: . nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median rd day ( - ). the involved organs were skin ( ), gut ( ), skin and gut combined ( ) and even lung in cases. the median time of msc's first infusion was days after the stem cell transplantation (hsct) and ( - ) days after the first episode of gvhd. of the cycles of msc-treatment led to complete remission ( . %) and resulted inpartial remission ( . %). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with % overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is million cd cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the million cells/kg goal. the ideal minimum cd cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from patients to evaluate the predictive value of the cd cells/ml level. data was collected over months from every patient who underwent a stem cell collection. four patients were allogenic donors and were autologous donors. the patients' weight, diagnosis, and pre-procedure cd cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd cells collected were recorded. the collection efficiency and the cd cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd cells/ml and post-procedure cd cells/kg (r . ). any patient who had a pre-procedure cd cells/ml count of or greater had a collection of at least million cells/kg. any patient who had a pre-procedure cd cells/ml count of or less collected less than conclusion: the pre-procedure cd cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd cells/kg level. to confidently know that a patient will be able to produce the desired million cells/kg, a pre-procedure cd cells/ml count of at least should be obtained. for any patient with a count below , they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between and cd cells/ml should be conducted. heidi elmoazzen , antonio giulivi , michael halpenny* , lisa martin , donna perron , chris bredeson , lin yang , locksley mcgann , paul birch and jason p. acker . canadian blood services, ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso ( % final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd , viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in % dmso and . % hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of . days for anc with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current % dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using % dmso and . % hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from % to %. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is . mg/kg, therefore patients weighing > kg would require a second vial, thus doubling the drug cost. in we implemented a policy of capping plerixafor at mg for patients weighing > kg. this retrospective study compares the mobilization of patients > kg who received capped doses ( ) ( ) ( ) ( ) , with historical control patients ( - ) who received full or uncapped doses. study design/method: patients weighing > kg with crcl > ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of and consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd /cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at mg for patients > kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (< yrs) have a higher pre-apheresis %cd level than any of the other groups, reaching statistical significance when comparing the %cd pre-apheresis between the youngest group (< yrs) and the oldest group (> yrs). hispanic donors show statistically similar %cd pre-apheresis levels over all age groups. moreover, the hispanic older age group (> yrs) had a statistically higher %cd pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last years was analyzed to determine the th percentile, median and th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc ( x cell / ml) and three low wbc ( x cells / ml) concentrations, each at high ( ml), low ( ml) and median ( ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax (pericell protocol, cs. . kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and -aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting -aad viability of % [range - ]% and a hematocrit of % [ - ] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was % [ - ] with a % [- - ] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of [ , ] % and a change in -aad viability of [ , ] % from the input product. the method was found to have a cv of . %. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating a transfusion clinical assessment. in the first phase, cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd -aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to hours post thaw. the second in vivo phase included use of an infusion pump for consecutive autologous patients, with comparison of infusion and transplant outcomes to previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than % within hour, while cd cell viability remained stable up to hours post thaw. small aggregates appeared after hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were . . and . . , respectively (p-value . ). platelet days to engraftment for pump and drip were . . and . . , respectively (p-value . ). infusion rates were slightly higher for the pump group. for control patients, required transfer of products to syringes due to slow infusion rate and others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the zikv ineligible cbus was: caucasian %, asian %, black/aa %, and multi-race %. racial distribution of all clinical cbu donors was caucasian %, asian %, black/aa %, and multi-race %, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: % of all ineligible cbus and % of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete , -diphosphoglycerate ( , -dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring , -dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n ), which were stored in sagm for days, to act as untreated controls. the remainder of each unit ($ ml) underwent treatment with the rejuvenation solution ( ml, minutes at o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros . pc were centrifuged at g in a sorvall rc c centrifuge (sorvall, usa) for min. the combination cryoprotectant dmso dextran (cryosure dex , germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at degrees c (barkey plasmatherm) for min. cpc osmolality was measured with an osmomat osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru u ). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso dextran (cryosure dex ) , as a cryoprotectant, to obtain a final concentration of % dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m were instrumental in automating that phase. pc to be frozen had an osmolality of no less than mosm/l. prp and ppp were frozen at a cooling rate of - c/min and stored at - in the chest freezer for up to months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru u ). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to mosm/l. freeze-thaw recovery of platelets was % or more of the original population. defrosted pc were stored at - with continuous gentle stirring from a helmer platelet agitator for no longer than hours before transfusion. it took no more than min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day and day evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day . a total of lrap units were tested over a -month period: were cultured and rapid tested on day ; were rapid tested on day . the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were true negatives (tn) and false positive (fp) on day when tested by bact/alert, with tns on day . bactx testing results showed tns on day and tns on day . testing using the pgd kit showed tns on day ; and tns and fps on day . fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in % plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day and day during the night shift to be accomplished without additional staffing and allows to extend outdate to day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for year jos lorinser , pieter f van der meer , hans van der heiden and dirk de korte* . department of product and process development, sanquin blood bank, mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers ( ml) is unknown. if these products can be stored at - c it will be feasible to store this product in -star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at - c or <- to - c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <- c for - months and controlled thawing, six different sera were used to fill a large number of mini ( ll) containers, which were refrozen and stored at either - c or <- c. during storage at months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <- c. growth factors tested were pdgf-aa&ab/bb, tgf-ß / / , vegf, a transfusion vol. supplement s egf, fgf . the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß were the most abundant growth factors, on average , resp. ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average ng/ml. tgf-ß , egf and vegf were detected at relatively low values, resp. ng/ml, . ng/ml and . ng/ml. average levels of fgf and tgf-ß were close to detection limit (< . ng/ml). the controls stored at <- c showed for all growth factors close to % of the initial values in samples at t (moment of filling mini containers). for serum stored at <- c for up to months, most factors showed less than % decrease, except for pdgf-aa and tgf-ß , showing % resp. % lower values. for serum stored at - c the values for tgf-ß , egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß showed a decrease of resp. , and %. conclusion: human serum eye drops can be stored in the new micro dose device at - c ( -star household freezers) or <- c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at - c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond year. ruqayyah almizraq* , heather inglis , phillip norris , , jennifer a muszynski , nicole juffermans , jelena holovati and jason p. acker , . university of alberta, blood systems research institute, university of california, san francisco, nationwide children's hospital, academic medical center, canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n per method). residual platelets and white blood cells (wbcs) were measured on day using flow cytometer (fc). on storage day and , number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day , apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p< . , wbd p< . ) and wbf (vs: apheresis p< . , wbd p< . ) methods. while rcf units yielded the lowest count of platelet-evs (cd a ) on day and , the highest number of platelet-evs were in apheresis (day ) and in wbd (day ). similarly, there was significant difference among methods in the number of wbc-evs (cd , cd , cd , cd , cd b ) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day vs day in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< nm) was greater than large evs (! nm) in all of the products on day and , and the highest level of evs < nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p< . ). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at centigrade maryanne c herzig* , crystal lafleur , chriselda g fedyk , sherrill j. slichter and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research, university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least weeks of storage at c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for , , , or days after collection. units were stored for days without agitation. units stored for , or days were agitated during storage with a model hybridization incubator at c set for end over end rotation at - rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at - c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai- ) as another fibrinolytic measure; and complement activation markers c a, c d, c a and c b- . data was analyzed by one way repeated measure anova. results/finding: only % of the platelets were recovered in units stored for days without agitation. these levels did not meet fda requirements of . x platelets per wb unit. subsequently, wb was agitated and platelet recovery was - %. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t (day of collection) and t , , , or measurements. significant elevations of pai- and scd l indicate activation of platelets and inhibition of fibrinolysis (p< . ). activated complement peptides c a, c a, and c d were all elevated over time (p< . ) while sc d- was not. however, only c a and c d levels at t were above normal reference ranges at . and . times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at c for - days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc d- reported, wb showed elevation of c a, a and c d and not sc d- . complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* and christian todd . cerus corporation, community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly . x . x . x . x rcf . x . x . x . x apheresis . x . x . x . x wbd . x . x . x . x platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following collection targets: . x in ml, . x in ml, . x in ml, and . x in ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of . x or . x was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: % of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was . . conclusion: it is possible to treat % of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward % while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* , andrey skripchenko , fei xu , ying li , stephen j wagner , pamela h whitley and jaroslav g vostal . fda/cber/ obrr/dbcd/lch, american red cross holland laboratory, american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature ( - o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts ( hrs ct: hr o c [tc]). autologous apheresis plts stored for -days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n ) and the same non-labeled plts were also infused into mice (n ). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts < % were considered background. results/finding: the mean recoveries of infused plts were . . % for rt, . . % for tc and . . % for ct in humans. in mice, mean recoveries of the same plts were . . % for rt, . . % for ct and . . for ct (mean sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $ % and ct was $ % of rt. in mice tc was $ % and ct was $ % of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to % for rt plts. human tc plts had % auc while ct plts had % auc compared to rt plts in humans. in comparison, the same tc plts had % auc and ct plts had % auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are . and . , respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* and geeta paranjape , . coastal bend blood center, carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g with the compomaster net software for data management. implementation was planned for a november go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g s and compomaster in june . training and validations were successfully completed and a full launch occurred mid-march . device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december . validation was completed and signed off in march of . manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g system. data points were collected from units bi and units ai. results/finding: upon initial implementation, staff training and use, the compomat g was found to be easy. plt weight spread was reduced from an average of gm to an average of gm. actual plt weights were reduced from an average of gm to gm, resulting in an average increase in recovered plasma of . ml per unit. plt count on average increased from a count of to ( /mm ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by . % after implementation of the compomat g and our plt concentrations increased on average by %. we were able to consistently produce a smaller volume plt (average gm), which gave us . ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell , angela hill , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: abo/rh matched lr sagm rccs were pooled and split to produce large ( ml) and small ( ml) rccs. the rccs were stored to d and glycerolized manually by mixing ml of glycerol with the rcc in a ml freezing bag. units were frozen at - c for ! h before being removed from frozen storage and thawed in a c water bath. large rccs and small rccs were deglycerolized using the organization's current procedure on the cobe cell processor prior to re-suspension in . % saline, . % dextrose. the remaining rccs were transferred into a l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of %, and deglycerolized in a ml centrifuge bowl on the acp- with re-suspension in as- . rbc quality was tested at h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p . , acp : p . ) and lower cell recovery (cobe: p . , acp : p< . ) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe had higher hemolysis (p< . ) and supernatant potassium (p . ) than did small volume rccs. large cobe rccs had higher hematocrits (p . ), hemoglobin (p . ), and recovery (p . ) than did large acp- rccs. however, all cobe rccs had higher (p< . ) hemolysis ( . . %) levels than did acp- rccs ( . . %). cobe rccs failed to meet regulatory hemolysis standards of . %. conclusion: addition of a ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as- and storage for h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp- cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using . % saline and centrifugation and the semi-automated washing method (sam) using the cobe blood cell processor. study design/method: in this study, units of single donor platelets were evaluated ( washed using the mm and washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas . results/finding: table shows that the average platelet recovery for the sam ( %) was significantly higher compared to the mm ( %). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took - minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas- ) for input platelet doses of . to . platelets in to ml of to % plasma and - % pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas- containing doses of . to . platelets in a volume of to ml. study design/methods: apheresis pcs (amicus v r ) were collected in % plasma and % pas- . one study was performed at the nominal dose ( . - . x platelets), volume ( - ml) in % pas/ % plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition ( . - . x platelets in - ml) using either single or pooled donations. input pcs (n ) were treated with the intercept ts set by the end of day post collection; the incubation time in the compound adsorption device (cad) container ranged from to hours and the intercept treated pcs were stored in containers (n ). day and post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas- treated in the intercept ts set demonstrated acceptable in vitro function (table ). all intercept treated pcs had ph( c) ! . . platelet dose and volume recovery post-treatment ranged from % to % and % to %, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing % plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n ). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n ). numerous in-vitro quality markers (plt concentration, atp, po , pco , ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days , , and for apheresis pcs, and on days , , and for wb-derived pcs. two flow cytometry assays were used to evaluate cd p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion vol. supplement s results/finding: platelet recovery was % and % for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells ( % (test), vs. . % . (ctl) on day ) and a higher rate of cd p expression than control pc units ( % (test), vs. % (ctl)) on day ). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* , lorraine blagg , christi e marshall , herman woodson , sean erony , krishna patel and eric gehrie . the johns hopkins hospital, johns hopkins hospital transfusion medicine dept, johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day . as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day . the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of lrap were tested. lraps initially tested negative by bactx, while lraps initially tested positive by bactx. all initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was . %. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only platelet units. the expected rate of bacterial contamination of platelets is less than per units. the . % initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as- and cp d/as- rbc alan d. gray* , matt landrigan , pamela whitley , michael wellington , sherrie sawyer , shalene hanley , emily rondeau , louise herschel , neeta rugg , patricia a.r. brunker , shawnagay nestheide , jose cancelas-perez , larry dumont and zbigniew m. szczepiorkowski . and , -dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for > hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood ( - ml) was collected and processed at sites into leukocyte-reduced rbc (a total of n cpd/ as- and n cp d/as- ). ml of rejuvenation solution (citra labs) was added to each rbc on day (d- ), incubated for minutes with agitation at c water bath (helmer dh ), washed (haemonetics acp ), and stored in as- at - oc for days (d- through d- ). in vitro recovery (%) was calculated and hemolysis, atp, and , -dpg were determined on day , d- , d- after rejuvenation and washing (postrjv), d- , d- , d- , and d- . all units were cultured on d- postrjv and on d- , and then concentrated by centrifugation on d- . results/finding: in vitro rbc recoveries were . % and . % (as- and as- , respectively) and no bacterial growth was observed. hemolysis on d- was maintained < % in / ( %) as- units and / ( . %) as- units. all as- and as- units ( %) had hemolysis < % following concentration by centrifugation. morphology score was reduced to % (as- ) and % (as- ) by d- , restored after rejuvenation ( %, %, respectively) and maintained through d- (> %). atp was restored and maintained above fresh levels after rejuvenation. , -dpg was restored above fresh levels and was maintained ! % of fresh levels through d- . all values were significantly different compared to d- except as noted (p< . , paired ttest) ( table ) . conclusion: rejuvenation of stored rbc restores atp and , -dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d- when compared to nonrejuvenated rbc on d- . this study is funded by zimmer biomet. storage > hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante , jason p. acker* , and jelena holovati . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during -day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and , -dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome rejuvesol-treated (l r). the prbcs were incubated for h at c with hepes-nacl (sham), liposomes (dopc:chol, : mol%, mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and , -dpg at day hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control ( . . %): l ( . . %, p . ), r ( . . %, p . ), l r ( . . %, p . ). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r ( . . , p . ) and l r ( . . , p . ) treatments compared to s ( . . ) but not l ( . . , p . ). rbc rigidity (kei) increased in all treatments compared to sham ( . . ): l ( . . , p . ), r ( . . , p . ) and r l ( . . , p . ). aggregation amplitude was significantly increased by r treatment only ( . . au vs. . . au, p . ). atp levels were significantly higher in all treatments compared to sham ( . . mmol/g hb): l ( . . mmol/g hb, p . ), r ( . . mmol/g hb, p . ), l r ( . . mmol/g hb, p . ). the levels of , -dpg were no longer detectable in s and l treatments at day . the combined treatment was comparable to r ( . . mmol/g hb vs. . . mmol/g hb, p . ). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer , eric ducas , patricia landry , nathalie dussault , jacques bernier , danny brouard* and anne maltais . h ema-qu ebec, institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the -ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < c) of one to six -ml whole blood units (wbu) within h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between c and c for h under extreme external conditions (- c to c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for h at - c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, -ml wb bags were filled with ml saline . % at t c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (- c) and summer ( c) conditions. shipping boxes were filled with either one or six bags (n ). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under c in . . h and maintain their internal temperature between c and c for h with final values ranging between . c and . c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the c threshold value in . . h and the bags' internal temperatures were within the acceptable range for h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than - c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* , christopher c. c silliman , beth shaz , marguerite kelher and claudia s. cohn . new york blood center, bonfils blood center, department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either % donor plasma (n ) or % pas- / % donor plasma (n ). within hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd ligand (scd l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c ( / products) compared to plasma platelets ( / products); however, the hla-antibody screen-positive supernatants of pas- a transfusion vol. supplement s abstract c platelets had fewer hla specificities ( specificities) compared to those of the plasma platelets ( specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd l levels were increased in the supernatant of pas-c compared to plasma platelets (table ) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd l and not bioactive lipids. although scd l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below x wbc in us and x wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n ) and leukoreduced (lr) rbc units (n ) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of wbc/ul. the spiked samples of , . , , , and wbc/ul were prepared from the source sample of wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations ( , . , , , , wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples ( , , , wbc/ul). tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of samples (plt and rbc) were run on both analyzers, repeated for days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt . , slope . ), (r-rbc . , slope . ). the %diff-plt at , , wbc/ul were . , . and , respectively. the %diff-rbc at , , wbc/ul were . , . and . , respectively. the average total testing time was similar on both instruments; min for the facsvia and min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated % ( of min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* and mickey koh . department of laboratory medicine and pathology, university of minnesota, st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of completed and partially completed surveys were received. results/findings: responses came from countries, but the majority of responses came from the united states (us). of the respondents, % reported aprp use in their hospital. aprp was used predominantly for outpatients, though > % of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by - mds; however, hospitals had > mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures ( %); however, respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, ) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n ) under blood bank conditions at day (d ), at day (d ), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from . - . % at d to . - . % at d . rejuvenation markedly reduced this storage-induced spherocytic shift ( . - . %) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell , angela hill , tracey turner , april xu , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units ( ) than in rcf units ( ), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as- additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: small rcf ( - ml) and large wbf ( - ml) rccs were stored for d before being glycerolized and frozen at - c for ! h. large rccs whose red cell mass exceeded the capacity of the ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a c water bath, deglycerolized and re-suspended in as- . rccs were stored d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p< . ) hematocrit, specific gravity, hemoglobin per unit, supernatant k and na concentration, deformability (ei max ), and higher (p< . ) recovery than did large wbf units. no significant differences in hemolysis, atp, , -dpg, p , rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table ) , however of large wbf units had rbc recoveries < % due to pre-glycerolization volume reduction, and of the small rcf units had hemoglobin values < g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above % and the hemoglobin failure rate would be below % of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. . . . . . . . . elongation index ( pa) . . . . . . . . this study is funded by zimmer biomet. (hasan ) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two ( ) rbc units (leukocyte-reduced), cpd/as- or cp d/as- , on day , day , and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o /g hb) and total releasable oxygen (tro) of the unit (ml o ). orc was determined by assessing the change in % o saturation from mm hg po (e.g., lung) to mm hg po (e.g., venous blood) multiplied by . ml o /g hb (li ). a simulated baseline pretransfusion vo of ml o /min was estimated using the day orc and assuming a g/dl transfusion trigger with a cardiac output of l/min and l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day restored orc and tro to levels greater than day ( table ) . orc of the rejuvenated unit was . . times and . . times greater than rbc on day and day , respectively (p< . ). vo increased after a simulated single unit transfusion of rbc (day , day , and pw) by . %, . %, and . % over the pre transfusion vo , respectively (p< . ). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release . times the volume of o compared to standard, untreated rbcs stored for days. inferior oxygen delivery to tissues (vo max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for days vs days which seem dependent on genetic variability and storage time (bennett-guerrero ). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* , jay srinivasan , gustaaf de ridder , alan d. gray , matt landrigan , keaton charles stoner , angela crabtree , jessica poisson and ian welsby . duke university school of medicine, duke health pathology, citra labs, a zimmer biomet company, zimmer biomet, duke university, department of pathology, durham veterans affairs medical center, duke university hospital, duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted , -diphosphoglycerate ( , -dpg). the loss of , -dpg increases the oxygen affinity of hemoglobin, resulting in lower p (partial pressure of oxygen at % hemoglobin saturation). decreased p may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and , -dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a , leukoreduced prbc stored in as- were obtained from our local blood center. after days of storage, units were divided into separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution ( ml) was added to the cr group, and all groups were then stored for another days at - c. on day of storage, the sr group was incubated for hour at c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps ) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a . . results/finding: significant differences in p were noticed between all groups (table ) . ei, ps , and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p ) seen over days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc ( ml) units stored in as- , obtained from a regional blood donor center at expiration ( days), were passed by gravity through sorbent-devices containing ml of multifunctional polymer bead, at a flow rate of ml/min. supernatants were analyzed for k removal as well as free hb, antibodies and cytokines ( -plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k ] from . . to . . meq/l; equivalent to an . % reduction. free hb was reduced by . % from . . to . . mg/ml. antibodies, specifically igg, iga, and igm decreased from . . to . . mg/ml ( . %), . . to . . mg/ml ( . %), and . . to . . mg/ml ( . %), respectively. inflammatory cytokines were significantly reduced, specifically: ip- from . . to . . pg/ml ( . %), mip- b from . . to . . pg/ml ( . %), and pdgf from . . to . pg/ml ( . %). filtration had no significant impact on cell surface markers of rbc viability (< . % decrease) or sensitivity to osmotic changes. values listed represent mean sem (p < . for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency ( . %) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in apheresis and whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and , -dpg profiles to fresh levels. the objective was to compare % hemoglobin-oxygen saturation (p ) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was . . %. hemolysis (%) was similar on day before and after dry-air incubation with the rejuvenation solution ( . . % vs . . %). percent hemolysis (%) decreased after washing ( . . %) and was maintained below < % for all units during storage for hr ( . . %). average atp and , -dpg were restored above the average fresh values. the morphology score decreased $ % by day , which was restored to near fresh values following rejuvenation and washing and storage hr ( . % and . %, respectively). rbc oxygen affinity, as assessed by p , was restored above fresh values. all values were significantly different compared to day (p< . , paired t-test) ( table ) . conclusion: rbc morphology was restored to near fresh and average atp, , -dpg, and p were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and , -dpg were maintained during storage hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d tetrahydrocannabinol (thc) and -oh-d -tetrahydrocannabinol ( -oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and -oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and -oh-thc to produce samples for lc-ms/ms analysis. lc used a c column. post-column detection by ms/ms used positive ion electrospray with q :q ion pairs of m/z . : . (internal standard (is), d -thc), m/ z . : . (thc), and m/z . : . ( -oh-thc). quantitative results for thc and -oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from - ng/ ml for both thc and -oh-thc. limits of quantitation, defined as standard deviations above background, were . ng/ml for thc and ng/ml for -oh-thc. results/finding: a total of donor plasma samples were tested for thc and -oh-thc. no samples tested positive for either thc or -oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a % probability of one or more positives at a prevalence of . % positive samples, and a % probability of one or more positives at a prevalence of . % positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than % among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than hours for post-exposure detection of thc and/or -oh-thc in plasma. conclusion: testing of donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than %. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* , ruqayyah almizraq , daniel millar and jason p. acker . university of british columbia, university of alberta, lightintegra technology inc., canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days , , and of storage. one rcf rcc was tested on days , , , , and and six ml aliquots were stored in parallel and tested on days , , and . all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days , and (p< . ) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the aliquots were consistent at each time point but statistically higher than in the original rcc on and after day of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow % screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n ) and paired ffp aliquots were stored for - days at - c and - c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! % levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c esterase and alpha -proteniase inhibitors). the level of factor xiii in odp was slightly lower, about % of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i ii and ddimer) and complement (c a and c a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion vol. supplement s abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to % in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to minutes. in addition, donations with collection times between and minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from - minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n ) were prepared from one - minutes bc and ml of autologous plasma in a ml pvc-dehp container. as a reference, spc from donations with collection times of < minutes were prepared (n ). in addition, pc were prepared from bc, of which at least bc were from - minutes donations (n ). after pooling of the bc, ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for days at c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume ( vs. ml) and platelet content ( vs. x ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day , ph( c): . . vs . . , other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day and/or in / pc (possibly because sometimes ab incompatibility was accepted). on day , plt showed low cd p expression ( . . %) and phosphatidylserine exposure (annexin v binding, . . %). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from - minutes whole blood donations had a normal composition and showed good in vitro quality during day storage. to substantiate that the exclusion of - minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* , jessie miller , ranee marie wannarka-farlinger , sandra bryant , scott a hammel , sherry stern and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as- rbcs, irradiated and non-irradiated, were selected for the study. the units varied in age, ranging from to days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< . ) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p . ). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after days of storage pei lun karen lim* , erma sofia sumardi , isamar eduardo ancheta , susan lim , christina yip , lip kun tan and shir ying lee . national university hospital singapore, national university hospital, national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within hours of processing and stored at - c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for days and kept at to c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than iu/dl. study design/method: randomly selected units of ffp (n ) were measured for fviii concentration based on clotting assay (stav r -deficient a transfusion vol. supplement s viii diagnostica stago). fviii levels were measured at five time points: prefreezing, , , and hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at to c for minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration ( hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at to c for days for subsequent testing. results/finding: results obtained were listed in table . units to were not tested for fviii at post thaw- hour due to operational issues. the overall fviii concentration decreased at an average of % from pre-freezing to post thaw hour. after further storage of tp post thaw- hour and - hour, residual fviii level remain to be above iu/dl except unit which had a lower initial fviii concentration. at post thaw- hour, out of units tested had residual fviii activity within the pre-set standard of iu/ dl. the average decline from -hour post-thaw to -hour, -hour and hour post-thaw was . %, . % and . % respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least iu/dl of fviii. typically patients with factor levels below iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi , ayman mohamad sabri , ali abdullah alajeafi , ashwaq hasan alhekri , saleem bin mahfouz , ali hasan alkhodari , rawya saeed shealy , marcus picard-maureau* and hussain bana almalki . king abdulaziz hospital and oncology center, cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in % plasma over a day storage period and the new "test" pathogen-reduced, pooled (pools of ) prp pc in % plasma over a day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of leucoreduced test pcs were assessed at day of storage and compared to leucoreduced control pc at day of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day ; the process was completed by day post-collection. samples were taken daily for quality analysis from test and control pc until day and day , respectively. for bacterial spiking, additional pc were spiked with each receiving ml of mcfarland ($ . x cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was . % . , the total average platelet loss at day was . % . . the average platelet loss in the control units at day was . % . . the average ph of the test units at day was . . and in the same range as the control pc, ph . . . glucose concentration in test pc at day ( . . mmol/l) was lower than in the day control units ( . . mmol/l). lactate levels increased during the course of storage; lactate levels at days and were outside the range of the assay (> mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* , jay srinivasan , jessica poisson and ian welsby . duke university, duke university school of medicine, duke university hospital, duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified proteins in cryo; of the most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf ( . mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial ( Á lm) filtration. cryoprecipitate mini-pools ( ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di( -ethylhexyl) phthalate (dehp)] adsorption device and a Á lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table ) . kit ensured bacterial sterility (table ) and most importantly, final product was free of hbv, hcv and hiv (table ) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase- , thereby blocking synthesis of thromboxane a from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n ) were prepared from a nsaid-bc and ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for days at c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n ) were investigated as a reference. values are expressed as mean sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume ( vs. ml) and plt content ( vs. x ) were similar in both groups. on day , both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day was significant higher in a subset of donors who had used ibuprofen (n ). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin ( , - , p< . ), diclofenac ( , - ) and naproxen ( , - , p< . ), compared to normal controls ( , . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (< hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, ] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type diabetes (t d). because of the strong rise of people with t d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t d, but accepted as donor. study design/method: twelve whole blood donors with t d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for days at c and sampled on day , or and . the diabetic marker hba c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 'good' (ph day > . ) and 'poor' (ph day < . ) storing spc were selected and analysed in more detail. results/finding: donors were of age year and primarily men ( %). donors with t d had a higher mean bmi ( . . vs. . . kg/m ) and higher hba c than controls. the spc of both groups had the same volume ( vs ml) and plt content ( vs x ) but on day glucose concentration was higher in the diabetic group ( . . vs . . mm, p< . ). on day , the average in vitro quality was comparable in both groups (data not shown). when combining a transfusion vol. supplement s the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed ( . . vs . . mmol/ day/ plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc- ) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day ('poor': . . vs 'good': . . , p< . ). conclusion: bc from donors with t d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table ). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* , , katharine a downes , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy month old unvaccinated girl presented with history of diarrhea for days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: x /l, platelets: x /l, bun: mg/dl, creatinine: . mg/dl, lactate dehydrogenase u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to x /l, adamts sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation ( x /l and a-ipc of . x /l). two consecutive tpe were completed which resulted in a platelet count decrease to x /l and a-ipc of . x /l. a-ipc ratio was . below the ratio of which has been reported for ttp patients. similarly a-ipc count was not below x /l threshold reported in setting of ttp with severe adamts deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o :h toxin. testing of c , c , factor h, factor h autoantibody, factor i and factor b were normal. adamts activity was %. patient was treated for the infection and platelet count improved within days to x /l, with resolution of her renal failure: bun: mg/dl, creatinine: . mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts activity. many patients with severe autoantibody-mediated adamts deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts activity < %) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value - days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column ), increased peak a-ipc value (results shown as percent increase, column ), delayed a-ipc peak, and delayed plt recovery (table ) . moreover, recurrent episodes required more procedures compared to initial presentation (table ) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february to march on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version . after taking consent from the donors. the target collection of each procedure was a dose of x platelets in - ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at consecutive donations within days were considered. data was analyzed by stata . within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the donors, repeated the plateletpheresis within a week (group i) and underwent nd plateletpheresis within - days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the groups (p . ). though above the eligibility cutoff of . lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at nd plateletpheresis in group ii donors. there were donors who presented to us for the rd time for plateletpheresis with a mean gap between st and rd plateletpheresis being days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p . ). plateletpheresis through all the cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type- receptor antibody (at rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at r ab are reviewed. results/findings: case : the patient is a currently -year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age , and a second deceased donor transplant due to a rejection of the transplanted kidney at age . three years post-transplant, her creatinine (cr) started to rise from . to . mg/dl and a biopsy showed banff criteria grade amr, grade a t-cell mediated rejection (tcmr) and grade interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at rab was identified at > u/ ml (high: > u/ml, intermediate: - u/ml, negative: < u/ml). she received tpe treatments every other day and started losartan. after a course of tpe, at rab decreased to u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to . ml/dl. in one month, her at rab increased again to > u/ml, therefore, she received more tpe treatments with a decrease in her at rab to u/ml. although at rab level increased slightly to u/ml after months, her cr has been stable at . - . ml/dl. case : the patient is a -year-old mean /-se - . /- . % * . % /- . %* * p< . a female with malignant hypertension who received a deceased donor kidney transplant at age . her cr started to rise weeks post-transplant from . to . mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at rab level at u/ml. she received tpe procedures every other day and at rab decreased to u/ml with a decrease of cr to . mg/dl and improved arteriopathy in histology. because her at rab level slightly increased to u/ml over the next weeks, she started weekly tpe treatment. after weekly tpe, tpe treatment was stopped because her at rab level remained relatively unchanged. her cr has been stable at around . ml/dl to date. conclusion: we present kidney transplant recipients who received tpe treatments for high at rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at rab levels; however, weekly tpe had no effect on reducing at rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < %], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a year old man (patient b) with a medical history of hypothyroidism (on synthroid for years), end stage renal disease and non-ischemic cardiomyopathy (ef of - %) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a - . plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within hours of the procedure completion. their total t , t and free t levels trended to normal or near normal range within hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy - weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* , laura martínez molina , cristina muniesa montserrat , octavio servitje bedate , silvia cosano navarro and maria isabel gonz alez medina . banc de sang i teixits, dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) , as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category . since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of patients diagnosed with ss and compare them in their first evaluation once the th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( -methoxypsoralen, -mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from . to total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in / patients) and with online system (therakos) just in . main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is ' % (partial remission . % and complete remission . % with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : u/l) requiring transfusions, mild thrombocytopenia ( x /l), acute kidney injury (bun mg/dl, creatinine . mg/dl). by the third hospitalization day hgb improved to g/dl, however with worsening thrombocytopenia ( x /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days - ). platelet count and a-ipc improved to x /l and . x /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count ( x /l) and a-ipc . x /l. these dynamics did not resemble those which had been described for ttp patients with adamts deficiency. adamts obtained prior to tpe initiation was resulted at this time and was %. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc ( . x /l) that preceded platelet count increase to x /l three days later when patient was discharged. other laboratory values at this time were ldh of u/l, hgb: . g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts deficiency eiman hussein* and jun teruya . department of clinical pathology, cairo university, texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts . since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts , the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts . the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january through march were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts activity of less than %. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts deficiency. eight patients ( %) were associated with suspected bacterial infection. four of the patients ( %) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than kilograms using a single apheresis procedure. study design/method: in october and june , two children with possible leukemia were submitted to tl procedure. they were and months old, and weighted , and , kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, % hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin ( ml of acd-a and , units of heparin), at a blood to anticoagulant ratio of : . a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were . and . / mm . the formula "collection pump flow , x inlet flow x preapheresis wbc count" was used with the goal of removing up to x leukocytes/ml. a single leukapheresis procedure was performed with total blood volume processed per patient. immediately after the -hour procedures, wbc count were . and . wbc/mm , and -hour post tl, wbc count were respectively . and . /mm . net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since . we report the data from the year . study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of , apheresis procedures were performed at hospitals. therapeutic plasmapheresis was the most frequent procedure ( . %) followed by autologous peripheral blood stem cell (pbsc) collection ( . %), allogeneic pbsc collection ( . %), donor leukapheresis ( . %), and therapeutic leukapheresis ( . %). cobe spectra ( . %) and amicus ( . %) were the most widely distributed instruments. centrifugation was the dominant technique ( . %) for therapeutic plasmapheresis. detailed information was given for , therapeutic plasmapheresis procedures performed on patients (some items were not completely filled out). spectra optia ( . %) and cobe spectra ( . %) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently ( . %) as the replacement fluid followed by % albumin ( . %), % albumin ( . %), and % albumin ffp ( . %). most of the procedures were performed for plasma volume ( . %). acd ( . %) and heparin ( . %) were used for anticoagulation. central venous catheter ( . %) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation ( . %), antibody mediated rejection in renal transplantation ( . %), thrombotic microangiopathy ( . %), desensitization for abo compatible renal transplantation ( . %), neuromyelitis optica spectrum disorders ( . %), and hyperviscosity in monoclonal gammopathies ( . %). adverse reactions were observed in . % of the procedures. allergic reaction ( . %), hypocalcemic symptom ( . %), and hypotension ( . %) were frequently reported. therapeutic effect was achieved in . % of the patients. our apheresis registry has been well run for years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride > - mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $ deaths/ , cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of pts who were diagnosed with hp from january, through january, , and referred for immunotherapy evaluation. / ( %) pts received conventional therapy (ct) and pe (pe group), and / ( %) pts received ct alone (ct group). mean age was years (range - ), and % were female. baseline mean triglyceride level (normal < mg/dl) for pe group was , mg/dl ( , - , ) versus , mg/dl ( , - , ) for ct group. baseline mean lipase level (normal < u/l) for pe group was , u/l ( - , ) versus u/l ( - , ) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving - medications. / ( %) of pe group and / ( %) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. / ( %) of pe group and / ( %) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of . pe treatments (txs) (median of , range - daily txs) using % albumin; / ( %) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were < - mg/dl and lipase < - u/l ( . - . x upper limit of normal). mean triglyceride levels after pe txs were , mg/dl ( - , ) for pe group (mean decrease %); mean triglyceride levels after additional hours of ongoing ct were , mg/dl ( - , ) for ct group (mean decrease %). while the pe group achieved a greater mean decrease in triglyceride levels after pe txs (compared to the ct group after hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p> . ). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a -year-old man with a chronic history of hypertriglyceridemia > mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa guidelines, in a patient unresponsive to optimal medical management. asfa guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely % albumin for exchange fluid ( % albumin procedures) or partial plasma replacement ( - units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while were performed with % albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* , , metha apiwattanakul , sompis santipong , jutaluk jaipian , jettawan siriaksorn and ponlapat rojnuckarin . chulalongkorn university, king chulalongkorn memorial hospital, prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june through february were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a % solution. before using, it was diluted to a % albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of tpes in patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was , (range , - , ) ml. although the corrected calcium level was low (< mg/dl) in . % ( / ) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in patients. the first patient had events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was . % ( / ). in , the incidence of tpe adverse effects was . % ( / ) when commercial albumin was used. the difference was not statistically different (p . ). median serum albumin levels pre-tpe and post-tpe were . ( . - . ) and . ( . - . ) g/dl. the increase in serum albumin after tpe was statistically significant (p< . ). eighty-two percent of pre-tpe serum albumin levels were lower than . g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether outpatients who underwent a total of la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer ( %) and ovarian cancer ( %). based on differences in the study protocols la was performed either one-time ( %), two-times ( %) or three-times ( %), with an interval of at least weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, out of patients ( %) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in % of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was . x wbc consisting of . x mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was % lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg -related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to , iu/mls (ref. range < ) and anti-cyclic citrulline peptide antibody was elevated to , units (ref. range < ) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were , and mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was . . peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be . centipoise (cp) at admission (ref. range . - . ). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated - total plasma volume; replacement fluid - % albumin and normal saline in a : ratio; replacement fluid volume: % of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to . cp. serum igg, igm and iga levels decreased to , and mg/dl respectively. her rf had decreased to , iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan -dec ). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with % human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between to years age (m: f; : ) underwent tpe procedures with an average of . per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three ( %) patients had only visual symptoms, ( %) had both visual as well as muscular symptoms whereas ( %) patients had muscular symptoms only. three ( %) out of the seven tested, were positive for aqp -igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in patients with grade- , in patient, and by grade- , in seven. adverse events were observed in % ( / ) of the procedures with allergic reactions to replacement fluid as most common event (n- ) followed by hypotension (n- ). follow up was available in % ( / ) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from . to . for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version . as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version . . the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to / / , plt collections were performed on nine trima accel machines operating with version . . upgrading and validating all nine machines to version . occurred from / / to / / . the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version . ( / / to / / ) was compared to version . ( / / to / / ). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version . and . , adjusting for multiple visits per donor, with significance defined as p-value < . . results/findings: following the upgrade to version . , staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version . of the trima accel showed a statistically significant increase in possible leukocyte contamination from % to % of collections as compared with version . . both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version . . conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version . software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version . currently does not provide added value over version . for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of ml % albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with acute episodes requiring prolong hospital admission of approximately months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of mg/dl, lipase u/l, glucose mg/dl, bicarbonate mmol/l, anion gap . ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day after admission. results/finding: the patient tg decreased by % ( mg/dl) with medical therapy, followed by additional % ( mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below mg/dl at days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from to , a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected , cases whose ferritin levels have been monitored more than twice with an interval of detection in - days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over lg/l. and the upper limit was set to be lg/ l in male and lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: times, to times, to times, to times and more than times. the high frequency (more than times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were , donors included in the study, of which , were male ( . %) and were female ( . %). the mean ferritin was . lg/l in male ( % ci: . - . lg/l) and . lg/l in female ( % ci: . - . lg/l). the result of anova indicates that the group with the highest frequency (more than times) has the significant lowest ferritin level (p< . ). the average change of ferritin if donation over times would up to . and . lg/l in younger and elder y/o male and and lg/l in female. and then for high frequency (half a year more than times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over times in $ days) was reduced from . lg/l in the first period to . lg/l in the third period ( period $ days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* , mary townsend and lizabeth rosenbaum . university of new mexico hospital, blood systems, inc., blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* , william korzun , teresa nadder , susan roseff and elizabeth ripley . virginia commonwealth university, virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for % of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a -hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from to could be a result of a change in the blood drive timing of the training schedule of that location. in , basic trainees at site a were scheduled at day of . in january , the blood drive date changed to day of . the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from to of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in . these observations support the hypothesis that the increase in hemoglobin deferrals in resulted from the implementation of the male hemoglobin standard change from . to . g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional % of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* , eva alonso , oscar bascuñana , monica romero , teresa vich , elena castaño , laura carbonell , eva palomas , saray almerge , francesc carpio and xavier curia . banc de sang i teixits, institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january to december we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, donors came to give blood, of these, ( %) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t but his hemoglobin levels were lower than our selection criteria. of the donors selected for donation ( . %) had sci lower than t and t . adverse reactions to donation ( . %) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of , automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an month period from january to november . the same information was assembled for the automated bp process for the month period of january to november . the automated bp process implemented in mid-december ; so the december data for both and has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < . . results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p . ). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < . ) with the automated bp while and reactions remained non-significantly lower (p . ). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below . g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < . . results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under . . statistically more visits with hgb less than . g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under . was . % higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from january to march . after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of donors who has donated blood in the blood bank's main branch were used as the baseline for this study. % of donors (n ) accepted automatic appointment booking, whereas some donors (n ) were not comfortable with it. % of those who declined still preferred walk-ins (n ) based on their own time schedule, the rest decided that variable situations (n ), donation frequency (n ) and choice of preferred donation locations (n ) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was %. a comparison was made and found that this study shown a significant increase of acceptance rate by %. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. ) , . . poisson distribution, normal distribution, logistic distribution, lognormal distribution a transfusion (p> . ) in donor and reference populations except in younger ( - yrs) male donors (p< . ; donor . %, reference . %). mean donor sbp, dbp, and pulse were . mmhg, . . mmhg, and . . bpm, respectively. screening blood pressure levels consistent with hypertension ( . % male; . % female) in the - year donor group, significantly (p< . ) higher than the reference population ( . % male; . % female). no differences were observed in the - year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in - year old females. developing blood donor educational materials gay wehrli* , susan rossmann , louis m. katz and dan a waxman . university of virginia health system, gulf coast regional blood center -sugar land, americas blood centers, indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, . page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table . results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level ( th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from to after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from . % to . % in and , respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from . % in to % in . moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately . million doses of ap transfused within . billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since . firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than times and had not donated for more than days or less than times with an interval of more than days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by . times from to and the doses of ap increased by . times from to within years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into groups: those who donated ap once, those who donated - times, - times, - times, and those who donated more than times, respectively. it was found that the number of permanent ap donors who donated ap more than times was only ( . %), but they denoted a total of doses of ap ( . %) from to . conclusion: aps increased at a rapid and steady pace in wuhan blood center from to , which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at sites on consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer < ng/ml and zpp levels > umol/mol heme) at hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp . % (r . ) at first and . % (r . ) at second donations. at first donation when compared to fs hb, only . % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. at second donation, when compared to fs hb, only % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p< . ) suggesting strong evidence against correlation. % ( ) responded to the survey of which % ( ) reported not feeling well after donation. it should be noted that noted that % ( ) female study participants reported feeling unwell after the first donation and had ferritin levels below ng/ml but the zpp levels were less than umol/mol heme. of the % ( ) male participants that reported not feeling well none had ferritin levels below ng/ml nor ven or fs zpp levels above umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from . to . gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from blood centers over two intervals, july-dec. and july-dec. (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male m, female f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab . , chicago il). p <. was considered significant. results/findings: data were provided by of centers invited, representing , , and , , wb donations and , and , ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from . % to . % in the two intervals among aggregated donation attempts (p<. ), and for m ap from . to . % (p<. ). the mean "by center" deferral rates (table) were similar to that and significant (p<. ). mean by center hb deferral rates among f donations during the two intervals were . and . % (p . ) for wb, . and . % (p . ) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only centers could provide specific high vs. low vs. irregular pulse deferrals; provided only a summary (i.e total pulse deferrals), and could provide none. for bp, provided detail (high vs. low), summary and none. p deferrals increased in the successive intervals among f wb donors from a center mean of . to . % (p . ) and for m wb donors from . to . % (p . ). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba c levels among those with rbs > mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from st march to st march . total of , blood donors were tested for rbs. those with rbs > mg/dl were further tested for hba c by gold standard hplc method using variant ii biorad. blood donors with > mg/dl rbs and hba c > . % were advised to consult a physician for further evaluation. results/findings: of the , donors tested, ( . %) donors showed a rbs of > mg/dl. forty two ( . %) were males and ( . %) females with a mean age of . years ( - years). of these, ( . %) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining , ( . %) of them had a family history of dm. of these donors, donors did not give a consent for testing for hba c. among the donors tested for hba c levels, ( . %) had hba c > . %. all the donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is . % ( of donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* , hwee huang tan and ai leen ang . health sciences authority blood services group, health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from . to . g/dl last may . the current minimum acceptable hemoglobin for male donors in singapore is . g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels ( . - . ) and in donors with hemoglobin g/dl and above. study design/method: during a month period, serum ferritin testing was performed on regular male whole blood and regular male apheresis donors who made at least donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin . - . ) group b (whole blood with hemoglobin ! , group c (apheresis with hemoglobin . - . ) and group d (apheresis with hemoglobin ! ). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below ug/l is considered low and levels below < ug/l are considered having absent iron stores. results/findings: . % of donors in the study have ferritin levels below ug/l. there were more donors with low ferritin in group a compared to group b, % and % respectively (p< . ). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, % and % respectively (p . ). ferritin results for the groups can be seen in table . conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or . % have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin . - . g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to . g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of % male and % female students in the age group of - years. only % of the students have heard about voluntary blood donation and % of the students have given blood once in their lifetime and among them % are blood donors at the moment. % of the participants believed that there is a specific reason why they don't donate blood and % believed that there is a risk involved for the donors, when donating blood. % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. ( ); miscellaneous effects were reported in courses. side effects led to interruption of supplementation in instances. ferritin levels (mgt sd) at entry into the program and at the last visit were . and . . mg/l in participants, vs . . and . . mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took ! % of the tablets. ferritin levels< mg/l were found in , % of participants and . % of controls. deferral for low hemoglobin was below % in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only % of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under cfr . and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § . to make a collection under this provision if the requirements set forth in § . (a)( ) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in , an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of - ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached , of whom ( %) are c y homozygotes. without active recruitment, accrual rate is about per quarter, with % of subjects qualifying as allogeneic donors. the mean current age is . years, % male, % caucasian. the majority of hh donors ( of an active cohort of ) are in the maintenance phase of therapy with an average of . donations/year and a % deferral rate. over the last years, hh donors contributed approximately - % of the hospital's allogeneic blood supply, averaging whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided - % of blood for in vitro research at our institution with an average of wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over years. since / / , with an increase in male hgb deferral threshold to g/dl, there has been only hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm- non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm- non-invasive occlusion spectroscopy device. over a span of days, eligible blood donors, both male and female, were first screened by the nbm- non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the blood donors for the performance of hb measurement on the sysmex hematology analyzer within - hours of collecting the venous samples. results/finding: the sd of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed . g/dl. the hb measurements obtained from the nbm- and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be . g/dl. the precision of the nbm- yielded a co-efficient of variation of . g/dl and a standard deviation of . g/dl. conclusion: the operators found the nbm- easy to install, maintain, and operate with minimal training. the nbm- non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder , ravi reddy , dhuly chowdhury , don brambilla and edward l. murphy* . sanbs, rti international, ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in and followed them for one year. within days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used -point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. ) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included , first-time black donors with median age and female predominance ( %). within one year, , donors ( %) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable ( strongly agree to strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) . , % ci . - . ), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or . , % ci . - . ). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or . , % ci . - . ) and "i wasn't treated well by the <blood center> staff" (or . , % ci . - . ). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or . , % ci . - . ). a secondary analysis treating the likert scales as -level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard , ramya ghantasala , obhijit d hazarika , nicole leonard , cori a polonski , zachary b wunrow , michelle heleba , jan k carney and mark k fung* . university of vermont larner college of medicine, american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" ( . %) and "traveling is a time for me to relax." ( . %). of the respondents who travel in the summer, very few reported donating while traveling ( . %). summer donation rates between summertime travelers ( . %) and non-travelers ( . %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website ( . %) and phone ( . %). willingness to use a regional blood donation smartphone app was highest among respondents ages of to ( - %) and lowest among ages and older ( - %). of respondents with no prior knowledge of summer seasonal shortages ( %), / rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv , and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin- (il- ) and il . others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent hard spins at rpm for minutes with separation after each spin on a compomatev r g . plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers ( m [ %]: [ %] f); median age years (range - ) donated a unit ( ml) blood from which buffy coats (average volume ml) were processed. the buffy coat process was previously validated on wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures and . all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by % within months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for days. results/findings: there were completed responses, of which ( %) indicated that their hospital had an msbos and ( %) did not. the majority of hospitals without an msbos were academic centers ( / , %) from oceania ( / , %) or europe ( / , %), had between - beds ( / , %); the majority of these hospitals transfused between , - , rbcs ( / , %) per year. / ( %) are going to implement an msbos in . of those with an msbos, the majority / ( %) were from north america. the majority were academic hospitals ( / , %) with - beds ( / , %) that transfused ! , rbc units per year ( / , %) offering a wide range of surgical services. on average there were procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of % of the procedures listed, a pre-operative type and screen for %, crossmatching rbc units for %, and for % of procedures a different recommendation was made. most ( / , %) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only / ( %) of msbos' were created solely by using procedure-specific data, and most ( / , %) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually ( / , %), and the hospital transfusion committee is often ( / , %) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents ( %) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, % of respondents felt that it was regularly used by all surgeons and anesthesiologists; % felt that it was not used at all at their hospital, % did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, % of the hospitals currently without one indicated that it would be implemented in suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a -bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see , hospital admissions and nearly , emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in was approximately $ . m. in nov. , an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $ . m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns ( and ), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee . development of evidence based transfusion triggers. . education on evidence based transfusion triggers across multiple campuses, specialties and resident programs . clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to " " unit instead of " " units. . updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in , we were still able to reduced blood product expenditures by $ , when compared to . conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* , anna w rains and christopher t clark . university of tennessee graduate school of medicine, univeristy of tennessee medical center, univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for week post-delivery, with cost of $ . per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc system. by using the blood volume values, and assigning a value of . ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in , there was a total of , infants delivered at our facility. out of all the deliveries, ( %) infants were transferred to the nicu. of those infants, % received at least one red blood cell transfusion and % received at least one platelet transfusion. of the infants transferred to the nicu, ( %) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to % (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from . % all the way up to . %. in those infants, the birth weight ranged from - grams, and the gestational age ranged from weeks to weeks and days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* , shannon davis , suzan new , vaishali patel and oren guttman . university of texas southwestern medical center, ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable : communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to % of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july ; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of randomized controlled trials ( , conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* and stephanie rogers . dignity health st joseph's medical center, dignity health background/case studies: over million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels > . g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of . g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of . g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to hospitals including cme presentations, online physician and nursing educational videos, communication tools including infographics and " is the new " buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb > . g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy to fytd , there was a % reduction in prbc units transfused to patients with hgb > . g/dl, starting at a baseline of % down to %. this represents an fy annualized savings of $ . m, from a baseline of units per , patients days down to an average units and approximately , fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels > . g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm - . ) reduced rbc units and two studies decreased the percentage of patients transfused (or . ). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or . ) and rbc units transfused (sdm - . ). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a- method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was g/dl/ % or g/dl/ %. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately % of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was units/ month. after the first months of buc activation the mean number of units was units/month a reduction of units/month or % of nonsurgical blood use (p . by t-test). non-surgical rbc use now represents approximately % of the total rbc use hospital wide a % reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital - month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital ( beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance ( q vs. q ): present and completed consents ( vs. %), present and completed nursing flow sheets ( vs. %), transfusion thresholds supported ( vs. %), discharge instructions provided ( vs. %); ( q vs. q ) vital sign compliance ( % vs. %). jwp increased from to ( / - / ). cost savings were realized by decreased utilization and implementation of bpa. (table - q ) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately - trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only percent of coolers fully used. it also consumed valuable staff time as technologists typically made - trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated - hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains - units of group o rhd negative, units of o rhd positive, and units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in , the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june -october were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a -month period, , cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table below. overall, ( %) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." ( ), "active bleeding" ( ), "platelet count of . . ." ( ), and "downtrend" ( ), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, ( %) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years - . study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of , g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey ( questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of complete responses ( %). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, % with physicians, and of these, % reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only % responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of - g/dl ( %), platelet count of - , ( %), and inr of greater than . ( %). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per , inhabitants may vary folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies ( . % and . %), for the remaining strategies weights varied between . % and . %. we estimate that , patients would be eligible for pbm strategies in one year time horizon, resulting in premature death avoided ( . % reduction) corresponding to a gain of approximately , life years and a reduction of , ( . %) disability adjusted life years (daly) relative to the current clinical practice. a decrease of , in-hospital days is expected mainly due to a . % reduction in hospital length of stay and a . % reduction in -day readmission rate. in this population the overall transfusion rate could decrease to . % from the current . % ( . % reduction) implying , blood transfusion avoided and , red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted < ml of rbcs, all cases with ml transfused were captured and only . % of the time > ml were used. if - ml rbcs were predicted to be transfused, > ml were used % of the time. if predicted usage was > ml, % of the time it exceeded ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for > ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than . is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a month period (jan to mar ). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the month period, there were patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of units of blood were requested. units were crossmatched, of which units were sent to the operating theatre (ot). only . % of blood issued to ot were transfused (n ) while the rest were unutilized. the observed ct ratio was . . conclusion: although only % of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of ! . , with almost % of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study patients who received tranexamic acid (txa) (study group) and patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < . g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were unique alert encounters. of these, ( . %) led to a crossmatch being ordered while ( . %) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds ( . g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds ( . or . g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at academic hospitals with data-derived msbos. study design/method: the hospitals were in groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if % of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if - % of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if ! % of the patients had been transfused. data were collected at each center over a month period between january to march and included a maximum of cases per hospital during that one month to ensure equal representation between centers results/finding: between these centers there were a total of cases analyzed. some of the more frequently performed surgeries included orthopedics ( % of cases), general surgery ( %) and cardiac surgery ( %). there were t&s ordered for these cases, of which were positive for antibodies on the day of surgery. of all the t&s ordered, % were ordered in accord with the msbos recommendation, % were ordered when the msbos did not recommend one, and in . % a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio . for prenatal hemoglobin (hgb) - . ). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of % among obstetric patients. study design/method: we studied a sample of anemic (hgb< . g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of women were enrolled, with median age (interquartile range - ) years, median gravida / para and median gestational age weeks. mean hgb before referral was . g/dl and most were already taking oral iron therapy. a total of women were hiv positive with mean cd lymphocytes counts of cells/ul; ( %) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and ( %) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in ( %) of women. there was concurrent chronic disease (n ), infection (n ), vitamin b deficiency (n ) and antenatal hemorrhage (n ); had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a month old boy presented to our institution after a month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige ( ku/l; rr: - . ). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp , while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp cd lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $ . %. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of . % over the last years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from to , with a peak of . % (range . %- . %). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for mtps. results/finding: in , mtp was activated times. in cases the patient did not receive any blood product and in cases plasma was already available at the time of rbc allocation/issue. this left cases to evaluate. the median time to plasma availability was minutes (range minutes - minutes). the mean plasma wastage for mtp activations was % (range - %). of the cha replies, were using thawed plasma and their wastage was </ %. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of minutes. our implementation began with thawing a single unit of plasma for "level neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are . %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated cases of gtx in pediatric patients with age from months to years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of granulocytes donations from to were collected from health donors ( male/ female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, mcg/kg/dose (donors up to kg, maximum of mcg) and oral dexamethasone ( mg), h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with gy. results/finding s: granulocytes number increased times from basal levels and the median of cells collected were . x (range . - . ). in general donors reported no significant adverse reactions. from donations, only in was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table . adverse reactions for patients were minor and not frequent ( in gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* , elena kurnikova and pavel trakhtman . russian institute for paediatric haematology, oncology and immunology, instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period / - / . donors underwent granulocyte mobilization with g-csf in a dose - mg/kg s.c. - hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed granulocytes collections in donors ( f, m), ages to . the mean increment of wbc after stimulation was , x /l ( , - , x /l; f- , x /l; m- . x /l); in the age groups - years (n ) and over years (n ), the mean increment of wbc was respectively , x /l ( , - , x /l) and , x /l ( , - x /l). the mean volume of treated blood was , a , ) . the mean number of total wbc in the product was , x /l ( . - x / l; f- , x /l, m- , x /l). only three donors ( , %) had apheresisrelated adverse effects the analysis included granulocyte transfusions in cases of infectious complications in patients. the mean number of transfusions was . ( - ). each patient received transfusions from mean donors ( - ); the granulocyte transfusions started at the mean days ( - ) after infection; the mean dose of wbc on the transfusion was . x /kg ( - . x /kg). wbc increment was able to estimate after transfusions. wbc increment was recorded after transfusions ( . %); the mean increment was , x /l ( , - , x /l). efficacy was determined by the number of patients who survived an infection episode and amounted to . %. in the cases of severe sepsis, the efficacy of transfusions was . %. the frequency of post-transfusion reactions was %, (n ): febrile nonhemolytic reactions , % (n ); pulmonary complications , % (n ); the anti-hla antibodies formation , % (n ) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp hemolytic disease of the newborn due to anti-rh keegan barry-holson* and jennifer andrews . stanford university, dept of pathology, stanford university, dept of pathology & pediatrics background/case studies: anti-rh is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g p > mother with a negative st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at hours of life because he was found to have anemia (hemoglobin . g/dl), severe hyperbilirubinemia (total bilirubin (t bili) . mg/dl), reticulocytosis ( %) and a positive direct antiglobulin test (igg ). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of . mg/dl on day of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin . g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* , cyril jacquot , valli criss , philippe p pary , jay greenberg , naomi lc luban and edward cc wong . children's national medical center, quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as - %. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of . g/dl/ . %. wbc counts ( x /l) were mildly elevated and platelet counts ( x /l) were within normal limits. her history was notable for upper respiratory tract infection days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total . mg/dl, direct . mg/dl), normal ldh ( u/l), and undetectable haptoglobin (< mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin ( - reactivity) with positive autocontrol. dat was positive for anti-igg and negative for c despite a positive cold antibody screen. the patient weighed . kg with an estimated total blood volume of ml. she initially received simple transfusions totaling ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of %, utilizing the central venous catheter. no adverse events took place over the course of the hour exchange. her one hour post-exchange hb was . g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin ( ). after initiation of steroid therapy (methylprednisolone, mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels ( mg/dl). at a subsequent follow-up visit months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a or gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, . /- . ml/ second) or with a mechanical syringe pump (slowly, ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the g catheter, the mean change in hct was - . /- . % with the one-way valve and . /- . % without (p< . ). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p< . ). during rapid manual transfusion with a g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse . ml when using a one-way valve (change in hct versus time: r - . , p< . ) compared to a significantly different (p . ) slight increase in hemolysis for samples that took less time to transfuse . ml when not using a one-way valve (change in hct versus time: r . , p . ). correlations between time and hemolysis were similar, but insignificant using g with washed rbcs and the g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in . % of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge . study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at weeks gestation with passive anti-d and an anti-ge titer of . she was d-and ge:- ,- , by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge . the birth hemoglobin (hgb) was . g/dl, reticulocyte (retic) was . %, bilirubin (bili) was . mg/dl; the infant was discharged. on day of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb . g/dl, retic . %, and bili . mg/dl. ge -blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh . obstetrics had to authorize maternal blood donation due to her hgb of . g/dl. maternal blood collection and rbc washing was expedited and the infant received ml of maternal rbcs within hours, at which time his hgb was . g/dl. post-transfusion hgb was . g/dl. one week later, the infant was symptomatic with hgb . g/dl, retic . %, bili . mg/dl. a nd aliquot of ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb . g/dl, retic . %, anti-ge titer , and needed another transfusion. the maternal blood stored for just weeks had hemolyzed necessitating a nd maternal donation for baby's rd transfusion. at weeks, the infant's anti-ge titer was , hgb . g/dl, retic . %; no transfusion was necessary. at weeks of life, hgb was . g/dl, retic was . %, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge hdfn. molecular analysis revealed that the mother was homozygous ge -negative ge* .- , the father had homozygous wild type ge* , and the infant was heterozygous ge* /ge* .- . conclusion: the infant had hdfn due to antibodies to the high prevalence ge antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge . hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results ( ) supporting a hemoglobin trigger of g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in , , rbc orders occurred and the top three patient groups were: % in congenital heart disease patients, % in hematology/oncology patients and % in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was . g/dl as measured in the hours prior to rbc order placement. in , ffp orders occurred and the top three patient groups were: % in neonates in the nicu, % in congenital heart disease patients and % in pediatric intensive care patients. average inr of every patient was . as measured in the hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates ( - months of age), infants (> - months of age) and children (> months- years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after months, pr-plt represented % of our platelet inventory (average daily plt inventory: units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among transfused neonates ( - m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased , dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of pediatric patients receiving prbc transfusion over a month period were retrospectively reviewed. a total of patients were identified as receiving allogeneic prbc transfusion. patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was . g/dl with post-transfusion hgb rising to . g/dl. the mean prbc volume transfused was . ml using a dose of ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of - ml/kg in a kg patient, for instance, would translate into full prbc units (about ml) in an average size adult. the current standard dose of - ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a month-old, previously healthy female infant who presented to the hospital with a -week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of . g/dl and . %, respectively, platelets of , , and a reticulocyte count of . %. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of . mg/dl with a direct fraction of . mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens ( total) were non-detectable. the patient was started on a -day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c . an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her -day hospital course, the patient received rbc transfusions on the day of admission and several rbc transfusions thereafter (see table ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day . her hemoglobin rose to . g/dl on hospital day and increased to . g/dl on hospital day . at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to . g/dl on day after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is or . platelet and leukocyte immunohematology, testing and genetics table . of pairs, pairs were complete match ( / ), pairs were partial match ( / ), pairs were complete mismatch ( / ). the matching rate of hla-dpb in our study is %. conclusion: the matching rate of hla-dpb in / hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china ( ) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts . therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a year old male with a -year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts < % and inhibitor of . . on day of admission platelet count was x /l which decreased within five days to x /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were x /l and x /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to . x /l achieving the ratio of previously shown to be diagnostic of ttp. on day his a-ipc and platelet counts had improved to . x /l and x /l respectively. absence of anti-pf antibodies ruled out heparin-induced thrombocytopenia at this time. on day he had an unexpected decrease in both a-ipc and platelet count to . x /l and x /l respectively, worsening by day to . x /l and x /l respectively despite daily tpe. patient received additional tpes that failed to improve a-ipc or platelets which on day were . x /l and x /l respectively. a-ipc had remained at this level for days suggesting that the observed decrease was irreversible. adamts activity remained < % low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa- a, hpa- a, and hpa- a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa- a, hpa- a, and hpa- a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p and gi , specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa- aa, hpa- ab and hpa- aa) were collected and reacted with anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex . the hpa- a serum was diluted to serial dilutions (from neat to / ) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa- a, hpa- a, hpa- a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( . vs . ), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa- a, anti-hpa- a, anti-hpa- a. furthermore, because the platelet was hpa- aa, the hpa- b serum did not react with the coupled beads with mfi was comparable to negative control ( . vs . ). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa- a was / ( . iu/ml) and / ( . iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa- a, hpa- a and hpa- a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in % of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from january to december . clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c , c bp, thbd, dgke, cfhr , cfhr , cfhr and cfhr ) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of patients tested, pathogenic mutations were detected in % ( / ) and vus in % ( / ). % ( / ) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and ( %) had vus. % ( / ) of patients had primary ahus; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. % ( / ) of patients had secondary tma; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. in patients with pathogenic mutations, % ( / ) were children, . % ( / ) had a positive family history of ahus and % ( / ) had recurrent disease. patients with primary ahus had a significantly lower age at presentation ( vs. yrs; p-value: . ) and a higher proportion of pathogenic mutations ( % vs. % p-value: . ) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl and sysmex xn compared with a flow cytometric method farshid ezligini , kjersti roen eriksen , annette vetlesen , thomas larsen titze and geir hetland* , . oslo university hospital, university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion ). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl (horiba abx, montpelier, france) and xn sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x /l sd were , (<) , (<) and for counting by sysmex toa, pentraxl , and the gallios flow cytometer, respectively. sysmex count was the very lowest a transfusion vol. supplement s abstract ( . % less than for flow cytometry), but all plt counts were significantly different (p< . ), although least so ( . %) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing , arishma lata , roland russnak , zachary antovich , heather dunckley and thierry viard* . new zealand blood service, linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of reactions that identify both variants of relevant snps located within hpa genes (hpa- through hpa- , and hpa ). genomic dna purified from blood samples, previously genotyped for hpa- ,- ,- ,- ,- and - by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were % concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late and to date has tested dna samples from blood donors ( donors were tested in duplicate). concordance between the sample replicates was %. there were occasions where the assay had to be repeated, giving a repeat rate of . %. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa- ( . %) and hpa- ( . %) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd antigen deficiency expression in jiangsu chinese han population qing chen* , jianyu xiao and chengyin huang . jiangsu province blood center, jiangsu province blood center background/case studies: cd has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd deficiency varies widely among different ethnic populations, with the frequency of - % in asians and . % of african americans, respectively. however, there is little information on the molecular basis of individuals with cd deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd expression levels and to determine the molecular basis of cd deficiency on the platelet surface of the han population in jiangsu region. cd expression levels on platelets were detected by flow cytometry among blood donors in jiangsu region. donors without cd antigen expression on their platelet surface were further to be determined the expression of cd antigen on their peripheral blood monocyte cells. the coding exons of cd gene and adjacent introns were amplified and sequenced in cd deficient individuals. results/finding: among these blood donors, cd -deficient and cd -expression individuals were . % ( / ) and . % ( / ), respectively. the frequencies of type i and type ii cd deficiency among the study population were . % ( / ) and . % ( / ), respectively. among individual with platelet cd expression, according to mean fluorescence intensity (mfi) value, , and individuals showed low, moderate and high expression levels of cd , respectively, and their mfis were . . , . . and . . (p< . ), respectively. the type i cd deficiency individual were heterozygous for - a>g and - c>g, respectively. among type ii cd deficiency individuals, two harbored a t insertion at position in exon which caused frameshift at codon ; one has a t>c exchange at position in exon which resulted in a tryptophan to arginine substitution at codon ; one has a a insertion before the th bp of the start codon atg in the promoter region; one were heterozygous for t>c and t>g, respectively. conclusion: platelet cd surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd deficiency was higher than that in type i. the study findings indicated that the frequency of cd deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd -deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd -deficient individuals widely varies among ethnic groups, with % to % in japanese, % in sub-saharan africans, . % in african americans, and . % in caucasians. although some studies of cd deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from unrelated platelet-apheresis donors in the eastern china. the expression of cd antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd and peanti-cd ). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd -nagtive phenotype. for those donors with cd -negative platelets, cd antigen expression on monocytes was analyzed further to distinguish between cd type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china ( ) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd in all samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd phenotypes using the (mean sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd deficiency on platelet, in which one sample was cd negative both on platelet and monocyte. the frequency of cd type i and type ii deficiency in the eastern chinese donors was . % and . %, respectively. the average mfi of cd deficiency samples was significantly lower than cd positive samples ( . . vs . . , p< . ). conclusion: the frequency of platelet cd deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd -deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd antibody. background/case studies: cd (gpiv, chromosome q . ) is an kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd deficiency (cd -n) is observed in - % of africans (t g) & is classified as either type i (cd -n plt, cd -n mono) or type ii (cd -n plt, cd mono). an acquired type ii cd -n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type cd -n individuals can develop anti-cd alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an year-old, group o african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin . g/dl & plt count k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, - % blasts & a complex karyotype with del( )(q q ) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < . hla antibody testing was negative (class i panel reactive antibody (pra) %). the patient was plt-xm-incompatible with most donors ( / ). a trial of group o, plt-xm-compatible plts was unsuccessful (cci ). subsequent testing for plt-specific alloantibodies identified anti-cd . fc-phenotyping showed no cd on patient's mono or plt, consistent with type i cd -n. preliminary dna results show that the patient is heterozygous for t g. because cd -n apheresis plt were unavailable from blood suppliers, the patient's children & grandson were screened as possible donors: all showed normal cd expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day ) demonstrated new class i alloantibodies (pra %) in response to transfusion ( apheresis plts, rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd -n & severe plt refractoriness in the setting of new mds, and q-chromosomal abnormalities. the absence of cd on plt & mono support congenital type cd -n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel , kristopher fernandez* , eric senaldi , pascal george , michael seul and ghazala hashmi . biomolecular analytics, central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur http://bit.ly/ q heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $ adult donors who had made ! donations in the previous months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for , , , , , , , , , and for hla class (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/ pdplf ) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a * : chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are , hla alleles documented according to the imgt / hla sequence database in janury , and more than % of them were identified in the last years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a * : allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a * : : : , but nucleotide substitution in exon , by nt c-a (codon acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a year-old pregnant woman typed as ab , who delivered a baby affected by severe hdfn. the newborn was typed as b and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted with the four monoclonal anti-d used (igm clones p x and rum and the blends clones th ms and d d ) and were typed as c-c e-e . an anti-d was identified in her serum. molecular analysis showed the c>t and a>c in exon , the snp t>c changes in exon and the t>g nucleotide change in exon . the set of snps found is similar to the molecular background of dol , except for a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of immuno hematological tests ( abo/d grouping (including newborn samples), extended erythrocytic phenotype, antibody screening, antibody identification, dat) and crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in . % of the abo/d tests (n ), , % of the antibody screening tests (n ), , % of the antibody identification tests (n ) and % of the dat tests (n ). there were discrepancies ( abo/d for the same patient, for antibody screening and antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than min). detection threshold of the d antibody was assessed at . ng/ml ( . ui/ml) whereas the french recommendations are ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a year old g p presented during her fifth pregnancy with anti-kpb with an initial titer measured of . by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams ( . moms) peaking at weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was . mg/dl with . g/dl hemoglobin. the baby typed as o positive, kp (b ) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by weeks of age. his hemoglobin recovered to . g/dl with an indirect bilirubin of . mg/dl at weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c d-specific dat may be too insensitive to detect low, but significant levels of c d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a " " reaction corresponds to about molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c d have been published. however, these are mainly designed to quantify the fraction of rbcs with c d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c d. study design/method: ten microliters (ul) of : (after documenting experimentally that this amount ensured maximum binding of anti-c d) mouse monoclonal anti-human anti-c d (abcam, clone c ) were added to ul of a . % rbc suspension. after incubation for minutes at c, samples were washed x , and ul of : diluted anti-mouse-f(ab) -pe (ro , dako) were added. after incubation at c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a rbcs stained with levels ( fold dilution, : - : ) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c d, edta-stabilized samples from healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c d (values ranging from to , abc) with level of -serum dilution (used to sensitize a rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r . , p < . ). compared with dc-screening , the sensitivity of the flow cytometric assay was superior. it detected c d sensitization at least dilution steps further. the median normal level of rbc-bound c d was abc (range - abc, n ). the assay enabled demonstration of specific c d-sensitization in the patient; the level of rbc-bound c d in the sample was significantly elevated ( , abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt gene. in humans, the abo and gbgt genetic loci are located on chromosome q , and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors ) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt genes have been constructed of vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion vol. supplement s the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c , e-, e and k -. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d , c-, e-, partial c , partial e . the probable rhce genotype, rhce*ce-jal/rhce*ce g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant woman and her newborn carolina bonet bub* , maria giselda aravechia , thiago costa , marilia sirianni , eduardo bastos , leandro santos , lilian castilho and jos e kutner . hospital israelita albert einstein, hemocentro unicamp background/case studies: rhd*weak d type is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion ) the c. g>c change (p.g a), which characterizes the rhd*weak d type allele is a splicing variant that induces skipping of the whole rhd exon . we report an altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- /ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d w c-c e e . the samples showed weak hemagglutination reactions ( / ) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon in both dna samples. sequencing showed the c. g>c change and the intronic c. - t>a and c. - t>c substitutions, which are associated to the rhd*weak d type allele. conclusion: our results showed that c. g>c associated with c. - t>a and c. - t>c variations had probably a functional impact on splicing inducing exclusion of exon in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type . background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than % of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with , patients and studies from other regions (brazil, egypt and france) with patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was to years and for the other countries to years. available data from us studies included a total of alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens ( . %, . %, . %, . %, . % and . % respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of . % ( . to . , % ci) vs. . % for non-us studies ( . - . , % ci) (p . ) and a transfusion vol. supplement s more alloantibodies per transfused patient ( . vs. . , p . ). similarly, the number of alloantibodies per transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort ( Á vs. . , p Á ). average number of rbc units transfused per patient in the us was also higher compared to data from france ( vs. , p . ). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* , melissa grohotolsky , lisa deblass , bala carver and kip kuttner . health network laboratories, miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: year old white mennonite female g ,p presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at . although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g (r g r) cells. both rhce*c and rhd genes encode ser which determines g expression. rare rhd variant antigens lacking ser are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e c e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r r , r'r, r g r and rr cells. this patient is predicted to be r r (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is a transfusion vol. supplement s excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* , christa voliva , kathy fletcher , heather vaught and tracie ingle . indiana university health, indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology ; : - ) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were > dat positive, were weakly dat positive and were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman . - . ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: bb, bg and bp. the ph of all eluates ranged from . - . with the highest percentage of eluates at a ph of . ( %). sixteen of the eluates tested yielded the same results in both automation platforms (concordance of %). four eluates with different results are summarized in table . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results ( ) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in led to the discovery of human blood groups. in the abo system > alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor ( units over years) who is actually a w . study design/methods: donations were tested with the pk instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns , and and exons and . specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table . tests with anti-a, -a , -b anti-a,b were negative as were the a cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table . the significant changes were found in exons and . in exon there was a nucleotide (nt) deletion of g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly ala. mutations in exon included a nt substitution causing a pro -leu change and a nt deletion c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o. . /abo*aw. ] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a and b cells. this abo discrepancy was caused by the presence of anti-a in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer > : . (schmidt, nacarrow et al. ) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. a transfusion vol. supplement s extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: edta blood tubes collected from random blood donors were used to extract dna from microliters of whole blood on day , and days post collection. blood samples were stored at - c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a /a using a nanodrop (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result ) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the samples, samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from . to . ng/ul. all readings with the exception of ( . ng/ ul) had concentrations > ng/ul. interestingly, the one that was < ng/ ul on day , yielded > ng/ul on day and post collection. over the next months, sets of samples were extracted and tested by hea. eighty-three ( . %) failed extraction and ( . %) failed hea. none of the samples that failed extraction were or days post collection; none of those that failed hea were days post collection; . % were > < days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas , carlos villa , rachel davis-rauser* , helen carpenter and vrunda patel . university of pennsylvania, hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in . communications suggest all patients receiving therapy would have a positive antibody screen because cd is a common antigen expressed on red blood cells. currently, patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at : dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for negative patients was observed up to a : dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a y/o g p at approximately weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [ , ] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m n-s s phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency > . ), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [ , ] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [ ] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays a transfusion vol. supplement s that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in dna samples from chronically transfused patients with scd, in patients with thalassemia and in dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in levels: ( ) rh and k matching; ( ) extended matching and ( ) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for % of our thalassemic and scd patients at level , % for scd patients and % for patients with thalassemia at level and % for patients with scd and % for patients with thalassemia at level . the patients were transfused with a median of . rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to - % with c e k matching and < % with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube ( drops plasma, minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to minutes and specimen volume to drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of clinically significant antibodies were detected using sprca technique, as well as warm autoantibodies and nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being % specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions ( versus ), it identified more clinically significant antibodies ( ) than liss ( ). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd on rbcs patricia a arndt* , anthony salazar and regina m. leger . american red cross blood services, long beach memorial medical center background/case studies: monoclonal anti-cd , e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or aminoethylisothiouronium bromide (aet). chapuy et al described ( ) and validated ( ) ), and % aet (ph . ) as per the aabb technical manual, th ed. these treated and untreated rbcs were stored in alsevers at c and tested on days , , , and by two methods: ) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from total dara patients were tested with reactivity - ), and ) flow cytometry using phycoerythrin (pe)labeled anti-cd . rbcs were also tested on days and or with a serum containing anti-k by peg iat. results/findings: the . m dtt in ph . pbs had a final ph of . and the ph of the commercial . m dtt was . . results are in table ; flow cytometry results from days , and (data not shown) were similar to those from days and . rbcs treated with . m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd by flow cytometry for up to days after treatment. rbcs treated with . m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening ( - %) of reactivity with pe anti-cd . background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon and ) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr ) in gestational week . the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of pregnant women were included. nip rhd was positive in / ( %), negative in / ( %) and inconclusive in / ( . %). compared to the postnatal rhd type, / ( . %) of nip rhd results were false positive (fp) and / ( . %) were false negative. in / ( %) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n ) at gestational week was . ( -and -percentiles: . - . ). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p , ). the fraction of ccfdna was calculated for randomly selected nip rhd true positive cases. median ccfdna ratio was . (the distribution had a highly positive skew, -and -percentiles: . - . ). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r , ; p . ). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of , suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only in , donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, lan-rbc units were transfused over days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day , the patient had symptomatic anemia with hemoglobin (hb) . g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of . g/dl was maintained for days. the antibody screen was negative on day post-transfusion, but strongly panreactive on day , with a positive dat (igg , c ) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from . g/dl on day to . g/dl by day with no bleeding identified, and increase in total bilirubin and ldh (peak . mg/dl and u/l on day ) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day with good response (hb . g/dl). the patient remained stable and was discharged to a skilled nursing facility days later. conclusion: transfusion of lan rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* , andrea gerner , lynne stewart , carol sostok , mollie bell and gregory r halverson . st. elizabeth healthcare, hoxworth blood center background/case studies: anti-f was first described in by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the th antigen assigned to the rh blood group system (isbt rh ). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused unit o-rbcs. two weeks later the patient received an additional o-rbc. within days the hgb had decreased from . to . g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused r r (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c e f phenotype. the rh phenotype and as was repeated on a sample collected days later. the c typing was micro positive, mixed field only after minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post hour hgb increment from the receipt of a standard unit of blood should be near g/dl (or % hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a . g/dl increase, and the second unit was only . g/dl. the last transfusion of units increased by only . g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of r r units was nearly complete. in a case from , ohto and kariyone (transf. ; vol , no. ) reported a cr Ásurvival study of f rbcs in a patient with anti-f. they showed that the initital survival of f cells was fairly normal, however, after days, there was a sudden increase of red cell destruction, and by day all f cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with . m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to days following the dtt treatment of rrbc. reactions were graded using standard serological grading of (negative) to (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to days. this suggests that dtttreated reagent red cells can be stored for at least days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with . m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract - c, and observed for hemolysis (none was seen) for up to days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted - with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* , monique scott , garcia curtis , ellice wong , alexa j siddon and christopher a tormey . yale-new haven hospital, va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately % to % of persons of african descent is characterized by neutrophil count of < . x /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p< . . results/finding: subjects who were clinically identified as having probable ben included patients (mean age . ; all self-identified as african-american; / were male) and controls included patients (mean age . ; self-identified as african american; ( / male). all of the cases ( %) diagnosed with ben had fy(a-b-) phenotype. mean anc ( . x /ul) and wbc counts ( . x /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p . and . , respectively) compared with controls (mean anc . x /ul ; mean wbc count . x /ul). there was no significant difference in mean platelet counts ( x /ul vs x /ul; p . ) or mean hemoglobin levels ( . g/dl vs . g/dl; p . ) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih- had % concordance for all blood grouping assays. for ahg assays, the ih- detected an anti-jka e, anti-fya warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih- identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih- with anti-igg,-c d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih- analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih- is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of tests were performed on , adult patient samples and donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in . % of samples tested. there were discrepancies, all antibody screening ( false positives, failure to detect a very weak prophylactic anti d and positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing - group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* , kathleen bensing , michael schanen , cindy piefer , randall w. velliquette , christine lomas-francis and connie m. westhoff . immunohematology reference laboratory, versiti/bloodcenter of wisconsin, immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new / / / cecf / / / rhce*ce or rhce*ce compound heterozygotes ce g ce g or ce c, g or ces or ceti / / / ce g ce c, g or c, g / / / ce g ces or cemo or ceek or ceek(var) or cern / / / ce c, g ce c, g or cemo or ceti / / / ce c, g/ce g/ g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi / / / total / / / a transfusion vol. supplement s reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd / and rd / , and a licensed comparator anti-e (p gd ms ), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n ) or edta blood from donors (n ) and were tested using a manual tube method or on a pk automated platform. a score ( ) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table ) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r r , r r , r r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: were rhce*ce that were in trans to rhce*ce; were various rhce*ce plus rhce*ce c compound heterozygotes; were rhce*ce or rhce*ce homozygotes; were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with rhce*cear/rhce*ce c compound heterozygote, and with rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and of rhce*cecf homozygotes were detected using the comparator reagent. rd / and rd / failed with and e variants, respectively (table ) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd / clone. none of the reagents detected e antigen variant expressed on example of rhce*cehar/rhce*ce. conclusion: rd / and rd / anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of blood group genes associated with the expression of blood group antigens from blood group systems. we used the illumina's hiseq / system to perform next generation sequencing first on sureselect-enriched genes from dna reference samples with average target design coverage of . %, and then on haloplex-enriched genes from dna reference samples with average target design coverage above . %. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for blood group genetic variants in these genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads ( . %) were mapped to the target regions relative to the sureselect reads ( . %). the mean sequence coverage depth of the targeted bases was around x for sureselect method and x for haloplex method. some exons, such as rhd exons and , , rhce exon , ermap exons and , cd exons and , cr gene (most exons) and gypb exon , are consistently covered with less than x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than % concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than % blood group genetic variants in selected genes. evidence rhce*cehar does not encode for rh (hr b ) antigen debra j bailey* , trina horn , paul mansfield , najmi qazi , pamela nickle , jessica keller , margaret a keller and jan r hamilton . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in and has a phenotype of c e c e w f w , g , hr w , hr , hr s , rh: , rh: with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh: (hr b ) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c. c>g and rhd c. c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d c e c e . her plasma contained an alloanti-s and an antibody that reacted strongly with all random e k s reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d and dc red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e s k red cells homozygous for the rhd*diiia-ce( - )-d, rhce*-ce c, g, t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce c, g, t/ rhd*diiia-ce( - )-d, rhce*ce c, g, t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr ( of sources) and hr b ( of sources) phenotype. conclusion: the rhd*diiia-ce( - )-d, rhce*ce c, g, t haplotype is one of the rh haplotypes expressed by the original hr b individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between / / and / / . all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately years, patients had htla established at least once by titration studies. serological investigations on a total of samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on samples was successful in rule out in % of cases. in an additional patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only % of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for % of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c. c>t (p.thr met). the dau allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion , : ) recently summarized serologic characteristics and associated anti-d alloimmunization for dau family alleles. we investigated two samples with the c. c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample was from a yo multiracial female. her rbcs reacted s at immediate spin (is) and in iat with immucor gammaclone and series and , and mi at is and in iat with ortho bioclone anti-d. rbcs did not react with of (lhm / & / ) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c. c>t characteristic of dau. rhd sequencing confirmed c. c>t and identified two adjacent changes, c. g>t and c. g>t (c. _ delinstt), in exon encoding p.gly leu. sample rbcs reacted w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series and , and quotient albaclone blend and alpha anti-d. papain treated rbcs were s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c. c>t. sequencing confirmed c. c>t and found a new c. c>t change (p.ser -leu) in exon . the c. t has not been reported, but c. g encodes a stop codon (p.ser stop) in japanese (vox sang , : ). conclusion: we report two new alleles: rhd with c. _ delinstt (p.gly leu) and rhd with c. c>t (p.ser leu), both also carrying the c. c>t (p.thr met) characteristic of the african dau cluster. d antigen associated with p. leu is a partial d antigen with a novel epitope pattern. the p. leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by / commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to . the number and diversity of alleles in the dau cluster supports that the c. c>t change is a major ancestral african background allele (wagner et al, blood , : ). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september to present day we got samples of repeated blood donors who are known to be d negative, c positive and/or e negative from blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon , exon and exon in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon to confirm the existence of c. g>a and c. t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the sample, we identified cases ( . %) of total rhd deletion, cases ( . %) of rhd-ce-d hybrid, and cases ( . %) of rhd variant. of rhd variant were determined to be asian type del with c. g>a variation. cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n ) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon and exon of the abo gene were amplified and sequenced. the abo*b . allele was confirmed by pcr-rflp analysis. results/finding: among subjects with b or ab phenotypes, were genotyped as abo*b . . the abo*b . group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage ( . . ) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other subjects with b or ab subjects, genotyped as abo*b . (n ), abo*bw. (n ), abo*bw. (n ), abo*bw. (n ) and abo*bw. (n ), displayed flow patterns differed from the abo*b . group. the abo*bw. , abo*bw. and abo*b . subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (< % in abo*bw. and abo*bw. subjects and < % in abo*b . subject). both abo*bw. and abo*bw. displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b . genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout , the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in % (n ) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in % (n ), % (n ), and % (n ) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n ), anti-jka (n ), anti-k (n ), anti-jkb (n ), both anti-e and anti-c (n ) (see table ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly in cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study volunteers in the gambela region (nct ). the whole ackr gene was amplified in one reaction covering , base pairs (bp). this primary amplicon was re-amplified using nested primers covering nucleotides. nucleotide sequence was obtained by sequencing reactions and manually annotated using ncbi refseq ng_ . . the sequencing covered bp of both exons, bp of intron, bp of '-flanking region, bp of '-utr, bp of '-utr and bp of '-flanking region and encompassed all the variations present in dbsnp and nhlbi esp databases. results/finding: among the samples, a total of snps, including one novel snp in '-utr were observed. snps occurred in the exons, in 'and 'flanking region, in '-utr and in the intron. all individuals carried the snp indicative of the common fy: phenotype; while individuals were homozygous and was heterozygous for the gata box mutation. no splice site mutation was detected. as individuals were observed as being homozygous or heterozygous for snp, we could unambiguously assign distinct alleles. in the remaining individuals with or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than . kb of the ackr gene and identified at least different alleles. the present study found that the vast majority of alleles ( / ) in the gambela population as defined by snps, were similar to the clinically relevant fy* n. allele, which in turn is defined by only snps at positions c. - t>c and c. g>a. out of the remaining alleles, were similar to fy* with the fy(b ) phenotype and was similar to fy* w. with the fy x phenotype. the high frequency of fy* n. ( %) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique ( %- %). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce( - )-d is the most common hybrid and is found in african blacks. it arose by conversion of exons - of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample (male) and sample (multiracial female), both c c e e (presumed r r ), presented with weaker than expected d typing; is and / at iat. rhd beadchip identified the common african rhd*diiia-ce( - )-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c. c>g and c. g>t (heterozygous) was also detected. as rhce*ce with g and t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons and replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce( - )-d were found in both samples. sample (scd male), d c e c e , by rh beadchip had one conventional rhd and rhd*diii type , and rhce*ce g/ces. as rhd*diiia type has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce( c) exons and had replaced those exons in the common hybrid rhd*diiia-ce( - )-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky and ky . we report two different and novel complex rh rearrangements: two samples thought to be r r had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia( - )-ce. in kind, a sample genotyped as diii type rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce c( - )-diiia( )-ces( - )-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples and have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a -year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at g/l. her pregnancy history was not provided. she had received units of packed red blood cells (rbcs) in the past including unit within the last months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r r , r r , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse polymorphisms which determine antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a ) and lu(b ). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of antigens encoded by the kel gene, is organized into exons. there are approximately kel alleles associated with a kell null phenotype (k ) in which no kell antigens are expressed, and alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons , , , and and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k kp(a-b ) js(a-b ). kel-cdna sequence analysis was performed and detected a single transcript species with c. c, c. c, t, and missing the sequences corresponding to exons , and . amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel* allele. this donor was presumed to have a k phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel* m. . here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a and a cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c. deleted g, characteristic of o alleles, c. t, characteristic of a and some uncommon o alleles, and c. a and c. a, characteristic of b alleles. genomic sequencing of exons and confirmed the presence of an o allele, abo*o del g, t, t), and the presence of a b allele ( g, g, t, a, a, c, and a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* , tatiane vendrame , janaína muniz , diana gazito , afonso cortez , lilian castilho and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the -bp deletion in smim in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting and in samples with reactivity of . dna was isolated from peripheral blood and smim was sequenced. results/finding: from donor samples studied, were serologically vel negative by gel-iat but positive by adsorption-elution, presented a reaction and the remained samples showed a reactivity of . genotyping results showed that the samples with negative results and of samples that presented reaction were heterozygous for the bp deletion and presented the a allele rs in homozygous status. from the of remaining samples with reactivity of , ( %) had the a allele of rs and ( . %) had the a allele of rs . in contrast, in the samples with stronger reactions we found the a allele of rs in ( . %) samples and the a allele of rs in ( . %) samples. conclusion: the molecular changes rs and rs are located in intron distancing nucleotides. this study reinforces the association of the a allele of rs with reduction of vel expression and suggests the involvement of a new rs change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as (rh negative), or ! on the neo or ! on the echo (rh positive) for both series and series anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series and series anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in of ( . %) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in of ( %) of samples. of ( . %) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. of ( %) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining of ( . %) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but < on the galileoneo or positive but < on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* , tatiane vendrame , janaína muniz , rosangela person , lilian castilho , afonso cortez and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of samples were included in the study, being previously genotyped as rhd*dar . , rhd*dar . and rhd*dau . the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from - corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon . rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that of samples ( dar . , dar . and dau ) had rhd genes, were phenotyped as c e-c e and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in dar . samples showed the rhce*-cear/ce s genotype, in dar . samples the rhce*cevs. /ce s genotype and in the dau sample the rhce*ce s /ce genotype. table describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized f antibody (hu f -g ) that binds human cd has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu f -g (anti-cd ) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a year old female with progressive follicular lymphoma who was enrolled in phase b/ trial of hu f g in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a year old male with refractory diffuse large b cell lymphoma enrolled in hu f -g clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing ( with anti-a, non-reactive with anti-b) and the reverse typing ( with both a cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase ( to ), at liss- c ( to ), at liss-polyspecific ahg (m ), and at peg-anti-igg (m to ). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at o c ahg phase. the abo typing of the second case performed after anti-cd administration showed a discrepancy between the forward ( with anti-a) and the reverse ( with both a and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is ( ), at c in liss ( ), and liss-polyspecific ahg (m ). the dat and autocontrol were negative. his genotype was determined to be a /o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o. . / o. . , consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n ) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt and a galt. papain-treated patient rbcs were used to screen donor plasmas (n ) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with polyclonal anti-a,b and a monoclonal anti-b (clone g / ) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors or nor antigens. the patient was le(a-b ) and thus a secretor. a positive crossmatch was seen with % of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type or ) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $ years earlier. the reactions are likely due to uptake of recipient-derived ble b (type ) antigen (isbt no. ), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b ). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of . shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive ( ) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from / / to / / . reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of , columns run as part of antibody screens, , ( . %) columns generated "?" results. assuming seconds of technologist time per "?", we estimate that . hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table ) . in cases, all three columns were visually negative but the analyzer reported reactivity with of cells. all cases had mts-gel tm antibody identification panels performed, of also had a mts-gel tm ficin panel. the yield for the panels performed was two routine panels with weak reactivity against hla cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; were negative. one patient newly demonstrated anti-jka. fifty percent ( / ) of visually negative but analyzer positive samples were tested with gel card lot number , % ( / ) with lot , and % ( / ) with lot . conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from patients ( cross-match samples, units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in / patients. further testing was performed in / . eight were tested for the presence of antibodies at c and confirmed in / . rouleaux formation was observed in / patients, / had reactivity detectable at c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* , sandra nance , david moolten and p. dayand borge . ( ): - ). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this . year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from random allogeneic and autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing . % bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* , anna burgos , virginia lew , sunitha vege , susan veneman , christopher j gresens , jonathan hughes and connie m. westhoff . immunohematology and genomics laboratory, new york blood center, blood centers of the pacific, bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons - , and long range sequencing of exon - ( . kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- / ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m n-individuals (meyer et al. br j haematol. ; : - ) . the st a allele, also described as gyp* , is a hybrid gypb-gypatranscript with the crossover in intron . we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a years old pregnant, african american female g p was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a like phenotype. genetic testing did not support the serological findings of a subgroup and a new abo allele, abo* c that has never been reported in correlation with an a like subgroup was detected study design/method: the patient rbcs were typed with anti-a (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a antibody work up was performed using three different lots of a cells and three lots of a cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and c for min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo* c found in the mother. the previously reported abo* a allele encoded an aspartic acid to asparagine change at position p. consistent with an a weak phenotype. also, at least five other alleles encoding an a phenoytpe consisted of polymorphisms at positions c. through c. , giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a / aweak phenotype is encoded by the variant allele abo* c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since ; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n ) were processed with the automated biorobot m robot using the magattract dna mini m extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of . ng/ml and . respectively. in the second phase of the study (n ), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was - ng/ll and the recommended purity was an absorbance ratio of . - . (a / ) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was % concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also % concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano , izaskun apraiz , maría azcarate , miguel angel vesga , montserrat rubia , mercedes piedrabuena , fernando puente , barbera veldhuisen , ellen van der schoot and m onica l opez* . progenika biopharma, a grifols company, centro vasco de transfusi on y tejidos humanos, banco de sangre y tejidos de arag on, sanquin blood supply research background/case studies: it is well established that weak d , and phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type , rhd*weak d type , rhd*weak d type , rhd deletion, rhd*pseudogene and rhd*diiia-ce( - )-d and itgb gene: hpa a and hpa b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa- blood group typing. study design/method: a cohort of previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications / /ce for a ivd product of list a (! % clinical samples, > % neonatal specimens and ! % weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n , %). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa- predicted phenotype were used for comparison. transfusion results/finding: no system failure, % call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a % concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was %. the following id rhd xt predicted phenotype results were obtained: d negative (n ), no amplification variant (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d types , or not detected (n ). regarding hpa- blood group, the predicted phenotype results obtained by id rhd xt were % concordant with bds results: hpa- a positive (n ) and hpa- a negative (n ), hpa- b positive (n ) and hpa- b negative (n ). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types ( % specificity and % sensitivity for d antigen, hpa- a and hpa- b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value < . (clinical analysis) or < x - (gwa) was considered statistically significant. results/finding: of the cohort patients, ( . %) transfused subjects were included with alloimmunized children < years ( . % of ) and alloimmunized adults ( . % of ). in multivariable logistic regression models, age (or . , p . , for age compared to - ), gender (or . , p . , for female compared to male), transfusion history (or . , p< . , for transfusions compared to - ), site, hemolysis (or . , p . , for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or . , p< . ) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from newborns less than , gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on sample pairs. dat test was negative on sample pairs and two were positive. there was % concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on placental blood samples and heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < , g birth weight newborns. o-( . %), ab ( . %), b-( . %), and a-( . %). among the tested donors, . % were d positive with r r being the most common rh phenotype. in the kell blood group system, . % of the donors were k positive, while the k antigen was found to be . %. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a b ) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a b ) and m n-s s at % and . % respectively. the le a and le b alleles were seen in . % and . % of donors respectively, while lu b -phenotype was found in . % of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a b ) and lu(a-b-) were . % , . % and . % respectively, while the m n-s-s-and m-n s-s-phenotypes were not found. the frequency of the p antigen was found to be at . % similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph . and the supernatant was discharged. a dilution buffer containing % human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with ll of washed red cells. the cell suspensions were incubated for min at c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional min at c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for - nm. events were recorded at a frequency of cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* , mark yazer , nancy m. dunbar and biomedical excellence for safer transfusion (best) collaborative . university of vermont medical center, university of pittsburgh, dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of . study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a : dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a -year and -year period, respectively. one center provided plasma and wb testing data for a -year period. results/findings: in total there were group a plasma units tested and ( . %) had a high titer anti-b. the range of high titer group a plasma units between these three centers was . %- . %. of the wb units tested, ( . %) units had a high titer; / ( . %) of the units had a high titer anti-a, / ( . %) had a high titer anti-b, and / ( %) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with . m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd on reagent rbcs and render them free from plasma anti-cd drug interference. procedures for the preparation of . m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on . m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of . m dtt, was adjusted to ph . , ph . , ph . using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n ) were treated with the . m dtt solutions in parallel by mixing : ratio of packed rbcs to . m dtt solution followed by incubation at c. for up to minutes during treatment, the expression of k antigens was measured every minutes by tube method using different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every minutes for each ph level. the reduction in average scores between different phs were also calculated at every minutes to measure the impact of . m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by ! ) after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . complete loss of k expression was seen after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . the reactivity patterns of k antigen tested with sources of anti-k correlate with each other. the reductions in average scores were seen between to minutes range of dtt treatment time when ph . was raised to ph . ; to minutes range when ph . was raised to ph . ; and to minutes range when ph . was raised to ph . . conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to minutes and/or beyond minutes of incubation. the ph of the . m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of . m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n , ) drawn between july , and june , . these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, , patients were found to possess clinically significant red cell antibodies for an overall incidence of . percent. the three most commonly encountered antibodies were anti-d (n ) . %, anti-m (n ) . %, and anti-e (n ) with a frequency of . %. a total of ( . %) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with instances ( . %) followed by anti-e and anti-c with ( . %), and anti-c, anti-e with ( . %). of the multiple antibodies identified, ( . %) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with or . % of the total. the west had ( . %), the midwest ( . %) and the northeast with ( . %). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of . and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the . thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p . ). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* , lindsy rich , sherry stern , sharon wangen and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was to days. results/finding: of the ntd specimens from the immucor neo, resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all results, it was determined that there was no reactivity and a valid result was present. the other samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the absc specimens that were resulted out as positive on the immucor neo, specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: - with ms (n ), - s with ms (n ), no reaction with ms , dgc , p x (n ). samples tested with a polyclonal anti-c showed a - reactivity. d c e c e cases tested with a polyclonal and monoclonal anti-e (ms , ms , ms , ms ) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c. g>a mutation in exon , predicted to encode the p.gly ser substitution. for apparent r r donors, a f-negative type allowed the prediction of a rhce*ce a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce a allele (c and e in cis) in all samples. d c e c e individuals were reactive s with the original source of anti-rh , slightly weaker when compared to rhce*ce a/rhce*ce rbc samples available from our cryobank ( ). conclusion: our results confirm that the c. g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh ). the locr reactivity appears to be rather similar when coded by rhce*ce a or rhce*ce a alleles. this was quite an unexpected finding, since the p.gly ser substitution is close to the critical amino-acid for c/c expression (p.pro ser). none of our cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce a was reported to code for a partial c (rh:- ), we consider that rhce*ce a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes , or . the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d , , or genotypes. study design/methods: between / and / samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types , , or genotypes, but a transfusion vol. supplement s had evidence of rhd genetic sequences in exon and/or intron in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons - to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients ( %) followed by transfusion patients ( %); % had no clinical indication provided. samples ( %) were found to be weak d type , , or ( , , and samples, respectively). samples ( %) appear to be genetically rhd negative. genetic sequencing was performed on samples; had rhd genetic variants that were not weak d types , , or (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples ( %) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types - . of the samples that had evidence of an rhd gene and did not carry the known weak d types - polymorphisms, ( %) of were found to have other rhd variants, and ( %) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types - variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel , curr opin hematol : ) , that individuals with weak d types , , and are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report months experience with rhd genotyping on samples referred with discrepant or weak d typing investigated from january to april . study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for samples ( . % caucasian, . % african american/african, . % multiracial, . % hispanic, % asian, and . % other). results/finding: rhd genotyping identified weak d types , , and in / ( %) and alleles known to encode partial d phenotypes in / ( . %) (table) . uncommon or rare weak d alleles including types , , , , , , (n ), , , , , and were found in ( . %). the partial d alleles found were diverse, but the largest number included partial rhd*d . (n ) and *dar ( conclusion: in a multiracial cohort of individuals with weaker than expected d typing % were due to weak d types , , or and would not be considered at risk of clinical significant anti-d, but for % there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp rhd* . allele causes discrepant genotyping results for rhce small c sabine scholz* , sandra schneider , sabrina k€ onig , susanne helmig and vicky van sandt . inno-train diagnostik gmbh, rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c ( c) and c ( t) is caused by the snp on position on the rhce gene. the rhd* . allele (also known as rhd cat vii type ) carries the snp t>c on the rhd gene and additionally the snp t>c. this rhd* . allele has been described to partially express rhc on the d polypeptide (faas, transfusion, ) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, ) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles ( t>c, t>c) confirming a rhd* . and one rhd* allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series and anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series and series , and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c. a>g change in exon encoding an amino acid change p.met val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c. a>g (p.met val) change in exon . several snps, deletions, and insertions have been reported with changes in exon . phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c. a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* , juan merayo-rodriguez , christopher lough and nancy eckert . lifesouth community blood centers, life south community blood centers, lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is % in most populations and greater than % in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is . / . and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal (k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to . / . the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient was a yo female, c e c e , whose rbcs reacted by echo and by neo with anti-d , and '?' with anti-d . testing with d and d by tube gave and w on initial spin (is) respectively and by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and w with immucor gammaclone anti-d, and all were at iat. rbcs did not react with two (lhm / & / ) of anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c. c>a change encoding p.his gln. patient was a yo pregnant female, c e c e , whose rbc were w at is and at iat with immucor series and and gammaclone, and moderately reactive, is and iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm / & / ) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon from c. to c. encoding amino acid changes p.ile leu and ser asn. conclusion: we found two previously reported rare alleles: rhd with a c. c>a (p.his gln), previously found in france (lefloch et al. genbank ku ), and rhd*dar with part of exon replaced by rhce, reported in sub-sahara africa (granier et al. transfusion : ) designated rhd*dar(ce :v v-s n) with an allele frequency of . to . . blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu f -g is a human monoclonal igg antibody recognizing cd that is in clinical trials to treat hematologic or solid malignancies. cd is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd is thought to enhance phagocytosis and promote anti-tumor responses. cd is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n ) from patients were tested over the course of month treatment. plasma was tested at immediate spin (is) and by iat with r r , rr, d--, rh mod and rh null rbcs, as cd expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg ) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd was observed in plasma as soon as hour post infusion. plasma reacted to at is and with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker ( and ) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was . in contrast, iat reactivity using gamma-clone anti-igg was only w to , and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd titer was at is and peg iat with gamma-clone anti-igg, but was ! with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n ) were reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after x allo-adsorption with papain treated rr cells, but in some samples low level (micro- ) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu f -g anti-cd therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu f -g is igg . reactivity was observed in all phases and with all test methods. cd is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg , can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in . in august , she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was with untreated and with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:- etc. had been excluded, k-phenotyping revealed a k -phenotype. a total of silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel* n. with c. t encoding p. ter (reported in an individual from austria in ). there are two known k -patients in our country, both homozygous for c. t. the daughter was a c. t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k -donors are available in our country. with the help of the isbt rare donor working party, a k o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* , kshitij srivastava , houda romdhane , saloua jemni yacoub and willy albert flegel . nih, dtm/cc/nih, regional blood transfusion center sousse, regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, a transfusion vol. supplement s since . the tunisian population has the largest known prevalence of weak d type . alleles, occurring in of rh haplotypes, compared to in , or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type . in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of , random blood donors were serologically screened for the d antigen using routine techniques. samples with weak reactivity were tested with a panel of monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of discrepant samples ( . %) were observed and expressed the serologic weak d phenotype. among them, carried an allele of the weak d type cluster ( . %), of which samples ( . %) showed the weak d type . allele. only sample each was found for the weak d types , and and the dvii, while samples showed the consensus rhd sequence. no mutation in any of the rhd exons was detected in another samples. the molecular analysis of the rhce gene showed that out of samples with serologic weak d phenotype ( . %) had a variant rhce allele and the most common associations were: weak d type . linked to rhce*cevs. . ; weak d type . . with cear; and weak d type . to rhce*cevs. , while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost % of the weak d phenotypes in tunisia were caused by alleles of the weak d type cluster, of which % represented the weak d type . allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type . in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type . phenotype. there is a possibility that the rhce*cevs. . allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* , heather simmons , christine lomas-francis , gayane shakarian , sunitha vege , lauren hutelmyer , sandra nance , jessica poisson , nicholas bandarenko and connie m. westhoff . duke university hospital, immunohematology and genomics laboratory, new york blood center, arc pennjersey, american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a , year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with . m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c , k , fy(a ),s ]. reactivity was detected to a titer of ; it was not removed by prewarm technique or by x peg alloadsorption. the adsorbed plasma reacted with . m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k , k , js(b ), kp(a b ) and sc: , . her plasma reacted with k o , mcleod, sc: , rbc samples and dtt-treated sc: rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused aliquots of crossmatch incompatible kp(b ), s rbcs. her post-transfusion dat was with anti-igg, with anti-c d. the eluate reacted with all rbc samples except kp(b ) sample. she tolerated additional aliquots from phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k , k , kp(a b ), js(a b ) and sc: , , discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c. a>t (p.glu val) (kel* . ) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a ). sc sequencing found heterozygosity for a '- g>a change (rs , to % prevalence) and conventional sc* , predicting sc: , , . kel and sc results on the mother were kel* /kel* . , heterozygous for the sc change '- g>a, and her rbcs typed k k kp(a b ), sc , ula , consistent with dna predictions. plasma collected months later was nonreactive at rt and in peg iat. her rbcs were dat and now typed k , kp(a b ), ul(a ) sc and sc ,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel* . homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc: , rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b ). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a -year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs - a)genotype, associated with a jkb null phenotype. anti jk was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a ) and jk(b ) . genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk . however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms a transfusion vol. supplement s (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a b-); kp(a b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon revealed a g>a mutation, fy *n. , known to silence fya. sequencing of kel exons - exposed a silent polymorphism in exon , g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy *n. mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly % of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have genetic variants not previously reported in those of african descent. only was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient . study design/methods: patient was a year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at years of age. anti-b changed from undetectable/weak to strong at the age of years. patient was a month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was / . both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient with - reactions up to years of age. thereafter, abo typing showed mainly strong anti-b. patient had / anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for children on long-term tpn. patient had absent/weak anti-b since birth up to years of age, then developed strong anti-b with no change in feeding regiment and medications. patient had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* and elizabeth hart . brigham and women's faulkner hospital, university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title , cfr part . (a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in . a total of samples were evaluated. each abid was subcategorized; ( ) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and ( ) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of abids were performed on new patient samples. of the new abid samples, ( %) had microscopic absc results. for the previously known antibody patients, there were which accounted for % of the total abids performed. when reviewing the total abid workups, a total of ( %) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the new antibody samples were: conclusion: a total of % of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: ( ) discontinue the use of the microscope, ( ) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or ( ) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than % in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a -yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a -year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only ( - ), and negative with anti-c b, c d reagent. the antibody showed a peak gel-igg iat titer of . results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w ), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from to %, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody anti-p anti-m anti-sd a anti-le b anti-jk a anti-k anti-e anti-c results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of % (type ), % (type ), % (type ), % (type ) and % other than , , or . further investigation was conducted to determine the molecular identity of the «others». out of samples, ( %) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon or both exons and . a surprising amount of samples were discovered to be normal rhd. conclusion: along with sandler et al. ( ) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types , , and obtained in serological weak d, years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was , %. no trali happened in the period. prophylaxis were used in % of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p < . ). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) ( . % vs . %, p < . ) or aes with a non-allergic type inflammation etiology ( . % vs . %, p < . ) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes ( . % vs . %, p . ) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for , , , and weeks were . %, . %, . % and . %, respectively (p . , logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week ( . %) and longer than a week ( . %) (p < . ). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found patients from - , who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these cases, only of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as out of transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was . g/dl but declined to . g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days and with poor responses. on day , routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from . mg/dl to . mg/dl (reference . - . mg/dl), and lactate dehydrogenase was above reportable linearity, > u/l (reference - u/l). testing revealed additional anti-e, anti-jkb, dat c , plasma free hb . mg/dl (reference - . mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b ), one of which was also e . one volume tpe was performed to remove free hb on days , , and using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at . mg/dl on day , decreased to . mg/dl before discharge on day results/findings: twenty three cases were identified, of which had medical records available for analysis. ten ( %) patients were male, the mean age was . years (range - years), ( %) had an underlying hematologic malignancy or bone marrow disorder, and ( %) had a history of coronary artery disease (cad). the implicated units included ( %) red blood cells and ( %) platelets; ( %) patients received a single unit, and ( %) received two or more within the previous hours; the mean volume transfused was . ml (range - ml). the mean time to onset of chest pain was . minutes (sd minutes), with % of patients presenting within . hours and % within hours of starting the transfusion. chest pain was present as the only symptom in % of the cases, and for the other cases the accompanying symptoms included dyspnea ( %), fever ( %), back pain ( %), and hypo-and hypertension ( %). a post-transfusion chest x-ray was performed in % of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in % of cases and showed no findings to suggest acute ischemia. three ( %) patients had a minimal increase in their troponin levels, although had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen ( %) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than minutes in the majority of patients ( %). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts . based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, degree, or anti-human globulin phase. check cells were found to be . these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o to a ) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a -year-old o man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha- antitrypsin deficiency who presented for olt (donor o ). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving units of o rbcs and units of o plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a plasma were transfused to wash out anti-a antibody prior to transfusion of a rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for hours post-transplant. the patient's total estimated blood loss was > l. he received a total of units of rbcs (including a ), units of plasma (including a ), units of cryoprecipitate, and units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o . on postoperative day (pod) , a ts showed predominantly a rbcs with trace o rbcs, as well as very low anti-a igm and igg titers (table ) . he received two additional o rbc units ( each on pod and pod ) with increasing o rbcs on ts and rising anti-a titers. his blood type was unequivocally o by pod . the patient showed recovery of liver synthetic function on pod (factor activity %) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod , the patient had reverted to o with recovery of anti-a titers. at months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* , alfred mingo , maria isabel gonzalez , antoni mena and juan pedro benitez . bst, at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h ) and an oncology center (h ). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of transfusion activity in both h and h shows pse and bca, out of and respectively, since the tss deployment in . retrospective analysis and classification of security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h , nm related to pse accounted for . % of all, being the mistake in concordance between patient identification and prescription order the most frequent ( . %). the nm detected in bcas were . % of all and mostly ( . %) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are . % of all and mostly ( %) the systems detects a not assigned bracelet. for h , nm related to pse accounted for the . % of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent ( . %). the nm detected in bcas accounts for . % of all and in . % occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are . % of all and in . % of them the blood components were assigned to another patient. ( , , , , , , , , , , , , , ) were analyzed via a commercially available elisa. comparison of adequate response to ppv , defined as ! mcg/ml for > serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in patients (alloimmunized, ); pre-and post-vaccine titers were available for patients (alloimmunized, ). of the patients, were on chronic transfusions, were on hydroxyurea, were surgical splenectomized, patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv in the previous years; / also reported previous history -valent sp conjugate vaccine within the last years. baseline pre-vaccination titers (n ) showed no difference between alloimmunized and non-alloimmunized patients (all p-values > . ). in the group with pre-and post-vaccination (n ) titers available, out of ( %) non alloimmunized patients had an adequate response versus out of in the alloimmunized group ( %, p ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from to ). patient harm events recorded within the veritas system from january to july were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of tres per month and per year were found over five years. % of tres are associated with pre-bb activities, % occur within bb, and % are post-bb events. sample collection and handling represent % of total tres. most tres ( %) were reported by bb staff, % were reported by non-bb staff. patient harm analysis revealed an average of four level (near miss), three level (no known harm), and . level (patient harm) per month. no deaths related to tre were detected over the seven month january to july period. patient harm was associated with tres occurring in the bb ( %) and post-bb ( %). these events were reported externally ( %) and by bb staff ( %). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january , and march , was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by and added to the number of individual whole blood plts; apheresis plasma units were multiplied by and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was . the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the -hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions ( [ /l vs. [ /l, p . ). there were no significant differences in the frequency of effective therapeutic ( % vs. %, p . ) and prophylactic ( % vs. %, p . ) transfusions between the prcs and gypcs. we did not find significant differences between prcs and gypcs in cci after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . . ) transfusions, in cc after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . .) transfusions. there were no significant differences between prcs and gypcs also in ma after prophylactic ( . . vs. . , p . ) and therapeutic ( . . vs. . . , p . ) pc transfusions. reduction of the severity of bleeds was obtained in ( %) of the cases after prpc transfusions and in ( %) of cases after gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, and cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in . it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were units administered using the emergency transfusion process in the months before the change was implemented. it was found that / ( %) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month ( components administered), / ( %) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: ) a stable anemic inpatient, ) a stable anemic inpatient to be discharged, and ) an asymptomatic post-operative inpatient. results/finding: we identified canadian tm experts: ( . %) provided a response and most had a primary place of practice in a laboratory setting ( / ; . %). for a stable, non-bleeding, anemic inpatient, . % of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with . % generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period ( - hours), a repeat hemoglobin > hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse ( . %) compared to an inpatient potentially symptomatic due to anemia ( . %). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium- ( cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n ) were divided into two ml aliquots, which were labeled with selected concentrations of s-nhsbiotin ( and lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of . . %). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that nm excitation of phycoerythrin (pe)-sa and high laser power ( mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with lg/ml of biotin resulted in $ , mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, % ci) of in , ( . %). the lld for rbc labeled with biotin at lg/ ml was $ in million ( . %). biotinylation was not associated with increased levels of hemolysis ( . . % before labeling versus . . % after labeling; p . ) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes ( ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer- (hboc- ) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: ( ) wb, ( ) wb % hboc volume (model of two units in an adult), ( ) wb % fdp, and ( ) wb % hboc % fdp. samples ( )-( ) simulated autoresuscitation by adding % plasmalyte to - . susceptibility to lysis was tested with ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of %, %, %, and % volume replacement with hboc and/or fdp, with or without prior % plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb hboc, and wb hboc fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb hboc in autodilution simulation (mean lysis . % vs. . %; p<. ). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc ( %) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even % hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc- , there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in / and / . we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of patients, were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery ( %). pcc was given for warfarin reversal in % of cases. a subset of patients received plasma within hours prior to pcc ( %) or hours after ( %). pcc was most frequently ordered in the or/perioperative service ( %). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in , several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; ) simplify and expedite the process; ) improve communication and expectations to decrease tat; ) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood ( units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of er episodes were received. the average tat from order to delivery at the bedside was reduced by % ( . minutes compared to minutes previously), while the compliance rate for er orders and physician documentation was % ( / ), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february -february . mt was defined as the transfusion of ! rbc units in a -hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at days. results/findings: thirty mts occurred during a one year period. a total of , blood products were transfused during that time period. gender distribution was / ( %) males, and the average age of all patients was with a range of to years of age. surgical patients accounted for / ( . %) mts, and / ( . %) were critical care patients. tumor categories included carcinomas ( / ), sarcomas ( / ), leukemias ( / ) and lymphoma ( / ). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer ( / ) was the most common disease seen followed by sacral chordoma ( / ). mtps were activated in only / ( . %) cases. thirty-day survival was seen in / ( . %) patients. only of mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage ( / ) or perisplenic hematoma ( / ). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a -year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a -year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a -year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* , , rebecca ross , christopher a tormey , , and amit gokhale , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: daratumumab (dara) is a igg monoclonal antibody therapy that specifically targets cd , a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, subjects were identified for analysis. their mean age was . years, with male and female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of % ( / ) prior to administration of dara. of these patients, were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these ( %; / ) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september , through april , were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in , , unique women receiving a total of , units of red cells (rc) in , transfusion episodes were identified. their median age was (range - ). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in ( . %) and ( . %). ( . %) transfusion episodes were associated with the use of units or more rc. as a result, ( . %) episodes resulted in a post transfusion hb ! g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given (< . %) and ( . %) women respectively. upon discharge, ( . %), ( . %) and , ( . %) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to ( . %) women. conclusion: in the present study, it is observed that . % transfusion episodes were given at hb ! g/dl. a substantial number of episodes ( . %) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! g/dl). for iron replenishment and bleeding control, up to . % transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes ( . - . x per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with gy and transfused over - hours within hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* , lee grabner , brenda herdman , robert fallis , amin kabani and charles musuka . canadian blood services, kenora rainy-river regional laboratory program, diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between and before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and % were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels ( . g/dl; p . ); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units ( . vs . g/dl; . vs. . g/dl; both p . ). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients ( . g/dl vs. . g/dl; p< . ). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p . ). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p . and p . , respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than % of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered times in total, affecting patients and users. stratified by location, the majority of triggers occurred in the perioperative areas ( times) and the liver icu ( times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin % iv solution, human albumin % iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* , , mahmut akgul , , hollie m reeves , , robert w maitta , , marcie pokorny , anne capetillo and katharine a downes , . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a -month retrospective study (january-july ) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was - mg/dl with critical value of mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were adult (> years) orders reviewed by tms out of which were approved. of the approved orders, ( . %) were in agreement with tms's estimated dose. of ( . %) orders that were not in agreement with the tms's estimate, ( %) were underestimated and ( %) were overestimated. seventeen of orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of . mg/dl (range . to mg/dl) and a median excess of . mg/dl (range . to mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was mg/dl above target, which is significantly higher with intervention than without (which could have been mg/dl below the target; p< . ). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention ( vs. . mg/dl, p . ). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention ( vs. - . mg/dl, p< . ). seven of ( ) ( ) orders were for critically low fibrinogen (< mg/dl) and of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were and units ( . % and %) i.e. and pools and the most frequent orders in the disagreement group were , , and units ( %, %, % and %). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was . and posttransfusion was . . only % of patients had their inr corrected to . , while % had no change, or had increased inr. (table) . the majority ( %) of patients received units of plasma. the mean plasma dose was ml/kg. there were transfusion reactions reported, non-hemolytic and transfusion associated circulatory overload reactions in which required admission to the icu. two patients experienced bleeding during ir procedures (tips) and developed a hematoma (tunneled central line). the median of inr correction in this study was . with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is . . randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. of the patients experienced bleeding complications indicating that inr of . may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately % of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population ( %) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately % of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july , as follows: units of group a plasma and units of group ab plasma is provided for the massive transfusion protocol (mtp) along with units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed mtps at our institution between july and march . twenty patients ( . %) were transfused with incompatible group a plasma ( group ab and group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april , our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in , bonfils immunohematology reference lab (irl) sent out approximately special platelets for patients with hla antibodies. by , hla platelet orders increased dramatically and the irl sent out over special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over , donors in the database with historical hla typing. however, only approximately of those donors actively donate. in the denver area, one of the most common hla types is a a b b . only of the , donors have this type ( . %). therefore, to fill an hla platelet order request for a common hla type, only donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a a b b , there is only out of , donors ( . %) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a for example, all of the a positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in , approximately % of these special order platelets were pra matched and the remaining % were hla matched by donor recruitment. by , approximately % of special platelets sent are pra matched. this change resulted in a . fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains packed red blood cells (prbcs), fresh frozen plasma (ffp) units, and plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august to march mtps were activated at ucm, of which orders could be traced to the origin: on inpatient floor (including icus), in the operating rooms, in the emergency department, in labor and delivery, and in other procedure rooms. of the prbcs that were issued, were transfused ( % utilized); of the units of ffp that were issued, were transfused ( % utilized); of the platelet packs that were issued, were transfused ( % utilized); of the units of cryoprecipitate that were issued, were transfused ( % utilized). since march , the time of first product issue after the initiation of an mtp has also been tracked. of the events that fall within this time period, ( %), had the first product issued in minutes or less. another ( %) were issued between - minutes, resulting in over % of patients being issued their first blood product within the first minutes. only of ( %) events had an initial time greater than minutes and none were greater than minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($ - %). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* , shailesh macwan , arline stein , jane fischman , nancy nikolis , matthew bank , lennart logdberg , alexander indrikovs , sherry shariatmadar and vishesh chhibber . north shore university hospital, northwell health background/case studies: massive bleeding is generally defined as any patient who requires blood volume replacement within hours and/or receives transfusion of greater than or equal to units in one hour with a transfusion vol. supplement s ongoing bleeding. our mtp was officially implemented in in preparation for an initial verification as a level trauma center by acs. our mtp has the following packages: st pack has a ratio of : : (rbcs, plasma & platelets) and subsequent packs a ratio of : : . our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march to add cryo and pcc at a defined point in the mtp (cryo is included in the rd pack and pcc in the th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received > rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had patients who received > rbc in and . mtp had been activated for all patients and all patients received between . to unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt > seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of mg/dl (table ) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of > rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's -day and -day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective -year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of published cases ( reports) of ta-pls, ( reports) were stem cell and ( reports) were organ transplants. all ( %) stem cell transplants and ( %) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the reports of stem cell ta-pls, actively screened for antibodies in the immediate post-transplant period, and of the reports of organ ta-pls, actively screened for antibodies. these screens detected cases of stem cell ta-pls before hemolysis became apparent and cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* , rosario mallari , marc de asis , elaine shu , jonathan hughes and tho pham , . stanford university, stanford health care, stanford blood center, bloodsource background/case studies: mur antigen is present in - % of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received - rbc units every - weeks to maintain a hemoglobin (hb) level of g/dl. the patient remained stable for months when his hb level acutely dropped to . g/dl. the antibody screen remained negative for an additional months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for of the rbc units he received. units were from an asian donor, and a unit transfused days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of , donors at a hospital-associated blood center located in a region where asians comprise approximately % of the population. results/finding: . % of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for of ( . %) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over % of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* , princess maynie , carol chandler , shelia garret and pampee young . vanderbilt, vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [ ] [ ] [ ] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of samples have been analyzed (table) , rh and non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being . (range - ) times higher. the average fold change for rhd/c/e antibody titers were . , whereas the average fold change for non rh titers was . (range - ). the range for anti d titers was particularly variable, - , whereas for c/e, it was - . the overall reproducibility/precision of the automated analyzer was $ %. to correlate the a transfusion vol. supplement s increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $ times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of . units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to % of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately % of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix . % suspension of a and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, group a, and group b. results/finding: of the whole blood edta samples tested, ( group o and group b) exceeded a high titer threshold of . when the pas samples of these donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of , a % decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the ( %) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these hospitals had a lower overall rate of wastage including their own donations than the other hospitals that did not collect in-house plt. the other hospitals had wastage rates ranging between - %. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* , , christopher j gresens , anne capetillo , hollie m reeves , and katharine a downes , . case western reserve university school of medicine, university hospitals cleveland medical center, bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of < . %. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a -year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure ( / mm hg to / mm hg) followed by hematuria ( ml). chills and rigors resolved; blood pressure stabilized after min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of (igm) and , (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* , leila patricia de sousa fontenele , isabel nagle reis , carolina bonet bub , araci sakashita , raffael zamper , cristiane nakazawa , tatiane almeida omura paula , patricia silva batista , marcio dias almeida , fernanda loureiro de andrade orsi and jose mauro kutner . hospital israelita albert einstein, hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of cases of olt performed between and in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of consecutive patients submitted to liver transplantation between and were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or , - % ci: , - , , p: , ), absence of hcc (or , - % ci: , - , , p: , ), cirrhosis of any cause (or , - % ci , - , -p: , ), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or , %ic , - , ) and retransplantation due to primary non function of the graft (or , %ci , - , , , p: , ) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a /b in group o platelet products. charles k. childers* , mark destree , ashley rose and theresa nester , . madigan army medical center, bloodworks northwest, bloodworks nw, dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a /b in group o platelet products is presented from a large regional blood center collected over - months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in ml edta sample tubes. a single : dilution of plasma was prepared using a hamilton microlab series dilutor using . ml saline diluent and . ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at rpm for seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of is used, approximately % of group o apheresis platelets will have a high titer, most commonly with anti-a . less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their day outdate. after months of testing pspp units and verifying that the products rarely had a high titer ( . %), the blood center stopped performing this testing for pspp units. rh ) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to % (from % on the previous day). his hematocrit did not increase ( %), and over the ensuing hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of % while reducing the number of a rbcs in his circulation by approximately %. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes - days for antibodies to develop and they are short-lived ( - weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the calendar year from hospitals. an additional hospital* provided data for august-december . rbc transfusions in patients < year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/ years was also determined. results/findings: see table . the fraction of all transfused rbcs that were oneg ranged from - % (row f). the percentage of oneg rbcs transfused to oneg patients ranged from - % (row g); thus, non-oneg patients received - % of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients ( %- %; row i). overall use of oneg rbcs could have been reduced by %- % if opos units had been given to all oneg patients >/ years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* , nehad mohammed , marwa aly and nashwa fahmy . national blood transfusion services, nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on multitransfused patients who received blood transfusion chronically at our central blood center. they were thalassemia patients ( bthalassemia patients, one patients with a thalassemia), sickle cell anemia patients and immune hemolytic anemia patients ( auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). oncology patients, chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: out of ( %) alloimmunized patients and out of ( %) non alloimunized patients(p< . ) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june . screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified articles for data abstraction, where articles were transfusion guidelines. there were guidelines ( %) that made a recommendation, for a single unit and for multiple unit transfusion strategy (table ) . review b identified retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or . , % ci . - . ), although heterogeneity was high (i %). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december and february was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the months, units of platelets were transfused to recipients. over the months, a median of units was given to each patient with a range of to . the overall distribution of products used was % plasma, % pr, % pas f and % pas c. thirty percent of patients (n ) received all of their products on a single day. single units were given to patients while , and received , , and units respectively. the distribution by product type was % plasma, % pr, % pas c and % pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the month period (p . ). the distribution by service was different for the groups receiving multiple units. for single units the distribution was % hematologic malignancy, % infusion clinic (nos), % solid tumor medicine, % surgery, and % pediatrics. for those receiving multiple units the distribution was % surgery and % each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of . . conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* , , yvemarie b.o. somsen , maike e. van hezel , marleen straat , robin van bruggen and nicole p juffermans . sanquin research and landsteiner laboratory, academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains mg of iron and % of the rbcs are cleared by macrophages within hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in icu patients who received one rbc transfusion, different iron parameters were measured before and hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il- levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion ( . vs. . mmol/l, p . ). also, the transfusion had no effect on transferrin saturation ( vs. %, p . ), ferritin ( . vs. . mg/l, p . ) and il- levels ( . vs. . pg/ml, p . ). hepcidin levels increased in these icu patients after rbc transfusion ( vs ng/ml, p . ). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (- . vs. . % change, p . ). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il- or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since and pathogen reduced platelets have been available since . in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between and . during this time pas c, pas f and pr went from % to % of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in patients received an average of . units/recipient/month and in the average was . units/recipient/month. the intervening data points for , , and were . , . , and . respectively. the year average was . . the slope of the graph for all points was y - . . . the two sample t-test showed that the plt/recipient/month from to was not statistically different with a p value of . . conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that % to % develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least k/ul. the analysis revealed that the mean of . k/ul (n ) had a percent lower bound confidence interval platelet increment of k/ul (p< . ) results/findings: (median range [ - ]) hla matched leucoreduced irradiated sda platelets were transfused to ( m/ f) patients, median age years (range - ). / ( %) patients showing broad alloimmunization to hla class i/class ii antigens. / ( %) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority / ( %) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); / ( %) female patients had prior exposure via pregnancy and / ( %) had a history of hsct. ( %) platelets were abo identical-platelet increment median k/ul (range - to ), ( %) were abo compatible -platelet increment median of k/ul (range - to ) and ( %) were abo incompatible with platelet increments median k/ul ( range - to ). platelet counts were performed within hours in ( %) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* , renee leblanc , dongfu xie , alice cabe and yanyun wu . overlake hospital, bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about for years (from to ) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than . % of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a -year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of . g/dl (baseline - g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was . g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin ( . to . mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to . g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october to march . patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were rbc products transfused to waiha patients. twenty-three ( . %) patients received at least incompatible unit. the mean age was . years (range - yrs) with % women. ethnic composition was % african-american, % caucasian, and % patients of mixed/other ethnicity. one hundred fourteen ( %) of these products were released as li products and ( %) were compatible. ninetythree ( . %) of the li product transfusions had a post-tfn hct change of < % whereas only ( %) of the compatible product transfusions resulted in a post-tfn hct change of < % (p . , v ( ), exact methods). the mean hct increase in the compatible group was . % per unit vs. a slightly lesser per-unit increase of . % in the li group (p . , t-test, -tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for units. units that were incompatible had a lower mean post-tfn hct rise compared to all other li units ( . % vs. . %); however, this difference was not statistically significant (p . ). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected % per unit more frequently than it was for waiha patients who received compatible products ( . % vs. %). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first -hours from mt onset) was calculated with % and . % control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were mt cases from hospitals ( tertiarylevel, smaller/medium sized acute-care and specialist women's). number of mt cases per hospital ranged from to . patient median age was years (iqr , ), % were male and % required admission to intensive care. the most common clinical groups were cardiac surgery ( % cases), trauma ( %) and gastrointestinal hemorrhage ( %); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the tertiary-level hospitals was % (range % to %) and / ( %) were within the % control limit. cb that required ! rbcs within -hours of mt onset occurred in % of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals ( % versus %; p . ). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of bcs and ml of platelet additive solution iii (pasiii) were used to produce pcs (n ). pcs were stored on a flatbed agitator ( cycles/min) in a temperature-controlled cabinet at c for - days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of mm hg was applied. using clamps, a flow velocity of - ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted - %, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration ( . - . x /l) and number (> x /unit). simulated transfusion had no effect on the percentages of cd p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* , , joan sevcik and joseph e. kiss , . university of pittsburgh, blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution : is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient and and (control). results/finding: all patients were ab blood type. patient , a year old female with recurrent adamts deficient ttp, received courses of tpe (total tpe procedures) for relapse and exacerbation. ten out of procedures were performed with ab and a plasma (average ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of tpe procedures. four out of procedures were performed with ab and a plasma (average . ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with adamts deficient ttp who served as a control, received a total of procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between patients. the trends of hemolysis laboratory data for patient and were comparable with patient . all patients had negative dat. only patient received rbc transfusions. all patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to % of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd negative platelet unit is not available for a patient with anti-cd antibodies sameer khatri* , charles harmon , brian r curtis and chisa yamada . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd ) is one of the identified plt surface ags and deficiency is rare, but found in asians ( - %), sub-saharan africans ( - %) and also in some people from mediterranean descent. two types of cd deficiency have been described. type deficiency is the complete lack of cd on both plts and monocyte-macrophages whereas type deficiency lacks cd on plts with variable expression ( - %) on monocytemacrophages. transfusing plts in a patient with cd deficiency is challenging given the rarity of cd negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than units of apheresis plt units over a week period without any significant increase in plt count. cross-match compatible plt unit found in of units and hla matched units were tried without success. at that point, a cd ab was identified in the serum and the patient's type cd deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was % due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd- negative (but blood type different and hla a transfusion vol. supplement s unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd antibody positive reactivity in serial dilutions has reduced from : to : dilutions and his hla class i pra has decreased to %. he is currently receiving apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to . k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec , , blood donations were screened for b microti by immunetics elisa. of those, ( . %) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between . % and . %. no patient babesia transmission has been reported since implementing this test, but we only had documented babesia ttd cases from - . donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of positive test results, lookback investigations were initiated representing % of positive donations. lookbacks were only performed when there was a donation within months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to % were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented . % loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only . % of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in > % of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for blood donors, were positive with an average load of . x copies/ml of plasma, a median value of . copies/ml of plasma, ranging from to . x copies/ml. pre-transplant viral loads were similar. for transplant candidates, were positive with an average of . x copies/ml of plasma, a median value of copies/ml of plasma, ranging from to . x copies/ml. post-transplant viral loads were remarkably different. for transplant recipients, all were positive with an average of . x copies/ml of plasma, a median value of . x copies/ml of plasma, ranging from to . x copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around - copies were present in > % of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt ( ) . post-ivig: all plt ( ) . post-ivig: cd -negative plt from relative ( ) . post-ivig: single donor apheresis ( ) . post-ivig: cross-match compatible ( ) . post-ivig: flow cross-match compatible plt ( ) . a transfusion vol. supplement s detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv ( / / / ), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows % of specificity, with no false positive results on the control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of tcid /ml for denv- , denv- and chikv and of tcid /ml for denv- , denv- and zikv. finally, the first results obtained on denv( ), zikv( ) and chikv( ) clinical samples show %, % and % correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of , , and were prepared from plasma or serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from : to : ( -fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of or higher and was not eliminated by the addition of a blocking step. pools of or samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of or samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $ cfu/ml of organisms associated with platelet contamination and incubated at room temperature for - hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher ( log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc , were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta d and virtuo the organism was recovered % . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc . however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of ml lrap demonstrated % recovery when loaded onto the virtuo and d ( table ) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus - , and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in , a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as- rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and hrs after amustaline addition, respectively, for titration by plaque assay on vero cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, > . log or log /ml of rrv was achieved, with > . log or > . log /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; - ml), large volume (lv; - ml) and dual storage containers (ds; - ml) designed to treat platelet doses between . and . x . the new triple storage (ts) set was designed to expand the dose range to . x and the maximum volume to ml, generating either or doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or % plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in % plasma/ % pas or % plasma with a final volume of $ ml and a dose of - platelets. these conditions represent inactivation using the lowest amotosalen concentration ( mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of - logs in respiratory secretions of mers patients, and with lower genomic titers of - logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log reduction/ml (log /ml) %plasma/ % pas e .coli . <- . > . e. cloacae . <- . > . k. pneumoniae . < . > . s. aureus . <- . > . blue tongue virus . <- . > . bovine viral diarrhea virus . <- . > . adenovirus- . <- . > . %plasma k. pneumoniae . - . > . s. aureus . <- . > . adenovirus- . <- . > . n a transfusion vol. supplement s abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged times up to days, assessing the infectious titer and genomic titer every rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of ! . log infectious titer. no viral replication was observed after days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* , jen-wei chen , chi-ling chen , sheng-nan lu and pei-jer chen . department of research, head office, taiwan blood services foundation, graduate institute of clinical medicine, college of medicine, national taiwan university, division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by , and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since ) and -sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since ) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during - and - , respectively. age-standardized prevalence and its % confidence interval ( % ci) were calculated with adjustment of who world standard population - . for the incidence study, donors who have donated blood two or more times during - and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its % confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ) during - , and the agestandardized prevalence was also decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ). the agestandardized prevalence of anti-hcv was generally higher in female donors before , but it was significantly higher in male donors at (p-value . ). a total of , hcv rna positive cases, . % of them were anti-hcv negative, identified from , first-time donors during - , and the crude and age-standardized prevalence of hcv rna was . per , ( % ci: . - . ) and . per , ( % ci: . - . ), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value < . ), but no significant difference was found after age standardization (p value . ). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend< . ). in the incidence study, a total of new hcv rna positive cases, . % of them were anti-hcv negative, found from , , donors followed for , , person-years. the incidence of hcv rna was . per , person-years ( % ci: . - . ), and no significant difference was observed between both genders (p-value . ) and between age groups (p for trend . ). conclusion: the prevalence of hcv infection has been dramatically decreased by . % during - . it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* , sahar muhmmad and dalia el dewi . national blood transfusion services, azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is - days, hiv from to days, and hbv from - days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from to , the total number of donor samples to be screened is , the age of the donors ranged from to years, and they were of both sexes (m: f : ).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v ) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of nat yield donations among ( . %) seronegative donors. among these nat yields cases, ( . %) were reactive for hbv, ( . %) were reactive for hcv and ( . %) were reactive for hiv- . we stratified the age of the donors into groups; group a ( - years), group b ( - years) and group c ( - years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p . ; with % confidence interval (ci) . - . & p . ; with % ci . - . respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p . ; with % ci . - . ). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p . ; with % ci . - . ). nat-hcv; did not differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups a and c & p . ; with % ci - . to . between groups b and c). nat-hiv; did not also differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p . ; with % ci . to . ). nat hbv was significantly higher in males (p . ; with % ci . - . ), but the prevalence of either hcv or hiv did not differ significantly between males and females (p . ; with % ci - . - . & p . ; with % ci - . - . ; respectively). conclusion: in this study the nat yield of in assumes more significance when one considers the fact that single donation is used for generating components that can be used by recipients. hence, in effect the nat yield becomes times that is, in . saving recipients from tti out of ( . %) is indeed very significant. results/finding: of the , donors who were tested by our donor center, , ( . %) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at : titer. the screening elia s/co of this donor was . . both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were - transfusion transmitted babesia cases per year from - (table ). in the months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed ( in , donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of years of additional nat testing at blood bank, dmch, ludhiana from september to december . results/finding: results . % ( of ) units were initially nat reactive. these units were further tested, of which . % were discriminated ( hiv, hcv, hbv and co-infections). the remaining . % ( ) were repeat non-reactive and . % ( ) could not be discriminated. overall, nat yield rate was one in , whereas virus-specific nat yield rates were one in , for hiv, one in for hcv, one in for hbv and one in , for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past years has increased the screening sensitivities to check viral load and prevented transmission of probable transfusion transmitted viral infections. assuming % component preparation it saved transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu , , wei mao , tao he , yashan yang , , zhan gao , , chunhong zhang , hongmei liao , jingxing wang , and miao he* , . institute of blood transfusion, chinese academy of medical sciences & peking union medical college, sichuan blood safety and blood substitute international science and technology cooperation base, chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from , voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as e - . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: . gb raw data with , , reads were generated in the dna library. meanwhile, . gb raw data with , , reads were generated in the rna library. after cleaning the human background, reads from bacteria, reads from viruses, and reads from parasites were identified (table ) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table ) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of february to february . thrombocyte concentrates are prepared out of single donation units by the buffycoat method. results/finding: over the period platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* , germ an leparc , phillip c williamson , lani palmer , ben reynolds , maria noedel and lindsey houghton . creative testing solutions, oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february , recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march , . with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february , and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february , , the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in , who reported $ million new cases worldwide, resulting in > , deaths. malaria prevalence is highest in sub-saharan africa, home to % of all infections accounting for % of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of . mm amustaline and mm glutathione (gsh) and a h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with % fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching > % parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at > . log or > . log /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from to days using an fda cleared rapid test (rt). in february , our hospital based transfusion service implemented the use of rt on day , and to routinely extend ap shelf life to days. prior to this, we tested aps by rt on day and transfused day or day units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of day platelets. study design/methods: data were obtained for two -month study periods: october -september (pre-implementation) and february -january (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table . the number of ap transfusions increased by % post-implementation, comparable to a % increase in inpatient admissions and an % increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different ( . pre; . post, p . ). the number of rts performed increased by %. the percentage of transfused units tested at least once by rt prior to transfusion increased by % (p< . ). the outdate rate decreased from % to % (p< . ). ad-hoc ordering decreased from % to % (p< . ). conclusion: use of an approved rt for routine ap outdate extension to day was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* , vito scalia , carla osiowy , michael carpenter , anton andonov and margaret fearon . canadian blood services, public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low ( . and . per , donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of units. hcv nat was in place since (using minipools of ) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at - o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the ' ntr-e and ns b regions. sanger sequencing of these regions represents approximately % of the genome. results/findings: all confirmed positive donations were whole blood donations. there were hbv positive donations. of these, had tested hbv nat positive. genotypes were type a, b, c, d and e. there were samples hbv nat negative but hbsag positive ( were anti-hbc reactive). of these, could not be sequenced and one was genotype a (also anti-hbc reactive). there were samples considered hcv positive. of these, samples were hcv nat positive. genotypes were type a, b, c, b and a. there were also samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert d microbial detection system (bta d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert d (bta d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels ( - cfu/ml) of bacterial species commonly associated with platelet contamination, and replicates ( per instrument) of ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta d and the other into virtuo and incubated until signaled positive by the instruments or for up to days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august . this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a % voluntary donor program since and is the only center in the country that has achieved this goal. results/findings: a total of , blood donations were studied from august to december . in the rbdc, where only voluntary blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of : , for hbv; : , for hiv and : , for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s system. hbv s region was also sequenced. results/finding: obi were found in the , donations. in the viral loads assay, samples were negative and samples' viral loads were lower iu/ml. the mean viral loads was . . (log ) iu/ml in other samples,while the mean viral loads with hbsag /hbv dna samples was . . (log ) iu/ml. samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype ( . %) and the other was hbv c genotype( . %). compared the samples with hbsag /hbv dna ,we found two obi samples carrying with t>c mutation, which could cause an amino acid s f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag /hbv dna , and some unique variation was identified in the obi individuals. a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat th days, month, months& months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may , we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may to dec was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s platform using pools of serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, seronegative donations were screened by nat and a total of hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group - years old showed a large proportion, who accounted for % of reported infections. most of the hbv dna cases (about . %) reached senior high school education. the average hbsag dna positive rate was . % ( / ). incidence among apheresis donors in this period for hbsag dna were . / . these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf with day stability at c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control ( ml) and test components ( ml ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within hr and wbd pf within hr. cryo was manufactured according to site sops and frozen at - c (test ml, control ml ). test and control cryos were thawed at c, and characterized immediately post thaw (t ), and after d storage at c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over d at c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through d storage at c. conclusion: pr cryo can be processed from plasma sources, including pf , and stored at rt for days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf with stability over days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , lynne fleischmann , mirjana sarac , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics, abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects - million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over days using a panel of positive and negative samples. sensitivity was evaluated on presumed antibody positive specimens and specificity was evaluated on random blood donor samples. results/finding: precision was % cv or less for positive samples over days. the overall specificity in a blood donor population was . % ( / ). sensitivity was . % for presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , lynn martin , daniela kaleve , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over days. sensitivity was evaluated using known positive samples, commercially available seroconversion panels, the who standard, hbsag mutants, and hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed positive samples. sensitivity was % for all genotypes. % of the mutants were detected vs % for the comparator assay. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. analytical sensitivity ranged from . to . iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , kevin callear , susan sullivan , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types and (anti-hiv- / ). blood centers require very high throughput anti-hiv- / assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv- / antibodies and hiv- p antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv- / antibodies and hiv- p antigen was evaluated on the abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv- , hiv- and hiv group o antibodies and hiv- p antigen. seroconversion sensitivity was evaluated with commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed antibody positive samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. also, sensitivity was % for antigen positive viral isolate samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , joyce siregar , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who st international standard. seroconversion sensitivity was evaluated using commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from . to . iu/ml. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* , anton vanweert , ed bakker , mark paradowski , jane bryant , tuan bui , joyce siregar , george chen , george schlauder and gregg williams . abbott laboratories, sanguin diagnostics, abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over days using htlv i and htlv ii positive samples. specificity was evaluated using , blood donor specimens from europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot . . results/finding: imprecision was less than . % for positive samples over days. clinical sensitivity was . % ( / ) on preselected htlv i and htlv ii positive samples. the specificity was . % ( , / , ) on a blood donor population and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez , michel garcia* , fernando palomino and guillermo orjuela-falla . national blood bank colombian red cross, universidad del rosario, fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than . ; abbott architect i sr) underwent supplemental testing by immunoblot (either chiron riba hcv . sia or hcv blot . test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: - . , - . , > . band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in . % ( / ) of samples, indeterminate in . % ( / ) and were positive in . % ( / ). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group - . ( . %) compared with the - . ( . %). in samples with indeterminate results, ns _ was the most frequent band detected ( , %). in contrast, the most frequent band in the group of positive results was core ( , %). only one sample from the indeterminate group ( . %) had a strong band intensity ( ), compared with samples from the positive group ( . %). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group ( - . ) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (> ). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns _ and ns _ cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* , ivanka mihaljevic , manuela miletic , miljana stojic vidovic , irena jukic , jane bryant , mark paradowski , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, croatian institute of transfusion medicine, abbott gmbh & co. kg, abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over days using positive samples. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than . % cv for positive samples over days. clinical sensitivity was . % ( / ) on preselected syphilis positive samples. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* , ed bakker , anton vanweert , jane bryant , mark paradowski , tuan bui , lynn martin , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over days evaluating positive samples. sensitivity was evaluated using preselected positive samples and seroconversion panels. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than . % cv for positive samples over days. overall clinical sensitivity was % on preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying more bleeds than the comparator assay. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of , was declared as the public health emergency of international concern by who in feb . in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where . % of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, . % of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately : to : . thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of lm and assayed after illumination with visible light from both sides for , , and min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was . log % tissue culture infectious dose (tcid )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for min, min or min and the losses of the infectivity were further demonstrated by repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial min of treatment whereby ct-value jumped from . (control) to . (mbpt for min) (table ) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* , margaret fearon , susan l stramer , megan l nguyen , france bernier , sheila o'brien , vito scalia , sakina smith , yves gr egoire and boris hogema . h ema-qu ebec, canadian blood services, american red cross, sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in , canadian blood donors in . in a subset of , donor samples the seroprevalence was . %. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately , canadian whole blood donors including , from canadian blood services (cbs) and , from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all a transfusion vol. supplement s donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test ( % lod . iu/ml, % ci . - . ) for use on the cobas v r / system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous months are destroyed. recipients will be traced in the event of any products transfused in the previous months. results/finding: as of april , , of , ( , cbs, , hq) tested samples with valid results have been found hev-nat reactive: donors have been confirmed by further testing to date. confirmation is pending in donor. of the donors, were from quebec, and one each from nova scotia and alberta ( male, female). ages ranged from to years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: ate pork (including who ate pork liver), ate shellfish, ate venison, and drank well water. one donor had no identifiable risk factor. viral loads ranged from to iu/ml, of which were < , were - , and were > iu/ml; were anti-hev igm positive and anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around / . the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about to million cases and to million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august . both thin and thick glass stained blood smears of blood samples with giemsa was examined using microscope. results/finding: of the donated blood samples, ( . %) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p> . ), majority of the blood donors that tested positive belonged to blood group o ( . %). the prevalence of malaria parasite in the study was . %. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert d (bta d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life ( , and days after collection). study design/method: pooled lrap were seeded with low levels of organisms commonly associated with platelet contamination at , and days post collection. the seeded lrap were inoculated into bpa and bpn bottles on different days (not consecutive) alternating between teams of people each. seeded bottles were loaded into a virtuo and a bta d and incubated until declared positive or negative (up to days). additionally, bpa and bpn bottles inoculated with ml of unseeded lrap were tested on the virtuo and the bta d ( and bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of . % for the virtuo and . % for the bta d. the virtuo demonstrated an average improved ttd of . hours, when compared to the bta d in the presence of ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for days barring continued zikv testing and nonreactive results. a total of , donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low ( . %). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn , felicia santa maria , yvette girard , peter bringmann , marion lanteri* and adonis stassinopoulos . microbiology department, cerus corporation, scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in . the rapidly increasing number of infections in brazil, with hundreds of fatalities since december , is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate d-yfv using amotosalen (s- ) and uva light prt of platelet components (pc). study design/method: pc in %pas (n ) or % plasma (n ) were spiked with high titers of d-yfv and treated with s- /uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were . . log pfu/ml for pc in % plasma and . log pfu/ml for pc in % plasma while titers in post-prt samples were <- . . log pfu/ml for pc in % plasma and <- . log pfu/ml for pc in % plasma. inactivation to the limit of detection of > . . log or inactivation of > . . log pfu/ml was achieved for pc in % plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples ( hiv- antibody and hiv- p antigen, and htlv-i antibody and htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were . for hcv, hbc, syphilis, cmv igg, and chagas, . for hiv ag/ab and htlv i/ii, and . for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were . % (antibody sample) and . % (antigen sample) for hiv ag/ab combo; . % (htlv i antibody sample) and . % (htlv ii antibody sample); - . % for anti-hcv, - . % for chagas, - . % for hbsag, - . % for anti-hbc, - . % for syphilis, and - . % for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of ng/ml. robustness of the abbott prism methods to biotin interference c fischer , r schneider , w leonard , m cobb , g schlauder , g williams , m zuske m janulis* . transfusion medicine, abbott diagnostics, wiesbaden, germany, add diagnostics, transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from to colony forming units (cfus)/bag (i.e. . to . cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of - cfu/ml over the days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, hours (day ) after collection a sampling volume of spiked platelets ( . - cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate ( . %). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann , frank tolksdorf , wiebke handke , thomas h. m€ uller and axel seltsam* . german red cross blood service nstob, maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from buffy coats using the additive solution ssp (macopharma) with a residual plasma content of %. for inactivation kinetics, pcs (n ) were spiked with bacteria to a final concentration of approx. colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately . cfu/ml. bacteria were allowed to grow for h in the pcs at c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log reduction factors ranged from to for enterobacter cloacae ( . . , pei-b-p- ), pseudomonas fluorescens ( . . , pei-b-p- ), staphylococcus aureus ( . . , pei-b-p- ), and streptococcus bovis ( . . , pei-b-p- ). pcs (n for each species) spiked with these different bacteria species were efficiently sterilized ( out of ). treated pcs remained sterile during storage for days, while bacteria in non-treated pcs grew to high titers of - cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july , to july , was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than . were considered significant. results/finding: a total of , consecutive blood donors were screened between and . the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was . %, . %, . % and . % respectively. the hiv-hbv co-infection was higher among blood donors ( . %) followed by hbv-hcv co-infection whish accounts about ( . %). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of - . in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was , . ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a month old infant patricia davenport* , geeta paranjape and laurie sutor , . carter bloodcare, ut southwestern medical center background/case studies: in at a large pediatric hospital, a month old infant was supported for days by extracorporeal membrane oxygenation (ecmo). over this time blood products were transfused. about days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of units of red blood cells. approximately weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of : . the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* , andrea j linscott and donny dumani . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from - , % of transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a -year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day of hospitalization showed no growth after five days. on day , the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the ml unit had been aliquoted via sterile connecting device days prior for a pediatric patient. all ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was . c. within minutes of starting the transfusion, the patient's temperature increased to . c and subsequently reached a maximum of . c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at - c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of determinations of a low positive control in run. inter-assay variability was determined by testing at least representative negative production pool samples, at least low positive sample (about s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of . and were . and . respectively. the hbsag assay detection limit was . iu/ ml for source plasma and . iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at - c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between : , to : , , depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than % for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than %. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on donors ( - days after the index donation) - donors in the follow up study and tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all donors. denv antibodies were negative in donations and equivocal in . our initial reactive rate is higher than that reported to date for the procleix zikv tma of per , [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since . over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late , for which the program consisted of types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june , bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with ml of plasma, vs. ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, ; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation # : updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation # : implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over , donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on - year olds and premenopausal women (ages - ) donors. on average, - year olds donate . times a year and premenopausal women donate . times a year. if both of these groups were limited to donating once a year, a total of , donations from - year olds and , donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin # - , the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for - year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a . hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: % ( / ) of the simulation group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of hybrid group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of online only students improved their post-test scores and had an average likert scale rating of . (good). the average changes in scores were statistically significant within all training groups (p value < . ). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p< . ) and the hybrid group (p< . ). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in . all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july through january were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a -month period, cases of complex antibody identification workups ( %), transfusion reactions ( %), consultations for blood component utilization ( %), and deviations from standard operating procedures and massive transfusion protocols ( %) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $ , of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* , david lancaster , dianne geary , robert scott , anh thu nguyen , adam garcia , raina shankar , leslie buchanan and tho pham . stanford health care, stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to ( ) streamline the ordering process to accurately reflect inventory status and transfusion practices and ( ) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a -month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a -month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: ( ) the ratio of units transfused per week to the number stocked (t:s), ( ) the number of products ordered as stat, and ( ) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. lines of code were written for both programs, including class modules and distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were . , . , and . before the pilot period compared with . , . , and . during the pilot period. these differences did not reach statistical significance (p . ). we also monitored the number of stat ordered products before and during the pilot period, which were and stat units per week, respectively (p . ). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were and units, respectively (p . ). an estimated hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to . fte and $ , per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* , yulia lin , troy thompson , allison collins and sheena scheuermann . ontario regional blood coordinating network, sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that of the province's hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in indicated that % plan to implement or already have implemented the ptqip and % of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in . publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* , rana hajjeh and cees th. smit sibinga . world health organization regional office for the eastern mediterranean, international quality management (iqm) consulting background/case studies: more than million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between and ; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may in tunisia. results/finding: we found publications on disaster from five countries in the region and publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries ( . %) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated - % of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. ( , , and for - ) . the number of collections per registered trt donor varied significantly, ranging from to therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from . to . between and . conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from through . it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of . after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a % response rate (n ). of these, . % have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. . % of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although . % of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only % of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate % ( units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in , apheresis red cells (arc) represented . % of total red cell collections at our center. hae mcs ln was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs operators first and then operators new to apheresis with a training goal of % of mobile staff. validation of the alyx began / / and took approximately days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs machines were removed from service / / . alyx go-live occurred / / . additional operator training continued through september . results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $ . each providing an estimated annual savings of $ , . conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately . % and decrease our kit costs by %. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october -march . background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! g/l) and for females (minimum interdonation interval increased from to days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october , changes in rebooking of donation appointments in december and culminating with eprogesa criteria changes on march , . both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by . percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from . % in the weeks pre-implementation to . % in the weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were . % in september, . % in october/november, and . % from december to march, . conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past years, , blood products, derived from , procedures, were distributed to different investigators in over laboratories. whole blood was the most common product ( . %), followed by unmanipulated mononuclear cell collections ( . %), and elutriated monocytes or lymphocytes ( . %). less common requests included platelets ( . %), plasma ( . %) and granulocytes ( . %). adverse donor reactions were infrequent ( . % of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable % collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in % plasma. the corresponding pathogen reduction system used for the study has kits with different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than . x , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than x /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of x / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of . x and platelet concentration of x /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of . x and . x at the volume of mls and platelet yield of . x , . x , and . x at the volume of mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* and steve cihura . bonfils blood center, bbc / bsi background/case studies: in , the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated devices with the following criteria in mind: ) device disposable costs, ) reagents/controls/quality control, ) objective hgb/hct measurement, ) portability and durability for a mobile environment, ) ease of use, ) donor experience, ) battery life, ) validation requirements plans, ) blood center suitability, and ) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february with a targeted implementation date of july . after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately %. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to . % in and . % in . during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in , the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may . conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* , ayumi araki , hiromi sanyoshi , hiromi kanai , hiroya kikuchi , katsushi tsukada and kazuhide mure . hokkaido red cross blood center, japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from individuals who donated platelets during the -month period between february and august , and data from the donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the men ( . %) and of the women ( . %) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the donors in the vvr group was . . %, which was significantly higher than the . . % in the non-vvr group. at a maximum dbf threshold of %, sensitivity for discriminating between vvr and non-vvr donors was . % and accuracy was . %. when % dbf was used as the alert level, alerts were issued for donors, including in the vvr group. therefore sensitivity for predicting vvr was . % and specificity was . %. mean time from alert to diagnosis in the vvr group was . . minutes, and accuracy of the alert was . %. some of the vvr could not be predicted even the value of maximum dbf exceeded %. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* , , salam abdus and shabrina shah . inova blood donor services, inova fairfax medical campus, inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every to weeks from december to march . blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of patients, females and males, who underwent a total of rbcx from october to march , using an average number of rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of healthy volunteers were collected before blood donation and after blood donation immediately, day, week, weeks, and weeks among men and weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component ( c ) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at weeks among men and weeks among women, while c significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover week after blood donated and reached their levels before blood donated within weeks among men and weeks among women. conclusion: the biomarkers mutually changed over the course of weeks among men and weeks among women. donating ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low % split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate percent and increased concurrent plasma collections by percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october . after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in and , a total number , rns and rns completed the questionnaires, giving a response rate of . % and . % respectively. the overall mean score in was . points (range to ). the mean score in was . points (range to ). the percentage of rns having perfect scores of increased from . % in to . % in . table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; purposively selected blood prescribing clinicians and nurses from hospitals in countries of the human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at . level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r . ; p . ; df ) and accessibility (r . ; p . ; df ) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f( , ) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, junior faculty co-investigators from west coast institutions each had months to create a minute powerpoint presentation on a fundamental tm topic, after which other members had months to review and edit. therefore, each member created and reviewed presentations (three total steps). during each step, members wrote multiple-choice questions for those particular topics. in the end, each topic would have quiz questions to assess learning. at completion, evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: ) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; ) pre and post-lecture question validated examination (best collaborative) to assess learning; ) resident in-service examination trends specific to tm. results/finding: six presentations were developed as of the abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables and . abo leaders pre-test data could not be obtained for institution b, and trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the minute vignette performance and the minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean %) when compared to the pre-test scores (mean %) ttest p< . . during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and % reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between . to . (in the range of - ). nps varies between and . according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased - from . to . . conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* , anne eder , beth a. dy and mary o'neill . american red cross, georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about in , apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. , a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to , hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the months before and after launching the educational outreach. results/finding: the web based course was completed by more than participants; were physicians. based on a review of the evaluations, the course was highly valued with % of participants rating it excellent or very good. the blood center physicians gave over presentations to hospital customers. reporting of suspected strs in increased by % compared to the prior year. the increased reporting came from specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required - minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, .) subscription-based e-learning utilizes - minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june , with a new equipment module assigned each month for the following five months. the series concluded in december with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of . on a -point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with positive and negative comment. level : learning the average score of users increased % from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of . and a t-stat value of . . level : results while equipment-related errors decreased by % after training, there is not enough data to demonstrate a statistical significance. conclusion: our level and evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october , a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n ) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was %. of those, % endorse that fbc creates a climate of respect within our transfusion practice, % believe it has improved communication between work units, and % feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only % responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: students of undergraduate semester and students of semester participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with % of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, / / from - am. there were attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, / / from - am. there were attendees, including regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) % as patients with fcr % may benefit from delaying the procedure for performance in the future. validation process included ( ) a deming regression to globally assess the predicted vs. actual results and ( ) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| %. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during days ( academic hours) the trainees can attend lectures, discuss the methodical materials, participate in seminars, interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, with the group capacity a transfusion vol. supplement s of up to people the number of medical specialists who have attended the training is nearly . results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of - ; being least satisfied/comfortable and being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of total technologists, technologists took the pre esd survey and technologists took the one year post esd implementation survey. table shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, ( . %) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were unplanned sop deviations; in the year after esd implementation there were only deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from to . the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in % plasma is broken up in to two days. on day platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for - hours. on day products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day and day . in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: of employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was . for the two month training period the daily average rose to . conclusion: our "flip the switch" training plan for implementing prt platelets in % plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly % of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as % of an activity. in we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of we performed a supply inventory on all departments to plan future purchases and control residual stocks. in , we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter (q / ) with last quarter (q / ). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q / blood bags were used compared to in q / , demonstrating an activity " . %. price negotiation resulted in . % readjustment. both indicated an estimated cost " . % with a possible impact of over us$ , . we have identified a real cost # . % in q / , representing an overall # . % and us$ , . (r$ , . ) savings. conclusion: economy had deteriorated in our country in with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih- tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v , statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in . in the - nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on % individual and % for the other four levels ( % for each). the bonus (%) is calculated based on the iis as follows; category a: % (iis > %), category b: % (iis: -< %), category c: % (iis: -< %) and category d: % (iis < %). on the pilot implementation, the individual scores for staff ranged from % to %. the iis were % to %. the number of staff in each bonus categories were , % (category a) and , % (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* , deborah r fludd and sandra j nance . background/case studies: rare donors are defined as a blood type occurring in less than in people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in , there were , active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the ( . % of total active donors) returned contact cards alerting ardp of changes in calendar year , ( . %) were donors moving from one ardp facility to another, ( . %) were donors no longer eligible to donate, and an additional ( . %) were address changes. other changes were ( . %) reactivated donors and ( . %) donors who we were notified were deceased, or did not want to be listed in the ardp. in , new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in was ( ), which would be . % of the new donors submitted. conclusion: with nearly a % response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in , donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* , barbara j bachman , mike leamy , susan olson and candace williams . university of florida college of medicine, bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih- (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two ( ) runs of one to six ( ) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing , types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. regardless of quality or speed metrics evaluated, the ih- demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < . ). ih- process steps and time studies addressed in the table below did not account for the ih- reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih- ( % reduction, a difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* , kimberly monnin , barbara j bachman , kyla warren , susan olson and candace williams . clinical pathology labs, bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih- tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of separate test runs of or samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately , type & screens (t&s). the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih- demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < . ). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih- (difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih- tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih- and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih- with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v . statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/findings: using automated result verification, only . % out of , samples evaluated for abo/rh testing would require visual verification, resulting in a % reduction in operator touchpoints (p < . ) and a labor saving of minutes ( : hh:mm) for abo/rh testing. for , antibody screens, automatic validation of results would result in . % reduction in operator touchpoints (p < . ) and a labor savings of minutes ( : hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by % annually and peaked at % in mid . to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an week period in late . hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than day shelf life remaining. units with tie tags attached were in hospital inventories for up to months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was % of the hospitals surveyed. mean percent o neg rbc gross sales for a month period before, during, and after the survey was . %, . % and . %, respectively. mean percent o neg net sales during the -month survey fell to . % compared to an average of . % in the months prior. during the -month survey period o neg rbc monthly return rate increased to an average of . % compared to an average of . % in the months prior. for the months after the survey the average o neg rbc return rate further increased to . % while mean percent o neg rbc net sales trended slightly upward to . %. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* , jeremie gachelin , veronique ollivier , thibaut mutin , xavier telot , benoit ho tin noe and sandra sanfilippo . aenitis technologies, hôpital bichat, inserm u background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac ) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than % (p< . ) and a purity of platelets close to . %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over . blood donations for an area with more than million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in , orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in , when we moved from this equipment to atreus c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in we changed to atreus c (terumo bct) and finally in , we moved to reveos system (terumo bct). since the changes in , our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in , bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during and . conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of - patients and an average, round-trip travel time of approximately -minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about % of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november , the hb has significantly improved the turnaround time of rbc issue -from -minutes to less than -seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately rbc per month out of the window for non-surgical patients. this has been reduced to approximately rbc per month, a % average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in , they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the st rescue squadron ( rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the rqs master sergeant in january . we asked what rqs's order and delivery expectations were. he said sporadic use and the blood order would be rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the rqs needs. staff was trained based on data from january meeting. we contacted the rqs in september to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the rqs association with a civilian blood center. based on his field experiences, he changed the blood order from to rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and rqs performed a mock run on october , and we felt prepared for any future events. results/finding: on november , , the rqs was deployed to a civilian aeromedical evacuation. we anticipated a rbc order. the actual order was rbcs and ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at : am by the rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november th , we established a maximum blood order of rbc and liquid plasma, noting future orders may request fewer products, yet meet the preferred rbc; plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table ). the highest mean fib concentration was mg/donor unit; lowest mean fib concentration was mg/donor unit. all sites had a mean fib concentration at least mg/donor unit above the fda minimum requirement of mg/donor unit. fifteen of blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; manufactured pooled donor cryoprecipitate. most froze plasma in a - c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every minutes for a hour period or until the temperature exceeded c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached > c in minutes as shown in table . the control thermometer recorded temperatures maintained at - c for the entire hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at - c for more than minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: % conventional (c-pc), % pr-pc, and mix of % c-pc/ % pr-pc. the model predicts a modest ($ %) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january . a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august , inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january . a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from to (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in , zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within weeks (sept. phase ) and nationwide within weeks (nov. phase ). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools ( -donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab had % and lab had % of clients requiring universal zkv testing. we evaluated a -month test result upload performance period to determine the impact of zkv test implementation. results/finding: during , lab upload time performance ranged from % to . % from january to july; upload time performance fell between august through november, returning to . % performance in december. lab upload time performance ranged from . % to . % january to august. performance fell september through december . % - . %. lab experienced a low of % upload time performance during phase when there was a rapid implementation; % clients required zkv nat. improved performance was observed during phase , with a % increase in zkv clients. for lab : phase experienced a modest decline of upload performance ranging from . % to . % with . % of clients implementing zkv nat. performance was . % in phase , when an additional . % of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* and colleen a. aronson . advocate lutheran general hospital, acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in of the hospital transfusion services. the sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the sites which implemented the verax pgd test perform testing on all day and day platelets to be issued for transfusion. this abstract summarizes the data collected for the first weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day , day , and those that were tested twice. inventory reports were reviewed for platelets issued on day or day that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february ( week of performing the test), . % of all platelets issued by the participating transfusion services were day or day platelets. in march that number dropped to . %. it is expected that this number will level off at some percentage at or below . % with further data collection. in february . % of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day or day ) will likely level off at some number at or below . % due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, of the sites performing testing are level trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed %. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n ). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days , , and of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was ( . . )%, relative change rate of hsr was ( . . )%, the residual leukocytes were ( . . ) . the ph, hsr, and the cd p expression of pooled platelet concentrates before and after filtering were ( . . ) vs ( . . ), ( . . )% vs ( . . )% and ( . . ) % vs ( . . )%. there is significant change for wbc after filtering (p< . ). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table . conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb - ) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of ml and ml. two users then evaluated the ss tubing segment types with ml or ml samples for a total of data points. samples were collected into the attached ml or ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january , a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by %. the platelet outdate rates dropped after implementing the platelet inventory tool from % ( units) to % ( units); a % decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of fte to . fte, lowering fte by %. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* , jaclyn mckay , jennifer curnes and rowena punzalan , . bloodcenter of wisconsin, children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: ( ) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, ( ) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within -hours after collection. this tight hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight -hour completion time constraint for cryo production (capacity expansion). in particular, during the th -quarter of , a blood processing region was able to process about more cryo units/month (an increase of %) at a slightly lower collection cost (cost avoidance), resulting in an approximately % reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the -the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after -day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from to days in the usa using an fda cleared rapid test (rt). in august , our hospital based transfusion service began using a rt on day and to routinely extend ap shelf life to days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of -day ap. study design/methods: data were obtained for two study periods: september -february (pre-implementation) and september -february (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from -day to -day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by % post-implementation while inpatient admissions and surgical volume increased by % and %, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by % post-implementation and the outdate rate decreased from % to % (p< . ). ad-hoc ordering was not statistically different between study periods (p . ). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different ( . and . , respectively, p . ). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years ( ) ( ) ( ) ( ) ( ) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was , , and units respectively. the mean number of discarded rbc units of the five years of the study exceeds % of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below mg/dl, and so the rejection threshold for lipaemia is level equal to or more than mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including rbcs, plasma products and apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from mg/dl to mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* , , rebecca ross , debra mraz , anne baker , zenna neal , melanie champion and edward l. snyder , . johns hopkins university school of medicine, yale university, yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, pr-sdp were transfused at our hospital (out of a total of platelets transfused). after months of scale-up, pr-sdp were approximately % of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($ mg per g) to the content in pr-sdp (< ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september , the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from donors was collected in k edta tubes. plt concentrations were determined at the qc department using the coulter act diff hematology analyzer (beckman coulter). sample tubes were stored at - c and measured at , and hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd ( ml; n ) or dpd units ( ml; n ). plt pools were stored at - c under mild agitation for seven days except for dpd, which were split in two -ml bags after h. samples were taken on days and . ph, po and pco , hypotonic shock response (hsr), extent of shape change (esc), cd p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at h ( pltx /l), h ( pltx /l) and h ( pltx /l) postdonation. dpd can be stored in the same collection bag for h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits ( - pltx / l) before splitting. on day , lactate and pco concentrations increased, and po decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march . there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted % increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* , colleen vincent and patricia kopko . university of california -san diego, american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only - % of double platelet collections meet requirements for pathogen reduction treatment. blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: , apheresis platelets annually), which includes two hospitals ( inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week ) and the bone marrow transplant (bmt) ward (week ). an e-mail communication explained the change to all physicians and nurses. in phase , we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase , we expanded usage to include the inpatient bmt ward. in phase , we lifted all restrictions so prp could be used throughout the health system, with the goal to reach % prp within months. results/finding: in phase (weeks - ), we requested prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the cancer center. in week , excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase . in phase (weeks - ), we formally expanded issuing of prp to include the inpatient bmt ward and requested prp products weekly. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the phased-in areas. in phase (weeks - ), we began issuing prp throughout the health system. our supplier provided an average of prp weekly (range - ), and prp constituted % of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table ) resulting in a cost savings of $ k. an additional cost savings of approximately $ k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as a transfusion vol. supplement s we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* , james r stubbs , scott a hammel and manish gandhi . mayo clinic, mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing % pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of . , . , and . x may end up below a . in the final storage bag and would need a post-processing sample to ensure the product met criteria at ! . x platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a . yield but ended with a yield below . . these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe- d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that of results were . or . x / mcl with the exception being the wbc failure with a count of . . further monitoring of the wbc counts discovered a result of . which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of . will be tested on the adam to confirm a leukoreduced product. we also discovered of ( %) of the . preprocessing yields products ended with a post processing yield < . . we decided to increase the yields requiring post processing samples to include the . . conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of ! . x platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from / - / were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need ) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in ; the revised prediction method (estimated clinical need ) was then evaluated retrospectively using the same data sources covering / - / and then compared to the prior method. results/finding: the average number of platelets transfused from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted amount and the number of units used was - . u/d. % days ( d/month) were under-predicted (average: u/d). % of days ( ) were under-predicted by ! u (average: u; max: u ( x)). the average number of platelets used from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted and units used was a . u/d. % days ( d/ month) were under-predicted (average: . u/d). one day ( %) over this period was under-predicted ! u ( u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages ( % a %), reduced the number of days under-predicted ( % a %), and decreased the discrepancy on those under-predicted days ( . u a . u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* , maria noedel , nancy haubert , kenneth hudson , larry morgan , robert shaw , tracy fickett , jamie jue , valerie winkelman , sally caglioti , german leparc , and phillip c williamson . background/case studies: on / / , fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within weeks; nationwide in weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on of manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for clients within weeks, and an additional clients within weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the -week period but overcome during the -week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* , hala samuel boules , fatemah saleh al matroud , rabab hussien ali dashti , hanan alawadhi and reem al radwan . kuwait central blood bank, kuwait central blood bank, kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of rbcs baby units were randomly chosen to be traced to their final deposition from the year till . half of them ( units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb ( units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is % efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (< days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march . results/findings: a total of products were tested. fifteen units ( %) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all by the vendor and none were true positives by re-culture. of the units that were successfully tested, were tested again on day for use on day ( %). there were platelets transfused ( %) and expired after day ( %). the cost to test the products including controls was $ , and our calculated cost to produce products would be $ , . if we had needed to import products to meet needs, the cost would be roughly $ , without shipping costs which are estimated at $ , . . we averaged expired platelet products per month (range - ) before verax testing and (range - ) after implementation. conclusion: using verax point-of-care testing saved platelet products from discard. the cost savings were $ , . from importing and $ , from producing a replacement for those products. the average discard rate per month went from to after verax implementation. extending platelet shelf life to days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for data, units were investigated without the use of secure texting. of these, units were identified as preventable wastage, and wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: ) product not released after procedure/ or when patient stabilized ( ) ) product returned outside of appropriate temperature range ( ) ) clinician unaware product was assigned ( ). thus far in , wastage records have identified units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, responses thanked the transfusion service for the information, and in instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: . % dmso, plas-a, % hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) . %dmso: plas-a : %hsa : : . plas-a and hsa are kept at room temperature ( - c, rt) and refrigerated at c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point . c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total tests. at least minutes cooling after dmso, before adding the next reagent. see table: ( ) after directly adding . % dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. ( ) in tube, autologous plasma first, dmso next, powder-like precipitates. ( ) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. ( ) & ( ) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at c. ( )&( ) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the c group is slightly milder/slower than rt group. so hsa should not be added first. ( )&( ) trace of hsa(< ml) was mixed into the plas-a bag ( ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive -month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from . % to . %. with an annual platelet usage of approximately , units, this reduction equates to approximately units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $ , to $ , , per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* , russell thorsen , rosaline ma , antonio g insigne , amy decourten , florence panganiban , patricia mckean , cyril jacquot , sara bakhtary and ashok nambiar . ucsf health, children's national medical center background/case studies: plasma (ffp, pf , pf rt ) stored at - c outdates hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (< months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited ( hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk and richard gammon* . oneblood, oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average - minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average - minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from % to %). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp- using the low glycerol ( %) freezing method and frozen at - c within six days of collection. thawing occurred in a c water bath in the following order: o positive and o negative on january ; o positive and o negative on february ; and o negative on february . deglycerolization occurred on site using the acp- with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $ - and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $ per unit to produce and distribute. drbcs have a shorter shelf life, days versus the days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp- 's and deglycerolize four units at a time. in january and february , it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c izekial butler* , karen leighton , scott jones and rachel beddard . qualtex laboratories, biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c instrument. precision of the new assay parameters was determined by testing replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from iu/ml to iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of . to . iu/ml. stability of samples was determined by testing samples stored at - c and - c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from . to . . so, precision was acceptable since the %cv for all samples tested was %. the mean values for the samples tested in the accuracy study were all % of the expected value which was much lower than the acceptance criteria which was % of the expected value. the linearity of the assay was acceptable with a r ! . %. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to days at - c and up to month at - c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for days at - c and stored up to one month at - c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, ) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april , to march , . local research ethics board approval was obtained. results/finding: patients received ivig for itp at smh over the study period for a total of unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding ( , %), pre-operative or antepartum care ( , %), a platelet count of less than and contraindication to corticosteroids ( , %). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between - x /l. patients received ivig for a likely diagnosis itp while a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. / ( %) of infusions consisted of g/kg over days; the remainder of infusions consisted of g/kg. of those who received g/kg, of patients ( %) had evidence of partial remission after a first g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to staff members in early february of . twenty-two technologists responded for a % response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, % indicated that workspace size was insufficient and % that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* , neil bangs and kimberly sanford . vcu health system, virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from / / to / / to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (< hours, > hours < hours), and if a second sample was required. results/finding: a total of t&s orders were initiated from the ed in this time period. ( . %) patients were not subsequently transfused any type of blood product within hours of tsd and ( . %) patients were not subsequently transfused any type of blood product within hours of tsd. a total of ( . %) patients required a second sample. of these patients requiring a second sample, ( . %) were not subsequently transfused any type of blood product within hours of tsd and ( %) were not subsequently transfused any type of blood product within hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within hours of tsd amounted to an estimated $ , in unnecessary patient charges and approximately . nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* , debra berry , yunchuan delores mo and gay wehrli . university of virginia health system, children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* , elizabeth halperin , sharon breining and mona papari . acl laboratories/ advocate hospitals, advocate health care, itxm background/case studies: a large midwest hospital system with level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and months of data were evaluated that contained events. results/finding: there was an equal number of events that were initiated in the ed and the or ( ). male patients were involved % of the time and % of time the patients expired. trauma of some type was the majority of the cause but . % of the cases involved gi bleed and only . % were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average . with the post hgb average of . . ratios of : for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of : . it was found that the rbc: plasma was . : , rbc: plt was . : and rbc to cryo was . : . use of txa was only . % and cacl was utilized in . % of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* , jody a barna , donald e ulinski and nancy m. dunbar . dartmouth hitchcock medical center, dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july , we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august and november (pre-implementation) , newly identified clinically significant antibodies were resulted for patients compared to patients between the months of august and november (post-implementation). the tat for allergy alert entry for both periods is shown in table . we observed that % of allergy comments were performed within hours in the post-implementation period versus only % pre-intervention (p . ). using the new process, nearly all of the alerts were entered into the emr within hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* , shailesh macwan , nancy nikolis , arline stein , janelle richardson , manju bagu , lennart logdberg , alexander indrikovs , vishesh chhibber and sherry shariatmadar . north shore university hospital, northwell health background/case studies: our institution is a tertiary care facility performing over cardiovascular surgeries (cvs) in , an increase of % after the healthcare system cvs integration in . transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december , the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after reported events in q that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: . open discussions and collaboration between blood bank and cvs nursing teams . mapping the process using flowcharts for additional blood orders from cvs. . identify bottlenecks and brainstorm solutions. . a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. . the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. . follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period / / - / / the blood bank has received verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* , tracie ingle and heather vaught . indiana university health, indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter - and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with - o, d negative red blood cells over days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series , anti-d series , a cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion ; : - who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for % and % of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february -june as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june -october . results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table ) . this led to a reduction in observed overall sli ( . . days vs . . days, p< . ) and odr ( . % vs . %). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. ( )) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january and december thirty-five ( ) notifications were sent to physicians using epic letters and of those, fourteen ( ) responded to the epic notification and five ( ) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* , denden benabdessadek , annu george and alexandra jimenez . westchester medical center, new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of orders were reviewed. approximately, % of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases ( %), but of the issued products, all were returned to the blood bank in % of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> type and screens (t&s) per day] blood banks , respectively. our laboratory which serves a large -bed multispecialty academic hospital and receives - t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of . . and . . minutes , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was % longer and had a larger standard deviation (s.d.) than the published trial result of . . . transfusion vol. supplement s abstract during phase i visionv r performed panels. during phase ii visionv r performed of the visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* , john roback , ronald arkin , michael bartlett , robert geiger and jaxk reeves . university of georgia, emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over , emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a % target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of participants responded to the survey ( . % response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n- - df) of the null hypothesis that the mean vs. the alternative that the true mean is> . overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on run cycles. ct was comprised of metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc ( : ), xm ( : ), cb ( : ) and dat ( : ). by implementing the future state, an average $ . min. lt and vt is saved on each sample loaded for ts equating to a % labor reduction over the current state. a % improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a % lt reduction. on average, a min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $ type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing -column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on patient samples for abo/d typing and antibody screening; of which at least had a positive antibody screen. out of the , had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which were d(-) and were d( ). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the patient and donor samples tested ( % concordance). all samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave % sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for samples at different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* , craig fletcher and peter millward . beaumont hospital, beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short -day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october and quality data was reviewed from august to december . the collected data was then analyzed using descriptive statistical methods. results/finding: data from indicates platelet wastage comprised % of total received platelets and % of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units months preimplementation of the report was units, compared to units months post-implementation and units months post-implementation. wastage rates improved from % (wasted yearly platelets/total received yearly platelet units) in , the year of report implementation, to post-implementation rates of % in and % in (see table) . importantly, this occurred despite a greater than % increase in platelet inventory between and and resulted in cost savings of over $ , in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* , lisa marie button , lori scanlan-hanson , karen koch , janet finley , deepi goyal and camille van buskirk . mayo clinic-rochester, mayo clinic, mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in and were / (events/ed transfusions - ). study design/method: electronic ordering for the ed was implemented march st . any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients < kg and pediatric patients > kg. a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for years (april -march ), and during that time there was instance of blood being ordered for an unintended patient . % ( / ). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in , / ( . %) units were transfused in the ed but not charted in the patient's medical record. in , / ( . %) transfusions were not charted. however, in , the first full year of electronic transfusion order capability, only / ( . %) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level trauma center, with over inpatient beds and over operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option (verbal) for verbal orders and coolers set up; and option (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by % with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* , steven zibrat , geoffrey wool and angela treml . university of chicago medicine, university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in , blood bank accounted for % of all rejected clinical laboratory samples, yet comprised only % of total laboratory volume; % of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was . %. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september . results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of . %, three times the national average of . %. under the new system, rejected blood bank samples decreased from an average of % to % of all rejected laboratory samples, a % decrease. implementation of the new process produced a net savings of $ . k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ). in the postimplementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ; p . ). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* , marcus holme , johnathan bakst , gunta musa and angela treml . university of chicago medicine, university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an minute turnaround time (tat). in april of , the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased %. tat analysis of a representative one week sampling per month showed an increase in outliers from per month to per month. average monthly tys samples performed is . these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was / / . results/finding: the average number of outliers decreased % from per month to . further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $ , for fiscal year . conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from - days with a mean of . days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of - days with a mean of days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* and randy levine . northwell health, lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and only of those units ( %) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, additional irradiated units were issued ( / ; %). post-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and all of those units ( %) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus of the ( %) total units were ordered as irradiated. again, additional irradiated units were issued ( / ; %) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* , charlotte van dyke , dina garza van hoose and rachel beddard . biobridge global, south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january , to april , , , total collections were flagged for additional qc by our trima accels and haemonetics instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in , the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: ) risk assessment, ) quality control plan and ) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* , rachel jug , kimberly ingersoll , nicholas bandarenko , nicole guinn and jessica poisson . duke health pathology, duke university hospital, duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced : : transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july -december . data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the : : ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* , nancy nikolis , linda benison , ruthmire thelusca , renee liberty , sherry shariatmadar , alexander indrikovs and vishesh chhibber . north shore university hospital, northwell health background/case studies: our blood bank (bb) processes approximately , specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june , a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed / . the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of . the implementation of this new process has led to a decrease in the number of unacceptable specimens up to % quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from % to %, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* , , morgan rockwell , joseph hagan , jun teruya , and shiu-ki hui . texas children's hospital, baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within hours pre and post transfusion were evaluated. patients ( - years) receiving prophylactic ptx from july to december admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < ml/kg for patients < kg and one apheresis unit (au) for patients > kg; therefore, patients were separated into groups: < kg and > kg. a significant proportion of orders for both < kg and > kg did not meet patient platelet threshold criteria (p< . ). conclusion: ptx threshold above ir for both groups were ( kg) and % (> kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than % of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p> . ) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only . % of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated ( x ) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately patients ( x ) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts ( . %) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by % with the communication category average rpn having the greatest reduction of %. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* , karen king and joseph sweeney . rhode island hospital, lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different ( minutes for the provuev r and for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over minutes are shown in the table. the results show a reduction in tat by minutes with a % reduction of tat greater than minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* , , lorna orengo , monique scott and christopher a tormey , , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as in , blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as % prior to interventions, but may potentially be reduced to as little as . %. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of < %, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged . %. reasons for specimen rejection were divided into groups: ) hemolysis, ) blood bank witness collection form errors, ) quantity not sufficient, abstract ) duplicate sample, and ) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table ) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a . % rejection rate with only rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s system christopher shahan* , christina dejesus , mosi mccall , fallon hampton , tangi herring , judy davis , anjali patel , sonya gomillion and bonnie maltby . qualtex laboratories, qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual - hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently % of whole blood donor testing turnaround time delays are due to issues and failed runs on the s system and we have technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s system. study design/method: the number of technician related failed runs on the s system were tracked from / / thru / / . a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s system. after counter measures were implemented, the number of technician related failed runs decreased from to . failures per week, which was a % decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by %. patients who were transfused for pre-transfusion hgb > g/dl with resulting post-transfusion hgb > g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: patient charts were reviewed. were excluded for bleeding and cardiovascular instability. / ( . %) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors ( . % vs . %, p . ) and anemia of chronic disease ( . % vs . %, p . ) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb . g/dl vs . g/dl, p< . ). inappropriately transfused patients also had higher median post-transfusion hemoglobin ( . g/dl vs . g/dl, p< . ). moreover, lab evalutions revealed association with lower folate levels (median . nmol/l vs . nmol/l, p . ). / ( . %) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. / providers were responsible for . % of all inappropriate transfusions. / appropriately-transfused patients experienced an fnhtr. deaths unrelated to transfusion occurred ( in appropriate, in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* , willem martin smid and ashley john duits . red cross blood bank foundation, sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* , dee dee cassidy , jed b gorlin , and nancy l van buren , . hennepin county medical center, innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant or more minutes may be required for transit of units often released in less than minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july . data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january -june , and post change included august -december . july data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of rbc/month in coolers. post change this dropped to rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of rbc/month were returned ( %). post change, the average rbc/ month returned was ( %), this represents an absolute % reduction in number of returned products. each rbc dispensed and returned takes approximately minutes to complete paperwork and transport, therefore this change saved an average of minutes per month. it was also noted that the average rbc/month transfused was for baseline and post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of - minutes (estimated) was reduced to - minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over patent-applications have been filed related to "transfusion medicine" and over related to "transfusion alarm", during the last years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an / inch color cmos sensor, providing effectively . mp, a . mm lens, ir-cut, day/night minimum illumination . lux/f and viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood a transfusion vol. supplement s abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h. , video frame rate (fps) - /s, refresh rate hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/ . /b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p p is provided. typical v power-supply, sized x x mm and weighing g. client software is required. the ir range is - m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a gb sd-card. pan/tilt-horizontal o and pan/tilt-vertical o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot ( cfu), multishot cfu or highdose k organism preparations at a low level (< cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < days and the fungal organisms in < days. the overall agreement was . % in bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* , diane schafer , debra brown , jesse cox , scott koepsell and sara shunkwiler . nebraska medicine, the nebraska medical center, university of nebraska medical center background/case studies: anticipating the implementation of the new ( th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september , ), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling in months post implementation compared to in the months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late ' s and in early , intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january , to june , . data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: % were female and % were less than weeks of gestational age. none had co-existing g pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn ( %) and rhesus-hdn ( %). antibodies most often implicated in rh-hdn were anti-d ( / ), anti-d and anti-c ( / ) and anti-c ( / ). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was g/kg (range from , g/kg to , g/kg). neonates received one to four ivig administrations. table shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, required an et for rh-hdn and for abo-hdn. forty-five ( %) patients needed top-up transfusions during hospitalisation and until three months of age: with abo-hdn and with rh-hdn. the mean number of transfusions was three (range: to ). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over % case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a -part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate ( each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, georgia institute of technology ap reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to a) all adult males (am), b) women of non-childbearing age (wncba), and c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to a) women of childbearing age (wcba) and b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed %) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap saving apheresis platelets through use of verax point of care testing jennifer rhamy* and rebecca wride . st. mary's regional blood donor center, st. mary's regional medical center background/case studies: our rural hospital-based blood center serves hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between and per day in ), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson and jun teruya , . texas children's hospital, baylor college of medicine ap vision titers --easier or problematic? (table ) . results/findings: post intercept, t had volumes of - ml, with % hemoglobin (hb) recovery. t had -fold less extracellular protein than c. after days of storage t had higher atp and na than c while lactate and hemolysis were lower. hct, ph, k and glucose were equivalent between t and c on d . d hemolysis for t was . - . %, while for c it was . - . %. t and c atp was > mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table ) . hematocrit (hct, %) . . * . . . . . . hemoglobin (g/unit) not measured hemolysis (%) . . * . . . . * . . ph ( c) . . * . . . . . . total atp (mmol/g hb) . . * . . . . * . . k (mm) . . * . . . total tested total plts issued feb mar totals table: . resident reports to the intranet "drop box" increased from . % to . % to %, each over month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from . per day or , dpmo to . per day or , dpmo. this is a statistically significant difference since the p-value calculated was . . conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately , specimens per month. since , the requirement of having a second blood type on record was met by: . utilizing the historical blood type and the current specimen, or . having second type performed on same specimen by different technologist, and . each type and screen specimen signed by staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards th edition, # . . . a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april , the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding: . there was a minor adjustment period with more phone calls made to blood bank to explain the process. . there was minimal impact on turn around times for release of components. . aborh retype workload decreased from to ( % to % of t&s volume) per month. . unnecessary blood draws minimized, improving patient experience. . no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as to %. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our bed hospital, a retrospective chart review was performed ( / / - / / ) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< grade" or "> grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples ( %) were > titer results higher, while the majority was titer results different ( %). the cost analysis is summarized in table . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated % decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc. samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another samples were measured with fc in ucc maribor. results/finding: samples ( rbc, plt, ffp-all leucocyte depleted and non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from , - , ; plt from , - , ; ffp from , - , ; and for non-leucocyte depleted ffp from , - , (table ) . samples ( rbc, plt, ffp -all leucocyte depleted and nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from , ; plt from , , ffp from , ; and for non-leucocyte depleted ffp from , - , (table ) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than x /unit for leucocyte depleted or x / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.