key: cord- - zthrgy authors: taylor, sylvia; lopez, pio; weckx, lily; borja-tabora, charissa; ulloa-gutierrez, rolando; lazcano-ponce, eduardo; kerdpanich, angkool; angel rodriguez weber, miguel; mascareñas de los santos, abiel; tinoco, juan-carlos; safadi, marco aurelio p.; lim, fong seng; hernandez-de mezerville, marcela; faingezicht, idis; cruz-valdez, aurelio; feng, yang; li, ping; durviaux, serge; haars, gerco; roy-ghanta, sumita; vaughn, david w.; nolan, terry title: respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged months to years in a multi-country population sample date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: zthrgy background: better population data on respiratory viruses in children in tropical and southern hemisphere countries is needed. methods: the epidemiology of respiratory viruses among healthy children ( months to < years) with influenza-like illness (ili) was determined in a population sample derived from an influenza vaccine trial (nct ) in centers in eight countries (australia, south east asia and latin america). active surveillance for ili was conducted for approximately year (between february and august ), with pcr analysis of nasal and throat swabs. results: children were included, of whom experienced ili episodes. rhinovirus/enterovirus had the highest prevalence ( . %), followed by influenza ( . %), adenovirus ( . %), parainfluenza and respiratory syncytial virus (rsv) (both . %), coronavirus ( . %), human metapneumovirus ( . %) and human bocavirus (hbov) ( . %). corresponding incidence per person-years was . , . , . , . , . , . , . and . . except for influenza, respiratory virus prevalence declined with age. the incidence of medically-attended ili associated with viral infection ranged from . (hbov) to . (rhinovirus/enterovirus). the percentage of children missing school or daycare ranged from . % (hbov) to . % (influenza). conclusions: active surveillance of healthy children provided evidence of respiratory illness burden associated with several viruses, with a substantial burden in older children. respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged months to years in a multi-country population sample introduction acute respiratory tract infections (artis) comprise the most common illnesses worldwide, and children often experience several episodes a year. in children, clinical presentation can range from mild, uncomplicated upper respiratory tract illness to severe lower respiratory tract infection (lrti) including pneumonia, bronchiolitis, croup and exacerbations of asthma or wheezing. the world health organization estimated that . million children died from arti in , % in africa and south east asia. in , pneumonia alone led to . million deaths worldwide in children less than years of age. most artis are caused by viruses, but until the advent of multiplex polymerase chain reaction (pcr) techniques, it was often not possible to identify accurately specific viral infections in clinical cases. , the pathogens considered responsible for most arti are respiratory syncytial virus (rsv), influenza a and b, parainfluenza viruses types , and , and adenovirus. several new pathogens associated with arti have been identified more recently, including human metapneumovirus (hmpv), rhinovirus, coronavirus, human bocavirus (hbov) and parainfluenza . , vaccine efficacy trials provide intensive, active followup of a well-defined population and can be used to evaluate viral epidemiology. as part of a trial of pandemic influenza vaccines, which included year of prospective, active, community-based surveillance for influenza-like illness (ili) in centers in eight countries, we evaluated the prevalence and incidence of respiratory viruses in children months to less than years of age at first vaccination. samples were obtained from an efficacy trial of two pandemic influenza h n vaccines (nct sponsored by gsk vaccines). an analysis estimating the prevalence of rsv in ili has been previously reported. here, we report data on all respiratory viruses evaluated, a secondary analysis objective. healthy children months to < years of age were enrolled in a randomized, observer-blind, parallel group, multi-country trial of as -adjuvanted versus nonadjuvanted monovalent pandemic h n vaccines. the trial was conducted in centers in australia, brazil, colombia, costa rica, mexico, the philippines, singapore, and thailand between february and august ; enrollment took up to months and timing varied by country. the trial was approved by an institutional review board for each center and written informed consent was obtained from parents/guardians. parents were instructed to contact the study center within h if the child became ill. active surveillance via scripted telephone contact was conducted from weeks after first vaccination, and contact made every e weeks to day for each child, regardless of time of enrollment. ili was defined as temperature ! . c by any route and at least one of: new/ worsening cough, sore throat, stuffy nose, or runny nose. study staff visited the child's home to collect one anterior nasal swab and one throat swab, ideally within h of ili onset, and at most within days. collection could also take place at a study center or hospital if necessary. swabs were transported in a single tube of m rt transport medium, stored at À c and maintained on dry ice during transport. a -day symptom-free period was required between new ili episodes. sample testing was performed by standard multiplex pcr techniques. , analysis of viral epidemiology the viruses evaluated were influenza (subtypes a-h , a-h and b), parainfluenza (subtypes , , and ), rsv (subtypes a and b), hmpv, rhinovirus/enterovirus (the assay did not distinguish between them), adenovirus, coronavirus (subtypes e, oc , nl and hku ) and hbov. first analyzed separately, influenza, parainfluenza and coronavirus subtypes were grouped in a post-hoc analysis. the main outcome variable was pcr-confirmed infection with the stated viruses in nasal/throat swabs in children with ili. this included infection with the virus under consideration alone (single infection) or with the virus under consideration plus one or more of the other viruses (co-infection). single and co-infections were also recorded separately. clinical characteristics of ili episodes were reported by the parents of the children, and any hospitalization or medical attendance (by a doctor or other healthcare professional, not including sample collection by study staff) were recorded. pneumonia was defined as acute illness (one or more of fever ! c, new or worsening cough, dyspnea, consistent auscultation findings [rales or diminished breath sounds], pain in the chest or abdomen when breathing, or purulent or blood-stained sputum production) and radiologic findings consistent with pneumonia. the total cohort included all children enrolled in the randomized trial. the total cohort with ili episodes tested by multiplex pcr included all children enrolled who experienced an ili and had an adequate nasal/throat sample tested by multiplex pcr. the analysis of prevalence of respiratory viruses in ili and clinical characteristics associated with ili was performed in the total cohort with ili episodes tested by multiplex pcr. the analysis of overall incidence of ili, medically-attended ili and hospitalized ili in which respiratory viruses were detected was performed in the total cohort. the prevalence of respiratory viruses among ili episodes was calculated as: where x is the number of ili episodes with nasal/throat samples positive for the virus and n is the total number of ili episodes with samples collected within days and tested. as there was at least days between two ili episodes, it was assumed that each episode was independent. exact % confidence intervals (ci) were computed. prevalence was stratified according to country, age at the time of the ili episode ( e , e , e , e , þ months) and whether the child was medically attended or hospitalized. the incidence per person-years (py) of virusassociated ili in the study population was calculated as: where n is the total number of children enrolled in the trial, ε i is the total number of virus-positive ili episodes for subject i, and d i is the follow-up period for subject i. incidence rates were stratified according to country and age group at the time of the ili episode. exact % poisson cis were calculated. observations with incomplete data for the outcome variable and ili episodes for which no nasal/ throat sample was taken were removed from the analysis. missing data were accounted for by calculating the missing proportion for each country and age group, then multiplying the py by ( minus missing proportion). the trial included children (total cohort). after excluding children with no ili or inadequate samples, children experienced ili episodes (total cohort with ili episodes tested by multiplex pcr). participant flow is shown in supplement fig. . demographics were similar in both cohorts, except that children in the total cohort were older (median versus months) (supplement table ). a respiratory virus was detected in of ili episodes ( . %). rhinovirus/enterovirus had the highest overall prevalence ( . %), followed by influenza ( . %), adenovirus ( . %), parainfluenza and rsv (both . %), coronavirus ( . %), hmpv ( . %) and hbov ( . %) ( table ]). co-infection was detected more often with adenovirus and hbov (fig. ). single infections with parainfluenza, hmpv and coronavirus were identified at approximately the same frequency as co-infections ( fig. ). however, parainfluenza and coronavirus e were identified more often as co-infections, whilst coronavirus nl was identified more often as a single infection (supplement table ). in all countries, rhinovirus/enterovirus was the most prevalent ( . e . %), whilst hbov was least prevalent ( . e . %) ( table ) . influenza prevalence ranged from . % to . %; parainfluenza prevalence was approximately % except in brazil ( . %) and singapore ( . %) ( table ) . (table ) . influenza was most prevalent ( . %) in the oldest children ( þ months), followed by e months ( . %) and the other age groups ( . e . %) ( table ). all other viruses were least prevalent in the oldest group (table ) . there was a less obvious pattern in the other age groups, but, in general, prevalence declined with age except for influenza ( table ) . the incidence of detected respiratory viruses associated with ili reflected their prevalence. the overall incidence per py (total cohort, all children randomized) was . for rhinovirus/enterovirus, . for influenza, . for adenovirus, . for parainfluenza, . for rsv, . for coronavirus, . for hmpv and . for hbov (table ) . australia had the highest incidence of hmpv ( . ) and the second highest of rsv ( . ), but low incidence of the other viruses relative to other countries ( table ). the philippines, singapore and thailand also had low incidences of most viruses in ili relative to the latin american countries ( table ) . detection of the respiratory viruses at different times during the year was highly variable across countries (fig. aeh) . the overall incidence of medically-attended ili associated with viral infection per py (total cohort, all children randomized) ranged from . for hbov to . for rhinovirus/enterovirus (table ) . corresponding values for incidence of hospitalized ili associated with viral infection were for hbov and . for rhinovirus/enterovirus (table ) . clinical characteristics of ili episodes associated with a single respiratory virus (i.e. no co-infection) are shown in table . median duration of ili episodes ranged from . to . days. few children were hospitalized but most were medically attended outside study procedures. the percentage of children missing school or daycare was highest with influenza-associated ili ( . %), followed by hmpv ( . %), adenovirus ( . %), rhinovirus/enterovirus ( . %), coronavirus ( . %), rsv ( . %), parainfluenza ( . %) and hbov ( . %) ( table ). sore throat was experienced by e % of children, cough by e %, stuffy nose by e %, and runny nose by e % (table ). fever was part of the ili definition and therefore experienced by all children. cough was reported in almost all children with influenza, parainfluenza, rsv, hmpv and coronavirus infections, but only in e % of children with rhinovirus/ enterovirus, adenovirus and hbov infections. there were no medically important differences in clinical characteristics between children with a single viral infection compared with children with multiple infections (supplement table ). a total of pneumonia cases were identified among the children enrolled in the overall clinical trial, corresponding to a detection rate of . %. of the cases, met the definition of ili and were therefore eligible for sample collection as per the clinical trial protocol. a sample was collected within days of onset of ili symptoms for of these cases: one case in thailand, three in the philippines, five in brazil, five in mexico and six in colombia (table ) . no virus was detected in three cases, a single infection was detected in cases (four rhinovirus/enterovirus, two parainfluenza, one influenza, two rsv and one hmpv), and co-infection was detected in seven cases (table ) . nine children were hospitalized. rhinovirus/enterovirus had the highest prevalence and incidence in ili of all respiratory viruses tested in all countries, followed by influenza, adenovirus, parainfluenza and rsv, coronavirus, hmpv and hbov. the burden of ili associated with respiratory viruses was considerable, with a high proportion of children being seen by a medical professional and many missing school or daycare. our analysis benefited from being part of a clinical trial, as previously described. most importantly, we conducted year of prospective, active community surveillance of healthy children in tropical and southern hemisphere countries where prospective data are lacking. most studies of viral epidemiology use hospital-based surveillance because community-based surveillance is difficult and expensive. however, hospital-based surveillance tends to capture only the most severe illness and many cases are missed in developing countries because of limited hospital access. our analysis avoided these limitations and allowed us to capture the burden of virus-associated ili in communities. understanding community epidemiology is essential to implement effective control measures. other advantages of being part of a clinical trial included a wellcharacterized population, wide age range up to years, samples taken from a high proportion of children, consistent methodology between countries, and use of sensitive and validated pcr assays. the trial was conducted in eight countries encompassing australia, south east asia and latin america. the exact timing of enrollment varied somewhat between countries, but was planned so that data collection was performed during the peak e influenza season for each individual country. as stated in the methods, all children were followed for days, with the complete period of surveillance for the study occurring between february and august . this allowed us to compare the distribution of viruses across the different countries. there was considerable variation in the incidence and prevalence of the viruses by country, although rhinovirus/enterovirus had by far the highest incidence and prevalence in all countries. hbov had consistently the lowest incidence and prevalence. several other studies have evaluated the prevalence of viruses in children with respiratory illness. the relative prevalence of the different circulating viruses varied by study. however, the main circulating viruses were similar between studies and with our study, and included picornaviruses (including rhinovirus), adenovirus, rsv, bocavirus, pivs, hmpv, influenza and coronavirus. e rhinoviruses are classified in the picornavirus family, of the enterovirus genus. a high prevalence of this family has been reported in other studies in different settings. , , e as in our study, an australian study with active community-based surveillance of healthy preschool-age children with arti found that picornaviruses (including rhinoviruses) were the most frequently detected ( . %). however, other viruses were detected less frequently than in our study: rsv ( . %), parainfluenza ( . %), influenza a and hmpv (both . %), adenovirus ( . %) and coronavirus nl ( . %). in another prospective australian study in children aged months to years reporting ili, rhinovirus was again the most commonly detected. however, in contrast to our results, adenovirus was detected at the same frequency as rhinovirus, followed by parainfluenza , polyomavirus, hmpv and hbov. influenza (a/h n ) and rsv were relatively uncommon; approximately % of children were fully or partially vaccinated against influenza. rhinovirus is not always the most commonly detected virus in children with respiratory disease. in children under years of age hospitalized for lrti in thailand, the most commonly detected viruses were rsv ( . %), rhinovirus ( . %), hbov ( . %) and influenza ( . %). a study of children aged < years hospitalized for lrti in brazil found that rsv was most prevalent ( . % of episodes), followed by hmpv ( . %), rhinovirus ( . %), influenza ( . %), hbov ( . %), parainfluenza and adenovirus (both . %) and coronavirus ( . %). in our analysis, influenza prevalence increased with age. the other viruses showed the opposite trend, with the lowest prevalence observed in the oldest children ( þ months). there was a less obvious pattern in younger ages, but, in general, prevalence of all viruses except influenza declined with age. despite this, the burden of illness remained considerable in older children. there was a clear seasonal pattern for influenza, rsv and hmpv in most countries, and to a lesser extent for rhinovirus/ enterovirus. a previous study found that, although there was no clear seasonal peak for rhinovirus/enterovirus, onset seemed to correspond with the start of the school year in the usa. a limited one year analysis of human rhinoviruses and enteroviruses in ili in latin america showed a year-round temporal distribution throughout central and south america. however, human rhinovirus c species displayed opposite seasonal trends on either side of the equator, accounting for a higher percentage of ili cases north of the equator between september and january, while south of the equator detection increased between april and july. as part of the study, all children received a monovalent influenza a/h n pandemic vaccine; one or two doses of an as -adjuvanted vaccine were administered or two doses of an unadjuvanted vaccine. trivalent seasonal influenza vaccination rate in the present study was approximately %. influenza a subtype h was not isolated in any children; influenza a subtype h was isolated in . % of children; and influenza b was isolated in . % of children. no difference between the study vaccine groups was observed. cases associated with influenza were least likely to be co-infected with other respiratory viruses. rhinovirus/ enterovirus was also more common as a single infection. adenovirus and hbov were found more often as a coinfection. bacterial co-infection was not measured as part of this study. in the us-based influenza incidence surveillance project, which evaluated the most commonly detected viruses in outpatients with arti or ili, threequarters of all co-infections involved adenovirus and rhinovirus/enterovirus. a uk-based analysis found negative associations between influenza a and hmpv, and between influenza a and rhinovirus. positive associations were found between parainfluenza and rhinovirus, rsv and rhinovirus, adenovirus and rhinovirus, and parainfluenza and rsv. no correlation was found between co-infection and clinical severity in a study in brazil evaluating children under years who sought medical care for respiratory tract infections. more research is needed to understand the interaction of respiratory viruses, and the host response to infection. there were no clear differences between viruses in the severity of illness. most ili episodes were medically attended. ili associated with influenza resulted in the highest proportion of children missing school or daycare ( %), although e % of children infected with the other viruses also missed school or daycare. there was no difference between viruses in the proportion of children hospitalized. clinical features were variable depending upon the viral infection associated with the ili episode. study limitations have been described previously. only healthy children participated in the trial, limiting generalizability. in addition, our study did not include any children aged < months and only a limited number of children aged e months, so our findings are mainly relevant to older children. fever was part of the ili definition, and therefore we would have missed cases in children with no fever. to put this into perspective, in the influenza incidence surveillance project, % and % of cases among children aged e years and e years, respectively, met the arti definition which did not require fever, but did not meet the ili definition which did require fever. however, our definition of ili was somewhat broader (except in children under years of age) and us data may not be generalizable to the tropical and southern hemisphere countries in our study. the inclusion of only healthy children in the study and the exclusion of cases with no fever would have underestimated the burden. we also could not discriminate between rhinovirus and enterovirus by pcr, therefore the exact prevalence and incidence of each one could not be determined. finally, our study included only a small number of pneumonia cases (n z ), limiting the conclusions that can be drawn regarding the distribution of viral infection in these cases. the overall pneumonia detection rate in the clinical trial ( . %) is higher than, but in line with, what has been reported in the us for hospitalized cases. however, our sample collection rate among pneumonia cases was only . % compared with . % for ili overall. in conclusion, our active surveillance of healthy children as part of a vaccine efficacy trial provided evidence of the burden of respiratory illness associated with a range of viruses. a substantial burden of illness occurs in older children. data on the epidemiology of respiratory viruses determined from active surveillance of healthy children are generally lacking, and are particularly sparse in the developing countries included in our study. a considerable amount of the burden would not be identified through hospital-based surveillance. these novel data fill an important gap in our knowledge of the epidemiology of viruses contributing to the substantial burden of respiratory disease in children, and may be useful in informing priorities for implementation of 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requiring hospitalization among us children all authors participated in the design, or implementation, or analysis and interpretation of the study results; as well as in the development of this manuscript. all authors had full access to the data and gave final approval before submission.terry nolan, charissa borja-tabora, pio lopez, lily weckx, rolando ulloa-gutierrez, eduardo lazcano-ponce, angkool kerdpanich, miguel angel rodriguez weber, abiel mascareñas de los santos, marco aurelio p safadi, aurelio cruz-valdez and juan-carlos tinoco were coordinating investigators, and together with sumita roy-ghanta, david w vaughn and ping li were responsible for the conduct of the flu q-pan h n - pri (nct ) trial. fong seng lim, marcela hernandez-de mezerville and idis faingezicht also contributed to study material and data collection.sylvia taylor led the epidemiology team in collaboration with gerco haars.yang feng was responsible for the statistical input; statistical expertise was also provided by gerco haars, sumita roy-ghanta, ping li and terry nolan. serge durviaux led the laboratory analysis.terry nolan, charissa borja-tabora, yang feng, david w vaughn and sylvia taylor were members of the core writing team. terry nolan and sylvia taylor contributed equally to this manuscript and the corresponding author was responsible for the submission of the publication. the the authors moreover thank magali ribot (gsk vaccines) for multiplex pcr testing, jean-yves pirçon (gsk vaccines) for critical review of the statistical analysis and mich ele seil (keyrus biopharma on behalf of gsk vaccines) for writing the study report.mary greenacre (independent medical writer) was paid by gsk vaccines to prepare the manuscript draft and sophie vanwetswinkel (xpe pharma and science on behalf of gsk vaccines) provided editorial assistance and manuscript coordination. lily weckx declares research grants from gsk to federal university of são paulo for conduct of three clinical trials and received payment from gsk, novartis, pfizer, and sanofi for board membership or lectures.rolando ulloa-gutierrez discloses having received honoraria from gsk for the original influenza a h n clinical trial discussed here, as well as from gsk, sanofi pasteur, pfizer/ wyeth and merck as a speaker in the past.marco aurelio p safadi has received grants to support research projects and consultancy fees from novartis, gsk, pfizer and sanofi pasteur.fong seng lim discloses having received travel grants from gsk as well as a grant from gsk to his institution to perform clinical trials.marcela hernandez-de mezerville declares having received honoraria from gsk for the original influenza a/ h n clinical trial discussed here, as well as travel support from gsk, sanofi pasteur and pfizer outside the submitted work in the past.idis faingezicht received payment from gsk as principal investigator in a previous vaccine clinical trial and as co-investigator in the influenza a/h n clinical trial.pio lopez, eduardo lazcano-ponce, angkool kerdpanich, miguel angel rodriguez weber, abiel mascareñas de los santos, juan-carlos tinoco, and aurelio cruz-valdez report having nothing to disclose. supplementary data related to this article can be found at http://dx.doi.org/ . /j.jinf. . . . key: cord- -b t a r authors: cheng, wei; yu, zhao; liu, shelan; zhang, xueying; wang, xiaoxiao; cai, jian; ling, feng; chen, enfu title: comparison of influenza epidemiological and virological characteristics between outpatients and inpatients in zhejiang province, china, march –june date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: b t a r given the rapid rate of global spread and consequently healthcare costs related to influenza, surveillance plays an important role in monitoring the emerging pandemics in china. however, the characteristics of influenza in southeast of china haven’t been fully studied. our study use the surveillance data collected from sentinel hospitals across zhejiang province during march through june , including the demographic information and respiratory specimens from influenza-like illness (ili) patients and severe acute respiratory illness (sari) patients. as analysis results, most sari and ili patients were in the age group of – years old ( . % of ili and . % of sari). the respiratory specimens have statistically significantly higher positive rate for influenza among ili patients than that among sari patients (p < . ). the comparison between ili patients and sari patients shows no statistically significantly difference in detecting influenza virus type and influenza a virus subtype. the sari and ili patients were found to be positively correlated for overall positive rate (r = . , p < . ), the weekly percentage of a(h n )pdm (r = . , p < . ), influenza b virus (r = . , p = . ), and a/h n (r = . , p < . ) among all the positive numbers. our study demonstrated that the activities of influenza virus, including its subtypes, had a similar temporal pattern between ili and sari cases. influenza virus is estimated to cause to million cases of severe illness and , to , deaths each year, while %- % of adults and %- % of children are infected with the influenza virus worldwide [ ] . in lower and middle-income countries, influenza could result in large economic burden encompassing direct costs to the health service and households, and indirect costs of productivity losses [ , ] . vaccination is a cost-effective way to reduce the public health and economic impacts caused by influenza. because of the antigenic shift and drift of the virus, the influenza vaccine composition needs regular updates. currently, the selection of strains for the annual influenza vaccine are primarily of based on the predominant strain of influenza virus detected in influenza-like illness (ili) [ ] . however, ili only represents a proportion of acute respiratory infections with mild clinical manifestations. severe acute respiratory illness (sari) surveillance is another type of surveillance for severe influenza-associated disease. it is recommended to be monitored with established surveillance systems with ili according to the guidelines of the world health organization (who) [ ] . in china, patients meeting with the definition of sari are required to have inpatient observation and necessary treatment. due to the emerging high healthcare cost and severe consequences of sari, effective vaccination is needed to reduce the incidence of sari caused by influenza [ ] [ ] [ ] . therefore, whether influenza epidemiological and virological characteristics among sari patients were consistent to those of ili patients is critical to the effectiveness of current vaccination plan. zhejiang province located in the southeastern china, featuring by its blooming economy and high density of population. it has over than million permanent residents, which are distributed in metropolitan areas. influenza surveillance in zhejiang province launched at the same time with national surveillance in the year of , and expanded to ili and four sari sentinel hospitals by the year of . both ili and sari are monitored in the surveillance system. although studies describing influenza surveillance with both ili and sari are well documented in the northern and southern hemispheres [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , few studies have fully compared the influenza epidemiological and virological characteristics between ili and sari cases. in this study, we used four-year continuous surveillance data to compare the epidemic and virological characteristics of influenza virus between ili cases and sari cases in zhejiang province. the influenza surveillance network in zhejiang province is a part of the national influenza surveillance system [ ] . the ili surveillance in zhejiang province was initially launched in . as to , the ili surveillance has been expanded into sentinel hospitals that cover all of the metropolitan areas in zhejiang province. the types of sentinel hospitals include general hospital, specialized clinic as well as maternal and children hospital. the sari surveillance started in . as to march , the surveillance has expended to four sentinel hospitals including three general hospitals and one children's hospital. all four sari sentinel hospitals were selected from the existing ili surveillance network ( figure ). ili is defined as any person with sudden onset of fever > • c and cough or sore throat in the absence of other diagnosis [ ] . the definition of sari is varied by ages. a patient > years old is defined as having sari if, upon or during admission, presenting an acute onset of elevated temperature (axillary temperature ≥ • c) and cough or sore throat, as well as tachypnea (respiratory rate ≥ /min) or dyspnea (difficulty breathing). a patient ≤ years old is defined as having sari if, upon or during admission, presenting with acute onset of cough or dyspnea, and at least one of the following six signs or symptoms: (a) tachypnea(respiratory rate > /min for ages < months, respiratory rate > /min for ages to < months, and respiratory rate > /min for ages to ≤ years); (b) inability to drink or breastfeed; (c) vomiting; (d) convulsions; (e) lethargy or unconsciousness; (f) chest in-drawing or stridor in a calm child [ ] . ili is defined as any person with sudden onset of fever > °c and cough or sore throat in the absence of other diagnosis [ ] . the definition of sari is varied by ages. a patient > years old is defined as having sari if, upon or during admission, presenting an acute onset of elevated temperature (axillary temperature ≥ °c ) and cough or sore throat, as well as tachypnea (respiratory rate ≥ /min) or dyspnea (difficulty breathing). a patient ≤ years old is defined as having sari if, upon or during admission, presenting with acute onset of cough or dyspnea, and at least one of the following six signs or symptoms: (a) tachypnea(respiratory rate > /min for ages < months, respiratory rate > /min for ages to < months, and respiratory rate > /min for ages to ≤ years); (b) inability to drink or breastfeed; (c) vomiting; (d) convulsions; (e) lethargy or unconsciousness; (f) chest in-drawing or stridor in a calm child [ ] . each week, physicians from departments of pediatrics and respiratory, as well as emergency rooms in all of the ili sentinel hospitals were required to report the number of total visits to outpatient, as well as the number of outpatients who presented with non-specific symptoms that meet a case definition of ili. as for four sari sentinel hospitals, the number of sari and total admissions from pediatrics ward, the respiratory medicine ward and the intensive care unit were also reported. all of the reported numbers in the above-mentioned departments were required to record by age classified as -, -, -, -, and -years old. nasopharyngeal or throat swabs were required to collect from all of sari cases and - ili cases of each sentinel hospital on every week. the collection of swabs was conducted by trained nurses from who had not received antiviral each week, physicians from departments of pediatrics and respiratory, as well as emergency rooms in all of the ili sentinel hospitals were required to report the number of total visits to outpatient, as well as the number of outpatients who presented with non-specific symptoms that meet a case definition of ili. as for four sari sentinel hospitals, the number of sari and total admissions from pediatrics ward, the respiratory medicine ward and the intensive care unit were also reported. all of the reported numbers in the above-mentioned departments were required to record by age classified as -, -, -, -, and -years old. nasopharyngeal or throat swabs were required to collect from all of sari cases and - ili cases of each sentinel hospital on every week. the collection of swabs was conducted by trained nurses from who had not received antiviral drugs. in addition, a standardized case report form containing demographic and sample information was also required to complete among whose biological samples had been collected. due to the outbreak of avian influenza a(h n ) in april , ili surveillance was strengthened by increasing the weekly number of the biological samples collected in each hospital from - to . the biological samples were collected and saved in cryovial tubes, stored at • c at the sentinel site, and then sent to regional center for disease control and prevention (cdc) within h after the sample collected. the regional cdc laboratory tested influenza using real-time reverse transcription polymerase chain reaction (rrt-pcr) assay following the standard protocols. specimens tested as positive for influenza a were further tested for subtypes (i.e., a(h n ), a(h n ), a(h n )pdm , and a(h n )) using specific rrt-pcr. once the laboratory complete, regional cdc submitted the laboratory results to the online surveillance system. data obtained from the surveillance system were reported weekly by the staff of the sentinel hospitals and laboratories. the mean and standard deviation or median and interquartile range (iqr) were calculated for continuous variables, and percentages were calculated for categorical variables. chi-squared test and fisher's exact test were used to assess the differences of age, sex, season, influenza virus type/subtype, and influenza positive rate between all the sampled ili and sari cases. the weekly number of positive influenza by subtype and the percentage of specimens tested positively were plotted to describe seasonality and circulation of influenza types/subtypes among sari and ili cases. spring, summer, autumn, and winter were defined from week to , to , to , and to of the next year, respectively [ ] . cochran-armitage trend test was used to analyze the trend change of influenza virus positivity with the increase of age. spearman correlation was applied to analyze the linear relationship of the influenza virus positive rate, weekly percentage of influenza virus subtypes accounted for all the positive numbers between sari and ili patients. ili percentage was calculated as the percentage of total outpatient visits that were due to ili and sari percentage was calculated as the percentage of total admissions that were due to sari. to compare ili percentage and sari percentage of which can better reflect the activity of influenza virus among outpatients and inpatients, spearman correlation analysis was used to assess the linear relationship between weekly ili influenza-positive rate and ili percentage, as well as weekly sari percentage and sari influenza-positive rate. a two-sided p-values were considered as statistically significant if it was found less than . . all the statistics were conducted using sas version . (sas institute, cary, nc, usa). verbal consent was obtained from all patients in prior to survey and specimen collection. for children aged under years old, verbal consent was obtained from at least one parent or legal guardian. the influenza surveillance were a national-wide, governmental public health activity. therefore, institutional review board approval was not required in china. in this study, the personal identifiers (e.g., names, address, occupations and so on) were not disclosed in order to maintain patient confidentiality, all the patient information was analyzed anonymously. during the study period, , patients completed both the standardized case report and laboratory sample test, of which , ( . %) were ili patients and ( . %) sari patients. the median age of the tested patients was years (iqr: - ), and the median age of ili patients was significantly older than it of sari patients (p < . ). in addition, the group of sari patients had higher proportion of children at - years old ( . % for sari versus . % for ili), male during the study period, , patients completed both the standardized case report and laboratory sample test, of which , ( . %) were ili patients and ( . %) sari patients. the median age of the tested patients was years (iqr: - ), and the median age of ili patients was significantly older than it of sari patients (p < . ). in addition, the group of sari patients had higher proportion of children at - years old ( . % for sari versus . % for ili), male ( . % for sari versus . % for ili), and patients enrolled in the winter ( . % for sari versus . % for ili) than those of ili patients (table ) . the most identified influenza virus were influenza a virus ( . % among ili, and . % among sari), followed by influenza b virus ( . % among ili, and . % among sari), and mixed type virus ( . % among ili; . % among sari), with no statistical significance between the two groups (p = . ). for influenza a virus in the ili group, a(h n ) was the most identified subtype ( . %), followed by a(h n )pdm ( . %), a(h n ) ( . %), and a(untype) ( . %). those proportions were correspondent to that in the sari group-a(h n ) ( . %), a(h n )pdm ( . %), a(h n ) ( . %), and a(untype) ( . %) with p-value . (table ) . influenza viruses were found in the specimen of of , ( . %) all patients, with of , ( . %) ili patients and of ( . %) sari patients. table shows the specific positive rate of influenza virus by age groups, genders, and seasons. for ili patients, the highest ( . %) rate is in the - years age-group, followed by > years age-group ( . %). meanwhile, the positive rate of influenza viruses of sari patients was highest in the > years age-group ( . %), followed by - years age-group ( . %). among both ili and sari cases, influenza virus was found in all age groups, and cochran-armitage trend test showed that the influenza virus positive rates tend to be higher at older ages (figure ) . the positive rate was highest in the winter, and lowest in the autumn. overall, the influenza virus positive rate among ili cases was significantly higher than that among sari cases across different groups of age, sex and season ( table ). mixed type virus ( . % among ili; . % among sari), with no statistical significance between the two groups (p = . ). for influenza a virus in the ili group, a(h n ) was the most identified subtype ( . %), followed by a(h n )pdm ( . %), a(h n ) ( . %), and a(untype) ( . %). those proportions were correspondent to that in the sari group-a(h n ) ( . %), a(h n )pdm ( . %), a(h n ) ( . %), and a(untype) ( . %) with p-value . (table ) . influenza viruses were found in the specimen of of , ( . %) all patients, with of , ( . %) ili patients and of ( . %) sari patients. table shows the specific positive rate of influenza virus by age groups, genders, and seasons. for ili patients, the highest ( . %) rate is in the - years age-group, followed by > years age-group ( . %). meanwhile, the positive rate of influenza viruses of sari patients was highest in the > years age-group ( . %), followed by - years age-group ( . %). among both ili and sari cases, influenza virus was found in all age groups, and cochran-armitage trend test showed that the influenza virus positive rates tend to be higher at older ages (figure ) . the positive rate was highest in the winter, and lowest in the autumn. overall, the influenza virus positive rate among ili cases was significantly higher than that among sari cases across different groups of age, sex and season ( table ). due to the outbreak of avian h n virus in april , ili surveillance was strengthened by increasing the number of samples for testing from - to . figure shows that although the weekly number of the samples tested has increased since the week of year , the weekly number of all ili patients remained at similar level from year to (figure ). due to the outbreak of avian h n virus in april , ili surveillance was strengthened by increasing the number of samples for testing from - to . figure shows that although the weekly number of the samples tested has increased since the week of year , the weekly number of all ili patients remained at similar level from year to (figure ) . figure a ). consistently, this influenza activity was also observed among sari patients ( figure b ). seven a(h n ) viruses (five in the ili group and two in the sari group) were detected during the early year of ( figure ). we found that the weekly percentage of influenza virus types/subtypes among all the identified influenza cases were significantly correlated between sari and ili patients, with a(h n )pdm (r = . , p < . ), influenza b virus (r = . , p = . ), and a(h n ) (r = . , p < . ) ( table ) . table . correlation analysis of weekly influenza virus type/subtype constitution among total positive numbers between influenza-like illness (ili) and severe acute respiratory illness (sari). figure a ). consistently, this influenza activity was also observed among sari patients ( figure b ). seven a(h n ) viruses (five in the ili group and two in the sari group) were detected during the early year of ( figure ). we found that the weekly percentage of influenza virus types/subtypes among all the identified influenza cases were significantly correlated between sari and ili patients, with a(h n )pdm (r = . , p < . ), influenza b virus (r = . , p = . ), and a(h n ) (r = . , p < . ) (table ). . . correlation analysis between weekly ili influenza-positive rate and sari influenza-positive rate, ili percentage and ili influenza-positive rate, sari percentage and sari influenza-positive rate table shows the results of spearman correlation analysis among influenza virus positive rate and percentage of people with ili or sari. the positive rate for influenza virus of ili cases and those of sari cases was statistically significantly correlated (r = . , p < . ). the percentage of ili cases and ili influenza-positive rate was statistically significantly correlated (r = . , p < . ), which was higher than it of sari (r = . ), whose coefficient was statistical significant though (p < . ). to our knowledge, this is the first study to compare the epidemic characteristics of influenza between outpatients and hospitalized inpatients in zhejiang province. children less than years of age were found to be the largest group of both ili and sari patients, which was consistent with those reported by other studies [ , , ] . besides, we found a good agreement between sari and ili patients for the weekly proportion of samples tested positively for influenza virus and the distribution of the influenza virus types/subtypes among all the identified patients. this demonstrated that the seasonal pattern and predominant circulation types of influenza were similar between ili and sari patients. finally, we found that the correlation between the weekly influenza positive rates and percentages of patients meeting the definition of ili and sari is higher among ili patients than it among sari patients, which indicated that compared to ili, influenza may less important in causing sari. although the largest age groups were same ( - years old) among ili patients and sari patients, this age group had higher proportion within sari patients than it within ili patients. this difference might be caused by the different behaviors when a child or an adult is found to be sick. compared to adults, children are more likely to be taken to hospital, especially for sari cases. therefore, children have sari were more likely to be involved in the surveillance. similar results have been obtained in mongolia [ ] , philippines [ ] , jordan [ ] . our findings further demonstrated that young children are vulnerable for both mild and severe respiratory infection, and the low influenza detection rate among - years age-group in both sari and ili patients foreshadow the need of expand the respiratory illness surveillance to more types of pathogens [ , ] . being consistent with other studies, influenza virus was detected in all age groups among both ili and sari cases [ , ] . and overall, the proportion of samples tested positively for influenza viruses in different age groups presented to be higher with the increase of age. therefore, all persons aged months and older are recommended for vaccination and elders should be considered with priority [ ] . to understand the temporal characteristics of influenza epidemics is essential for planning influenza vaccination programmes because vaccine effectiveness wanes over time, a boost of vaccine is essential to prevent the spread of diseases [ ] . the simple and preferred measure to assess and compare seasonality patterns was the proportion of influenza positives [ ] . the highly correlated influenza virus positive rate between sari and ili patients demonstrated the similar temporal pattern of influenza activity in the two groups. similar findings were recently reported in a description of influenza surveillance in egypt, which showed that the seasonality of influenza among ili cases and sari cases was consisted in november-february [ ] . moreover, the comparison between ili patients and sari patients shows no statistically significantly difference in detecting influenza virus type and influenza a virus subtype, which was similar to the findings from nigeria [ ] . finally, the correlation of weekly percentage of influenza virus type/subtypes accounted for all the positive numbers between sari and ili patients indicated that the predominant influenza types/subtypes among ili and sari cases was corresponded. these findings are essential for planning influenza vaccination programmes for those severe cases given recommendations for strain inclusion within the vaccine are based on the ili surveillance system. our results were consistent to the study conducted by peng et al. in the year of [ ] . but in that study, the detailed analyses such as correlation analysis between weekly ili influenza-positive rate and sari influenza-positive rate, ili percentage and ili influenza-positive rate, sari percentage and sari influenza-positive rate were not performed. therefore, we believe the present study can provide more comprehensive information on the comparison of influenza epidemiological and virological characteristics between outpatients and inpatients. in line with the findings of other studies [ , , ] , we also found higher influenza detection rate among ili patients compared to that among sari patients. moreover, we also found the correlation coefficient of ili percentage and ili influenza-positive rate was higher than that of sari percentage and sari influenza-positive rate. these phenomena may due to the higher specificity of the ili diagnosis compared to the sari diagnosis [ ] . some studies have demonstrated that influenza played an important role in the viral aetiologies of ili cases [ ] [ ] [ ] , while other respiratory virus such as respiratory syncytial virus, rhinovirus, human bocavirus were essential for the cause of sari, especially in children [ , ] . this indicated that it is more necessary to conduct pathogen spectrum test for sari cases so as to accurately understand the cause of those severe cases. of note, we detected seven cases of a(h n ) viruses (five in the ili group and two in the sari group) during early year of , when this virus was outbreak in zhejiang province [ ] . the detection rate of a(h n ) virus in the sari patients was higher than that in the ili patients because this strain frequently cause severe syndromes. however, our study found that the surveillance network has low sensitivity on the capture of patients infected with a(h n ) virus. although one of the critical functions of influenza surveillance is to detect novel strains of influenza, the rapid detection of emerging novel influenza strains or outbreaks of respiratory disease calls for other surveillance with standardized methodology [ ] . this study has several limitations. first, although sari surveillance was required to catch all patients in accordance with the definition, sometimes the cases may not be fully recorded because of physicians' oversights and absenteeism. in the ili surveillance, to get all ili patients surveyed was impossible due to the limited amount of resources and personnel. therefore, the subject chosen for ili sampled may prone to sicker patients or younger patients. second, compared to ili surveillance, sari surveillance may be less representative due to its sparse surveillance sites. in the future, we should consider to expand and to enhance the coverage of the surveillance network, with priority to choose hospitals from ili surveillance. third, we did not test for pathogens other than influenza, which made us unable to exclude other viral, bacterial, and fungal pathogens that could be the causes of ili and sari. this study demonstrated circulating types/subtypes of influenza strains and seasonality pattern of ili cases were similar to that of sari cases in zhejiang providence. this reassured the effectiveness of influenza vaccine as strain selection based upon ili surveillance. our study results suggest that compared to ili patients, it is more necessary to conduct pathogen spectrum detection among sari patients. in the future, the expanded and enhanced ili and sari surveillance in the province may contribute to identify the novel virus, detect pandemics at early stage, and then improve the understanding of the etiology of ili and sari. a systematic review of the social and economic burden of 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illness in seven teaching hospitals viral etiology and clinical profiles of children with severe acute respiratory infections in china identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia epidemiology of human infections with avian influenza a(h n ) virus in the two waves before and after october in zhejiang province acknowledgments: this work was financially supported by grants from zhejiang province (major science and technology programme, grant number c ), (medical research programme, grant number rcb , rca ). we would like to thank physicians and staff in the influenza surveillance hospitals across zhejiang province for their dedication in completing the countless numbers of data forms that made this work possible. we also want to thank physicians and staff of hangzhou, ningbo, huzhou, shaoxing, jiaxing, quzhou, taizhou, zhoushan, wenzhou, jinhua, lishui and yiwu centers for disease control and prevention for their invaluable assistance with samples collection and detection. the authors declare no conflict of interest. key: cord- -j sqs q authors: koetsier, antonie; van asten, liselotte; dijkstra, frederika; van der hoek, wim; snijders, bianca e.; van den wijngaard, cees c.; boshuizen, hendriek c.; donker, gé a.; de lange, dylan w.; de keizer, nicolette f.; peek, niels title: do intensive care data on respiratory infections reflect influenza epidemics? date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: j sqs q objectives: severe influenza can lead to intensive care unit (icu) admission. we explored whether icu data reflect influenza like illness (ili) activity in the general population, and whether icu respiratory infections can predict influenza epidemics. methods: we calculated the time lag and correlation between ili incidence (from ili sentinel surveillance, based on general practitioners (gp) consultations) and percentages of icu admissions with a respiratory infection (from the dutch national intensive care registry) over the years – . in addition, icu data of the first three years was used to build three regression models to predict the start and end of influenza epidemics in the years thereafter, one to three weeks ahead. the predicted start and end of influenza epidemics were compared with observed start and end of such epidemics according to the incidence of ili. results: peaks in respiratory icu admissions lasted longer than peaks in ili incidence rates. increases in icu admissions occurred on average two days earlier compared to ili. predicting influenza epidemics one, two, or three weeks ahead yielded positive predictive values ranging from . to . , and sensitivities from . to . . conclusions: icu data was associated with ili activity, with increases in icu data often occurring earlier and for a longer time period. however, in the netherlands, predicting influenza epidemics in the general population using icu data was imprecise, with low positive predictive values and sensitivities. a limitation of current influenza surveillance systems is that timely information on severe influenza illness requiring hospital admission is not available. influenza surveillance in most countries is based upon sentinel general practitioners (gp) networks and the collected information on influenza like illness (ili) is dependent on the health care seeking behavior of the general population, which can fluctuate with for example media attention. the implementation of a hospital based surveillance system for severe acute respiratory infection (sari) is now promoted by the world health organization (who) and european centre for disease prevention and control (ecdc) as a public health priority worldwide, both for routine surveillance and for preparedness [ , ] , such as in the case of the middle east respiratory syndrome coronavirus (mers-cov). sari surveillance can focus on admissions for respiratory infections in general hospital wards or in intensive care units (icu) . during the pandemic period, hospitalization for laboratory confirmed influenza a(h n )pdm infection was notifiable in the netherlands. in the season / as well as in the season / , ili incidence as measured by gp sentinel practices, reached the epidemic threshold of . consultations per . enlisted patients at a time when already more than patients had been hospitalized, with several icu admissions and deaths from laboratory confirmed influenza (national institute for public health and the environment, unpublished surveillance data). hospital admission for influenza is not notifiable anymore and in the netherlands sari cases are not routinely collected. an alternative source of information could be the dutch national intensive care evaluation registry (nice) [ ], wherein diagnostic, and physiologic information from the first hours of adult icu admissions, as well as length of stay and in-hospital mortality of all icu patients are registered. patients are admitted to the icu if they have a high severity of illness, and require constant monitoring of their vital functions, regardless of their expected outcome. respiratory infections, such as pneumonia, are among the most common conditions for which patients are admitted to the icu. in the early onset of an influenza epidemic, patients with multiple comorbidities are more likely to develop more severe influenza related diseases like pneumonia. they possibly get submitted to the icu before there is an epidemic in the general population [ ] . therefore, increases in the number of admissions at the icu with a respiratory infection can possibly occur before a detectable increase in ili incidence in the gp sentinel network. in this study we explore whether icu data on respiratory infections reflect ili activity in the general population, and which relevant time lag exists between both data sources. additionally we assessed whether icu data can predict ili defined influenza epidemics (further referred to as influenza epidemics). in the netherlands, patients that consult the gps with symptoms of ili are reported on a weekly basis to the continuous morbidity registration sentinel general practice network (further referred to as sentinel gp registry), covering . % of the dutch population being nationally representative by age, gender, regional distribution, and population density [ ] . the sentinel gp data consists of the weekly number of patients presenting with ili at the gp. in , the registry had participating gp practices with gps covering a population of approximately , patients [ ] . ili was defined according to the criteria of pel [ ] , by ( ) acute onset, with prodromal stadium of three to four days, ( ) a temperature increase to at least degrees celsius, and ( ) at least one of the following symptoms: cough, nasal congestion, raw throat, frontal headache, retrosternal pain, and body aches. incidence of ili is calculated on a weekly basis, using number of patients registered at the reporting gp practices as the denominator. this is acceptable as almost every person in the netherlands is registered with a gp. in the netherlands, an influenza epidemic is defined as more than . patients with ili per , inhabitants per week consulting the gp for at least two consecutive weeks [ ] , combined with influenza a virus isolation from laboratory samples. data quality is assured by training of gps for data entry, and a pop-up appearing in the system when an ili diagnosis is entered reminding the gp to register the ili case in the sentinel gp registry. the nice registry was founded in , with initially six participating icus. in this number had grown to and in icus participated covering approximately % of the dutch adult icus. for each admission, among other items, the acute physiology and chronic health evaluation ii (apache ii) [ ] reason for icu admission diagnosis is registered and sent to the nice registry on a monthly basis. upon receipt, the data is usually entered into the nice registry database and available for participants within one day. there is currently no distinct variable describing whether an icu patient has influenza like illness or was diagnosed with an influenza virus infection, or receives antiviral drugs. therefore, we defined an icu admission with a possible sari (further referred to as icu admission with respiratory infection) when the following four criteria were met: ( ) the patient was admitted to the hospital less than two days before icu admission, ( ) there was a medical (non-surgical) reason for admittance, ( ) the icu admission was not a readmission to the icu within the hospitalized period, and ( ) the apache ii reason for admission was 'respiratory infection. we used the percentage of icu admissions with a respiratory infection (relative to the total number of medical icu admissions), instead of the absolute number of icu admissions to adjust for the growing number of nice registry participants throughout the study period. thus our study dataset included study year, week number, number of patients with ili, population size of reporting gps, number of icu admissions with respiratory infection, and number of medical icu admissions. data quality is assured by regular on site visits of the icus [ ] . in this study, we used weekly time series of patients presenting with ili from the sentinel gp registry and icu admissions of respiratory nature from the nice registry. in the nice registry, few icus were participating in the years until , leading to large variations in the percentage of icu admissions with respiratory infections. therefore, from both registries, we used data from through . as influenza generally occurs between week and week of the subsequent year [ ] , we defined an influenza year (i.e. season) from july st until june th the next year, thereby having ten influenza years in our dataset. to explore the association between the weekly incidence of ili patients and the percentage of icu admissions with a respiratory infection, we plotted per week the percentage of icu admissions with a respiratory infection and the incidence of ili over the period january , , through december , . in addition, we performed a generalized estimating equations (gee) [ ] additive poisson regression analysis [ ] using an autoregressive working correlation matrix over this time period. the dependent variable was weekly incidence of patients with ili, and the independent variables were chronological week number, percentage of icu admissions with respiratory infection in the current week, and one to five weeks before the current week and one to five weeks after the current week, and sine and cosine terms to adjust for seasonality [ ] . the sine term was sin(k pt/t) and the cosine was cos(k pt/t), where k is a constant with values (yearly seasonality) or (half year seasonality), t is current week number, and t is total number of weeks in the specific influenza year, e.g. weeks (years and had weeks, and the cases were added to week ). we adjusted for autocorrelation in the residuals, where the unit of clustering was influenza year. we calculated the average time lag between the sentinel gp and icu data by computing the weighted average of the time lag in weeks ( , …, , …, + ), using the corresponding regression coefficients as weighting factors. the r-squared (r ) value based on the deviance residuals [ ] was also calculated. to assess the possibility of using icu data for predicting influenza epidemics, we used a subset of three years of training data to develop three gee models with the same characteristics as the aforementioned model, to predict the incidence of ili patients one to three weeks ahead. in each of these models, independent variables were removed by pseudo stepwise selection [ ] , using a fixed scheme for removal. we first considered the time trend (chronological week number) for removal, then seasonal terms, and finally time lagged percentage of icu admissions with respiratory infection. in order for the final models to be useful in surveillance, the following restrictions also applied: ( ) the percentages of icu admissions with respiratory infection one to five weeks after the current week were not included as they are unavailable and useless for prospective surveillance, ( ) the percentage of icu admissions with respiratory infection in the current week cannot be removed from the model, ( ) additional variables of lagged icu admissions should correspond to a range of subsequent weeks (e.g., one and two weeks before the current week, not one and three weeks before the current week), and ( ) a seasonal term is always a combination of a sine and cosine function. we used data of the first three influenza years (january , , , through june , to generate the final model for predicting the incidence of ili one week ahead. to accomplish this, we varied the decay factor l of the full model, giving weeks further back in time exponentially lower weights, from . to . with increments of . , resulting in seven candidate models. variable selection for these seven models was performed with pseudo stepwise selection, with the quasi-likelihood information criterion (qic) [ ] as performance measure. to determine the optimal value of l, -fold cross validation [ ] was performed for the seven candidate models using the same three influenza years that they were built with. the model with the best r value was selected as final model. the above steps were repeated for variable selection of the models predicting ili two and three weeks ahead. using the final models based on the training years of data we started predicting the incidence of ili patients week by week starting from the fourth influenza year in our dataset (july , ) onward. before predicting each successive week, the model parameters were recalculated with an updated dataset that included the data of the previous week to make the model dynamic. we continued updating the model parameters week by week, until all remaining seven influenza years were predicted. from the fourth influenza year onward, the predicted incidence of ili patients was plotted together with the observed incidence of ili patients. we used the same threshold as the ili sentinel surveillance to define an influenza epidemic in the predicted ili numbers (incidence . = . ili patients per , inhabitants for at least two consecutive weeks). we compared the predicted epidemic weeks using icu data with observed epidemic weeks based on ili data. accordingly, we calculated the positive predictive value (ppv), and sensitivity of the predicted epidemic weeks on a weekly basis. for comparison, we also predicted the weekly incidence of ili with models that used only seasonal terms and auto-regressive ili variables, but excluded icu data. these models were also created with pseudo stepwise selection. the resulting ppv and sensitivity were also calculated. the statistical analyses were performed using the statistical package r, version . . (http://www.r-project.org/; vienna, austria). in the period january , , through december , there were a total of , icu admissions, of which , ( . %) had a medical (non-surgical) reason for admittance. of the medical icu admissions, , ( . %) were for a respiratory infection ( table ). the incidence of ili was on average . per , , with a standard deviation of . , and a minimum of . per , and a maximum of . per , . there were nine epidemics, consisting of weeks in total, and an average length of eight weeks. on average, gps supplied data on ili. both the incidence in ili and percentage of icu admissions with a respiratory infection show a similar timing of seasonal peaks (figure ). the amplitude of the yearly peaks in the icu data were relatively lower than ili peaks, and often lasted for a longer time period. increases in the incidence of ili showed a yearly pattern and increased in a smoother pattern compared to icu data. while trends were roughly comparable, they differed in some instances since peaks in icu data occasionally occurred when ili increases were absent in the general population. the association between the different lags of icu admissions and ili incidence is shown in table (assessed using gee additive poisson regression analysis). the r value of the full model was . . of each variable, the contribution to the r of the full model is also shown, which is the r value of the full model minus the r of the model with the corresponding variable omitted. figure also shows the predicted ili incidence (black line) according to the full model. statistically significant time lags were percentage of icu admissions with respiratory infection were one week before, current week, one week after, two weeks after, and four weeks after current ili incidence. the time lags mostly associated with increases in ili incidence one week before and in the current week, with coefficients of . and . . for example, if the percentage of icu admissions with a respiratory infection in the current week increased by one percent, then the incidence of ili in the general population increased by . per , population. according to the contribution to r , also icu admissions one week later was strongly associated with current ili (coefficient of . ). there is no linear time trend present, but seasonality exists in the data reflected by a half year sine function (sine term with k = ) with a p-value of , . and a large contribution to r . looking at figure , a yearly time trend would be expected but is now partly reflected in the different icu time lagged variables. using the coefficients of the time lags in table , the average of the weighted relative week numbers was . weeks implying that the increase in percentage of icu admissions with a respiratory infection was on average . days earlier than the increase in ili incidence. for our second research question, whether icu data can predict ili incidence ahead in time, we generated three gee models predicting the incidence of ili patients one to three weeks ahead using icu data. table shows these three different gee models, and their ppv and sensitivity. predicting two weeks ahead yields the largest sensitivity of . and predicting one week ahead has the largest ppv of . . for comparison, models using only auto-regressive ili variables and seasonal terms, showed the sensitivity to range between . - . and the ppv between . - . . figure shows three figures plotting the predicted incidence of ili patients one to three weeks ahead versus the actually observed ili incidence. the epidemic threshold of . patients (or more) with ili per , population is plotted and the weeks in which an influenza epidemic occurred according to the predicted versus the actual data is shown. predicting one week ahead detected three of the six epidemics, of which two were longer and one shorter according to the icubased predictions. one epidemic was predicted earlier, one at the same time and one later. using icu data to predict two weeks ahead resulted in five of the six epidemics detected, one is missed, and one false epidemic is predicted in the autumn of (which was not present in the ili data). according to the icu data, the predicted epidemics were longer in time except one. besides, most predictions were shifted in time compared to the actual occurrence: two epidemics were predicted earlier and three later. when predicting three weeks ahead all six epidemics were detected, however four were shorter in length, one longer, and one had the same length, again shifted in time: two epidemics were predicted earlier, three later, and one at the same time with icu data. the study showed that the percentage of medical icu admissions for respiratory infection was associated with weekly incidence of ili in the current week, and with one week positive and negative time lag. an increase in the percentage of icu admissions for respiratory infections on average preceded the increase in the incidence of ili (gp data) by . days, implying that before an epidemic the severely ill influenza cases get admitted to the icu. despite this precedence, our analyses showed that with the current models icu data do not accurately predict influenza epidemics in the general population, but including icu data showed an improvement in sensitivity and ppv compared to only including auto-regressive ili variables and seasonal terms. in our study we built three additive poisson gee regression models with icu data to predict the incidence of ili patients, thereby detecting influenza epidemics and aimed at detecting opportunities for enhancing the current national surveillance method. previous studies also aimed at enhancing their current surveillance of influenza epidemics, using laboratory or hospital data. steiner et al. [ ] used an exponentially weighted moving average control chart to enhance and automate influenza epidemic detection. weekly laboratory notifications data of seven years were used instead of the ili data that we studied. the predicted influenza epidemics were compared to retrospective inspection of the same notification data by epidemiologists. the predictions were, just like our study, not the same as their reference data. however in their study there was a maximum of one week difference only, except for one year where there was a difference of eight weeks. a study by closas et al. [ ] used a kolmogorov-smirnov test with virologic laboratory data of five years to detect influenza epidemics. the test provides a binary signal indicating epidemic activity and a quantitative measure of its confidence. they sequentially updated the test as new data became available. the results differed one to nine weeks with the retrospective data of the sentinel network, which is comparable to our results. google flu trends also aimed to detect influenza epidemics, but overestimated peak influenza levels [ ] whilst our study underestimates peak influenza levels. these methods complement the current surveillance networks, but cannot replace them. a study by van den wijngaard et al. [ ] did not aim to predict influenza epidemics, but instead explored whether excesses in influenza severity per season can be detected by combining gp, hospital, laboratory, and mortality data ( years of data). their finding was that combining these data sources is of added value, allowing for better understanding of increases in severe morbidity and mortality due to influenza infections. also from our data we see that trends in icu related sari differ from the trends of ili in the general population and may thus be of value in offering additional information on severity of influenza seasons which need to be explored further. however, both respiratory icu admissions and ili in the general population are not necessarily caused by influenza alone. microbiological laboratory results would provide better insight but to date, these data are not available at the icu patient level. the major strength of our study is that we had access to two large historical datasets from the nice registry and the sentinel gp registry. this allowed us to retrospectively analyze ten influenza years of data, which, to our knowledge, is a longer time period than in comparable studies. a second strength of our study is that we used gee in our additive poisson model, thereby correcting for correlations between weeks. the last strength of our method is that for each additional week we sequentially updated the coefficients of the covariates in the model used for prediction of ili, adding a decay factor giving historic data less weight, and adjusted our models for seasonal changes. with these adjustments, our models always incorporated the most recent information on icu and ili trends. a limitation of the nice registry data is that there is no distinct variable describing whether a patient has an influenza like illness or whether an patients has been diagnosed with an influenza virus infection. furthermore it only contains adult patients thus representing an older population compared to the ili surveillance which also includes children. we extracted admissions with a medical respiratory infection, admitted to the icu within two days after hospital admission, and excluding readmissions. these admissions represent community-acquired respiratory infections and, therefore, included influenza virus infections. additionally, the data of the sentinel gp registry is weekly updated, whereas the nice registry is updated on a monthly basis. this frequency is developed because outcome data, e.g. mortality, is measured at hospital discharge. for sentinel purposes this delay is too long and more frequent updates are needed. however our results can give incentives to set up an additional registry of near real-time surveillance of sari cases at the icu. our statistical analysis also has some limitations. due to the weekly scope of the ili data, we aggregated the icu data on a weekly basis, losing detail as they are available on a daily basis. with regard to our chosen models to predict ili, in the ideal situation stepwise variable selection is combined with -fold cross validation. since automating this process is not possible in gee, we first performed stepwise selection and then -fold cross validation on the seven remaining candidate models. additionally, we used three years of data as training set to determine the best models, whereas a longer period would also have been an option but not necessarily better, since we continuously added data to the baseline data. another limitation is that during the - pandemic, the ili peaks were not detected or later. this means that our models were not sensitive to large or unexpected changes. apparently the association between icu admissions and ili in the general population can change greatly from season to season. icu related sari might occur at a very different rate (compared to symptoms in the general population) during a pandemic or unexpected seasons [ ] . a probable explanation is that the influenza pandemic caused by the a(h n )pmd virus targeted another patient population than the previous epidemics with severe illness in younger patients, and fewer elderly with a severe infection. this could explain why increases in icu admissions during the pandemic were later than usual. additionally, the icu data reflects only sari cases. therefore, we do not know if the icu data reflect an influenza epidemic in the general population or possibly very different influenza dynamics in the icu population alone. icu data on respiratory infections was associated with ili incidence, with highest association in the same week and in the week before and the week after. increases in icu data on average occur two days sooner and for a longer time period than increases in ili. icu data thus contains additional information on icu related sari cases during a specific influenza epidemic. predicting influenza epidemics one, two or three weeks ahead in the general population using icu data was imprecise, reflected by the low ppvs and sensitivities. thus, icu data cannot improve the current surveillance method to detect influenza epidemics. due to the association between both data sources, a next step is to investigate the possibility of using icu data in combination with microbiological laboratory results for surveillance of severity of illness, and icu capacity prediction when an (severe) influenza epidemic is present. the performance of the gee models in predicting the start, end and length of an influenza epidemic is expressed by the positive predictive value (ppv), and sensitivity (n = weeks) based on comparing the signals for an epidemic predicted with intensive care unit (icu) data with the reference standard from the observed influenza like illness data. doi: . /journal.pone. .t surveillance trends of the influenza a(h n ) pandemic in europe responding to new severe diseases-the case for routine hospital surveillance and clinical networks in europe continuous morbidity registration sentinels: netherlands proefonderzoek naar de frequentie en de aetiologie van griepachtige ziekten in de winter - 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tnv lp authors: santillana, mauricio; nsoesie, elaine o.; mekaru, sumiko r.; scales, david; brownstein, john s. title: using clinicians’ search query data to monitor influenza epidemics date: - - journal: clinical infectious diseases doi: . /cid/ciu sha: doc_id: cord_uid: tnv lp search query information from a clinician's database, uptodate, is shown to predict influenza epidemics in the united states in a timely manner. our results show that digital disease surveillance tools based on experts' databases may be able to provide an alternative, reliable, and stable signal for accurate predictions of influenza outbreaks. the discovery of unusual outbreaks often depends on individual health practitioners who can promptly identify abnormal circumstances and then report those concerns to the greater community [ , ] . although the impact of these reports cannot be overstated, recent developments in internet technologies have demonstrated the power of the crowd as well. for example, crowdsourcing approaches allow members of the public to complete tasks relevant to a larger goal [ ] . search activity on diseases such as influenza and dengue has been shown to correlate with traditional surveillance data in multiple instances [ ] [ ] [ ] [ ] [ ] . google flu trends (gft) demonstrated a link between influenzarelated search query data and the centers for disease control and prevention's (cdc) influenza-like illness (ili) index [ ] . other examples include the use of search query data from yahoo! [ ] and from baidu [ ] to track influenza epidemics. internet search queries are available much earlier than data from validated traditional surveillance systems and have the potential to provide timely epidemiologic intelligence to inform prevention messaging and healthcare facility staffing decisions. the potential for the public's search activity to be influenced by anxiety, fears, and rumors raises concerns regarding reliability [ ] [ ] [ ] [ ] . although recent revisions to gft have shown that these concerns can be partially mitigated [ ] [ ] [ ] , shifting internet-based surveillance from the entire public to subjectmatter experts may maintain timeliness while generating a more reliable and stable signal requiring much less data. a recent small retrospective study using data on queries to a finnish primary care guidelines database demonstrated, for example, that disease-specific queries for lyme disease, tularemia, and other infectious diseases correlated well with concurrent confirmed cases [ ] . here, we show that uptodate (www.uptodate.com), a physician-authored clinical decision support internet resource that is used by clinicians in countries and almost % of academic medical centers in the united states, can be used for syndromic surveillance of influenza. specifically, we use upto-date's search query activity related to ili to design a timely sentinel of influenza incidence in the united states. uptodate is a professional database utilized by healthcare practitioners for point-of-care decisions. the information provided is rigorously authored and edited by experienced physicians. also, uptodate topics are accessed > million times monthly, and studies suggest that information provided through the site helps improve healthcare outcomes in hospitals [ ] [ ] [ ] . in collaboration with uptodate, we obtained search volume of search terms related to ili, as well as overall search activity from november to november for us accounts only. the search terms were as follows: influenza, haemophilus influenzae, flu, parainfluenza, h n , h n , h n , h n , grippe, gripe, adenovirus, rhinovirus, respiratory syncytial virus, metapneumovirus, coronavirus, bordetella pertussis, mycoplasma pneumoniae, pneumonia, bronchitis, h n , sinusitis, upper respiratory tract infection, and tamiflu. we obtained a weekly search fraction for each search term, at any given point in time, by dividing the number of searches for a given phrase by the total number of searches in the uptodate database, thus minimizing the effects of variation in the overall use of the uptodate database through time. we also obtained the national ili weekly index from the cdc for the same time period to use as a comparator (available at: http://www.cdc.gov/flu/weekly/pastreports.htm). we built a collection of multivariate linear models using the z scores of the aforementioned search terms' weekly search fraction as explanatory variables and the cdc ili index as our dependent variable. the multiplicative coefficients associated with each search term in each multivariate linear model were updated weekly as the cdc ili index was updated. our multivariate models can be expressed as where i(t) is the percentage of national ili physician visits, q i (t) is the search fraction associated with term i at time t, α i (t) is the multiplicative coefficient associated with each term at time t, and e is the normally distributed error term. model selection was performed using a least absolute shrinkage and selection operator (lasso) technique [ ] at every single week incorporating new cdc ili information as it became available. therefore, our approach recalibrated weekly the relevance of the search activity for each individual term according to its historical prediction ability. the lasso technique uses an optimization algorithm that favors models that minimize the mean squared error between the observations and predictions, while penalizing models containing many variables by simultaneously minimizing the sum of the absolute size of the regression coefficients. we produced real-time estimates of ili activity at time t, assuming that ( ) we only had access to cdc-reported ili data up to weeks prior (ie, up to t- weeks), and ( ) assuming that we had access to the real-time (time = t) number of searches in the uptodate database. our dynamic approach is similar to the one presented in santillana et al [ ] , and inspired by data assimilation techniques widely used in weather forecasting and oceanography [ , ] and supervised machine-learning techniques [ ] . our methodology was implemented in matlab version r a. the lasso routine was obtained from (available at: http://www. stanford.edu/~hastie/glmnet_matlab/) in november [ ] . the training period for our first prediction comprised weeks ( november - april ). thus, our first real-time figure shows our real-time estimates and the cdc-reported ili visits. gft estimates are included for context. our estimates predict very well the cdc-reported ili visits and outperform gft estimates during the prediction period. moreover, our approach estimates accurately the peak of the - influenza season (in the week of december ) and produces a slight overestimation of the influenza epidemic curve in the second week of january (overestimating the flu activity by approximately % in relative terms-ie, . % of ili as opposed to the actual . %). this overestimation is minimal when compared to the gft estimates (overestimating the influenza activity by % in relative terms-ie, . % of ili as opposed to the actual . %). our methodology has strong predictive power (pearson correlation of . ; a root mean square error [rmse] of . %) during the prediction period starting in the week of may and ending in the last week of november . although gft has a very high pearson correlation ( . ) during this same time period, it clearly fails to produce reliable estimates for the peak of the - influenza season. this mismatch is better captured by the rmse, which shows that gft estimates are on average off by . % of the national population (ie, almost times larger than our rmse). in figure we present a heatmap representing the relevance of each search term in predicting influenza activity as a function of time, during the validation time period. the term tamiflu is the strongest predictor, whereas sinusitis, influenza, h n , and coronavirus display relevance as predictors during different time periods. our findings demonstrate that combining a robust dynamic methodology and subject-matter experts' search activity more accurately predicts influenza activity than the well-established internet-based tool google flu trends. specifically, the model presented here has numerous strengths compared to gft. first, the model does not require expert supervision to adjust the search terms over the course of the influenza season. our clinicians' tamiflu search activity among clinicians is highly correlated with centers for disease control and prevention-reported influenza-like illness and thus is found to be the strongest predictor by our algorithm. sinusitis, influenza, h n , and coronavirus display significant relevance as predictors during different time periods. approach can also accommodate and identify changes in clinicians' selection of search terms over time while retaining the model's predictive power as demonstrated in figure . not only does this strength address evolving medical vocabulary, it also avoids "model drift" (static models typically match the training data well; however, as time progresses its deviation from truth may cause its predictions to drift farther and farther from truth (as seen in cook et al [ ] with gft). the success of our approach suggests that low volumes of queries (in the order of - seconds) in relevant subject-matter experts' databases, such as uptodate, provide a promising way to identify meaningful signals to track influenza activity. this will motivate the need for future research aimed at testing the accuracy of our methodology at state and city levels, and potentially in the prediction of other diseases. moreover, our findings in combination with those shown in jormanainen et al [ ] suggest that data acquired from specialized databases may have an improved signal-to-noise ratio and may be less likely to be impacted by public disruption resulting from anxiety or media reports on increased morbidity and mortality during (novel) outbreaks of influenza. limitations in this data source include those inherent in most novel data sources advanced for monitoring infectious diseases. although timely, these data sources lack the specificity observed in traditional surveillance systems, which rely on hierarchical reporting procedures. these data streams therefore supplement traditional disease surveillance provided by organizations such as the cdc. finally, uptodate data is not publicly available and thus not ready to be used as an alternative disease detection sentinel. in this study, we demonstrate that search queries from the up-todate database in conjunction with a dynamic multivariate methodology can be successfully utilized to obtain real-time estimates of influenza incidence in the united states before the release of official reports. clinicians can use outcomes from the model to monitor estimated levels of influenza in the united states. we also discuss the potential usefulness and limitations of digital data sources for infectious disease surveillance based on search query data [ , , , [ ] [ ] [ ] [ ] [ ] . future work may include analysis of smaller geographic units. promed-mail: an early warning system for emerging diseases evaluation of promedmail as an electronic early warning system for emerging animal diseases: to crowdsourcing-harnessing the masses to advance health and medicine, a systematic review using internet searches for influenza surveillance detecting influenza epidemics using search engine query data a new approach to monitoring dengue activity using web search query data to monitor dengue epidemics: a new model for neglected tropical disease surveillance monitoring influenza epidemics in china with search query from baidu use of prediction markets to forecast infectious disease activity assessing google flu trends performance in the united states during the influenza virus a (h n ) pandemic when google got flu wrong reassessing google flu trends data for detection of seasonal and pandemic influenza: a comparative epidemiological study at three geographic scales the parable of google flu: traps in big data analysis google disease trends: an update what can digital disease detection learn from (an external revision to) google flu trends? physicians' database searches as a tool for early detection of epidemics the value of library and information services in patient care: canadian results from an international multisite study how doctors make use of online, point-of-care clinical decision support systems: a case study of uptodate association of a clinical knowledge support system with improved patient safety, reduced complications and shorter length of stay among medicare beneficiaries in acute care hospitals in the united states regression shrinkage and selection via the lasso data assimilation in meteorology and oceanography data assimilation and its applications glmnet for matlab influenza forecasting with google flu trends twitter catches the flu: detecting influenza epidemics using twitter separating fact from fear: tracking flu infections on twitter surveillance sans frontieres: internet-based emerging infectious disease intelligence and the healthmap project healthmap: global infectious disease monitoring through automated classification and visualization of internet media reports acknowledgments. we thank the analytics team at uptodate for sharing the data used in this manuscript.disclaimer. the funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.financial support. financial support for this study was provided by the national library of medicine (grant number r lm - ).potential conflicts of interest. all authors: no potential conflicts of interest.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- - cxzc z authors: tam, clarence c.; anderson, kathryn b.; offeddu, vittoria; weg, alden; macareo, louis r.; ellison, damon w.; rangsin, ram; fernandez, stefan; gibbons, robert v.; yoon, in-kyu; simasathien, sriluck title: epidemiology and transmission of respiratory infections in thai army recruits: a prospective cohort study date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: cxzc z military recruits are at high risk of respiratory infections. however, limited data exist on military populations in tropical settings, where the epidemiology of respiratory infections differs substantially from temperate settings. we enrolled recruits undertaking a -week military training at two royal thai army barracks between may and july . we used a multiplex respiratory panel to analyze nose and throat swabs collected at the start and end of the training period, and from participants experiencing respiratory symptoms during follow-up. paired sera were tested for influenza seroconversion using a hemagglutinin inhibition assay. overall rates of upper respiratory illness and influenza-like illness were . and . episodes per person-weeks, respectively. a pathogen was detected in % of samples. the most commonly detected microbes were haemophilus influenzae type b ( . %) or non–type b ( . %) and rhinovirus ( . %). at baseline, bacterial colonization was high and included h. influenzae type b ( . %), h. influenzae non–type b ( . %), klebsiella pneumoniae ( . %), staphylococcus aureus ( . %), and streptococcus pneumoniae ( . %). at the end of follow-up, colonization with h. influenzae non–type b had increased to . %, and s. pneumoniae to . %. in the serology subset, the rate of influenza infection was . per person-months; % of influenza infections resulted in clinical disease. our study provides key data on the epidemiology and transmission of respiratory pathogens in tropical settings. our results emphasize the need for improved infection prevention and control in military environments, given the high burden of illness and potential for intense transmission of respiratory pathogens. military recruits are at high risk of respiratory infections. the congregation of individuals from diverse geographic locations in semi-closed settings, together with high levels of close contact, provide conditions that favor the introduction and transmission of respiratory pathogens. studies among military recruits have found high rates of illness and infection with respiratory viruses. a longitudinal study in singapore found that . % of military personnel had evidence of seroconversion against influenza a/h n during the pandemic, compared with . % of individuals in the community and . % of hospital staff. numerous outbreaks of respiratory infections among military recruits have been reported in the literature, including outbreaks of influenza, adenovirus, - and pertussis. resumption of vaccination against adenovirus types and in the u.s. military is estimated to have resulted in a -fold decrease in acute respiratory disease and a shift in the predominant adenovirus types from types , , and to types and . the increasing availability of multiplex molecular diagnostic assays can yield data on multiple pathogens that would not routinely be detected. , studies in military populations can provide valuable information regarding the epidemiology and transmission of respiratory infections in adults because of the availability of well-defined populations that can be followed up over time. despite this, data from tropical settings, in which the epidemiology of influenza and other respiratory infections differs substantially from temperate settings, are limited. increased understanding of the etiology of respiratory infections in these settings is important to identify opportunities for disease control in high-risk military populations and to identify risks of pathogen emergence with potential for spread into the wider community. we present the results of a longitudinal study of the burden and etiology of respiratory infections in thai military recruits. the study setting and procedures have been previously described. between may and may , we enrolled participants from consecutive cohorts of recruits undertaking basic military training at two royal thai army (rta) barracks in bangkok. trainees entered the camps at the start of may and november each year and remained in the camps for weeks. the camps consist of large, common sleeping quarters with beds arranged foot-to-foot in long rows. trainees share meals in a common canteen and train in a large field in the center of the barracks and a number of ancillary buildings. each camp has its own medical unit. individuals were eligible for enrollment if they were aged ³ years and were entering one of the two army barracks involved in the study. suspected tuberculosis cases or individuals with immune deficiencies, such as acquired immune deficiency syndrome, leukemia, or lymphoma, were excluded. individuals providing informed consent were enrolled in the study and a demographic and clinical questionnaire was administered. participants were then followed up for symptoms of respiratory illness until the end of the -week training period. at one camp, nasal and throat swabs were obtained at the time of enrolment and at the end of the follow-up period ( weeks later) from consenting participants. participants experiencing respiratory symptoms were asked to consult the medical unit, where medical staff took a history, conducted a medical examination, and recorded symptoms of upper respiratory illness (uri) or influenza-like illness (ili). upper respiratory illness was defined as an illness with at least two of the following: ) runny nose or sneezing; ) nasal congestion; ) sore throat, hoarseness, or difficulty swallowing; ) cough; ) swollen or tender glands in the neck; and ) fever (oral temperature > °c) at the time of presentation. influenza-like illness was defined as a respiratory illness with acute onset presenting with fever and cough or sore throat. all ili cases also met the case definition for uri. non-ili cases were defined as uri cases not meeting the case definition for ili. additional nasal and throat swabs were requested from participants presenting with acute ili or uri. laboratory investigations. nasal and throat swabs were placed in viral transport media and stored at − °c until transfer to the armed forces research institute of medical sciences for further testing. we tested acute swabs for influenza virus using reverse transcription polymerase chain reaction (rt-pcr) following the u.s. centers for disease control protocol. , we also tested acute samples (from both camps) and the routine enrolment and follow-up specimens (from one camp) using a multiplex real-time pcr assay comprising bacterial, viral, and fungal targets (ftd kit; fast track diagnostics, esch-sur-alzette, luxembourg). a cycle threshold value of < was considered a positive result. previous studies have shown high agreement between ftd and other commercial multiplex assays with specificities > % for most viral targets. , sensitivity for influenza a and b, and human coronaviruses is high (> %), but is lower and more variable for other viral targets, including human bocavirus ( - %), rhinovirus ( - %), respiratory syncytial virus ( - %), and human metapneumovirus ( - %). , in a subset of recruits, we measured influenza antibody titers in paired sera taken at enrollment and at the end of followup using a hemagglutinin inhibition assay. seroconversion was defined as a -fold rise in influenza subtype-specific antibody titers. statistical analysis. rates of illness. individuals were considered at risk from the date they entered the camp for a period of weeks. we computed the rates of uri and ili as the number of cases of each outcome divided by the total person-time at risk, using robust standard errors in the calculation of % confidence intervals (cis) to allow for clustering of illness episodes by camp and cohort. disease etiology. we determined the percentage of uri and ili cases positive for each target pathogen, the percentage positive for more than one pathogen, and the percentage with no pathogen identified. influenza seroconversion. we calculated the percentage of recruits who seroconverted, overall and by influenza subtype, based on a -fold rise in antibody titer between baseline and end of follow-up samples. bacterial colonization. we determined the percentage of recruits with a positive identification of a bacterial pathogen at recruitment, the overall change in colonization prevalence between baseline and end of follow-up for each bacterial species, and the risk of acquisition of bacterial pathogens over the follow-up period among those initially negative. antibiotic use. we calculated the percentage of uri and ili cases that were prescribed antibiotics and investigated clinical signs and symptoms associated with antibiotic prescription using logistic regression. among those prescribed antibiotics, we determined the fraction of cases with a likely viral etiology based on the available fast track multiplex pcr results. analyses were conducted using stata (statacorp, college station, tx) and r . . . ethical approval. the study was approved by the institutional review boards of the rta in bangkok, thailand, the walter reed army institute of research, and the london school of hygiene & tropical medicine. all participants provided written informed consent. three cohorts of army recruits undertook basic military training at the two camps during the study period, in may -july , november -january , and may -july . in total, recruits undertook training during these three periods. of these, ( %) were enrolled in the study. of the participants, five participants withdrew from the study and participants recruited in november were transferred out of the camp to complete their training elsewhere. the remaining ( %) completed the -week follow-up and were included in the analysis. all participants were male and the median age was years (range: - years). details of recruitment and follow-up completion for each cohort are given in supplemental figure . rates of illness. there were a total of episodes of uri and , person-weeks of follow-up, yielding an overall uri rate of . per person-weeks. upper respiratory illness rates ranged from . to . per person-weeks across cohorts, with the exception of the camp a may cohort, in which no uri episodes were reported ( figure ). there were ili episodes, giving an overall ili rate of . per person-weeks, ranging from to . per person-weeks across cohorts. symptoms and time off work. fever was present in nearly two-thirds of uri episodes. cough and sore throat occurred in > % of uri episodes, nasal congestion and headache in > % of episodes, and malaise and breathing difficulty in > % of episodes. all these symptoms were significantly more common in ili compared with non-ili episodes ( table ) . influenza-like illness cases were also more likely to take time off work than non-ili cases ( . % versus . %, p < . ), although the length of time taken off work was short (median day). there was one hospitalization in an individual diagnosed with pneumonia who was off work for days. disease etiology. among the uri and ili samples tested by multiplex pcr, a pathogen was detected in . a single pathogen was detected in % of samples, two pathogens in %, three pathogens in %, and four pathogens in %. among uri pathogens, the most common viruses detected were rhinoviruses ( . %), influenza b ( . %), coronavirus ( . %), adenovirus ( . %), and influenza a/h ( . %). other viruses accounted for < % of uri cases each. of samples tested for influenza by both rt-pcr and the fast track multiplex assay, ( %) tested positive by at least one assay, and all of these were also positive by rt-pcr. of the rt-pcr positives, four were positive for influenza a/h , of which one was also positive by the multiplex assay. of the remaining positive for influenza b by rt-pcr, were also positive by the multiplex assay. there were no samples that tested positive by the multiplex assay and negative by rt-pcr. among the bacteria, the most commonly detected species were haemophilus influenzae type b ( . %), h. influenzae non-type b ( . %), streptococcus pneumoniae ( . %), and klebsiella pneumoniae ( . %). however, these were commonly found in combination with other pathogens. when considering only samples in which a single organism was found, h. influenzae type b was found in . % of samples, h. influenzae non-type b in . %, and s. pneumoniae, k. pneumoniae, and legionella pneumophila in . % each. there was little evidence to suggest that etiological agents differed between ili and non-ili cases, with the exception of h. influenzae non-type b and coronavirus , which occurred more frequently in non-ili cases, and influenza a/h and coronavirus hku , which occurred more frequently in ili cases ( table ) . analysis of uri cases by time since the start of follow-up indicated strong temporal clustering of cases, with each cluster involving several respiratory pathogens (figure ). for example, the single cluster in camp a during the november cohort resulted in identification of both rhinovirus and coronavirus , as well as numerous colonizing bacteria. in other clusters, rhinovirus was also identified in combination with parainfluenza viruses, adenovirus, and influenza b virus ( figure ) . influenza vaccination and seroconversion. at enrollment, . % of recruits reported having received influenza vaccination in the previous months. in the serology subset, table ) . thirteen of ( %) recruits seroconverted over the -week training period. seroconversion was most common against influenza b/massachusetts (n = ) and influenza a/h (n = ) (table ) colonization. at baseline, . % of recruits were colonized with h. influenzae type b and . % with h. influenzae non-type b. other common colonizing bacteria were k. pneumoniae ( . %), staphylococcus aureus ( . %), and s. pneumoniae ( . %). at the end of follow-up, colonization with h. influenzae non-type b had increased to . %, and s. pneumoniae to . %, whereas colonization with h. influenzae type b and k. pneumoniae had decreased to . % and . %, respectively. in addition, moraxella catarrhalis was found in four ( . %) and l. pneumophila in two ( . %) end of follow-up samples. no change in s. aureus colonization was found. in a subset of paired baseline and end of follow-up samples initially negative for h. influenzae non-type b (n = ), the risk of h. influenzae non-type b acquisition was . % ( % ci: . - . %). among those negative for s. pneumoniae at baseline (n = ), the risk of acquiring s. pneumoniae during the follow-up period was . % ( % ci: . - . %). use of medications. of uri episodes, . % were prescribed antibiotics. of these, all but one were prescribed amoxicillin or azithromycin. in multivariable logistic regression, cases were more likely to be prescribed antibiotics if they presented with tonsillitis (or = . , % ci: . - . ), chills (or = . , % ci: . - . ), and swollen lymph nodes (or = . , % ci: . - . ). antibiotic prescription was less likely if patients presented with nasal congestion (or = . , % ci: . - . ). there was no difference in prescribing between ili and non-ili patients. of uri cases prescribed antibiotics and with available multiplex pcr results, ( . %) had viral pathogens detected and eight ( . %) had a bacterial target detected in the absence of other pathogens. we found a high incidence of uri in thai army recruits, indicating that approximately % of recruits experienced a uri episode over the -week training period. comparison with other military cohorts is not straightforward because of differences between settings in terms of vaccination policies and seasonality of key pathogens such as influenza. the uri and ili rates in our study are nonetheless comparable with those reported by other studies in the united states. [ ] [ ] [ ] despite this, we found low rates of clinical influenza a in this minimally vaccinated population. possible reasons for this could be exposure to circulating influenza strains before recruits entering the training camps. thai national influenza surveillance data indicate high influenza activity in the first half of , with influenza a/h n and influenza b predominating. concomitantly, our data indicated that % of recruits with available serological information had baseline influenza a/h n titers ³ : . titers greater than this value have been shown to be associated with a % reduction in risk of clinical influenza from homologous strains, for both influenza a and b viruses. [ ] [ ] [ ] [ ] of interest, % of recruits also had baseline titers against influenza b/massachusetts greater than this level. the higher baseline titers against influenza b/massachusetts are consistent with the frequent detection of influenza b isolates from the yamagata lineage in clinical specimens from thailand in the first half of . in our study, the fast track multiplex assay appeared to have lower sensitivity for influenza compared with the cdc rt-pcr assay. we did not specifically investigate reasons for these discrepancies, although it should be noted that the two assays were not performed at the same time, as the multiplex assays were all performed at the end of data collection. we therefore cannot discount the possibility that sample degradation might have influenced the relative performance of the fast track assay. although influenza incidence was low, pathogen detection with sensitive multiplex diagnostics indicated high frequencies of respiratory pathogens, with co-circulation of multiple viruses including rhinovirus, adenovirus, and coronaviruses as well as influenza. in addition, we found high levels of colonization with numerous bacterial species, including both type b and non-type b h. influenzae, k. pneumoniae, and s. pneumoniae. although we did not specifically study transmission chains or epidemiologic links between cases of illness, in the context of our study, recruits did not leave the camps during the -week training periods, such that the occurrence of respiratory pathogens is most likely to have resulted from transmission within the camps rather than from external sources. intense transmission of bacterial species was apparent, as the risks of acquiring h. influenzae non-type b and s. pneumoniae among initially non-colonized individuals were % and %, respectively. bacterial species were also commonly found in samples from uri cases. the colonizing nature of these bacterial species makes it difficult to determine the clinical relevance of these detections. however, h. influenzae type b and non-type b were identified in % and % of clinical samples in the absence of any other pathogens, suggesting that these pathogens could be responsible for illness in a fraction of adult uri cases. interestingly, h. influenzae type b has not been previously associated with a significant burden of uri in adults. it is possible, however, that these cases were caused by other pathogens not included in the multiplex assay. high levels of colonization with bacterial species also point to the need for judicious use of antibiotics to treat uri in military populations. a quarter of uri cases in our study were prescribed broad-spectrum antibiotics, of whom % were more likely to have infections caused by viral pathogens based on multiplex pcr results. although we lacked additional information to determine whether this resulted in adverse antibiotic resistance patterns, high levels of antibiotic use in a population with a high background of bacterial colonization presents a risk for development of antibiotic resistance, and highlights the importance of improved rapid diagnostics to rule out viral etiologies to aid clinical management. in addition, it points to the need for increased antibiotic stewardship and health-care provider education to reduce unnecessary use of antibiotics in military settings. our study also emphasizes the importance of systematic microbiological surveillance of uri in military populations, to allow prompt detection and control of outbreaks and early identification of emerging pathogens that may be circulating or could be introduced in the wider population. this is particularly important as infection control options in such closed settings are limited and vaccination coverage for key pathogens is low. limited resources meant it was not possible to conduct multiplex diagnostics for all baseline, acute, and end of followup samples, as well as influenza serology. as a result, there was limited power to investigate whether colonization influenced risk of illness, or factors associated with colonization and acquisition of bacteria. in addition, co-circulation of multiple pathogens makes it difficult to establish chains of transmission, which would require more detailed microbial sequencing information. clinical signs and symptoms were available at presentation only, and we did not have information regarding the duration of individual symptoms or additional clinical investigations conducted longitudinally during the course of illness, as these were not routinely performed. the specific barracks setting and population characteristics limit the generalizability of our findings to other military settings with similar conditions. nevertheless, our study provides key data regarding the epidemiology and transmission of respiratory pathogens in military populations in tropical settings. our results emphasize the need for improved infection prevention and control strategies in military settings, given the potential for intense transmission of a wide range of respiratory pathogens. financial support: this work was supported by the united states department of defense-global emerging infectious disease surveillance (dod-geis), protocol a. disclosures: the authors declare that they have no competing interests. material has been reviewed by the walter reed army institute of research. there is no objection to its presentation and/or publication. the opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. the investigators have adhered to the policies for protection of human subjects as prescribed in ar - . respiratory tract infections in the military environment environmental factors, immune changes and respiratory diseases in troops during military activities influenza a(h n ) seroconversion rates and risk factors 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man estimation of the association between antibody titers and protection against confirmed influenza virus infection in children relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model haemophilus influenzae infections in the h. influenzae type b conjugate vaccine era acknowledgments: we are grateful to the military recruits who participated in the study, the royal thai army, and the clinical, laboratory, and administrative personnel at afrims for their support. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- - wao a authors: dia, ndongo; richard, vincent; kiori, davy; cisse, el hadj abdoul khadir; sarr, fatoumata diène; faye, abdourahmane; goudiaby, déborah g; diop, ousmane m; niang, mbayame n title: respiratory viruses associated with patients older than years presenting with ili in senegal, to date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: wao a background: in africa, especially in west africa, studies about the prevalence and diversity of respiratory viruses (influenza and others) in elderly people are largely lacking. in studies done elsewhere, it is well established that older people, when compared with younger adults, are at greater risk of significant morbidity and mortality from complications arising from influenza. the main aim of this study was to determine the prevalence and the diversity of respiratory viruses associated with ili cases in adults over years old in senegal. methods: the recruitment period of this study was from january to december . patients aged years and above presenting ili cases were enrolled. nasal-pharyngeal and/or oral pharyngeal swabs were collected from patients. rna was extracted from μl of each sample followed by a two-step real-time rt-pcr. the anyplex™ ii rv detection kit was used for viral detection. the kit enabled the simultaneous detection of the presence of respiratory viruses. results: viruses were detected: influenza viruses ( . %) and rhinoviruses ( . %) were the most prevalent. we detected human parainfluenza viruses ( . %), human respiratory syncytial viruses ( . %), coronaviruses ( %), human metapneumoviruses ( . %), human adenoviruses ( . %) and human bocavirus ( . %). cases ( %) of dual virus infections and one triple viral detection case were encountered. ( . %) viruses detected were found in the - year old age group, ( . %; p < . ) from – year old age group and ( . %) were detected in the ≥ year old age group. the viral co-infections were more frequent in the - age group ( / ). conclusions: this pilot study demonstrates a variety of respiratory viruses in the elderly. it also highlights a high prevalence of these viruses in this age group. we speculate from these results that the impact of respiratory viruses other than influenza on the elderly has been considerably underestimated. a more exhaustive study seems necessary in order to provide a more complete picture of the burden of respiratory viruses on morbidity among adults over years old in the sub-saharan context. viral aetiology, prevalence and diversity data in people with influenza like illness (ili) and/or acute respiratory illness (ari) in africa, (especially in west africa), are scarce and often limited to the influenza viruses' infection. following the last influenza pandemic episode [ ] , few global and pediatric studies were conducted in some countries of the sub-region [ ] [ ] [ ] , and only a limited number of studies have described the etiology of ili due to viruses including non-influenza respiratory virus [ ] [ ] [ ] . however, no study has been conducted to describe the prevalence and the diversity of respiratory viruses (influenza and others) in west african elderly people. in studies done elsewhere, it is well established that older people, when compared with younger adults, are at greater risk of significant morbidity and mortality from complications arising from influenza [ , ] . for example in the united states alone, up to % of non-pneumonic lower respiratory illnesses in the elderly have been associated with respiratory viral infection [ ] , and an estimated , deaths annually have been attributed to the influenza and respiratory syncytial viruses (rsv) [ ] . it should be highlighted that in senegal the number of elderly people in consultation in healthcare centers for influenza like illness (ili) is very low. indeed, routine influenza monitoring in senegal showed that samples from people above years old represent only . % of the total, over a year surveillance period [ ] . some practices such as auto-medication and the use of traditional medicine to treat ili largely explain this situation with the socio-economic situation being another contributing factor. thus the main aim of this study was to determine the prevalence and the diversity of respiratory viruses associated with ili cases in adults over years old. the recruitment period of this prospective observational study was from january to december inclusive. all patients aged years and above presenting with ili during this period were enrolled in the study. it should be noted that samples were collected in the context of flu monitoring. an influenza sentinel surveillance system for outpatients with ili was established in in senegal and became part of the who global influenza surveillance and response system (gisrs). it is coordinated locally by the national influenza center (nic) at the institut pasteur de dakar. trained medical personnel were asked to screen all outpatients who were attended at the sentinel sites for signs and symptoms of ili. the symptoms of influenza are similar to those arising from other viral respiratory pathogens. the inclusion criteria, according to the cdc case definition, were sudden onset of fever (≥ °c) with cough or sore throat fewer than days in duration. nasal-pharyngeal and/or oral-pharyngeal swabs were collected from each enrolled ili case, placed in cryovials containing ml of viral transport medium (universal transport medium, copan diagnostics inc., murrieta, ca, usa) and stored at °c on site. if nasal-pharyngeal and oral-pharyngeal swab specimens were collected from the same patient, both swabs were placed in the same cryovial. upon arrival at the laboratory the specimens were separated into aliquots for analyses. the first aliquot was used for molecular analysis for the detection of influenza viruses (real-time reverse transcription polymerase chain reaction or rrt-pcr detection), the second was used for influenza virus isolation, and the third was stored at − °c for further analysis. the latter was used in the present study. for each patient who met the case definition criteria, a form collecting demographic and clinical data was completed.the questions included information on date of enrollment and symptom onset, sex, age, clinical symptoms, previous treatments, vaccination status for influenza, and whether or not the patient was hospitalized. ribonucleic acid (rna) extraction was performed from μl of each sample using the qiaamp viral rna kit (qiagen, valencia, ca, usa) according to the manufacturer's instructions. each rna sample was eluted with μl nuclease-free water before rna quantification with a nanodrop apparatus (nanodrop lite, thermo scientific). a two-step real-time rt-pcr was performed using the cfx real-time pcr detection system (bio-rad). the revertaid first strand cdna synthesis kit (thermo scientific) was used. first ng of rna was mixed with μl of random hexamer primer and nuclease free water for a final volume of μl. it was then incubated at °c for minutes and immediately put on ice in order to remove the secondary structures in gc-rich rna. for the cdna synthesis step, μl of x reaction buffer, μl of rnase inhibitor ( u/μl), μl of dntp mix ( mm) and μl of revertaid m-mulv reverse transcriptase ( u/μl) were added and incubated for minutes at °c followed by minutes at °c and °c for minutes. the cdna product could be used directly for the next step (pcr amplification) or stored at − °c until use. for viral detection, the anyplex™ ii rv detection kit (seegene) was used. the kit enabled simultaneous detection of influenza a virus, influenza b virus, human respiratory syncytial virus a, human respiratory syncytial virus b, human adenovirus, human metapneumovirus, human coronavirus e, human coronavirus nl , human coronavirus oc , human parainfluenza virus − , − , − , − , human rhinovirus a/b/c, human enterovirus and human bocavirus. reactions are duplicated in two panels (a and b) for detection of the viruses. the total reaction volume was μl for each sample (for each panel), containing μl x rv a (or x rv b), μl of -mop solution, μl of x anyplex pcr master mix (mix well by inverting times) and μl of cdna product. pcr was assessed after °c for minutes for transcriptase reverse enzyme inactivation, cycles of °c for seconds, °c for seconds and °c for seconds. additional cycle of °c for seconds was added for completion. the fluorescence is detected with a melting curve step, °c- °c ( seconds/ . °c). fisher's exact test was used to verify whether the associated proportions were statistically supported and a p-value < . was considered statistically significant. we used the - year' old group as the reference. the r. . tool was used to perform the analyses. the senegalese national ethical committee of the ministry of health approved the surveillance protocol as less than minimal risk research, and written consent forms were not required. throughout the study, the database was shared with the epidemiology department at the senegalese ministry of health and prevention for appropriate public health action. a total of patients above years old were enrolled into the study, ( . %) were women and ( . %) were men (table ). patients' ages ranged from to years, with a mean age of years. ninety-nine ( . %) enrolled patients were between and years old, ( . %) between and years old and ( . %) were years old or older. fever was the most reported clinical symptom, in ( . %; / ) of the enrolled patients, followed by cough ( . %; / ), rhinitis ( . %; / ), myalgia ( . %; / ) and pharyngitis ( %; / ). in all, ( . %) out of the patients were found to be infected with at least one of the viruses of interest. a total of ( . % of patients) viruses were detected. of these viruses, influenza viruses ( . %; / ) and rhinoviruses ( . %; / ) were the most prevalent viruses detected (table ) . we detected human parainfluenza viruses ( piv , piv , piv ) ( . %), human respiratory syncytial viruses ( rsv a and rsv b) ( . %), coronaviruses ( coronaviruses nl , one coronaviruses e and one coronavirus oc ) ( %), human metapneumoviruses ( . %), human adenoviruses ( . %) and one human bocavirus ( . %). a total of cases ( %) of dual virus infections and one triple viral detection case were encountered. influenza viruses ( cases) and rhinoviruses ( cases) were the most common type of virus found in samples with coinfections. regarding the number of viruses detected per age group, ( . %; / ) were from the - age group, ( . %; / ; p < . ) from the - year old age group and ( . %; / ) were detected in the older than year old age group ( table ) . the viral coinfections are more frequent in the - year old age group ( / ) followed by the ≥ years group ( / ). taking into account the clinical symptoms and viral detection, cough, rhinitis, pharyngitis or headache were in similar proportion in viral positive and non-positive patients: cough was observed in . % of positive patients and % in the negative patients group (p = . ), rhinitis . % and % (p = . ), pharyngitis in . % and % (p = . ) respectively. in contrast myalgia symptoms are significantly higher among viral-positive patients: . % versus % (p < . ). the pattern of the virus detection throughout the study period is showed in the figure . influenza viruses (a and b) were mostly detected from july to august (between weeks and ), which corresponds to the rainy season in senegal. a minor detection peak is also registered at the beginning of the year. rhinoviruses and parainfluenza viruses showed homogeneous detection levels throughout the study period. grouped, the remaining viruses seemed to have a similar temporal pattern to that of influenza viruses. the gap observed between weeks and correspond with a lack of samples from patients from our targeted age group. the present study is the first description of the etiology of respiratory viruses associated with patients with ili in a cohort of elderly people in the west african context. the results obtained showed that samples of the study population out of contained at least one of the targeted respiratory viruses. the frequency of virus detection ( . %) among the elderly with ili in our study is consistent with that of several studies already conducted. huo et al. ( ) [ ] , in a similar study in china detected at least one respiratory virus in % of patients years old or older, and munoz et al. ( ) [ ] % in elders in a long term care facilities in ontario during the - period. hasman et al. ( ) [ ] detected at least one respiratory virus in % patients in a study conducted in usa, without any precision about the ages of the adults. in others studies frequencies are lower. for example nicholson et al. ( ) [ ] % ( / ) of viral detection among the elderly between and years of old, % ( / ) detected in the elderly over years old in a study in china [ ] and . % in a recent study in japan [ ] . it is important to note that the technical approach used explains some discrepancies in rates of detection: primarily in their sensitivity and secondly in the number of targeted viruses. alternatively differences in rates of detection could be due to true geographical differences in overall burden, differences in study populations (outpatients or hospitalized patients) and to the studies sample collection periods. overall, the viral detection rate in the present study is very high as elderly people are often protected by pre-existing antibodies from previous illnesses, maybe illnesses suffered even decades back [ ] [ ] [ ] . indeed, because of pre-existing systemic and mucosal antibodies, elderly adults have been observed to have lower amounts of respiratory secretions and lower viral loads compared to children [ ] . consistent [ ] noted that the age distribution differed significantly between positive and negative patients, with positive patients being younger than negative patients (or = . , ic . - . ; p = . ). of the viruses detected in the elderly, influenza a virus was the most common viral pathogen. combined with influenza b viruses, influenza viruses represented % ( / ) of viruses detected. this influenza detection rate was expected as the enrollment of patients was directed towards patients with ili. these results are in agreement with previous findings in the elderly [ , , ] . our study revealed a high detection rate of rhinoviruses ( / ; . %). rhinovirus is the most common respiratory pathogen in all age groups [ ] . in a previous study, nicholson et al. ( ) [ ] showed that rhinoviruses were responsible for a greater disease burden (activities restriction, duration of illness) than that of influenza in elderly subjects representing % of detected viruses. in another study published by greenberg ( ) [ ] , rhinovirus was the most prevalent pathogen ( isolates; %) of the identified in upper respiratory episodes. these findings are in concordance with the high rhinovirus detection rate in the present study. with lower prevalence, piv, rsv, hcov, hmpv, enteroviruses, adenoviruses and bocaviruses were identified from elderly patient' specimens and contributed collectively to . % of all ili cases in our study. these results show the high diversity of respiratory viruses circulating in the elderly population. this viral diversity supports previous results [ , , ] and often in similar distributions with those of the present study. co-infections were relatively common in this study especially in the - years old age group ( . %; / ). the rate found in this age group was in line with the findings of hasman et al. ( ) [ ] ( %) and huo et al. ( ) [ ] , . %. huo and colleagues, in agreement with our results noted that co-infections were found most commonly in adults older than years of age. focusing on clinical symptoms, with the exception of myalgia, our study showed no significant differences between viral-positive and viral-negative patients with ili. viral circulation observed during the study period showed different patterns depending on the viral types. if we consider influenza viruses, we observed a circulation peak during the period starting in week and ending in week . this period corresponds to the middle of the rainy season in senegal. this result is further supported by a recent study conducted by mbayame and colleagues [ ] . these authors established clearly the seasonality of influenza viruses in senegal after many years of surveillance with a regular circulation during the year and a peak in the middle of the rainy season (july-august-september). the slight peak of influenza observed at the beginning of the year (february) is the result of the shift caused by the recent pandemic episode. the pandemic occurred in early in senegal with a peak in february [ ] . rhinoviruses showed a regular yearly circulation with peaks along the year corresponding to any rain season influence. the remaining respiratory viruses (piv, rsv, hcov, hmpv, enterovirus, adenovirus and bocavirus) were more likely associated with ili peak during the rainy season. this co-circulation with influenza viruses was also seen in a previous pediatric study in senegal [ ] . further studies (multiple year surveillance) are needed in order to properly define the temporal patterns of non-influenza virus circulation in senegal. our study did have several limitations. the first weakness is the small number of samples treated in this study. a more exhaustive sampling would give a better representation of the different targeted viruses in the ili cases among the elderly population in senegal. unfortunately after years of influenza sentinel monitoring we noted that the number of elderly presenting at healthcare centers for ili consultation is rather low compared to other age groups (children and young adults). the absence of nursing home services as in industrial countries, the use of traditional medicine (especially among the elderly) and economic constraints do not facilitate such studies in the west african context. it is worth noting that this was a retrospective study, the database contained limited information on disease outcome and atypical clinical symptoms in ili patients which were not reported. thus the association between viral infections (or co-infections) and severe signs could not be established. as in previous studies it appears that co-infections were associated with more severe signs than mono-infections [ , ] . without such data we could not measure the burden of targeted respiratory viruses in older patients with ili. another limitation is that our study is only focused on outpatient' cases; it would be interesting to investigate hospitalized patient cases (severe cases). a final limitation was that the study included mainly one geographic location, dakar, the capital city of senegal. despite the small number of samples included, the present pilot study demonstrates a variety of respiratory viruses in the elderly. it also highlights a high prevalence of these viruses in this cohort. from these results, it appears that the impact of respiratory viruses other than influenza was considerably underestimated. a more exhaustive study (increasing the number of elderly patients, with a better clinical picture and better documentation including disease outcomes, illness duration, hospitalizations etc.), relying on the new sentinel surveillance system (extension of sentinel sites in others geographical areas), seems necessary in order to provide a more complete picture of the burden of respiratory viruses on morbidity among adults over years old in the sub-saharan context. influenza-like illness; rrt-pcr: real-time reverse transcription polymerase chain reaction; who: world health organization; cdna: complementary deoxyribonucleic acid; rna: ribonuleic acid; gisn: global influenza surveillance network; nic: national influenza center rsv: respiratory syncytial virus; hcov: human coronavirus; hmpv: human metapneumovirus emergence of a novel swine-origin influenza a (h n ) virus in humans virological surveillance of influenza-like illness among children in ghana influenza viruses in nigeria, - : results from the first months of a national influenza sentinel surveillance system sentinel surveillance for influenza in senegal viral etiology of influenza-like illnesses in antananarivo viral etiology of respiratory infections in children under years old living in tropical rural areas of senegal: the evira project viral etiology of influenza-like illnesses in cameroon the impact of influenza on the health and health care utilisation of elderly people influenza-related hospitalisation and death in australians aged years and older new respiratory viruses and the elderly mortality associated with influenza and respiratory syncytial virus in the united states surveillance of respiratory viruses in patients with influenza-like illness in nanjing current research on influenza and other respiratory viruses: ii international symposium aetiology of influenza-like illness in adults includes parainfluenzavirus type acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden influenza a/h n pandemic and respiratory virus infections the post-infection outcomes of influenza and acute respiratory infection in patients above years of age in japan: an observational study characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness epidemiology of pandemic influenza a (h n ) deaths in the united states outbreaks of pandemic influenza a (h n ) among long-term-care facility residents-three states nationwide surveillance of respiratory viruses in patients with influenza-like illnesses: a pilot feasibility study in the french sentinel network rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults fields virology viral respiratory infections in elderly patients and patients with chronic obstructive pulmonary disease a subregional analysis of epidemiologic and genetic characteristics of influenza a(h n )pdm in africa respiratory viruses in children admitted to hospital intensive care units: evaluating the clart pneumovir dna array comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children respiratory viruses associated with patients older than years presenting with ili in senegal the authors thank dr abdou salam gueye, associate director for science at the us centers for disease control (cdc) in ivory coast, for his great help in the revisions of this paper, especially for the english language. many thanks to oumy niass (phd student, immunology unit, ipd) for her help in the statistical analyses. this work was funded by institut pasteur de dakar, senegal. the authors declare that they have no competing interests. the work presented here was carried out in collaboration between all authors. mnn and omd, defined the research and revised the manuscript; nd performed and coordinated technical work, wrote the draft and revisions of the paper; dk performed the main technical part of this work; eakc and af participated in the technical work; dg and eakc participated in data management and analysis; vr revised the manuscript and participated in the monitoring of the surveillance sites; fds participated in the monitoring of the surveillance sites. all authors have contributed to, seen and approved the manuscript. key: cord- - g lu y authors: wertheim, heiman f l; nadjm, behzad; thomas, sherine; malik, suhud; nguyen, diep ngoc thi; vu, dung viet tien; van nguyen, kinh; van nguyen, chau vinh; nguyen, liem thanh; tran, sinh thi; phung, thuy bich thi; nguyen, trung vu; hien, tran tinh; nguyen, uyen hanh; taylor, walter; truong, khanh huu; ha, tuan manh; chokephaibulkit, kulkanya; farrar, jeremy; wolbers, marcel; de jong, menno d; van doorn, h rogier; puthavathana, pilaipan title: viral and atypical bacterial aetiologies of infection in hospitalised patients admitted with clinical suspicion of influenza in thailand, vietnam and indonesia date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: g lu y background: influenza constitutes a leading cause of morbidity and mortality worldwide. there is limited information about the aetiology of infection presenting clinically as influenza in hospitalised adults and children in south-east asia. such data are important for future management of respiratory infections. objectives: to describe the aetiology of infection presenting clinically as influenza in those hospitalised in south-east asia. methods: respiratory specimens archived from july to june from patients hospitalised with suspected influenza from indonesia, thailand and vietnam were tested for respiratory viruses and atypical bacteria by polymerase chain reaction. results: a total of patients’ samples were tested. of , patients ( · %) were under the age of . viruses detected included rhinoviruses in of patients ( · %), bocaviruses in ( · %), respiratory syncytial viruses in ( · %), parainfluenza viruses in ( · %; piv : ; piv : ; piv : ; piv : ), adenovirus in ( · %), influenza viruses in ( · %; influenza a: ; influenza b: ) and coronaviruses in ( · %; oc : ; e : ). bacterial pathogens were mycoplasma pneumoniae (n = , · %), chlamydophila psittaci (n = ), c. pneumoniae (n = ), bordetella pertussis (n = ) and legionella pneumophila (n = ). overall, in-hospital case fatality rate was of ( · %). conclusion: respiratory viruses were the most commonly detected pathogens in patients hospitalised with a clinical suspicion of influenza. rhinovirus was the most frequently detected virus, and m. pneumoniae, the most common atypical bacterium. the low number of detected influenza viruses demonstrates a low benefit for empirical oseltamivir therapy, unless during an influenza outbreak. influenza is a common reason for primary care consultation and constitutes a leading cause of hospitalisation, morbidity and mortality worldwide. non-influenza viruses are the most common causes of illness that clinically resemble influenza, particularly in children. data on the epidemiology and disease burden of influenza-related disease in south-east asia (sea) are emerging, and this, in turn, is shedding more light on the epidemiology of other viral and bacterial aetiologies of influenza-like illnesses (ilis) in hospitalised adults and children in this region. the insight provided by this information is important not only for future prevention strategies, treatment and clinical management of respiratory infections, but also to guide future studies in this region. recently, many diagnostic advances have been made in order to help to determine the aetiology of acute respiratory infections. these advances have been made possible by molecular diagnostic techniques that are now in widespread use. furthermore, research in this field has gained much attention and funding due to the outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) and avian influenza viruses a/h n and a/h n in sea and middle eastern respiratory syndrome coronavirus (mers-cov) in the middle east. generally, the causative agents of ili remain undiagnosed in routine clinical practice due to the expense of testing and the slow turnaround time for most diagnostic polymerase chain reactions (pcrs), considered too long to impact the management of usually self-limiting respiratory tract infections. however, with the increasing use of antiviral drugs, such as neuraminidase inhibitors for influenza, testing for ili pathogens may become more important for rational antiviral drug treatment and to prevent overuse of antibiotics, thereby helping to control costs and prevent the emergence and spread of antimicrobial drug resistance. during the past few years, several novel causative agents of ili have been identified in respiratory specimens, including human metapneumovirus, new human coronaviruses (nl , hku , sars-cov, mers-cov), rhinovirus c and continuously emerging novel lineages and subtypes of influenza virus a capable of infecting humans (h n , h n , h n pdm , h n v, h n , h n , h n , h n , h n ). viruses for which disease causation has not (yet) been established have also been identified, including human bocavirus and respiratory polyomaviruses, and likely there are many more to come. [ ] [ ] [ ] [ ] the most comprehensive data on the causes of ili originate from developed countries, with some data available from sea. recently, studies have been published from asian countries, [ ] [ ] [ ] [ ] [ ] [ ] providing much needed, valuable information and showing a similar spectrum of viral pathogens. this study was designed to determine the viral and atypical bacterial aetiologies of ilis in patients in sea using molecular techniques. the detection of influenza viruses and respiratory pathogens other than influenza viruses in specimens that were sent for routine influenza testing can provide valuable insights into the epidemiology of ili across all age groups in three sea countries over the same time period. furthermore, this study could address the potential occurrence of multiple simultaneous infections in the same patient. we performed a laboratory-based surveillance study, in which we tested all archived respiratory specimens from hospitalised patients over year (july to june ) with clinically diagnosed ili meeting the following criteria: age ≥ year, signs of ili according to treating physician, duration of ili symptoms ≤ days and respiratory specimens sent for influenza testing. clinicians at all participating hospitals received regular training in who protocols for the management and recognition of influenza/ili. archived respiratory specimens (nose swab, throat swab, nasopharyngeal aspirate, nasal wash, tracheal aspirate, bronchoalveolar lavage) were obtained from sites in three countries across sea (three sites in indonesia, four sites in thailand, five sites in vietnam). influenza testing capacity was created for a randomised clinical study comparing two dosages of oseltamivir for severe influenza by sites in the south east asia infectious diseases clinical research network, as described elsewhere. the specimens used for this study were stored at À °c until further testing. where more than one specimen type was available for each patient, they were prioritised in the sequence endotracheal aspirate/bronchoalveolar lavage > nasopharyngeal aspirate > throat swab > nasal swab/ washing. this retrospective study for additional testing on archived specimens after full anonymisation of specimens was approved by the institutional review boards of each site. commercially available multiplex polymerase chain reaction (pcr)/gel electrophoresis assays (seeplex â rv ace detection and seeplex â pneumobacter ace; seegene, south korea [ ] [ ] [ ] ) were used to detect major respiratory viruses and six respiratory bacterial pathogens: influenza viruses a and b, respiratory syncytial viruses (rsvs) a and b, rhinoviruses a/b, coronaviruses oc /hku and e/ nl , adenovirus, parainfluenza viruses - , human metapneumovirus, mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumophila serotype and bordetella pertussis. additional in-house real-time [reverse-transcriptase (rt)] pcrs were used to detect the following viruses: rhinoviruses a, b and c, enteroviruses, parainfluenza virus , bocavirus, parechoviruses and chlamydophila psittaci, as described elsewhere. [ ] [ ] [ ] basic demographic data (country, sex, age) were collected as well as date of disease onset, date of admission, type of specimen and intensive care unit (icu) admission. for the analysis results of streptococcus pneumoniae and haemophilus influenzae, pcrs were not taken into account, because of the high positivity rates resulting from nasopharyngeal carriage of Á % and Á %, respectively, in this study (data not shown). the baseline demographic characteristics (age category, sex, country of admission and proportion of icu admissions) were summarised as frequency and proportion. the frequencies of different pathogens were summarised per country, sex and age category to determine the most frequent viruses and bacteria in each stratum. disease outcomes including severity (severe defined as icu admission), death, duration of hospitalisation and duration of illness were examined by regression analysis. the dependence of binary outcomes (severe disease and death) on the presence of pathogens was analysed by logistic regression, adjusted for age and sex. kaplan-meier survival analysis was used to visualise the number of hospitalisation days between each age category (patient deaths were censored). cox regression was used to analyse whether viruses were found more often if the duration of illness before presentation to hospital was shorter. regression analysis was based on the data from vietnam and thailand only as relevant clinical data were missing from indonesia. the statistical software sas version . (sas institute inc., cary, nc, usa) was used for all computations. a two-sided p value of Á or less was interpreted as statistically significant. a total of patient samples were collected at participating sites. table summarises the demographic data. the median number of days from illness onset to specimen collection was (iqr: - days). for patients, no exact admission date was known. the majority of patients in this study were under the age of ( , Á %). most patients were enrolled in vietnam (n = , Á %). the median age was higher in indonesian patients as compared to those from vietnam and thailand ( year, versus year and year, respectively). one hundred and patients ( Á %) were admitted to the intensive care unit (icu); icu admission data from patients were unknown (indonesia: ; thailand: ; vietnam: ). sixtyseven patients ( Á %) were mechanically ventilated and the overall reported in-hospital mortality was Á % (n = ). detected bacterial pathogens were m. pneumoniae (n = , Á %), c. psittaci (n = ), c. pneumoniae (n = ), b. pertussis (n = ) and l. pneumophila (n = ). fluctuations in frequencies were found in particular months for several viruses, with rsv occurring from july to november and rhinovirus peaking in march ( figure ). due to differences in age group representation, the number of viral agents detected per patient was lower in indonesia ( / , Á ) than in vietnam ( / , Á ) and thailand ( / , Á ). likewise, the proportion of identified viral agents detected that were rsv was higher in thailand and vietnam than in indonesia ( / , Á %; / , Á %, respectively, compared with / , %) whilst coronavirus was a greater proportion of identified viral agents in indonesia ( / , Á %) than thailand ( / , Á %) and vietnam ( / , Á %). see table s for pathogens detected by country. there were of patients ( Á %) who had at least two pathogens detected (bacterial and/or viral). of these, patients were found to have two pathogens, patients had three pathogens, nine patients had four pathogens, and one patient had five pathogens. the frequency of each pathogen is shown in table s . the pathogens most commonly detected in patients with two or more pathogens were bocavirus (n = ) and rhinovirus (n = ). the proportion of patients who had either a virus or a bacterium detected appeared to decrease with age, with the highest proportion of pathogens detected in the - age group, but increased again in those older than . most viral pathogens were more common in children, with parechovirus and parainfluenza virus only found in children (table ) . m. pneumoniae was found only in patients younger than years, whilst both cases of c. psittacii occurred in patients years and above. the icu admissions for each country are shown in table . the number of patients who were mechanically ventilated was of ( Á %) in vietnam and of ( Á %) in thailand (data not available for indonesia). for the vietnamese data set, where data were most complete, icu admission was related to age; of children ( Á %) aged under years, three of ( Á %) aged - years, of ( Á %) aged - years, of ( Á %) aged - years and of ( %) aged over years were admitted to icu (v for trend p < Á ). logistic regression was performed on this data set including the covariates: pathogen type (virus, bacterial, both and neither), age and sex. the analysis showed that only higher age was associated with icu admission (adjusted or for icu admission in those aged > years compared to those aged < years: Á , p < Á , table ). no specific pathogen was associated with icu admission. there were only sufficient data available to analyse the duration of hospitalisation for patients in vietnam. the median duration of hospitalisation was days (iqr: - days). multivariate cox regression analysis for hospitalisation days by pathogen type (virus, bacterial, both and neither), sex and age was performed, and both pathogen type (p = Á ) and age category (p = Á ) had a significant effect. it was found that patients in the > -year group were hospitalised longer, but did not differ by sex. influenza a virus-positive patients were admitted for a median of days (iqr: - days) versus days (iqr: - days) for patients where influenza a was not detected. a similar pattern was seen for coronavirus e : median of days (iqr: - days) for positive patients versus days (iqr: - days) in the negative group. patients who were m. pneumoniae-positive were admitted longer: a median of days (iqr: - days) versus a median of days (iqr: - days). there were deaths and a viral pathogen was detected in ( %) of these: influenza virus a (n = ), rhinovirus (n = ), bocavirus (n = ), coronavirus (n = ), parainfluenza virus (n = ), rsv (n = ) and one combination of influenza a with adenovirus and rhinovirus. data concerning the cause of death were not collected in this study; consequently, conclusions about causation cannot be drawn. influenza-like illnesses are a major cause of mortality and morbidity worldwide, especially in younger and elderly age groups. it has been suggested that a large proportion of iliattributable deaths globally occur in africa and sea. data regarding the aetiology of respiratory infections in sea are limited, with some of the available data suggesting that viruses account for a large proportion of these infections. in this -year study, we used molecular techniques to detect viruses and atypical bacteria from samples collected from patients hospitalised with ili. the majority of patients enrolled in this study were enrolled in vietnam and were under the age of . for ili diagnostics and surveillance, nose and throat swabs are considered the sampling methods of choice, whereas for the diagnostics of h. influenzae and s. pneumoniae, gram-staining and bacterial culture of representative purulent sputum, blood culture or urinary antigen tests (for s. pneumoniae) would be the testing method of choice, which were not done. detection rates of these two pathogens were therefore not included in the analysis. in this study, a potential pathogen was identified in Á % of patients. this rate is higher than the previously published rates of between % and % of organisms identified, in studies looking at the aetiology of respiratory tract infections in different asian countries. , a total of Á % of the study cohort were found to be positive for viruses compared to just Á % of the cohort who had an atypical bacterial pathogen alone detected. more recent studies form asian countries using molecular techniques have shown the rates of virus detection reaching %. the most common viruses detected in our study were rhinoviruses, accounting for % of the patients in whom viral pathogens were detected and Á % of the cohort as a whole, similar to other studies. , variation between the three countries in the proportion of patients in whom viral pathogens were detected, and variations in the relative importance of different viruses, largely represents the differing age groups sampled, with the younger patients in vietnam and thailand having higher a proportion of rsv detected. bocaviruses were first described in , and since then, numerous studies have described their detection in the human respiratory tract and their possible association with disease, but causation has not convincingly been proven. in this study, this virus was detected in Á % of patients, which is similar to other studies where detection rates ranged from Á % to Á %. , these previous studies as well as this study have detected bocavirus, predominantly from young children rather than from adults. in this study as in other studies, bocaviruses were commonly ( / , % bocavirus isolates in our study, table s ) detected in combination with other pathogens, most commonly rhinovirus. atypical bacteria were rarely ( Á %) detected. this rate of detection is lower than previously published data, where detection varied from % to %. , however, as we did not include serological diagnostic methods for atypical bacteria, the true prevalence in our patients may have been higher. the most commonly detected atypical bacteria in this study were m. pneumoniae, followed by c. psittaci and l. pneumophila. the results from our study are similar to the aetiological studies of community-acquired pneumonia in singapore and malaysia, in which m. pneumoniae was the commonest atypical pathogen detected. very little is known about the epidemiology of c. psittaci in sea, and indeed this pathogen has only very recently been described as causing human disease in vietnam. the type of pathogen detected was significantly associated with the duration of hospitalisation. atypical bacteria and in particular infection with m. pneumoniae was associated with longer duration of hospitalisation. the age of the patients was also significantly associated with longer duration of hospitalisation, with the > years age group requiring longer admissions. this probably corresponds with increasing comorbidities and decreasing immunity with age, similar to other regions in the world. no clear seasonality was seen with the various pathogens detected, but rates of infection appeared to be higher in the winter and spring months and a study over a single year may miss seasonal trends. rates of influenza virus a increased in indonesia and vietnam in april coinciding with an epidemic of seasonal h n influenza in vietnam, prior to the first sporadic cases of h n pdm detected at the end of may . the strengths of this type of study include the large numbers of patients who were enrolled prospectively from three different countries in south-east asia allowing an overview of the viral and atypical bacterial causes of ilis in the region. this study also used rt-pcr techniques; however, an important limitation was the lack of microscopy and sputum cultures for common bacteria associated with ilis such as s. pneumoniae and h. influenzae type b and serology for atypical pathogens. also, limited clinical data were recorded for these cases, making it difficult to assess the clinical significance of the various pathogens detected and the virulence of each pathogen. although swabs from all patients meeting our eligibility criteria were analysed, we have no data concerning the number of patients who may have been admitted with ili but not swabbed. the lack of clinical data also limits comparison between sites as differing admission criteria may impact on the distribution of pathogens. similarly, both the lack of clinical data and the absence of data concerning overall admission limit our ability to interpret the findings relating to icu admission; the finding that adult patients were overrepresented amongst those admitted to icu could represent differential routine sampling of adults and children on such units; however, we feel this is unlikely. there was a lack of data related to patients and co-morbidities, such as immunosuppression and copd, which could also have had an impact in the severity of infection seen. furthermore, a lack of suitable control patients also limits our ability to determine the attributable fraction of ili that is likely to be caused by each virus and also prevents useful analysis of the relevance of the high rates of nasopharyngeal carriage of s. pneumoniae and h. influenzae type b. establishing the aetiology of ili is becoming increasingly important, not only to establish the burden of various pathogens in order to improve management and prevention, but also to guide antiviral treatment and to try to limit the inappropriate use of antimicrobials to reduce the emergence and spread of antibiotic resistance. our study, along with overwhelming evidence from many other studies from these and other countries, suggests that viral causes of ili are much more common than bacterial causes, but that the viral aetiology may vary in space and time. in recent years, the emergence of sars-cov, mers-cov and avian influenza viruses a/h n and a/h n has highlighted the risks posed by respiratory viral infections in humans and sea has become an area of increasing interest for monitoring pathogens. very little has been published from this region regarding the incidence and prevalence of viral or atypical organisms that can contribute to ilis, making studies looking at this increasingly important. additional supporting information may be found in the online version of this article: table s . frequency of detected pathogens by country. table s . frequency of pathogens found in a combination with another pathogen. prospective study of the incidence, aetiology and outcome of adult lower respiratory tract illness in the community etiology and management of community-acquired pneumonia in asia cloning of a human parvovirus by molecular screening of respiratory tract samples viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis respiratory infections unique to asia masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during viral pathogens associated with acute respiratory infections in central vietnamese children association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in vietnamese children identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city a prospective three-year cohort study of the epidemiology and virology of acute respiratory infections of children in rural india fatal respiratory infections associated with rhinovirus outbreak effect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe influenza: double blind randomised controlled trial use of the seeplex rv detection kit for surveillance of respiratory viral outbreaks in comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses detection of respiratory viruses with two-set multiplex reverse transcriptase-pcr assay using a dual priming oligonucleotide system rapid detection of human parechoviruses in clinical samples by realtime pcr development of an internally controlled real-time pcr assay for detection of chlamydophila psittaci in the lightcycler . system development and evaluation of a four-tube real time multiplex pcr assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts epidemiology and etiology of childhood pneumonia non-flu aetiology of ili in se asian countries ª the authors. influenza and other respiratory viruses published by viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care human bocavirus-the first years high frequency of human bocavirus dna in infants and adults with lower acute respiratory infection human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand atypical bacterial pathogen infection in children with acute bronchiolitis in northeast thailand acute bronchitis in the community: clinical features, infective factors, changes in pulmonary function and bronchial reactivity to histamine an asian study on the prevalence of atypical respiratory pathogens in communityacquired pneumonia first report of human psittacosis in vietnam a clinical virological and epidemiological analysis the investigators were involved in all aspects including protocol design, study execution and oversight and the writing of this publication. richard molenkamp and bob de wever, dept of medical microbiology, academic medical center, amsterdam, the netherlands, are kindly acknowledged for sharing of protocols and positive controls for setting up in-house real-time (rt-) pcrs for respiratory viruses and atypical bacteria. the authors declare that they have no conflict of interests. the study was conducted by the seaicrn (http://www.seaicrn.org/) and sponsored by the national institute of allergy and infectious diseases. site support was also provided by the wellcome trust (london, uk) through its major overseas programmes. h wertheim, md de jong, hr van doorn and p puthavathana contributed to study design, study implementation, analysis and interpretation of the data, writing of the manuscript, and reading and approval of the final version. b nadjm involved in analysis and interpretation of the data, writing of the manuscript, and reading and approval of the final version. s thomas, dung vtv and uyen hn participated in analysis and interpretation of the data and reading and approval of the final version. n agustiningsih and diep ntn contributed to study implementation, analysis and interpretation of the data, and reading and approval of the final version. s malik, kinh vn, chau vvn, liem tn, sinh tt, thuy btp, trung vn, tran th, khanh ht, tuan mh and kulkanya c involved in study implementation and reading and approval of the final version. w taylor and j farrar contributed to study design, study implementation and reading and approval of the final version. m wolbers involved in study design, study implementation, analysis and interpretation of the data, and reading and approval of the final version. key: cord- - oqv jom authors: rguig, ahmed; cherkaoui, imad; mccarron, margaret; oumzil, hicham; triki, soumia; elmbarki, houria; bimouhen, abderrahman; el falaki, fatima; regragui, zakia; ihazmad, hassan; nejjari, chakib; youbi, mohammed title: establishing seasonal and alert influenza thresholds in morocco date: - - journal: bmc public health doi: . /s - - -y sha: doc_id: cord_uid: oqv jom background: several statistical methods of variable complexity have been developed to establish thresholds for influenza activity that may be used to inform public health guidance. we compared the results of two methods and explored how they worked to characterize the influenza season performance– season. methods: historical data from the / to / influenza season performance seasons were provided by a network of primary health centers in charge of influenza like illness (ili) sentinel surveillance. we used the who averages and the moving epidemic method (mem) to evaluate the proportion of ili visits among all outpatient consultations (ili%) as a proxy for influenza activity. we also used the mem method to evaluate three seasons of composite data (ili% multiplied by percent of ili with laboratory-confirmed influenza) as recommended by who. results: the who method estimated the seasonal ili% threshold at . %. the annual epidemic period began on average at week and lasted an average of weeks. the mem model estimated the epidemic threshold (corresponding to the who seasonal threshold) at . % of ili visits among all outpatient consultations. the annual epidemic period began on week and lasted on average weeks. intensity thresholds were similar using both methods. when using the composite measure, the mem method showed a clearer estimate of the beginning of the influenza epidemic, which was coincident with a sharp increase in confirmed ili cases. conclusions: we found that the threshold methodology presented in the who manual is simple to implement and easy to adopt for use by the moroccan influenza surveillance system. the mem method is more statistically sophisticated and may allow a better detection of the start of seasonal epidemics. incorporation of virologic data into the composite parameter as recommended by who has the potential to increase the accuracy of seasonal threshold estimation. seasonal influenza epidemics result in considerable annual morbidity and mortality, with an estimated , to , deaths per year globally [ ] . associated with these seasonal epidemics are substantial economic losses due to absenteeism, lost wages and increased utilization of health care services [ ] . the influenza-associated respiratory annual mortality rate for people aged and older in morocco has been recently estimated by the us centers for disease control and prevention (us cdc) at . per , ( % credible interval of . - . ) [ ] . the risk of hospitalization due to influenza is to times greater in high-risk populations in morocco (e.g., the elderly and people with chronic disease) than in the general population [ ] . the most effective ways to prevent or mitigate these effects are through vaccination combined with appropriate clinical management of persons infected with influenza. optimal impact of vaccination campaigns is achieved by timing them prior to the beginning of the influenza season to ensure maximum coverage and protection among the population. likewise, a timely signal to healthcare providers that the influenza season is underway helps to guide their patient management decisions and to mitigate the effects of illness in the individual and in the community. local patterns of influenza virus circulation and seasonality may differ geographically, necessitating national estimates of seasonal influenza activity to inform public health guidance. national surveillance data is essential for understanding those patterns and establishing signals for the beginning of the influenza season and epidemic periods. establishing baseline activity, epidemic and alert thresholds is a useful tool to inform recommendations for timely influenza vaccination to lessen the burden of seasonal epidemics [ ] . while several statistical methods are commonly used, there is no gold standard for calculating influenza epidemic thresholds. the methods developed to date vary in their complexity and calculate either time-varying or fixed thresholds. the simplest ones use visual inspection of historical data to create a fixed threshold indicating the expected level of activity throughout the year [ , ] . statistical methods include regression models [ ] [ ] [ ] [ ] , time series methods [ ] , adaptation of industrial control processes such as shewart charts [ ] , cumulative sum (cusum) [ ] and rate difference models [ ] . methods that involve calculation of means and medians are of medium complexity but are practical as they may be simple to implement. the objective of this study was to evaluate the performance of two methods using means and medians to establish thresholds using data from the moroccan national influenza-like illness (ili) syndromic surveillance system. we compare the results of the world health organization averages method (who method) with the moving epidemics method (mem) which is recommended by both the who and the european centre for disease prevention and control (ecdc). as a complement to the thresholds using syndromic data, we also calculated a threshold using a composite parameter integrating both syndromic and virologic surveillance data. following these direct comparisons of the methodologies, we explored the best method for characterizing the / influenza activity. in , the epidemiology department of the ministry of health of morocco launched a year-round public sector syndromic surveillance system for ili comprised of primary health centers, with a catchment population of almost million people. sites report weekly ili activity to the regional and central levels, where health officials aggregate the surveillance data. a case definition similar to the who ili case definition recommended for public health surveillance, defined as "a sudden onset of fever, a temperature > °c and cough or sore throat in the absence of another diagnosis" was used from to [ , ] . in , morocco adopted the updated who standard ili case definition [ ] developed in as "an acute respiratory illness with a measured temperature of ≥ °c and cough, with onset within the past days" [ ] . reporting includes the total number of ili consultations aggregated by gender and age group, as well as total outpatient consultations. the proportion of ili visits among all outpatient consultations is used as a proxy for influenza activity. in , the moroccan national influenza center (nic) began a virologic surveillance system in both ambulatory and hospital sites to complement the syndromic system and provide data on laboratory-confirmed influenza activity [ ] . after an interruption in data collection beginning in , virologic surveillance was resumed in sentinel sites in . specimens were collected and characterized between september and june. enrolling patients from both out-and in-patient facilities allowed the integration of epidemiologic and virologic data representing the spectrum of illness from mild (ili) to severe (e.g. severe acute respiratory infection or sari) [ ] . we used seasons of syndromic surveillance data ( / to / , excluding the / pandemic year from analysis as influenza activity was not reflective of a typical season); this was described elsewhere [ ] . we compared two methodologies for establishing seasonal baseline activity and epidemic thresholds. we also compared the calculated thresholds with the observed weeks for the start and end of the / season. using three seasons of virologic ili surveillance data ( / to / ), we used the mem method to make calculations using the composite parameter recommended by who [ ] ; this method estimates the proportion of laboratory-confirmed influenza ili consultations among all outpatient consultations, or the product of weekly ili consultations of total outpatient visits and weekly percentage of influenzapositive specimens among respiratory tests. the methods discussed in order to standardize country information on influenza activity, have raised basic concepts summarized in table . the who global epidemiological surveillance standards for influenza (who manual) [ ] included a simple method to establish an average epidemic curve to identify the beginning of the influenza season using national influenza surveillance data. this method characterizes the intensity of influenza activity each year and may be used to describe the seasonality of influenza virus circulation. using ili as a proxy for influenza virologic activity [ , ] , we used weekly proportion of ili among all outpatient consultations as our indicator of influenza activity. with this method, we were able to produce an average epidemic curve. using data from the average epidemic curve, we used statistical measures of variance to establish an alert threshold. we determined the flat baseline for expected influenza activity throughout the year in order to develop an indicator for the onset of influenza season (seasonal threshold). sustained influenza activity (i.e., three consecutive weeks) above this baseline indicated the start of the influenza season or the epidemic period [ ] . in the final step, moderate, high, and extraordinary intensity thresholds were estimated as described in the who pandemic influenza severity assessment manual [ ] , (fig. ) . the moving epidemic method (mem) [ ] [ ] [ ] [ ] [ ] [ ] is an alternative tool developed to help model influenza epidemics also using retrospective national surveillance data. it may be described as a combination ratedifference model that uses cumulative differences in mem software produces an average curve, lower interval, and higher interval. calculate the mean and standard deviation (sd) of the average epidemic curve. for each week, the alert threshold is . sd above the weekly ili% mean. ili% > . sd indicates high ili activity or outbreaks and may be used to characterize a severe season. a graph consisting of the alert thresholds for each epidemic week. median weekly ili% over all weeks (i.e., the average epidemic curve is not used). indicates the level of influenza activity that signals the start and end of the annual influenza season(s). for prospective surveillance: upper limit of the % onesided confidence interval of the arithmetic mean of the highest pre-epidemic weekly ili% values. parameter value which marks the start of the epidemic period. for prospective surveillance: upper limit of the % onesided confidence interval of the arithmetic mean of the highest post-epidemic weekly ili% values. the third of three consecutive weeks with ili% above seasonal threshold. indicates that influenza activity occurs consistently. for retrospective analysis of individual season data: see "length of epidemic period". the third of three consecutive weeks with ili% below seasonal threshold for retrospective analysis of individual season data: see "length of epidemic period". weeks from epidemic start to end. for retrospective analysis of individual season data: mem software uses a "maximum accumulated proportions percentage (map)" algorithm to split the season into three periods: a pre-epidemic, an epidemic, and a post-epidemic period. proportion of total cases that occurred during the epidemic period upper % limit of -sided ci of mean of all peak values. upper % limit of the one-sided confidence interval of the geometric mean of the highest epidemic weekly ili% values. upper % limit of -sided ci of mean of all peak values. upper % limit of the one-sided confidence interval of the geometric mean of the highest epidemic weekly ili% values. upper . % limit of -sided ci of mean of all peak values. upper % limit of the one-sided confidence interval of the geometric mean of the highest epidemic weekly ili% values. rates to determine epidemic periods and intensity of activity [ , ] . using the free software r for statistical computing and graphics [ ] and its open source user interface rstudio [ ] , we uploaded our surveillance data via the mem application [ ] , and fit the model using three steps. we first visually compared activity over the seasons in order to compare the timing of peak activity and activity trends across seasons. the mem procedure has three main steps: first, the length, start and the end of the annual epidemics are determined, splitting the season in three periods: a pre-epidemic, an epidemic and a postepidemic period [ , ] . in the second step, we built the model by using retrospective data from all seasons. the mem app calculated the pre-epidemic threshold that marks the start of the epidemic period (analogous to the seasonal threshold in the who method). in the third step, medium, high, and very high intensity thresholds were estimated ( table ) . using the app, we produced graphs of each season showing the preepidemic, epidemic and post-epidemic periods (fig. ). in addition, as the assumption that ili activity is reflecting influenza virus circulation has limitations, we created a second seasonal threshold with this methodology using the composite parameter recommended by who for three seasons of virologic ili surveillance (fig. ) . lastly, we calculated indicators of performance of the app to detect epidemics, using values from the model for sensitivity, specificity, positive predictive value, negative predictive value, percent agreement and the matthew correlation coefficient ( table ). the application allowed us to optimize the model by searching the optimum slope of the map curve to optimize the goodness-of-fit of the model for detecting epidemics. the mem app calculates goodness-of-fit indicators in an iterative process using a cross-validation procedure [ ] . true positives (tp) were then defined as values of epidemic period above the threshold, true negatives (tn) as values of the non-epidemic period below the threshold, false positives (fp) as values of the non-epidemic period above the threshold and false negatives (fn) as values of epidemic period below the threshold. the process was repeated for each season in the dataset and all tp, tn, fp and fn were pooled. to measure the performance of the threshold, the following statistics and definitions were used [ ] : . sensitivity: the number of epidemic weeks above the pre-epidemic threshold and above the postepidemic threshold divided by the number of epidemic weeks (epidemic length). . specificity: the number of non-epidemic weeks below the pre-epidemic threshold and below the post-epidemic threshold divided by the number of non-epidemic weeks. . positive predictive value (ppv): the number of epidemic weeks above the threshold divided by the number of weeks above the threshold. the ili sentinel surveillance system is a public health activity organized by the ministry of health of morocco. personally identifiable data is excluded from this surveillance system; as a result, no request for authorization from the national ethics committees was required. indeed, the royal dahir n° - - dated august , , promulgating the law n° - relating to the protection of persons participating in biomedical research, provides for special provisions for non-interventional or observational researches as stipulated in its articles and . when applying the who method to our years of surveillance data, we estimated that the seasonal threshold was the point at which more than . % of outpatient consultations were due to ili (table ) . influenza activity crossed this threshold on average at week and the beginning of the epidemic period would be declared after three consecutive weeks of activity above this threshold, on average at week . the typical epidemic period lasted weeks, finishing at week , when activity was below the seasonal threshold for three consecutive weeks. the average peak activity occurred during week . seasons where ili activity regularly crossed the alert threshold may be characterized as severe ( fig. and table ). intensity thresholds were ili% of . , . and . % for moderate, high and extraordinary intensity thresholds) ( fig. and table ). the mem model produced an estimate that the average annual influenza epidemic period began on week , and that the epidemic period lasted on average weeks. the epidemic threshold (corresponding to the who seasonal threshold) was higher, at . % of ili patients among all outpatients. the average peak activity occurred during week , consistent with the estimate using the who method. intensity thresholds were of . , . and . % of ili patients among all outpatients for respectively medium, high and very high intensity thresholds ( fig. and table ). indicators related to the goodness-of-fit of the mem model for detecting the epidemics, using these retrospective data showed that the sensitivity of the mem epidemic threshold was . whereas the specificity was . . positive predictive value was . and negative predictive value was . (table ) . using three seasons of virologic data, we established a third seasonal baseline based on the composite parameter recommended by who, which integrated both laboratory-confirmed influenza and syndromic ili reporting (fig. ) . this method allowed us to compare the results of characterizing seasonality using these data types to identify the beginning of the influenza season. applying the mem methodology to our combined data, we determined that the average epidemic began at week , average peak activity occurred at week and the average epidemic period lasted weeks. using this method, medium, high and very high intensity thresholds were set at . , . and . % of laboratory-confirmed ili patients among all outpatients ( fig. and table ). goodness-offit indicators showed a sensitivity of %, specificity of %, positive predictive value of % and negative predictive value of % (table ) . ili data with the who/ ili data with the who thresholds, the curve overlapped the average epidemic curve and activity crossed the seasonal threshold during week of and was sustained after this time, confirming that this was the start of the epidemic period (fig. ) . the season peaked during the second week of , week earlier than the average identified by the who methodology (week ); we observed peak activity of . % of ili patients among all outpatients (fig. ) . when using the mem method with ili proportions, the epidemic period began at week , or the end of november . this finding indicated an early season, beginning weeks before the average epidemic start week of . the season peaked at week of (beginning of january), week before the average peak week determined by mem (week ), with peak activity above % of ili patients among all outpatients. this season was characterized as one of medium intensity (fig. ) . when considering the composite parameter, the mem method showed that the epidemic period began at week , or the end of november , with a sharp increase of the epidemic curve weeks prior to the average start (week ). the seasonal peak occurred at week of , week before the average peak week (week ), with peak activity above . % of confirmed ili patients among all outpatients. this season almost reached the threshold for high intensity (fig. ). the occurrence of the h n pandemic highlighted the need for a robust and standardized method to make timely assessments of the severity of influenza activity that may be used as an indicator of an unusual event. who developed and began implementing a framework on pandemic influenza severity assessment (pisa) [ ] in march . member states are encouraged to establish influenza baseline and epidemic alert thresholds from surveillance data and to monitor and describe the severity of each influenza season (seasonal, epidemic or pandemic influenza) using these thresholds. for this purpose, a simple method proposed by the who was used [ , , ] . who is now recommending mem, which is a more sophisticated method of reporting influenza activity adopted by the european centre for disease prevention and control [ ] [ ] [ ] [ ] and adopted by several countries from other regions [ , ] . the analysis using the mem application with seasons of syndromic surveillance data showed clear seasonality to ili activity and visual inspection of graphed data revealed a single seasonal peak per year. the data show seasonal peaks between december and march, varying by year, as described by barakat et al. based on visual analysis [ ] , matching trends observed in other northern hemisphere countries [ ] . the average seasonal peak in morocco occurs at week (mid-january) using either method. the seasonal threshold established using the method described in the who influenza surveillance guidelines was lower than the epidemic threshold calculated by the mem method when ili proportions are considered ( . % versus . % of ili patients among all outpatients). the average epidemic start week was estimated to be earlier when using the who method, with an average start at week versus week or by using respectively ili proportions or the composite parameter with the mem method. there is a three-to four-week difference between these methods when describing the typical start to a season; the optimal timing of a seasonal influenza vaccination campaign might vary accordingly. public health officials must weigh the costs and benefits of the optimal campaign period. influenza vaccine administration is ideally timed at least several weeks prior to influenza virus circulation as antibody response is achieved on average weeks post vaccination [ ] . the average epidemic period estimated by the who method was longer compared with the mem method ( weeks vs. or weeks respectively). there are few publications with estimates of the typical duration of an influenza season [ ] . according to the available evidence, the duration of the influenza season in the temperate zone of the northern hemisphere, ranges - weeks in europe [ ] . the goodness-of-fit calculations from the mem application indicate that the mem capacity for detecting epidemic activity had a sensitivity of % and a specificity of % when using ili proportions, implying that it is better for eliminating false signals than it is for detecting a true signal. our finding is similar to that of vega et al., who also found the sensitivity to be significantly lower than the specificity [ ] . using cambodian surveillance data, ly et al. [ ] also found that the who methodology appeared to have a higher sensitivity for detecting early epidemic activity, but a lower specificity than mem, implying a greater risk of signalling false starts to the season. timely detection of the start of seasonal epidemics may be important to alert health services and to mitigate morbidity, mortality and economic costs by allowing resource allocation and adjusting response measures to face the seasonal overload in the healthcare system. the public health implications for this difference between methodologies are that using the mem method without applying the seasonal threshold established using the who method, there is a risk of missing the beginning of the epidemic period and not providing timely guidance to clinicians to indicate influenza season has begun, and to manage patient treatment accordingly. using the lower who threshold for public health messaging regarding the beginning of the influenza season may pose the risk of a false alert and perhaps overprescribing antiviral medications. from another point of view, using a low seasonal threshold could influence decision-makers to recommend earlier vaccination. as our results showed that the seasonal threshold typically occurs between mid-november and mid-december in morocco, appropriate timing for vaccination could be about month before this date. of note, the us advisory committee on immunization practices (acip) recommends that vaccination should be offered by the end of october, considering the unpredictability of timing of onset of the influenza season and concerns that vaccineinduced immunity might wane over the course of a season [ ] . low seasonal thresholds may be crossed multiple times as was the case in our application of the who threshold for several seasons ( / , / , / , / and / [not shown]), due perhaps to variability in reporting by the surveillance sites. because of this variability, it is possible that declaring the start of the influenza season after two or three sustained weeks of activity above the threshold as recommended by who, is a prudent option for considering influenza transmission as epidemic. the mem methodology, however, calculates the length of the epidemic period during each season separately in order to determine the average length. thus, the epidemic threshold calculated with the mem method could be preferable to that established with the who method. mem was first used in in the who european region to estimate epidemic period and intensity using a minimum of five historical seasons for the calculations and the target season [ ] . despite the availability of only years of virologic data in morocco, we followed a who recommendation to use the composite parameter with mem [ ] . this allowed a clearer cut estimation of the beginning of the influenza epidemic period, characterized by a sharp increase in influenza-confirmed ili cases. when ili proportions are used, the two methods produce similar values for each intensity threshold considered in the pisa assessment of seasonal transmissibility; who has adopted the mem for this purpose. when comparing the highest weekly activity per season (the seasonal peak) to the intensity thresholds established by who and mem procedures, the / season was of moderate intensity ( figs. and ) . using the composite parameter, the / seasonal peak nearly reached the high intensity threshold, whereas this curve didnot cross the medium intensity threshold when using only ili proportions. our study has several limitations. first, the assumption that ili activity reflects influenza virus circulation is limited because of possible concurrent circulation of other respiratory viruses (e.g., rsv) [ , ] . who recommends using a composite parameter defined as the product of the ili or ari proportion and the percentage positive for the transmissibility indicator of the pisa tools [ ] . unfortunately, virologic data collected prior to was not consistently available for the period of our study as virologic surveillance was disrupted between and . despite this limitation, our laboratory-confirmed data showed something different than the syndromic data as the start of the virologic activity occurs suddenly and is therefore clearly identified. it is obvious that the inclusion of virologic data increases the specificity of seasonal threshold estimation. according to the who guidelines [ ] , "a combination of parameters may be preferable. for example, a seasonal threshold could be defined as the week in which the ili rate crosses a certain value and the percentage of specimens testing positive reaches a certain point". given the long life of our surveillance system, our data were limited by changes in data collection practices, inconsistency of reporting by surveillance sites, and variable access to primary health care. these problems are not unique to the morocco ili surveillance system, and we believe they are the nature of routine, sentinel surveillance. another limitation was the adoption of a new case definition in , at which point we also relaunched our surveillance system using a new protocol. these changes may have affected the trends that we observed in ili activity from that year forward. since both methods we used to establish thresholds recommend using a minimum of three to five seasons of data, we would not have enough data to run the models if we used only data from onward. determining a gold standard for influenza epidemic and intensity thresholds has been a long-standing research question for both international organizations and country-level public health authorities, and there is no consensus on the best method [ , , , , [ ] [ ] [ ] . both the who method and the moving epidemic method translate quantitative trend data into standardized qualitative intensity levels, which permit countries to determine if the current season is atypical or to assess country or regional differences in activity and intensity. both methods identified that the / season was the most active in morocco, excluding the / pandemic season according to non-published observations. both methods are coherent to identify excess activity or high intensity thresholds even though with adequate laboratory data mem with the use of the composite parameter, gives a theoretically better qualitative measure of the level of activity. this comparative study has shown that the threshold methodology presented in the who manual is simple to implement and easy to adopt for use by the influenza surveillance system in morocco or the national surveillance systems of other similar countries. mem is more statistically sophisticated and may provide a more accurate detection of the start of seasonal epidemics in temperate countries with clear seasonal circulation of influenza viruses, especially if virologic data are considered. whichever method is used, analysis of surveillance data will provide information about seasonal thresholds and epidemic curves that may help health care personnel in the clinical management of respiratory illness after the start of influenza season. establishing a seasonal threshold for influenza helps health authorities to identify suitable periods for annual vaccination campaigns and for health practitioners to administer influenza vaccines or prescribe influenza antiviral drugs. computerization of the influenza surveillance system improves timeliness and assessment of the intensity of the influenza epidemic early in its course will guide policymakers in ensuring the appropriate allocation of resources to control seasonal epidemics. estimates of global seasonal influenza-associated respiratory mortality: a modeling study influenza (seasonal) fact sheet epidémiologie de la grippe et facteurs de risque d'infection respiratoire aiguë sévère au maroc influenza seasonality: timing and formulation of vaccines world health organization. global epidemiological surveillance standards for influenza overview of 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method establishing thresholds and parameters for pandemic influenza severity assessment evaluating tools to define influenza baseline and threshold values using surveillance data, egypt, season / seasonality, timing, and climate drivers of influenza activity worldwide kinetics and humoral antibody response to trivalent inactivated split influenza vaccine in subjects previously vaccinated for the first time influenza activity in europe during eight seasons ( - ): an evaluation of the indicators used to measure activity and an assessment of the timing, length and course of peak activity (spread) across europe prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices -united states, - influenza season influenza interaction with cocirculating pathogens and its impact on surveillance, pathogenesis, and epidemic profile: a key role for mathematical modelling possible interference between seasonal epidemics of influenza and other respiratory viruses in hong kong establishing thresholds for influenza surveillance in victoria european centre for disease prevention and control. indicators of influenza activity detecting the start of an influenza outbreak using exponentially weighted moving average charts publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank dr. amgad elkholy and dr. mohamed elhakim from infectious hazard management (ihm)/emro at world health organization and dr. henry laurenson-schafer for organizing training sessions on statistical methods for analyzing data provided by influenza surveillance systems as well as pr. abderrahmane maaroufi, former director of epidemiology at the ministry of health of morocco. a pilot study to assess the historical surveillance data of influenza in morocco and to compare the who method authors' contributions ar and ic designed the study. ic performed data analysis, interpretation of results and drafted the manuscript. mmc helped with study design, data analysis, interpretation of results, and drafting of the manuscript. ar and my assisted with study implementation and provided oversight of study personnel. ho, ab, fef, zr and hi assisted with access to and interpretation of laboratory testing results. st and he helped with data collection and study design and implementation. cn read and approved the final manuscript. all authors have read and approved the manuscript. none.availability of data and materials datasets were collected by each participating site including the national influenza center and gathered on a pooled database at the direction of epidemiology and disease control of the ministry of health of morocco. data cannot be publicly shared due to internal regulations of the ministry of health of morocco. the datasets analyzed during the current study could be available from the corresponding author on reasonable request and with special authorization of the ministry of health of morocco. the ili sentinel surveillance system is a public health activity organized by the ministry of health of morocco. personally identifiable data is excluded from this surveillance system; as a result, no request for authorization from the national ethics committees was required. indeed, the royal dahir n° - - dated august , , promulgating the law n° - relating to the protection of persons participating in biomedical research, provides for special provisions for non-interventional or observational researches as stipulated in its articles and . not applicable. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention. the authors declare that they have no competing interests. key: cord- -bs hti authors: niang, mbayame ndiaye; diop, ndeye sokhna; fall, amary; kiori, davy e.; sarr, fatoumata diene; sy, sara; goudiaby, déborah; barry, mamadou aliou; fall, malick; dia, ndongo title: respiratory viruses in patients with influenza-like illness in senegal: focus on human respiratory adenoviruses date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: bs hti background: human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory tract. in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after consecutive years of sentinel surveillance of influenza-like illness cases. methodology and results: from january to december swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex(™) ii rv detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. samples were collected. more than half of patients ( . %; / ) were children of ≤ years. ( . %) were positive for hadv with ( . %) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses ( . %; / ), rhinoviruses ( %; / ), enteroviruses ( . %; / ) and rsv ( . %; / ). children under were the most infected group ( . %; / ; p < . ). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv- genome type. the hadv-b species like strains from senegal grouped with genome types hadv- , hadv- and hadv- as shown by a phylogenetic branch with a high bootstrap value of ( %). conclusion: in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b species. the circulation of though hadv- and hadv- genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. from january to december swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex™ ii rv detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. samples were collected. more than half of patients ( . %; / ) were children of years. ( . %) were positive for hadv with ( . %) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses ( . %; / ), rhinoviruses ( %; / ), enteroviruses ( . %; / ) and rsv ( . %; / ). children under were the most infected group ( . %; / ; p < . ). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv- genome type. the hadv-b species like strains from senegal grouped with genome types hadv- , hadv- and hadv- as shown by a phylogenetic branch with a high bootstrap value of ( %). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b a a a a a species. the circulation of though hadv- and hadv- genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory, ocular, gastrointestinal, and genitourinary systems [ ] . they belong to the family adenoviridae, genus mastadenovirus with seven species (a-g), including each various types [ ] . ubiquitous in the environment, hadvs are non-enveloped, double stranded dna viruses that vary in size from to nm [ ] . hadvs are recognized as a common cause of respiratory infection in persons of all ages. the illnesses range from influenza-like fever and discomfort to pneumonia and death [ ] . indeed, hadvs infections are usely mild but some groups such as very young children, elderly, immunocompromised persons, or persons with underlying pulmonary or cardiac disease, might be at higher risk degree for severe disease [ , , , ] . the most common hadvs species that cause respiratory tract infections in children are b (hadv-b and b ) and c (hadv-c , c , and c ). serotypes b , b , and b are the most frequent strains responsible for epidemics of acute febrile respiratory disease [ ] . circulating hadvs can vary temporally and geographically with possibility of emergent genomic variants which can be associated with more severe illness [ , ] . in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after consecutive years of sentinel surveillance of influenzalike illness cases. from january to december we collected specimens (nasal-pharyngeal and oral-pharyngeal swabs) and surveillance data for influenza and other viral respiratory pathogens from outpatients presenting with influenza-like-illness (ili) at different sentinel sites in senegal. once collected, swabs are placed in -ml cryovials with viral transport medium (universal transport medium; copan diagnostics inc., murrieta, ca), and transported at a controlled temperature of ˚c- ˚c to the laboratory. an ili patient was defined as a person presenting with sudden onset of fever (> ˚c) or history of sudden onset of fever in the recent past ( days) and either cough or sore throat and/or rhinorrhea in the absence of other diagnosis, according to the cdc case definition. each sample is accompanied by a case report form collecting demographic and clinical data. the questions included information on date of enrollment and symptom onset, sex, age, clinical symptoms, previous treatments, travelling history, vaccination status for influenza, and whether or not the patient was hospitalized. upon arrival at the laboratory, the specimens were processed immediately for virus diagnosis. aliquots of samples were also stored at − ˚c for additional analysis (isolation and/or molecular characterization). the data obtained daily were entered into an epi info database (centers for disease control and prevention, atlanta, ga) and analyzed using epi info. total viral nucleic acid (dna and rna) was extracted from μl of each clinical specimen using the purelink™ viral rna/dna mini kit (invitrogen, carlsbad ca, usa) according to the manufacturer's recommendation. dna/rna are eluted with μl nuclease-free water and stored at − ˚c until use. a two-step multiplex real-time rt-pcr was performed with a bio-rad cfx- thermocycler (bio-rad laboratories) and the anyplex™ ii rv detection kit (seegene) for a simultaneous testing of influenza viruses (flua and flub), human respiratory syncytial virus (rsva and rsvb), human adenoviruses (hadv), human metapneumovirus (hmpv), human coronavirus ( e, nl , oc ), human parainfluenza virus (piv , - , - and - ), human rhinovirus (hrv), human enterovirus (hev) and human bocavirus (hbov), as previously described [ ] . in consideration with low ct-values, hadv positives samples ( per year) were selected using a random number generator on ms excel for further molecular characterization using classical pcr and sequencing. viral dna was extracted as previously described and eluted with μl water nuclease-free. dnas were stored at − ˚c until pcr reactions. for hadv molecular characterization the last base pairs (bp) of the hexon gene were amplified with the following specific primers: adeno ( '-cctttggcgcatcccattct- ') and adeno ( '-tgggcacctatgacaagcgc- ') previously used by garcia et al, [ ] . the phusion high-fidelity pcr master mix with hf buffer (new england biolabs, ipswich ma, usa) was used for amplifications. for each sample, pcr was carried out in a total reaction volume of μl consisting of μl h o rnase free, . μl of each primer (diluted at μm), μl of x phusion master mix and μl of dna template. cycling conditions were as follows: denaturation step of min at ˚c, pcr cycles including s at ˚c, s at ˚c, s at ˚c followed by an extension step of min of ˚c. five microliters of the pcr product was then mixed with μl of x prime loading dye and loaded on to a % agarose gel along with an appropriated molecular weight markers ( bp ladder, new england biolabs), and gels were stained with ethidium bromide ( . μg/ml) before visualization under uv. for positive samples ( bp size band), amplicons were cut and purified using the gene-jet gel extraction kit (thermo scientific). purified products are then sent for sequencing to beckman coulter services. sequencing was performed in both directions with the same pcr primers (adeno and adeno ) on an abi prism bigdye terminator v . ready reaction cycle sequencing kit (applied biosystems) on a -capillary abi prism -xl (applied biosystems). data in fasta format were then sent to the laboratory for analysis. sequences successfully obtained were aligned with representative genbank sequences of previously published genotypes using the bioedit sequence alignment editor [ ] . the search for sequence similarities were carried out using the basic local alignment search tool (blastn) from ncbi blast web portal. phylogenetic trees were performed in mega software [ ] using the neighbor-joining method, and the statistical significance of the tree topology tested by bootstrapping ( , replicates). the evolutionary distances were derived using the tamura-nei method. bootstrap replicates with values ! are shown on the trees. statistical analysis. regarding hadv infection comparisons between age groups were performed using the fisher's exact test. p value < . was considered statistically significant and the - year age group was used as reference group. hadv mono-infections were also compared to hadv co-infections. the r. . . tool was used to perform the analyses. this study is a component of the s network syndromic surveillance [ ] . the principles of the s network were approved by the ministry of health in its guidelines for influenza surveillance policy, finalized with the support of pasteur institute in dakar and the strengthening influenza sentinel surveillance in africa (sisa) project funded by the who. the protocol and oral consent were determined as routine surveillance activity, and therefore non-research by the senegalese national ethics committee and the steering committee for s network, an entity representing moh, ipd, who and clinicians in compliance with all applicable national regulations governing the protection of human subjects. data were collected in an objective of surveillance and are anonymous. the information provided to participants was an informal description of the study. respiratory specimens were collected, only after informed consent was granted, verbally, to local health care workers by the patients or parents in the case of minors. oral consent was documented in the patient form with two questions about received information and about oral consent. patients could refuse to participate, no specimen will be taken. for the surveillance activities, written consent is judged not necessary by the senegalese national ethics committee, which has also previously approved the work of the national influenza center. collections of non-sensitive data or an observation from normal care in which participants remain anonymous do not require ethics committee review. the patients included in this study were of all ages and consulted the sentinel sites due to influenza-like symptoms; the patients, or parents in the case of minors, accept the tests for respiratory viruses largely because they are free and safe. of specimens tested, ( . %) were positive for hadv (table ) . detection rates over the study period are almost similar in the first years ( , and ) while in there is a marked decrease in adenoviral infections. the mean age of infected patients was years months and median age was years. regarding the viral detection per age group, most of hadv infected cases ( . %; / ) were under years patients, a statistically significant finding (p < . ). however, the detection rates in the other groups including the elderly (above years old) remain high. no significantly gender distribution of adenoviral infection was observed. the comparison of symptoms prevalence between ili patients with adenoviral infection and patients without adenoviral infection showed that cases of myalgia (p = . ), cough (p = . ), diarrhea (p < . ), rhinitis (p < . ) and headache (p = . ) are significantly higher in patients infected by adenoviruses ( table ). the fig shows the temporal distribution of hadv positivity rate per month in senegal from to . we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitude. the highest peak, with % of detection rate, was recorded on december . hadv circulation pattern shows no seasonality even if results suggest a higher activity of these viruses during cold periods. it should be pointed out that the cold periods (between december and february) experience some instability in senegal with possibilities of shifting. for phylogenetic analysis, we were able to obtain the partial hexon gene sequence from hadv-positive samples: were from samples in , from , from , from and from . unfortunately, some samples showed no amplification or poor-quality sequences. the low sensitivity of conventional pcr compared with real time pcr on samples with low viral load, and certainly non-specific amplifications could be the cause of these failures. the nucleotide sequence alignment clustered the majority of senegalese isolates into hadv-c species ( / ). isolates grouped with hadv-b species and the remaining isolate, from , seems close to the hadv- genome type belonging to the hadv-e species. in all cases bootstrap values are high (more than %). within the hadv-c species, senegalese isolates are grouped with the type hadv- ( . %); isolates with hadv- type ( . %), with hadv- type ( %), and isolates formed a subcluster with hadv- and types (fig ) . the hadv-b like species from senegal grouped with genome types hadv- , hadv- and hadv- as shown by a phylogenetic branch with a high bootstrap value of ( %). we also noted that this dominance of species c and b is confirmed over the years. in this four-year retrospective study, we characterized hadv isolates derived from an ili surveillance program conducted as collaboration between pasteur institute of dakar and the senegalese ministry of health between and . it is the first nationally molecular epidemiology investigation of hadvs and even in west africa. our study suggests that hadv are strongly associated with ili syndrome in senegal with an overall detection rate of . % among patients. this rate seems very higher in comparison with available data. indeed, much lower rates are reported in similar other studies conducted in other countries. a study conducted in kenya [ ] on refugees from different countries (somalia, sudan, ethiopia and kenya) yielded a detection rate of . %, in gabon douki et al [ ] detected hadv in . % of outpatients with ili. these detection rates are still lower in other geographical regions: south korea with . % or . % [ , ] , china with . % or . % [ , ] , philippines with . % [ ] , malaysia less than % [ ], usa with . % or . % reported [ , ] , canada in the ontario provence with . % [ ] , peru with . % [ ] , venezuela with . % [ ] , england with . % [ ] . the analysis of these data tends to confirm a higher prevalence of adenoviruses in the respiratory sphere in african populations. this trend was largely confirmed when we investigated the importance of hadv in children with acute respiratory infections. indeed we observed that proportions in cameroon ( . %) [ ] and senegal ( . %) (fall et al, on submission) were considerably higher than those found in other geographical areas: nascimento-carvalho et al., [ ] in brazil with %, moe et al., [ ] in norway ( . %), wansaula et al., [ ] in usa ( %) or lu et al., [ ] in china. however, these discrepancies in hadv detection rates can be also due to differences in technical approaches, virus burden geographical differences, the number of patients tested, the periods during which samples were collected and even the duration of the study. it should be also noted that adenoviral detection does not necessarily prove disease causation as coincidental upper airway infection, asymptomatic viral carrier state [ ] , or prolonged shedding [ ] in a previous infection could explain adenoviral detection. regarding the group age, as expected, results showed that most patients with hadv infection were younger than years ( . %), a statistically significant finding. these results are in concordance with those of other studies which findings concluded that most children are infected by adenovirus at an early age [ , , [ ] [ ] [ ] [ ] . indeed, it is well established that by years of age, % to % of children demonstrate antibodies to at least one serotype [ ] . additionally more than % of diagnosed hadv infections occur in children < years old (due to lack of humoral immunity) [ ] . although most cases exhibit low to mild symptoms are and indistinguishable from other viral causes, acute respiratory infections caused by hadv can be severe [ ] , or even fatal [ , ] , and are associated with the highest risk of long term respiratory sequelae [ ] . consistent with the report from many other studies, results here showed that . % of hadv infected participants were co-infected with one or more other respiratory tract viruses. the most frequently co-detected viruses were influenza viruses ( . %), rhinoviruses ( %), enteroviruses ( . %) and rsv ( . %). however, we noted no significant differences in clinical characteristics and laboratory findings between patients with single hadv infection and those co-infected. the same observation was reported in studies conducted in diverse geographical contexts [ , ] . a previous study conducted in chilean children stated that the clinical severity in patients with single hadv infection and those with mixed infections was the same [ ] . the overall finding is that the clinical value of such co-infections is not clear and still requires independent investigations in order to assess the association between co-infection and severe illness or symptoms. regarding the four years of surveillance, hadv circulation pattern shows no clear seasonality even if results suggest a higher activity of these viruses during cold periods. this lack of seasonality of hadv infection has been largely reported elsewhere [ , , ] . however, seasonal peaks for hadv infection were noted in summer in some china areas [ ] or in spring in northern china [ ] , mexico [ ] and taiwan [ ] . in our study, the last bp region of the hexon gene were used for molecular studies of the different hadv isolates. phylogenetic analysis showed that among the sequenced strains hadv-c species were the most common hadv detected ( . %) in patients with ili in senegal from to . despite some divergences, the strains from senegal were close to types , , , and . this hadv-c species predominance was reported in malaysia [ ], in italy [ ] , in many latina america countries [ , , ] in contrast with studies done in the united states of america [ ] , united kingdom [ ] , korea [ ] , in argentina [ ] and china [ ] , where hadv-b species were the most commonly isolated hadv. hadv-b species were the second most common in senegal with strains, and only one type belonging to hadv-e species was sequenced. hadv-b species from senegal clustered with genome types hadv- , hadv- d, hadv- and hadv- ( % bootstrap value). hadv- d serotype, firstly identified in in beijing [ ] , is of particular concern as it was often associated with illnesses presenting with more severe and higher levels of morbidity than other respiratory hadv pathogens, and also may result in higher levels of fatalities [ ] [ ] [ ] . the hadv- genome type, formerly known as hadv- a, is a genotype resulting from recombination between hadv- and hadv- [ ] . the serotype has recently reemerged as a highly virulent pathogen, causing severe [ ] and sometimes fatal pneumonia among immunocompetent adults, particularly in asia [ ] [ ] [ ] . so the circulation of such hadv genome types in senegal emphasizes the need to reinforce hadv surveillance, especially in hospitalized patients, by including hadv genome detection and genotyping in the documentation of severe respiratory infections. the single hadv-e species strain was typed as hadv- , the unique human type in this species, which is more commonly associated with high rates of febrile respiratory illness in us military recruits [ ] though associated with viral conjunctivitis outbreak in australia [ ] for example. we observed some limitations in our study. first, considering the vast number of hadv positive samples, only a small number of hadv were typed. so the sequencing results do not reflect the full spectrum of hadv strains that may be circulating in ili patients in senegal, and even for selected samples it may have a bias toward samples with a high viral load. another limitation concerned the molecular methods used for typing hadvs in this study, a method which targeted a short hexon hypervariable region that has been shown to correlate closely with serotype. this method does not provide genomic detail and might miss recombination events located in other regions of the genome. therefore, full-genome sequencing would be more informative on senegalese strains, especially for hadv-b and hadv-b types. the results of this study should also be interpreted with caution especially for hadv ili causality (carriage in healthy or asymptomatic individuals). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c, hadv-b species. the circulation of though hadv- d and hadv- genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. so in the interest of global public health we strongly suggest molecular surveillance and genotyping of newly detected hadv strains in senegal and even by whole genome sequencing for some especial strains. our study offers also an important perspective on the burden of adenovirus-associated respiratory illness in senegal. such a perspective, especially among children, should include asymptomatic controls, sari cases, information on disease outcome, atypical clinical signs, duration of symptoms, and treatment. data regarding viral load, shedding, and other possible etiologies (e.g., bacterial and other viruses) would also enable a more thorough assessment of the viral effective disease (or symptom) causality. adenovirus infections in immunocompetent and immunocompromised patients family adenoviridae structural studies on adenoviruses worldwide epidemiology of human adenovirus 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with adenovirus type infection outbreak of febrile respiratory illness associated with adenovirus a infection in a singapore military training camp epidemiology of human adenovirus and molecular characterization of human adenovirus in china fatal pneumonia cases caused by human adenovirus in immunocompetent adults respiratory diseases among u.s. military personnel: countering emerging threats outbreak of adenovirus type conjunctivitis in south australia this study would not have been possible without the excellent support from all the health-care workers of the s network who contributes, every day, to the surveillance network. we convey our special thanks to kathleen victoir from the international network of pasteur institutes for her unwavering support to the s network. we acknowledge the senegalese ministry of health for their help in implementing the s network. key: cord- -w hsr m authors: jiang, lili; lee, vernon jian ming; cui, lin; lin, raymond; tan, chyi lin; tan, linda wei lin; lim, wei-yen; leo, yee-sin; low, louie; hibberd, martin; chen, mark i-cheng title: detection of viral respiratory pathogens in mild and severe acute respiratory infections in singapore date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: w hsr m to investigate the performance of laboratory methods and clinical case definitions in detecting the viral pathogens for acute respiratory infections (aris) from a prospective community cohort and hospital inpatients, nasopharyngeal swabs from cohort members reporting aris (community-ari) and inpatients admitted with aris (inpatient-ari) were tested by singleplex real time-polymerase chain reaction (srt-pcr), multiplex rt-pcr (mrt-pcr) and pathogen-chip system (pathchip) between april and december . community-ari and inpatient-ari was also combined with mild and severe cases of influenza from a historical prospective study as mild-ari and severe-ari respectively to evaluate the performance of clinical case definitions. we analysed community-ari and inpatient-ari episodes ( inpatient-ari excluded because multiple pathogens were detected), involving and samples respectively. detection by pcr declined with days post-onset for influenza virus; decrease was faster for community-ari than for inpatient-ari. no such patterns were observed for non-influenza respiratory virus infections. pathchip added substantially to viruses detected for community-ari only. clinical case definitions discriminated influenza from other mild-ari but performed poorly for severe-ari and for older participants. rational strategies for diagnosis and surveillance of influenza and other respiratory virus must acknowledge the differences between aris presenting in community and hospital settings. influenza and other respiratory viruses such as respiratory syncytial virus, rhinovirus, parainfluenza virus, adenovirus and human metapneumovirus are common causes of respiratory infections . while often manifesting as a mild illness, these viruses can result in serious complications , , hospitalisations and deaths . identifying the viral aetiology of respiratory infections has applications in both clinical management and surveillance. early diagnosis may allow timely initiation of appropriate treatment , , and where warranted, rapid confirmation of an outbreak can lend itself to control and mitigation efforts for influenza , . and while specific therapeutic or preventive measures for most viral respiratory agents (other than influenza) are lacking, diagnosis can still help in ruling out other causes of respiratory illness and facilitate implementation of appropriate infection control measures in healthcare settings . moreover, with increasing concerns about the spread of new and dangerous viral respiratory pathogens such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) viruses, we need to better appreciate how we can optimally combine clinical information with routine as well more complex and expensive laboratory testing technologies to diagnose and conduct surveillance for unusual pathogens . current approaches to diagnosis and surveillance rely heavily on clinical case definitions and a variety of laboratory assays. however, overlapping symptoms makes existing clinical case definitions for respiratory infections inadequate for diagnosis of specific infections , . reverse transcriptase-polymerase chain reaction (rt-pcr), which detects viral nucleic acid by use of amplification techniques, is now considered as the gold standard assay for detection of respiratory viruses, and also has the advantage of short turn-around times as compared to scientific reports | : | doi: . /srep methods based on virus culture and isolation . moreover, multiplex rt-pcr (mrt-pcr) assays, which allow for rapid detection of multiple types of known viral agents, are now widely available . studies have evaluated the performance of pcr-based assays for diagnosing respiratory viruses [ ] [ ] [ ] , but were mostly restricted to either outpatients or inpatients, or to at-risk groups such as the elderly and young children. moreover, large-scale pathogen detection technologies, like the genome institute of singapore (gis) pathchip, have recently been developed , and there is also a need to rationalize how these could be integrated with routinely available assays. our study had two key objectives. firstly, we investigated the performance of the singleplex rt-pcr (srt-pcr) and mrt-pcr, as well as explored the use of the gis pathchip for the detection of viral respiratory pathogens for mild and severe acute respiratory illness (ari). secondly, we evaluated how well clinical case definitions might differentiate influenza from other causes for mild and severe aris. we collected samples and clinical data from both a community cohort and adult inpatients to address the first objective while we combined this with data from mild and severe influenza cases prospectively recruited during the influenza a(h n )pdm pandemic of to support the second objective. of participants from the community enrolled into a cohort, reported having ari episodes. we also enrolled inpatients from tan tock seng hospital (ttsh), and retrieved influenza cases from a previous study (historical-flu). community participants with ari episodes did not differ significantly from the underlying cohort (table ) . compared to community participants with ari, inpatients were older, more likely to be male and to have chronic medical conditions. similarly, historical-flu participants with medical indications were older, and more likely to have chronic medical conditions as compared to those who were admitted for public health indications. historical-flu participants with medical indications were fairly similar to the inpatients although they were slightly younger and less likely to have copd and heart disease. one hundred and thirty episodes were reported from community participants (community-ari) with , , and individuals reporting , , and episodes respectively. for the inpatients, there were episodes with only individual having and another having episodes (inpatient-ari). viruses detected by srt-pcr and mrt-pcr in community-ari and inpatient-ari episodes. on using srt-pcr with mrt-pcr, . % of the community-ari episodes were diagnosed as panel virus positive (i.e. positive for at least one of the viruses on the mrt-pcr panel used), including ( . %) influenza virus positive episodes ( a(h n ), a(h n )pdm and influenza b) and ( . %) non-influenza panel virus positive episodes. two viruses were more common than influenza in community-ari, with ( . %) episodes positive for rhinoviruses and ( . %) for coronaviruses (table ). there were no co-infections detected by mrt-pcr in either community-ari episodes or influenza negative inpatient-ari. however, influenza positive inpatient-ari episodes were positive for more than one pathogen, including dual-pathogen (influenza a + influenza b [ ] , influenza a + rhinovirus, and influenza b + respiratory syncytial virus) of the and samples from community-ari and inpatient-ari episodes, and respectively were for influenza positive episodes (where multiple samples were collected for each episode). the time between episode onset and the first sample for inpatient-ari was longer than for community-ari (median of days vs day respectively, p < . ); however, no such difference was observed between influenza versus other ari episodes either for community-ari or inpatient-ari (p = . and p = . respectively). we compared the detection of influenza and non-influenza panel viruses by days post-onset. mrt-pcr detected influenza in only . % of the samples that were positive for influenza by srt-pcr. for influenza positive episodes, the proportion positive by either assay decreased as days post-onset increased for both the community and inpatient samples (fig. a ,b). the srt-pcr ct values also increased with days post-onset in both groups, but the ct value for community samples increased more sharply than for the inpatients. the gee model suggested that inpatient-ari had a higher starting ct value than community-ari (p = . , table ). ct value increased as days post-onset increased (p < . ), but the increase in ct value with every additional day post-onset for inpatient-ari was lesser than that for community-ari (p < . for interaction term between participant type and days post episode onset). for non-influenza panel virus episodes, changes in the proportion of positive samples showed no consistent pattern by days post-onset in both community-ari and inpatient-ari (fig. c ,d). amongst (no samples were sent for pathchip analysis for pcr negative inpatient-ari episodes) pcr negative episodes, pathchip did not detect any viruses in episodes. in episodes, multiple viruses (both respiratory and non-respiratory) were detected; these were excluded from further analysis. episodes were positive to mrt-pcr panel virus; all were from community-ari, with positive for rhinovirus, for influenza a and another for metapneumovirus. more episodes ( community-ari, inpatient-ari) were positive for viruses including coxsackievirus ( ), human enterovirus( ), human parainfluenza virus ( ) and influenza c virus ( ) where ari is a recognised presentation . episodes had only non-ari viruses detected (human herpesvirus , human papillomavirus, molluscum contagiosum virus and human t-lymphotropic virus ). the pathchip results increased the proportion of community-ari episodes positive for respiratory viral pathogens from . % to . %, and from . % to . % for episodes meeting febrile respiratory illness (fri) criteria (i.e. ari with self-reported fever, regardless of body temperature measurement). due to the small number of influenza positive episodes in community-ari, we combined community-ari and inpatient-ari episodes with influenza episodes identified from a previous study (described further in supplementary material). clinical features of historical-flu admitted for public health indications were fairly similar to influenza cases identified amongst community-ari, which justified grouping them together as mild-ari (i.e. community-ari + historical-flu, public health indications); likewise historical-flu with medical indications for admission was fairly similar to table . non-influenza mono-infection episodes in community-ari and inpatient-ari episodesvirus identified. a as proportion of all community-ari episodes. b as proportion of community-ari episodes positive for non-influenza viruses. c for inpatient-ari, this excludes episodes where there was a co-infection between influenza and non-influenza viruses on mrt-pcr. d as proportion of inpatient-ari monoinfection episodes positive for non-influenza viruses. e includes hcov e/nl and hcov oc . influenza from inpatient-ari, and were designated severe ari (i.e. inpatient-ari + historical-flu, medical indications, also see supplementary table s ). none of the respiratory symptoms assessed were significantly more common in influenza, and for mild-ari, sore throat and runny nose were significantly less common in those testing positive for influenza (table ). however, for both mild and severe ari, fri and influenza-like illness (ili) table . outcome of the gee model a evaluating the factors on the dynamics of ct values. a generalized estimating equation model which included participant type (community cohort or inpatient), days post episode onset, age, gender, comorbidities (comparing those who reported any comorbidities with those who didn't report any comorbidities, with the comorbidities included being diabetes, asthma, copd, heart disease, cancer and other significant conditions), as well as the interaction term between participant type and days post episode onset. case definitions as well as temperature cut-off points showed significant discriminatory value for influenza over non-influenza episodes or any panel viruses positive over viruses negative episodes. figure a shows that in mild-ari, while only . % was influenza positive by srt-pcr, this rose to . %, . % and . % for fri, ili-u (us centers for disease control ili definition) and ili-w (world health organisation ili definition) respectively, with lr+ > for ili case definitions and temperature cut-off points ≥ . °c. while using ili-w increased the proportion positive for any mrt-pcr panel viruses to . %, this represented a relatively small improvement over the . % positive in all mild-ari, with lr+ of only . (fig. b) . for severe-ari, use of more specific case definitions also improved the likelihood of being positive for influenza and viral respiratory infection, but the lr+ only ranged from to . . notably, for severe-ari, ili-w performed best, with . % and . % of such episodes positive for influenza and any panel viruses respectively (fig. c,d) . we performed a sensitivity analysis excluding the historical-flu cases and the results were essentially the same other than for the wider confidence intervals (due to the reduced sample size, see supplementary fig. s ). there were insufficient numbers of mild-ari with age ≥ years for age-stratified analysis, but influenza was significantly more likely than non-influenza episodes to meet fri, ili-u and ili-w criteria for both mild-ari and severe-ari in those aged < years (table ). however, case definitions had poorer discriminatory value in severe-ari and older ages. for instance, in those aged < years, severe influenza was significantly more likely than mild influenza to meet ili-w criteria ( . % vs . % respectively, p = . ), but non-influenza causes of severe-ari were also more likely to have febrile presentations, with . % meeting ili-w criteria (vs . % for non-influenza mild-ari, p < . ). in addition, comparing severe influenza between age groups shows that episodes in older individuals were also significantly less likely than to meet ili criteria (e.g. for ili-u, . % for severe-ari in those aged < years versus only . % for those aged ≥ years, p = . ). our study concurrently assessed the role of routine laboratory diagnostics, and usefulness of the novel pathchip platform as well as ili case definitions in identifying respiratory virus infection in a community cohort and hospital inpatients from a broad range of age groups ( to , and to years respectively), to reflect what may be encountered in either community or primary care (mild-ari) as well as tertiary care settings (severe-ari) in a tropical environment with less distinct seasonal patterns. our study clarifies the role of singleplex and multiplex rt-pcr in the respective populations. we observed imperfect sensitivity of mrt-pcr for influenza ( . %) as compared to srt-pcr, but better performance was reported in two outpatient studies ( - %) , , possibly related to the longer time between onset and sample collection for inpatients in our study, as the recovery of samples positive for influenza and sensitivity of mrt-pcr for influenza was dependent on the time between onset and sample collection. post episode onset, the proportion positive for influenza decreased (ct value increased) faster in community-ari as compared to inpatient-ari, but no such pattern was observed for non-influenza panel viruses; likewise, others have found slower viral clearance in inpatients compared to outpatients for influenza a(h n )pdm , and a lack of association between days post-onset and ct values for adenovirus, human metapneumovirus, parainfluenza virus - . pcr-based assays may thus retain greater value for detecting influenza amongst inpatients that present late than in the community where episodes presenting more than days post-onset would likely be influenza negative. our study also explored the application of newer platforms like the pathchip for detecting respiratory virus infections. in contrast to multiplexed pcr-based technologies which rely on a set of primers that target a finite number of specific pathogens, the pathchip is designed to detect a wide array of pathogens simultaneously by detecting signatures in the pathogen genome sequences. while costly, our results suggests that it has the potential to complement existing technologies as a diagnostic tool. simões et al previously evaluated the diagnostic value of the pathchip using paediatric nasal wash samples and reported variable sensitivity ranging from . % to . % and reasonable specificity from . % to % . in our study, the pathchip was a valuable addition in community-ari where it substantially increased the proportion positive for respiratory etiological agents, so that only about % of febrile episodes did not have a viral aetiology identified. notably, the vast majority of additional viruses detected were those known to cause ari. it may thus help in ruling out more sinister causes of ari if added to mrt-pcr for surveillance in community settings; it may also help detect unexpected but dangerous infections such as sars and mers-cov following further validation on actual patient samples with such infections. however, due to the high cost of the assay (about four times mrt-pcr) and the imperfect sensitivity for some pathogens (as low as . % for adenovirus), we opted to test only the rt-pcr negative samples with the pathchip. as such, we are unable to comment on its sensitivity for samples from adults, which may be inferior to the previously published results which used samples from paediatric subjects, who are known to have higher viral loads for some viruses , . however, given its current cost, variable sensitivity and slower turn-around time as compared to mrt-pcr (about hours for pathchip), we believe this technology is most appropriately used in the way we designed our study, which is to detect additional respiratory viral pathogen positive episodes as an adjunct to more widely available mrt-pcr panels designed to cover the most common pathogens from community-based samples. finally, our study highlights how the same clinical case definitions, which have been used in both community and inpatient settings , may actually perform differently in the two settings for distinguishing influenza from other respiratory infections, identifying influenza cases, and increasing the yield of diagnostic assays. we found that, while respiratory symptoms themselves are poor in distinguishing influenza from other ari causes, clinical case definitions using fever or temperature cut-off points demonstrated good discriminatory value in our mild-ari episodes, which should reflect what can be anticipated in primary care settings. however, for ari severe enough to require hospitalisation, the case definitions had poorer discriminatory value. this was partly because, while a good majority of older individuals ( . %) with severe influenza had self-reported fever (as in fri), only about half met the temperature criteria used for ili case definitions ( table ). the use of such high temperature cut-off points would hence substantially reduce sensitivity for detecting influenza in the elderly. also, non-influenza episodes in ari severe enough to require hospitalization are more likely to also have a higher temperature than non-influenza episodes in mild ari; this further reduces the ability of ili case definitions to distinguish influenza from other respiratory causes in the inpatient setting. the choice of an appropriate case definition hence depends on the objectives and setting of the application. if the aim is to diagnose as many influenza cases as possible (e.g. for identifying patients for antiviral treatment or outbreak management), ili case definitions have inadequate sensitivity, particularly in older age groups. however, the discriminatory value of ili criteria finds application when aiming to reduce background noise during syndromic surveillance, either in community type settings or even a healthcare worker population . the ili case definitions are also useful for optimizing the yield of influenza viruses from samples tested, and particularly efficient in settings where milder ari presentations are encountered (fig. a) ; and if the aim is to identify all types of respiratory viruses for surveillance, ili criteria would also modestly improve the probability of obtaining a positive result as compared to sampling all ari in both mild as well as severe presentations (fig. b,d) . a key limitation we acknowledge is the less than ideal number of influenza infections identified through our community cohort, which led us to supplement our concurrent community and inpatient studies with data from our older prospective study of influenza cases. while not ideal, supplementary table s suggests that influenza cases from this historical dataset which were classified as mild were reasonably similar to those from the community cohort, and likewise those classified as severe were similar to those from the later inpatient study. a sensitivity analysis without the influenza cases from our historical dataset gave a similar result. however, even with these additional influenza cases, the numbers remained inadequate to assess the performance of the case definitions for mild influenza in older age groups, and we are also unable to assess performance in paediatric subjects. secondly, the panel of viruses tested on the multiplex pcr assay was not comprehensive; while this was supplemented by the pathchip, there is no means of ascertaining what proportion of the remaining ari episodes are truly not due to an infectious viral aetiology. we also recognise that our study population, in particular the community cohort, may not be representative of the general community, which was further complicated by fluctuations in the rate at which participants notified us of ari episodes, as well as intra-seasonal variations within each year and sporadic outbreaks of particular respiratory pathogens . scientific reports | : | doi: . /srep the performance of various technologies and case definitions for viral respiratory pathogens presenting with ari differs substantially between community and hospital-based settings. pcr-based assays may still be relevant for detecting influenza amongst inpatients that present late but less so for community ari episodes with delayed presentations. the pathchip may add value for respiratory virus detection in samples negative by multiplex rt-pcr, but only for community-ari. finally, us-cdc and who ili case definitions had similar performance for mild ari, but performed less adequately amongst presentations severe enough to require hospitalisation, including in older individuals. rational strategies for diagnosis and surveillance of influenza and other respiratory viruses must acknowledge the differences between these two populations. study population. we recruited a community cohort and inpatients from ttsh between april and december . the community cohort was recruited by contacting community-dwelling adults who participated in pre-existing prospective cohort studies conducted by the national university of singapore (nus) saw swee hock school of public health. consenting individuals were then enrolled through home visits, where we also opportunistically recruited other members of the household, including children. at enrolment, participants contributed demographic and health information through a baseline interview at enrolment, and then followed-up for up to . years, with instructions to notify the study team within hours on developing ari symptoms (community-ari). once notified, research staff would obtain nasopharyngeal swabs and symptom data from the participant on the next working day. for inpatients, on each working day, we would screen through the list of adults admitted to ttsh within the last hrs who had undergone routine diagnostic testing (by srt-pcr) for influenza. we then enrolled and collected nasopharyngeal swabs from consenting inpatients fulfilling the same ari criteria (inpatient-ari) used for the community cohort. to better characterise influenza, we intentionally oversampled influenza to obtain a ratio of approximately influenza positive to influenza negative patients. additional swabs were taken on days to and days to post-onset (community-ari) or post-enrolment (inpatient-ari) for influenza positive episodes where possible. in addition, we retrieved historical influenza data from a prospective study of admissions to ttsh testing positive for influenza between may and september (historical-flu). this included ari patients with medical indications who were admitted alongside ari cases referred to ttsh for public health indications (clinically suspected to have influenza a(h n )pdm based on epidemiological risk factors like travel and contact history). ethics approvals were obtained from nus institutional review board and the national healthcare group (singapore) domain specific review board, in accordance with relevant guidelines and regulations. a written informed consent was signed by each study participant. laboratory analysis. three assays were performed. srt-pcr and mrt-pcr assays were conducted at the nphl on all available samples while pathchip assays were performed at gis using rt-pcr negative samples. srt-pcr. this assay was only used to detect influenza virus types a and b at a higher sensitivity than might be achieved using multiplexed pcr assays, and to further subtype influenza a positive samples. the protocols had been adopted from the studies by spackman et al. and krafft et al. and, respectively, with cycle threshold (ct) values documented for positive samples (samples with a ct value less than were considered as positive). influenza a positive specimens were then subtyped with subtype specific primers and probes that targeted at haemagglutinin (ha) gene of a(h n ) , and ha and nucleoprotein (np) genes of a(h n )pdm pathchip. this assay was used to detect additional viruses beyond what was covered by the primers within the mrt-pcr panel used. for each sample, the cdna was amplified from extracted nucleic acid. the novel platform from the genome institute of singapore (gis) then automatically detects which pathogens' recognition signatures are present, based on a proprietary algorithm constructed based on genetic sequences of viruses clinically relevant to humans (downloaded from the ncbi taxonomy data-base, http://www.ncbi.nlm.nih.gov/taxonomy/ taxonomyhome.html/) to evaluate how the gis-pathchip might add to detection above routinely available mrt-pcr assays, viruses were grouped into panel viruses (i.e. virus group represented in seegene rv ), non-panel viruses for which ari is a common presentation and non-ari viruses. in our analysis on the combined performance of clinical case definitions and laboratory assays, we grouped all community-ari episodes (none of which required hospitalisation) with historical-flu admitted for public health indications; these were designated as "mild-ari" since these would ordinarily not be sufficiently severe as to require hospitalisation. inpatient-ari was grouped with historical-flu cases admitted for medical indications ("severe-ari"). this increased the number of influenza cases available for evaluating the discriminatory value of clinical parameters and case definitions, which were: • ari: episode with acute onset with any key respiratory symptoms including cough, shortness of breath, sore throat, or runny nose. • febrile respiratory illness (fri): ari with self-reported fever, regardless of body temperature (t) measurement. • influenza-like illness defined by centers for disease control and prevention of the united states of america (ili-u): fever ≥ . °c together with cough and ⁄or sore throat in the absence of a known cause other than influenza . • influenza-like illness defined by world health organization (ili-w): fever of ≥ °c plus cough with onset within the last days . the proportion of episodes fulfilling different criteria was compared by viral pathogen status (influenza viruses, non-influenza panel viruses and negative), and we then investigated the discriminatory performance by calculating the positive likelihood ratio (lr+ ) and its % confidence intervals (ci) for positive detection of influenza and any viral infection. this was also done to investigate the effect of age (age < vs ≥ years). estimates of lr+ requires assumptions on the anticipated prevalence of influenza. for community-ari, this was assumed to be what was obtained in ari samples from the community cohort. for inpatient-ari, since influenza positive inpatients were intentionally oversampled (crude_flu), the proportion positive for influenza was estimated based on the proportion positive for influenza from routinely ordered tests of ari admissions to ttsh (adjusted_flu), which was then applied to derive the adjusted (adjusted_non-flu) from the crude proportion (crude_non-flu) positive for non-influenza viruses detected in mrt-pcr using the following formula: adjusted non flu crude non flu ( adjusted flu)/( crude flu) all analyses were conducted using r version . . 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). supplementary information accompanies this paper at http://www.nature.com/srep the authors declare no competing financial interests. key: cord- -eqf ame authors: van beek, josine; veenhoven, reinier h; bruin, jacob p; van boxtel, renée a j; de lange, marit m a; meijer, adam; sanders, elisabeth a m; rots, nynke y; luytjes, willem title: influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections date: - - journal: j infect dis doi: . /infdis/jix sha: doc_id: cord_uid: eqf ame background: data on the relative contribution of influenza virus and other respiratory pathogens to respiratory infections in community-dwelling older adults (≥ years) are needed. methods: a prospective observational cohort study was performed in the netherlands during winters. nasopharyngeal and oropharyngeal swabs were collected during influenza-like illness (ili) episodes and from controls. viruses and bacteria were identified by multiplex ligation–dependent probe amplification assay and conventional bacterial culture. results: the ili incidence in the consecutive seasons was . % and . %, and influenza virus caused . % and . % of ili episodes. potential pathogen were detected in % of the ili events with influenza virus, coronaviruses, rhinoviruses, human metapneumovirus, respiratory syncytial virus, parainfluenza viruses, and haemophilus influenzae being the most common. influenza vaccination reduced influenza virus infection by % ( % confidence interval [ci], %– %) and % ( % ci, %– %) in ili patients. however, ili incidence was similar between vaccinated ( . % and . %) and nonvaccinated ( . % and . %) participants in – and – , respectively (p > . ). conclusions: influenza virus is a frequent pathogen in older adults with ili. vaccination reduces the number of influenza virus infections but not the overall number of ili episodes: other pathogens fill the gap. we suggest the existence of a pool of individuals with high susceptibility to respiratory infections. clinical trials registration: ntr . influenza virus causes seasonal epidemics, resulting in - million severe cases and - deaths globally each year [ ] . elderly persons, individuals with certain medical conditions and children aged < years have the highest risk for complications. vaccination is an important tool to prevent infection and to reduce morbidity and mortality [ ] . in the netherlands, individuals aged ≥ years are offered the annual influenza vaccination. however, public acceptance of vaccination is moderate [ ] . vaccine effectiveness (ve) varies per season and depends on age and health of the recipients, and the antigenic match of vaccine strains with circulating strains [ ] [ ] [ ] . furthermore, there is scientific debate about the methodology of determining ve [ , ] . these discussions reach the media and influence the general opinion on influenza vaccine benefit. moreover, to the public, flu as caused by influenza virus is the same as influenza-like illness (ili) caused by respiratory pathogens, against which influenza vaccination will not protect. consequently, influenza vaccination is perceived to be ineffective and vaccine uptake is reduced. to counter this trend, data on the relative contribution of influenza virus and other respiratory infections to ili in older community-dwelling adults are lacking and needed [ ] [ ] [ ] [ ] . this group is underrepresented in the dutch primary care sentinel surveillance system [ , ] and by definition absent in the dutch sentinel nursing home surveillance network [ ] . this is also the case in many other studies worldwide [ ] [ ] [ ] . the aim of this prospective observational study was to determine the relative contribution of influenza virus and other respiratory pathogens to ili in older adults (aged ≥ years) in consecutive seasons in the netherlands. in addition, influenza ve was estimated in both seasons. upon ili, we determined the presence of potential pathogens in the pharynx of the participant within hours of symptom onset. as a control, we analyzed samples of the same individuals taken after recovery ( weeks) and in a subset of asymptomatic controls for the same potential pathogens. this prospective observational study was conducted during consecutive influenza seasons (from december to april [ ] [ ] and from october to may [ ] [ ] ) in the netherlands. adults aged ≥ years were recruited through their general practitioner or through the civil registry. for the second season, participants were reinvited and additional participants were recruited through the civil registry. we also started earlier to use the same monitoring period as in the dutch sentinel surveillance system [ ] . there were no exclusion criteria for the study. influenza vaccination status was recorded ( ) ( ) ( ) ( ) . participants were part of the study for the entire duration of each season and contacted at the end of season to verify participation. written informed consent was obtained from all participants. all trial-related activities were conducted according to good clinical practice, which includes the provisions of the declaration of helsinki. the study was approved by the acknowledged ethical committee metc noord holland (http://www.trialregister.nl; ntr ). participants were instructed about ili symptoms according to the dutch pel criteria, defined by fever (≥ . °c) with at least other symptom of headache, myalgia, sore throat, coughing, rhinitis, or chest pain [ ] and to report ili as soon as possible after onset. a research nurse performed a home visit within hours of fever onset. during this acute phase, nasopharyngeal and oropharyngeal swabs were obtained and additional information on demographics and comorbidities was recorded. a second visit (recovery phase) was performed weeks (± week) later, during which the same samples were collected. if a new ili episode was reported, participants were visited again. in the second season, a control group of asymptomatic participants, equally distributed over the different age groups and season, was sampled and questioned. nasopharyngeal and oropharyngeal samples were obtained with a sterile swab with a flocked nylon tip and stored separately in ml modified liquid amies transport medium (eswab, copan, brescia, italy). samples were transported at room temperature to the laboratory and processed and stored at - °c within hours after sampling. dna and rna were isolated from µl of both swabs by easy-mag isolation and eluted in µl of buffer (biomérieux, the netherlands). five microliters was used for the detection of a panel of respiratory viruses and bacteria by a real-time polymerase chain reaction (pcr)-based multiplex ligation-dependent probe amplification (mlpa) assay (respifinder smart kit; pathofinder, the netherlands). all analyses were performed on a roche lightcycler . mlpa analysis was performed on both swabs separately. a participant was excluded from the analysis and considered missing if either of the swabs or data for a swab were missing. for mlpa data analysis, a participant was considered positive for a target if at least swab of the participant was positive. influenza virus-positive samples were subtyped by real-time reverse-transcription pcr using the roche lightcycler system with slightly modified protocols as described previously [ , ] . conventional culture of oropharyngeal swabs was performed for streptococcus pneumoniae and haemophilus influenzae according to standard procedures [ ] . discrimination of h. influenzae and haemophilus haemolyticus was performed by matrix-assisted laser desorption/ionization-time of flight (maldi-tof) [ ] . participants without available oropharyngeal swabs were excluded from analysis and designated as missing. for analysis of the contribution of influenza virus, a sample size of ili cases was estimated to be required. based on an expected ili incidence of . % and a drop-out rate of %, a cohort size of participants was calculated. based on results of the first season, participants were included in the second season. pearson χ testing and independent samples t test of the means was applied to analyze participant characteristics with spss . for windows software. a p value ≤. was considered significant. the incidence of different viruses or bacteria was calculated as the percentage of swabs positive with the potential pathogen of the number of ili events during the season. attack rates were calculated as percentage of detected pathogens per number of monitored participants. vaccine effectiveness was determined by test-negative design analysis of ili positive participants in the influenza-active period [ ] . the analysis is restricted to the period that influenza virus was circulating in the netherlands for that particular season, defined by the national influenza surveillance weekly reports. participants with < days between the date of vaccination and the date of home visit were excluded from the ve analysis, as it is uncertain whether the vaccine already had any effect in this period. second and third ili periods were included in the analysis only if an earlier ili period was influenza virus negative. for ili participants, influenza virus positive was considered "case" and influenza negative "control. " the ve is calculated as ( -odds ratio [or]) × % with % confidence interval (ci) and is calculated per influenza virus subtype or lineage. the following factors were regarded as potential confounders: period in the season (early and late season), sex, smoking, comorbidity, and age (natural smoothing spline, degrees of freedom). the association between the potential confounders and influenza virus positivity (any subtype) was analyzed with univariate logistic regression. variables with p < . were considered in the multivariable analysis. variables that changed the or by at least % are included in the final multivariable logistic regression model for any influenza subtype (backward selection). all analyses were performed with sas version . software. sensitivity analyses were performed in the ve analyses for other control groups (supplementary materials). in this prospective study, we observed an ili incidence of . % ( / ) and . % ( / ) in consecutive seasons ( - and - , respectively) ( figure a ; table ). the average age of vaccinated individuals was significantly higher than that of unvaccinated individuals (respectively, . vs . years in - and . vs . years in - ; table ). the asymptomatic controls from the second season were older and more often vaccinated compared with the overall cohort ( figure b ; table ). furthermore, participants who reported comorbidities were vaccinated significantly more often than participants who did not report comorbidities (table ) . no significant differences were found between individuals with or without ili with respect to sex, age, and chronic illnesses (tables and ) . importantly, no differences were found in the incidence of ili episodes between vaccinated and unvaccinated participants (table ) . in . % and . % of the acute ili samples from the seasons, at least potential pathogen could be identified by mlpa or bacterial culture ( in . % and . % of the samples from the acute phase, > potential pathogen was detected, but no specific combinations of viruses and/or bacteria were observed (data not shown). in recovery samples, weeks after acute ili, potential pathogens were detected in . % and . % of cases in - and - , respectively. in asymptomatic control samples, similar potential pathogens were observed as in recovery samples ( . %) ( figure c ). in - , influenza virus was detected in . % of the acute ili samples, predominantly of the a(h n ) subtype ( . %) ( figure ; supplementary table a) . influenza virus was not detected in the corresponding recovery samples in this season, suggesting that influenza virus was the actual cause of ili. in - , influenza virus was detected in . % of the acute ili samples, and all circulating subtypes were detected: . % a(h n ), . % a(h n )pdm , . % b/yamagata lineage, and . % b/victoria lineage. in addition, influenza virus was detected at a very low level in both the recovery samples and in samples of asymptomatic controls ( . % and . %, respectively). we investigated which other viruses and bacteria were detectable during ili episodes. in . % ( - ) and . % ( - ) of ili samples, potential pathogens other than influenza virus were detected (figure ; supplementary table ). coronaviruses of all common human subtypes ( . % in - and . % in - ), human metapneumovirus (hmpv) ( . % and . %), rhinoviruses ( . % and . %), respiratory syncytial virus (rsv) ( . % and . %), and parainfluenza viruses ( . % and . %) were detected in > % of the ili samples in at least season. hmpv and rhinovirus varied most between the seasons. all viruses were detectable at low levels in recovery and control samples. rarely, the same virus was observed during both the ili event and the recovery sampling, except for rhinoviruses, which were detected frequently at both visits ( . % in - and . % in - ), but as our test only detected rhinovirus in general, we cannot exclude that these are different serotypes. the attack rate of influenza virus was significantly higher in the second season compared to the first season (p < . ; table ). interestingly, the increased attack rate could be attributed to significant increases in the attack rates of h n , the b/victoria-like subtype, and the b/yamagata-like subtype (p < . , p = . , and p < . , respectively), whereas the attack rate of the h n subtype was not significantly different between these seasons. for the other viruses, we observed significant increased attack rates in the second season for rhinovirus (p < . ) and parainfluenza viruses (p = . ), whereas hmpv attack rates were significantly lower in the second season (p = . ). the other viruses had similar attack rates during the seasons. the only bacterial species detected by conventional culture in a significant number of acute ili cases was h. influenzae ( . % in - and . % in - ), frequently as the sole pathogen, while presence of other bacteria such as h. haemolyticus and s. pneumoniae was low. haemophilus influenzae was also detected frequently in recovery and control samples. we evaluated whether influenza vaccination reduced the overall influenza virus infection incidence and whether this influenced the incidence of ili. although influenza virus lost to follow-up n= no ili reported n= per protocol no ili reported n= a subject could have multiple ili episodes per season. an ili visit (v ) was considered "out of window" if the sample was taken > hours after start of fever. for the recovery visit (v ), the window was - weeks after ili onset. subjects were considered lost to follow-up if they did not respond to the end of study mailing and had no ili visit (a). after the baseline visit had been performed, a subject could have an ili event. subjects were considered lost to follow-up if they did not respond to the end of study mailing and had no ili visit (b). infection incidence was significantly lower in influenza vaccinated than in unvaccinated individuals (table ) , the incidence of ili cases was not reduced by vaccination (table ) . among participants with ili, we observed a high ve of % ( % ci, %- %) in - and a moderate ve of % ( % ci, %- %) in - against influenza virus during the influenza-active period (table ). furthermore, the ve for the predominant influenza virus subtype a(h n ) in - was % ( % ci, %- %). in - , the ve against influenza virus type a(h n ) was % ( % ci, %- %). additional sensitivity analyses for multiple ilis, households with ili, and the presence or absence of other virus infections did not affect this conclusion (supplementary table ). in this study in a cohort of community-dwelling older adults in the netherlands, we show that influenza virus was present in . % and . % of ili cases in consecutive seasons and that influenza vaccination significantly reduced laboratory-confirmed influenza virus infection. in . % and . % of the acute ili cases, potential pathogens other than influenza virus were detected. in addition, these pathogens were more often present during ili than after recovery or in asymptomatic elderly persons. in % of the ili cases, no potential pathogen was detected. the incidence of ili cases was expected to decrease by a reduction in influenza virus-caused ili through vaccination; however, this effect was not observed. instead, the incidence of c individuals in the asymptomatic subset were selected to be evenly distributed over the different age groups; therefore, the overall vaccination level was higher in the asymptomatic subset compared to the ili and non-ili groups. ili remained the same between the vaccinated and nonvaccinated individuals. influenza vaccination is offered to individuals years and older in the netherlands, as older adults are at increased risk for morbidity and mortality from influenza virus infections due to an aging immune system and age-related chronic illnesses [ , ] . however, this policy is based on studies mostly performed in elderly persons in nursing homes, who are substantially older and more frail compared with community-dwelling elderly individuals. studies in community-dwelling older adults are scarce [ ] [ ] [ ] [ ] , and only a restricted number of pathogens have been analyzed in these surveillance studies. to fill the gap in knowledge, we recruited participants through general practitioners and the civil registry, resulting in > % of the participants living in the community. in line with the risk-based vaccination strategy in the western world, older participants and participants with chronic conditions were more likely to be vaccinated [ ] . the age distribution in the cohort was similar to that observed for those ≥ years in the general dutch population; only the oldest age group (> years) was underrepresented (table ) . these elderly persons are often less likely to participate in studies. "healthy user effect" or "frailty selection bias" is a well-known issue in observational influenza vaccine studies, as the very frail elderly persons are difficult to reach [ , ] . we found that influenza virus is involved in . %- . % of ili cases in the seasons studied. in addition, coronaviruses, rhinoviruses, hmpv, rsv, parainfluenza viruses, and h. influenzae were frequently observed as the sole detected pathogen in acute ili cases, whereas they were low to absent in most of the recovery samples. however, rhinoviruses and the bacterium h. influenzae were also commonly detected in asymptomatic controls. the rhinoviruses may be of different subtypes, as we did not type these viruses. viruses can often be detected in individuals without clinical manifestations [ ] , but when they are found as the sole agent in the context of disease, they are commonly considered to be the cause of the disease. for bacteria, this is less clear as they are commonly carried in asymptomatic persons but may expand during respiratory viral infection or new acquisition. haemophilus influenzae may induce an enhanced inflammatory state in the presence of other pathogens, as was shown in children [ ] . this may lead to ili. however, in most ili cases where we found h. influenzae, we did not find a second pathogen that could also explain the ili symptoms. influenza virus incidence in our cohort broadly matched the incidence reported in the dutch sentinel surveillance system [ ] , reported a high ili incidence and mortality in the - season [ , ] . this underlines a potential difference between the generally healthy community-dwelling elderly persons and that of the generally more frail institutionalized elderly persons, who may overlap in age. it also shows that data acquired in one group do not necessarily apply to the other, including exposure and susceptibility to infection, as well as influenza ve. we show that the incidence of influenza virus infection was reduced in individuals who received influenza vaccination and that ve to laboratory-confirmed influenza virus infection was high to moderate in the seasons. the % cis of the data are wide, probably due to the relatively small sample size, a common problem in similar studies. ve estimates are notoriously variable between studies [ ], but the ve data from this study are in line with those reported by van der hoek et al [ ] . the most striking finding in this study was the similar incidence in ili cases observed between vaccinated and the nonvaccinated individuals. this may be explained by assuming that a pool of people exists that is highly susceptible to respiratory infections. the reduction by vaccination in the number of cases caused by influenza virus infections is offset by a rise in the number of cases caused by infections by other pathogens. cowling et al have described a similar increased risk of noninfluenza respiratory virus infection in influenza-vaccinated children [ ] . however, we cannot attribute this effect to a specific pathogen. influenza virus may take preference over other viruses and prevent them from filling the niche, possibly by inducing a prolonged antiviral state, as has been described for other viruses [ , ] . when vaccination reduces influenza virus infections, the other pathogens can fill the gap. it needs to be confirmed in other studies whether influenza virus vaccination has no effect on the total number of ili cases. it would have important consequences for decisions on implementation of vaccination for community-dwelling older adults when looking at the overall disease burden and cost-effectiveness. however, a limitation of our study is that we did not directly monitor the duration and severity of disease in the ili cases: difference in disease or hospitalization was only registered in post-ili questionnaires, and no significant events were reported. more detailed data on severity and duration of symptoms would allow assessing the relative risk posed by different pathogens, taking into account that influenza virus, unlike most other potential pathogens, can vary in pathogenicity between seasons. in combination with other medical information, it may then be possible to assemble a profile of individuals potentially at risk for ili or worse. such a profile would be of great value to public health professionals. a limitation of this study is the definition of ili used, which differs between the world health organization, the european centre for disease prevention and control, and different countries, although most include fever and cough [ ] [ ] [ ] . in this study we used the dutch pel criteria [ ] , which includes fever. it has been described that this specific ili definition can affect the number and type of pathogens detected. falsey et al showed that fever is more frequently associated with influenza virus infection in the elderly persons compared with other respiratory infections [ ] . cough is not a prerequisite in the pel criteria, but as > % of the participants with ili and > % of the participants with influenza virus-positive ili reported coughing (data not shown), it is unlikely that cases were missed due to this difference in definition. in summary, we show that influenza virus caused between . % and . % of ili cases in community-dwelling older adults aged ≥ years in influenza seasons in the netherlands, leaving the remainder caused by other pathogens. we also show that influenza vaccination was effective in reducing the incidence of influenza virus infections but did not reduce the ili incidence, which may have important public health and healthcare consequences. our data will also help to better for more details, see supplementary table . abbreviations: ci, confidence interval; ve, vaccine effectiveness. a corrected for the possible confounders age group, comorbidity, sex, and smoking. data were calculated for the influenza-active period in the netherlands as defined by the netherlands institute for health services research [ ] . inform the public what to expect from influenza vaccination and how it will not protect against all cases of ili, popularly seen as "flu. " supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. notes influenza (seasonal) social determinants of health and seasonal influenza vaccination in adults ≥ years: a systematic review of qualitative and quantitative data effectiveness of seasonal influenza vaccine in community-dwelling elderly people: a meta-analysis of test-negative design case-control studies the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial vaccines for preventing influenza in the elderly cochrane re-arranged: support for policies to vaccinate elderly people against influenza respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden influenza vaccine effectiveness among elderly persons living in the community during the - season a case-control study of acute respiratory tract infection in general practice patients in the netherlands nivel influenza surveillance sentinel surveillance network on infectious diseases in nursing homes study group. absence of influenza a(h n ) during seasonal and pandemic seasons in a sentinel nursing home surveillance network in the netherlands proefonderzoek naar de frequentie en de aetiologie van griepachtige ziekten in de winter - preparing the outbreak assistance laboratory network in the netherlands for the detection of the influenza virus a(h n ) variant differentiation of influenza b virus lineages yamagata and victoria by real-time pcr effect of -valent pneumococcal conjugate vaccine on nasopharyngeal carriage with haemophilus influenzae and moraxella catarrhalis in a randomized controlled trial identification of haemophilus influenzae and haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry letter to the editor: influenza vaccine effectiveness: heterogeneity in estimates for the / season frailty, inflammation, and immunity vaccines for the twenty-first century society mortality benefits of influenza vaccination in elderly people: an ongoing controversy effectiveness of influenza vaccine in aging and older adults: comprehensive analysis of the evidence dutch rsv neonatal network. respiratory syncytial virus and recurrent wheeze in healthy preterm infants excess mortality among the elderly in european countries increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine does viral interference affect spread of influenza? world health organization. ili sari surveillance case definition overview of influenza surveillance in the united states european centre for disease prevention and control. influenza case definitions we gratefully acknowledge all participants for their time and commitment to the study. we thank the study staff at the spaarne hospital and the laboratory staff members at the regional laboratory kennermerland and the centre for infectious disease control at the national institute for public health and the environment.financial support. this work was supported by the dutch ministry of health, welfare and sport.potential conflicts of interest. e. a. s. has received research grants from pfizer and gsk, in addition to fees paid to wilhemina children's hospital/university medical center for advisory boards and participation in independent data monitoring committees for pfizer and gsk. r. v. has received research support from gsk and pfizer for vaccine studies and consulting fees for gsk. all other authors report no potential conflicts of interest. 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- -qwe ndvb authors: qian, yan‐hua; su, jing; shi, ping; he, en‐qi; shao, jie; sun, na; zu, rong‐qiang; yu, rong‐bin title: attempted early detection of influenza a (h n ) pandemic with surveillance data of influenza‐like illness and unexplained pneumonia date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: qwe ndvb please cite this paper as: qian et al. ( ) attempted early detection of influenza a (h n ) pandemic with surveillance data of influenza‐like illness and unexplained pneumonia. influenza and other respiratory viruses ( ), e –e . background to collect disease information and provide data for early detection of epidemics, two surveillance systems were established for influenza‐like illness (ili) and unexplained pneumonia (up) in wuxi, people’s republic of china. objectives the current study aims to describe the performance of these surveillance systems during – and to evaluate the value of surveillance data in detection of influenza epidemics. methods two national ili sentinel hospitals and three up sentinel hospitals provided data to the surveillance systems. the surveillance data from hospital‐based outpatient clinics and emergency rooms were compared by year. the ili data of were further modeled based on previous data using both a control chart method and a moving average regression method. alarms of potential epidemics would be raised when the input surveillance data surpassed a threshold. results in , the proportions of ili and respiratory illness with fever (one surveillance syndrome of the up system) to total patient visits ( · % and · %, respectively) were higher than the previous years. the surveillance data of both systems also showed developing trends similar to the influenza a (h n ) pandemic in . when the surveillance data of were fitted in the two detection models, alarms were produced on the occurrence of the first local case of influenza a (h n ), outbreaks in schools and in general populations. conclusions the results indicated the potential for using ili and up surveillance data as syndromic indicators to detect and provide an early warning for influenza epidemics. influenza, commonly known as the flu, is a severe infectious disease accounting for between and deaths worldwide every year. millions of people died in the influenza pandemic of , one of the worst epidemic disasters in recorded human history. the life style of modern society, including overcrowded cities and rapid global transportation, has resulted in dissemination of influenza and generation of novel virus strains. in the past year, the world health organization (who) announced a level- alert for the influenza a (h n ) pandemic, the highest level of disease outbreak, because of the sustained spread of this new virus on a global scale. the world was fortunate not to have experienced another devastating influenza epidemic, because the fatality rate of the new virus strain was similar to seasonal influenza virus. however, there remains an inevitability of a new influenza pandemic, which could be highly infectious and as deadly as the influenza pandemic. thus, it is necessary, although challenging to detect the onset of a new influenza epidemic, to allow the government to make timely and proper interventions to reduce the social and economic impact from influenza epidemics. the influenza surveillance system long has been playing an essential role in collecting disease information and providing evidence for prevention and control strategies and measures. two surveillance systems were established in wuxi for influenza-like illness (ili) and unexplained pneumonia (up) after the severe acute respiratory syndrome (sars) outbreak. the ili system was implemented for influenza surveillance, and the up system was designed to track lower respiratory illness with pneumonia symptoms. although the disease surveillance systems appeared decades later than in developed countries, their performance is by no means inferior to define the distribution of circulating strains in the community and detect disease aberration. here we described and compared the accumulated ili data from to and up data from to . to further evaluate the effectiveness of these surveillance systems in early warning of influenza epidemics, we monitored ili data between and by both a control chart method and the serfling method and tested goodness of fit using influenza a (h n ) data of . wuxi is a prefecture-level city of jiangsu province, located in eastern china, with a population of ae million. wuxi has seven districts and two county-level cities (jiangyin and yixing). wuxi has a typical subtropical monsoon climate and seasonal influenza usually peaks once a year. there are two surveillance systems in wuxi. one is ili surveillance system and the other is up surveillance systems. both systems collected data from sentinel hospital-based outpatient clinics and emergency rooms. all the selected sentinel hospitals are general hospitals and available to every resident in wuxi city including jiangyin and yixing county. the participating doctors in referral sentinel hospitals were provided with detailed diagnosis instructions for sample selection. as required by the chinese center for disease control and prevention (cdc), two hospitals (children's hospital and the no. people's hospital of wuxi) were selected as the national sentinel hospitals for ili surveillance. the number of patient visits ranged from to ⁄ week. ili was defined as follows: fever ‡ °c, either cough or sore throat, and lack of evidence of other laboratory-confirmed diagnosis. pharyngeal swab specimens collected from patients with ili were sent to cdc laboratories for subsequent isolation and identification. the proportion of patient visits for ili was calculated as the number of patients with ili divided by the total number of patients and was reported through the influenza surveillance system of china. the up surveillance was performed in three hospitals (the no. people's hospital of wuxi, the people's hospital of jiangyin county, and the people's hospital of yixing county). this system was built with special attention to lower respiratory illness, especially pneumonia caused by unknown reagent like sars. routinely collected data included respiratory illness with fever (riwf, also known as acute respiratory illness) and classified pneumonia. emergency measures would be taken in case of any up. pneumonia was diagnosed according to international classification of diseases ( th version, j -j ). unexplained pneumonia was defined as follows: fever ‡ °c, pneumonia-like characteristics in diagnostic imaging, decreased white blood cell count or decreased lymphocyte differential count in early clinical stage, and no improvement in or even worsening of patient's condition after regular antibiotic treatment for - days. the total number of patient visits ranged from to ⁄ week, among whom an average of ae - ae % were diagnosed as classified pneumonia. weekly numbers of riwf and classified pneumonia were reported to wuxi center for disease prevention and control. we retrospectively studied ili data from to and up data from to , respectively. all the data were organized by microsoft office excel . statistical analysis was performed by using spss software (version . , spss inc., chicago, il, usa). the kolmogorov-smirnov test was used to check the normality of the data. the chisquare test was used for the comparison of different ratios and proportions. the ili data during - were used in statistical modeling for influenza detection. both a control chart method - and a moving average regression method , were applied to produce the threshold value. alarms of potential epidemics were raised when the input surveillance data of surpassed the threshold. the control chart method was originally developed to determine and control the situation for manufacturing processes and was later implicated in surveillance analysis. in this study, a long-term average weekly proportion of ili (p) and its standard deviation (sp) were calibrated using all the surveillance data. the data higher than p + sp were referred to as being out of statistical control and were excluded. such data were usually observed in epidemics of seasonal flu and could not generally represent a stable and predictable situation over time. adjusted average weekly proportion of ili (p') and standard deviation (sp') were calculated with those data in the state of statistical control. the threshold was defined as p' + sp'. the moving average regression method was originally proposed by serfling and was used in our study to generate a time-varying threshold for influenza. , [ ] [ ] [ ] [ ] a linear secular trend was estimated from the surveillance data through - by using the regression analysis tool of excel. the secular trend was then removed from the original surveillance data, so the adjusted data represented only seasonal change and could be compared by year. the time-varying threshold was calibrated as the -year moving average plus ae sd (standard deviation) (p = ae ) or ae sd (p = ae ) from the mean. the first imported case of the influenza a (h n ) was reported on june , (the th week of the year). the first local case was reported on august (week ). the first school case was reported on september (week ), and transmission spread quickly when students came back to school in september. the influenza epidemic spread widely during september to december in the general population, with confirmed cases in september, cases in october, cases in november, and cases in december. the last identified patient was reported on february , . the epidemic timeline is shown in figure . the surveillance data collected from the two sentinel hospitals showed an earlier peak of the proportion of ili in when compared with the same period in and ( figure ). the ili proportion aberrantly increased to ae % in the nd week of , higher than ae % in and ae % in . this increase in ili proportion appeared weeks earlier before the first imported case of influenza a (h n ) and started to decline after week . simultaneously with the appearance of the first local case, the proportion of ili started to increase rapidly beginning in week , indicating the early stage of the influenza epi-demic. the proportion of ili peaked in the general population at week ( ae %), when the schools also experienced a peak of illness. figure ). the proportion of riwf to total patient visits ( ae %) was also significantly higher than that in the previous years ( ae % and ae %, respectively), while the proportion of pneumonia to riwf was similar in all years ( ae - ae %) ( table and figure ). the proportion of riwf began to increase in week , around the time of the first imported influenza a (h n ) case. when we examined the epidemic season, the trend of up surveillance data was similar to the trend of ili data (figures and ) . therefore, the data observed from up surveillance system may also reflect the epidemic situation of influenza. the basic design of outbreak detection is that an alert is generated when the current data surpass a threshold. therefore, determining a threshold is essential and crucial for the performance of an early warning system. to obtain the optimal sensitivity and specificity, both control chart method and moving average regression method were used to calibrate the threshold. the control chart method was used to determine an influenza threshold by week. a centerline threshold value was drawn based on the weekly proportion of ili from to . when the data were fitted into the chart, alarms of aberrant higher proportions of illness appeared in week , week to week , week to week , week to week , and week (figure ) . these results were consistent with the true observations, including the first imported case in week , the first local case in week , the outbreak in school beginning in week , and the outbreak peak in general population in week . in the moving average method, a time-varying threshold for influenza was generated. the ili counts for were higher than the moving average baseline in week , week to week , week , and week to week ( figure ). more precisely, the ili counts were higher than mean + ae sd in week to week , week , and week to week , and the periods from week to week and week to week showed the highest ili counts (above mean + ae sd). the aberration points were consistent with the reported date of the first local case, the outbreak in schools, and the peak in the general population. the early detection of disease outbreaks has long been important to public health. the importance of a surveillance network at the global level has been unprecedentedly emphasized because of the emergence of newly infectious diseases, pandemics, and the threats of bioterrorism. the monitoring of surveillance data plays an essential role in detecting signals for a potential outbreak. for diseases or illnesses with enormous social impact, timeliness of detection is extremely valuable. with these types of diseases or illnesses, indicators like hospital consultations rather than mortality or morbidity are used in modeling, because they tend to provide earlier outbreak indication. , for influenza, ili is commonly used as an indicator to predict the occurrence of epidemics. , , unlike other diseases, the value of routinely collected ili data for influenza detection has been carefully studied and confirmed. , , in this current study, weekly counts of consultations for ili and up were monitored for influenza detection. the distribution of sentinel ili data in ( figure ) were consistent with the real spread of the pandemic and showed aberrant increase close to or earlier than the reference data. these results confirmed the capability of ili surveillance system to detect influenza. although the up surveillance system was designed to detect sars instead of influenza, the data are useful because ili and pneumonia usually have similar symptoms such as fever, cough, and breathing difficulty. compared with the reference data (figure ), the sentinel up data (table and figure ) also showed similar epidemic trend. however, the efficiency of early detection was not high using the up surveillance system as significantly increased proportion of riwf was observed in sentinel hospitals only after the first local case was reported. a reasonable explanation might be because of the diagnostic criteria. ili was used specifically to indicate influenza, while riwf includes pneumonia and many other kinds of respiratory illnesses besides ili. the nature of the up system could not distinguish between the respiratory complaints related to influenza and those caused by other pathogens. the population at risk could be another reason for not being able to detect outbreaks because the ili sentinel sites and up sentinel sites cover different areas. nevertheless, the sentinel up data provide additional information to detect influenza epidemic and can add confidence to declare an epidemic alarm. at the beginning of the pandemic flu period, asia was in especially high alert of epidemic influenza and china implemented aggressive policies trying to delay illness spread by early identification and isolation of every confirmed h n patient. the increased proportion of ili and riwf before the first imported h n case could be explained as a surveillance artifact because of the heightened concern of individuals. with the development of the epidemic, control strategies were changed to intensive treatment of the seriously ill and self-isolation of mild cases. people who had mild flu symptoms were encouraged to take anti-viral medicines and stay at home instead of seeking medical care in hospitals. all the sentinel hospitals collected data as usual without any strengthened effort. the higher proportion of ili and riwf observed after week was more likely due to the high incidence of h n influenza in general population rather than surveillance artifact. a variety of statistical methods have been developed to monitor surveillance data, including time series, regression, cumulative sum (cusum), and hidden markov models. , , , [ ] [ ] [ ] [ ] the performance of these models is usually evaluated from the following characteristics: sensitivity, specificity, and timeliness. , sensitivity is the ability to find a real outbreak alert; specificity is the ability to exclude false alarm; timeliness is the ability to raise an alarm within the shortest amount of time from the onset of the peak of the season. most approaches of early detection produce alerts based on the theory of signal detection. the optimal sensitivity and specificity depend on a proper level of threshold, but there is always a trade-off between a high threshold for good specificity and a low threshold for good sensitivity. the calibration of a threshold is a complex determination for each system. both the control chart method and the serfling method are time series approaches and have their own advantages in reducing the variance caused by epidemic data or secular trend. , the control chart method optimizes the threshold calibration by excluding the unexpected data that are beyond the controlled scope. the serfling method provides reasonably accurate estimates by adjusting the threshold line with secular regression trend. the us centers for disease control and prevention (atlanta, ga, usa) developed an early aberration reporting system (ears) based on cusum method, which can use only recent data and is supposed to incorporate the threshold calculation by regression method. , however, a study in hong kong showed that the regression method and the time series method are superior to the cusum method in a small city with fewer sentinel sites, more variable data, and a more abrupt peak in activity. as a result, the cusum method was not used in our study. the control chart method detected all four remarkable signals, namely the occurrence of the first imported case, the first local case, outbreaks in schools, and the peak in the general population ( figure ) . it also produced two false-positive alarms, one in the beginning and the other at the end of the epidemic period. when using mean + -ae sd as the threshold, the serfling method detected the outbreaks in schools and the peak in the general population, with a false-positive signal in the interval; when raising the threshold to mean + ae sd, only the outbreaks in schools could be detected in a timely manner, and the second alarm was week after the peak in the general population ( figure ). the comparison of the two methods indicated that the control chart method had better sensitivity and timeliness and the serfling method had better specificity. nevertheless, considering that the false-positive alerts of the control method appeared outside the epidemic period, this fact might be taken in favor of a more sensitive detection. the same opinion was seen in gault's paper. a final decision probably should be made combining the results of both methods. the control chart method can generate sensitive and timely alerts, and serfling method can offer secular trend to increase specificity and avoid wasting medical resources. in developing countries like china, surveillance systems like the ili and up systems have only recently been implemented. we performed this study after the pandemic flu in attempt to make full use of these routinely collected background data. the results are theoretically helpful for healthcare workers to take prevention and control measures when influenza epidemic occur, including preparing antiviral drugs and other medical supply needs, and sending health education messages. so far no such actions, which we already took during the epidemic period, have been applied based on our study. future studies will be carried out to improve the performance of these surveillance systems, such as adding more sentinel sites, covering larger population, improving the quality of indicators, and trying different detection algorithms. studies of decision making are also needed before these approaches could be used in practice. the decision theory provides mathematical methods to estimate the benefits of true alarms and the costs of false alarms for getting an optimal threshold. this analysis was beyond the scope of our current study, and we will investigate such methods in our future work. in conclusion, this study described the surveillance data of ili and up during - and the epidemic progress of influenza a (h n ) pandemic in wuxi, china. the surveillance data of both systems were correlated with the influenza epidemic and therefore valuable in monitoring influenza activity and generating early warning methods. the control chart method and serfling method had their respective merits and faults in data analysis. further studies could be considered to optimize the threshold of warning. the principle is to improve performance of surveillance systems and allow timely precautionary measures to be implemented in vulnerable populations. emerging infections: pandemic influenza world now at the start of influenza pandemic mortality from pandemic a ⁄ h n influenza in england: public health surveillance study guideline for prevention and control of influenza statistical quality control methods in infection control and hospital epidemiology, part ii: chart use, statistical properties, and research issues statistical quality control methods in infection control and hospital epidemiology, part i: introduction and basic theory study on early warning based on influenza surveillance data in guangdong province methods for current statistical analysis of excess pneumonia-influenza deaths time-series forecasting use and interpretation of statistical quality control charts influenza-associated hospitalizations in the united states impact of influenza vaccination on seasonal mortality in the us elderly population can syndromic thresholds provide early warning of national influenza outbreaks? methods for monitoring influenza surveillance data trends and directions of global public health surveillance the impact of influenza epidemics on hospitalizations value of icd- -coded chief complaints for detection of epidemics performance of a syndromic system for influenza based on the activity of general practitioners, france situational uses of syndromic surveillance sensitivity, specificity and predictive values of health service based indicators for the surveillance of influenza a epidemics comparing aberration detection methods with simulated data a simulation model for assessing aberration detection methods used in public health surveillance for systems with limited baselines review of an influenza surveillance system, beijing, people's republic of china disease surveillance using a hidden markov model the emerging science of very early detection of disease outbreaks key: cord- - fb xnil authors: weaver, anne m.; khatun‐e‐jannat, kaniz; cercone, emily; krytus, kimberly; sohel, badrul munir; ahmed, makhdum; rahman, mustafizur; azziz‐baumgartner, eduardo; yu, jihnhee; fry, alicia m.; luby, stephen p.; ram, pavani k. title: household‐level risk factors for secondary influenza‐like illness in a rural area of bangladesh date: - - journal: trop med int health doi: . /tmi. sha: doc_id: cord_uid: fb xnil objective: to describe household‐level risk factors for secondary influenza‐like illness (ili), an important public health concern in the low‐income population of bangladesh. methods: secondary analysis of control participants in a randomised controlled trial evaluating the effect of handwashing to prevent household ili transmission. we recruited index‐case patients with ili – fever (< years); fever, cough or sore throat (≥ years) – from health facilities, collected information on household factors and conducted syndromic surveillance among household contacts for days after resolution of index‐case patients’ symptoms. we evaluated the associations between household factors at baseline and secondary ili among household contacts using negative binomial regression, accounting for clustering by household. results: our sample was household contacts of index‐case patients. seventy‐one percentage reported that smoking occurred in their home, % shared a latrine with one other household and % shared a latrine with > other household. a total of household contacts ( . %) had symptoms of ili during follow‐up. smoking in the home (rr (adj) . , % ci: . , . ) and sharing a latrine with one household (rr (adj) . , % ci: . , . ) or > household (rr (adj) . , % ci: . – . ) were independently associated with increased risk of secondary ili. conclusion: tobacco use in homes could increase respiratory illness in bangladesh. the mechanism between use of shared latrines and household ili transmission is not clear. it is possible that respiratory pathogens could be transmitted through faecal contact or contaminated fomites in shared latrines. annual influenza epidemics occur worldwide with sporadic pandemics. influenza is an important aetiological agent for febrile illness and pneumonia among children in urban dhaka, bangladesh [ ] [ ] [ ] , where influenza incidence is approximately episodes per childyears, and an estimated % of childhood pneumonia episodes are influenza-associated [ ] . influenza-like illness (ili) refers to a syndrome with symptoms typical of influenza virus infection: fever with sore throat and/or cough [ ] . in community-based surveillance in bangladesh, % of all people who died during , excluding those who died from injury, suicide or homicide, had symptoms of ili within days before death [ ] . although was a pandemic year, which may have influenced mortality from influenza, hospital-based surveillance indicates a similar incidence of influenza-associated ili in ( cases per person-years), ( . cases of seasonal influenza and . cases of pandemic influenza per person-years) and ( cases per person-years) [ ] . in bangladesh, influenza and ili result in a high economic burden for families of ill individuals. families of individuals with influenza identified during surveillance paid a median of % of monthly household income in out-of-pocket costs for treatment of influenza-associated illness [ ] . many families reported reducing monthly food expenditures and/or borrowing money in order to pay for treatment [ , ] . ill individuals may be unable to work and/or attend school for several days, further increasing the financial burden on families [ , ] . annual vaccination is a key strategy for the prevention of influenza in high-and middle-income countries [ ] . in bangladesh, as in many low-income countries, vaccination against influenza viruses has not been widely promoted, likely due to high costs and competing priorities of the healthcare system [ ] . non-pharmaceutical interventions that modify influenza transmission risk factors would be particularly useful in such a setting. respiratory virus transmission has been demonstrated in hong kong and the united states to be common among household contacts [ , ] . household contacts are in frequent contact with infected individuals and have similar risk factors to infected household members [ , ] . crowding and poor hand hygiene, which are prevalent in low-income settings, facilitate transmission of influenza and other respiratory viruses [ ] [ ] [ ] [ ] . handwashing has been associated with a reduced risk of acute respiratory infections in children [ , ] and influenza transmission [ , ] in high-and low-income settings. exposure to indoor and ambient air pollution has been associated with an increased risk of all-cause acute respiratory infections [ ] [ ] [ ] [ ] . exposure to air pollution may damage lung tissue and compromise immunity, increasing susceptibility to respiratory infection [ , ] . air pollution concentrations in a home can be affected by tobacco smoking, biomass fuel use for cooking and proximity to biomass cookstoves [ , ] . influenza and ili carry a high disease burden and subsequent economic burden in bangladesh, a lower middle-income country where widespread pharmaceutical interventions may not be currently feasible or affordable for patients. however, most studies on non-pharmaceutical interventions for influenza have been conducted in high-income settings. it is, therefore, important to identify and address modifiable factors associated with secondary ili, defined as ili in another household compound member after the first patient has been identified, at the household level in bangladesh and other highburden, low-income settings in order to design interventions to reduce transmission. for this study, we aimed to identify household-level risk factors associated with secondary ili in rural bangladesh. we conducted this analysis using the control group of a randomised controlled trial, bangladesh interruption of secondary transmission of influenza study (bistis) [ ] . during the and influenza seasons, patients who sought care for respiratory symptoms at jahurul islam medical college hospital, two district health complexes, and six local pharmacies in rural kishoreganj district, bangladesh, were recruited as index-case patients. study physicians screened patients for the presence of influenzalike illness (ili), which was defined as fever in those less than years of age and fever with cough or sore throat in those years or older. as this study was investigating transmission of influenza at the household level, patients who were admitted to the hospital were ineligible to participate. consenting index-case patients were accompanied to their home by study staff. typically, residents of this area live with extended family members in compounds of several households, sometimes with a shared cooking space and a latrine. if at least two people other than the index-case patient intended to reside in the compound for the subsequent days, we sought to enumerate and enrol all members of the compound ( figure ). eligibility requirements of index-case patients varied during the study period [ ] . briefly, in , we recruited index-case patients who experienced symptom onset in the prior days, who lived within min travel time to the health facility, and had no ili among household compound members in the prior days (n = ). due to a lack of recruitment, after one month, we expanded this criteria to include those living within two hours' travel time and those with ili in other household compound members (n = ). in , in response to literature indicating that handwashing was effective against influenza transmission within h of symptom onset [ ] , we limited enrolment to index-case patients with symptom onset within h. we allowed recruitment of those compounds where individuals who did not live in the indexcase patient's home had ili (n = ). full details of the eligibility requirements are described elsewhere [ ] . household contacts who had fever at enrolment (n = ) were excluded from these analyses. randomisation to an intensive handwashing intervention or control arm was carried out at the compound level. details of the handwashing intervention are described elsewhere [ ] . the following analyses were conducted among participants randomised to the control group to reflect household-level risk factors for ili. at the initial healthcare facility visit of the index-case patient, a trained study physician procured specimens using a nasal swab and an oropharyngeal swab, which were batched and tested by rt-pcr for influenza viral rna using the world health organization protocol [ ] . after index-case patients were recruited and tested for influenza virus infection, study staff accompanied index-case patients to their homes and recruited members of their compounds into the study. a staff member then administered a questionnaire to the male or female head of each household in the compound to assess demographics, socio-economic factors and individual-and household-level characteristics. the staff member observed each household for certain factors such as presence of a handwashing station with soap and water, location of cooking area, type of fuel used, water source and latrine facilities. study staff visited each household compound daily until the tenth day after resolution of the index-case patient's symptoms to conduct surveillance for ili symptoms. any member of the compound with new ili symptoms during the follow-up period was considered a secondary ili case. after consent was obtained, the secondary ili case patients were tested for influenza in the same manner as the index-case patient. written informed consent for specimen collection was obtained from all adult index-case patients and secondary ili cases. if the index-case patient or secondary ili case was a child, written informed consent for specimen collection was obtained from a parent or guardian. written informed consent was obtained from the head of the compound (typically the eldest male) for all household data collection activities. all study procedures were approved by the international centre for diarrhoeal disease research, bangladesh (icddr,b) research and ethics review committees. as few (n = ) index-case patients had laboratory-confirmed influenza in the control arm, we included all index-case patients with ili and conducted analyses to determine household-level risk factors associated with secondary ili in household members. we examined the following household-level characteristics as potential risk factors for secondary ili: crowding, building materials of homes, exposure to indoor air pollution, presence of handwashing materials, water source, latrine quality and sharing, education of the household respondent and household wealth status. crowding was assessed as number of people per room (the number of people in the household divided by the number of rooms in the home, excluding latrine and kitchen). we assessed indicators of exposure to indoor air pollution, such as frequency of index-case patients with influenza-like illness (ili) identified at hospitals, health complexes, pharmacies, tested for influenza (n = ) household compound members of index-case patients recruited, baseline questionnaire given (n = ) handwashing intervention given at repeated visits household compound members with ili tested for influenza (n = ) all household compound members followed for ili for days after resolution of index-case patient's symptoms (n = household compound members) all household compound members followed for ili for days after resolution of index-case patient's symptoms (n = household compound members) household compound members with ili tested for influenza (n = ) exclude those with missing questionnaire data from final analysis (n = ) smoking in the home, cooking fuel use, building material of the home and the distance between the cooking and sleeping spaces. we observed handwashing materials, soap and/or water at a handwashing station [ ] . we defined latrine quality as improved (flush/pour flush to piped sewer system, septic tank or pit latrine; or pit latrine with slab) or unimproved (flush/pour flush to elsewhere, open pit latrine, bucket, hanging latrine or no facility/bush/field), according to the who/unicef joint monitoring programme for water supply and sanitation. for socio-economic status, we examined education level of the household respondent and developed a wealth index using principal component analysis of household assets [ ] . we used the first principal component as our wealth index and categorised it into quintiles. we also examined each household asset that weighed on the wealth index in principal components analysis as indicators of wealth. we reported household-level factors potentially associated with ili transmission at the household and individual levels. those factors with - % variability among all households were considered for multivariable analysis. we adjusted multivariable models for age of the indexcase patient (< years, ≥ years), as previous analyses showed age to be associated with ili transmission in bistis [ ] . we examined age of the susceptible contact as a potential confounder, both as a continuous variable and defined in the following categories: very young child (less than years), young child ( - years), older child ( - years), adult ( - years) and older adult ( years and older). we examined sex and wealth status of the susceptible household contact, as well as any factors associated with risk of ili in the bivariate models (p < . ) as potential confounders. since case definition varied by age, we conducted a sensitivity analysis in which we stratified analyses by age of the index-case patient (< years, ≥ years). we also examined bivariate associations between household factors associated with secondary ili and multiple daily interactions with the index-case patient (collected in ), as this was shown in our prior study to be associated with ili [ ] . we conducted mixed-effects log-binomial regression to evaluate the relationship between household-level factors and identification of a secondary case of ili, adjusting for age of the index-case patient and the susceptible household contact, and we accounted for clustering at the household level. in order to evaluate independent associations, we adjusted models for all other householdlevel factors associated with secondary ili in bivariate analyses (p < . ). we estimated the adjusted risk ratios of developing a secondary ili case among those who lived in households with factors of interest compared with those who lived in households without the factors of interest. among susceptible household contacts of index-case patients, seven individuals ( . %) from two households were excluded due to missing data. a total of ( . %) susceptible contacts developed ili symptoms during follow-up. among household contacts included in this analysis, household members were from index-case patient households and from households in the compound other than the indexcase patient's household (table ) . houses typically consisted of one ( %) or two ( %) rooms, were made of brick or concrete ( %) and had a separate cooking space outside of the main living area ( %). almost all households cooked with biomass fuels and used tube wells for drinking water. smoking occurred in approximately % of homes. of household contacts, ( %) reported smoking; ( %) of adult men were smokers vs. ( . %) of adult women (results not shown). most ( %) household respondents had eight or fewer years of education. our wealth index accounted for % of the variance in asset ownership. a total of ( %) of the secondary ili cases lived in the index-case patient's household (table ). in our final negative binomial regression models, we evaluated the independent associations between ever smoking in the home or sharing a latrine with one other household or more than one other household, and secondary ili, adjusting for age category of the index-case patient (< , ≥ years). models examining smoking in the home were also adjusted for shared latrine use, and models examining shared latrine use were also adjusted for smoking in the home. all other models adjusted for both smoking in the home and shared latrine use. sex and age of secondary contacts were not included as model covariates, as sex was not associated with risk of secondary ili in bivariate analysis, and addition of age of the secondary contact did not substantially change model estimates. addition of further covariates resulted in unstable models. in our final models, the risk of developing secondary ili was % ( % ci . - . ) greater in those who lived in a household in which smoking ever occurred, compared with those who lived in a household with no smoking. additional adjustment for education, wealth quintile and each individual asset that weighed on the wealth measure (ownership of a chair, table, mobile phone, watch or clock, sewing machine and electricity in the home) did not substantially change the estimates of the relative risk for ili among those who lived in a household where smoking occurred compared with those who did not (rr adj between . and . ). those who lived in a household with water at a handwashing station had a % lower risk of developing secondary ili compared with those without water at a handwashing station, but this association was not statistically significant ( % ci . - . ). after adjustment, having soap and water at a handwashing station was not associated with risk of secondary ili. compared with those living in a household with a private latrine, those who lived in households sharing their latrine with one other household were at a . -fold increased risk of developing secondary ili ( % ci: . , . ) and those who shared their latrine with more than one other household had a . -fold increased risk of developing secondary ili ( % ci: . , . ). additional adjustment for education, wealth quintile and each individual asset that weighed on the wealth measure did not substantially change the estimates of the relative risk for ili among those sharing a latrine with one other household (rr adj between . and . ) or among those sharing a latrine with more than one other household (rr adj between . and . ). living in the same household as an index-case patient, crowding (number of people per room), building material of home, water source and improved latrine use were not associated with risk of secondary ili. in stratified analysis, associations between household-level risk factors and risk of secondary ili did not substantially differ by age of index-case patient. sex of the secondary contact and relationship of the secondary contact to the index-case patient were not associated with risk of developing secondary ili in this analysis or in prior bistis analyses (results not shown) [ ] . multiple interactions with the index-case patient were not associated with shared latrine use or smoking in the home (results not shown). in this study of household-level risk factors for ili, we found that smoking in the home and sharing a latrine with other households were associated with increased risk of secondary ili among household contacts. these results suggest that exposure to environmental tobacco smoke increases the risk of secondary ili; there are several potential mechanisms for the increased risk of ili due to shared latrine use. both factors are potentially modifiable. our results support exposure to indoor air pollution from environmental tobacco smoke as a potential risk factor for ili. exposure to indoor air pollution is a wellestablished risk factor for all-cause acute respiratory infections, due to its detrimental effects on respiratory tissue and immune functioning in the respiratory tract [ ] [ ] [ ] . exposure to environmental tobacco smoke is also a well-established risk factor for numerous other conditions, including low birthweight, various cancers and chronic respiratory and cardiovascular diseases [ ] . the prevalence of smoking in the home was high in this study, highlighting the need for tobacco control measures in bangladesh. greater use of effective tobacco control measures, such as taxation, could help to reduce tobacco smoking prevalence in bangladesh [ ] . the global adult tobacco survey estimated that % of adult men in bangladesh smoke tobacco products [ ] . in contrast, only . % of adult women in bangladesh smoke. our study showed a lower proportion of men who smoke ( %) compared with the global adult tobacco survey. in our study, the household head reported tobacco smoking for all members of the household; it is possible that respondents may underreport smoking habits of other household members. although biomass fuels are considered to be the major source of indoor air pollution in low-and middle-income countries [ , , ] , we were unable to assess the effect of biomass fuel use on secondary ili, as nearly every participant ( . %) reported using biomass fuels for cooking. sharing a latrine with at least one other household was the strongest exposure associated with secondary ili observed in this study. although shared latrines have not previously been shown to be associated with respiratory infections, there is some evidence of an association between shared latrines and diarrhoeal disease [ , ] . shared latrines may not be cleaned as frequently as private latrines [ ] , so it is possible that pathogens remain present longer on surfaces in shared latrines compared with private latrines. contact transmission, by either direct contact with infected fluids or indirect contact via fomites, may be an important route of transmission for influenza and other respiratory pathogens [ , ] as well as diarrhoeal pathogens [ ] . contaminated fomites in shared latrines, such as doors and traditional pots used for anal washing after defecation, may provide a route of transmission for pathogens in bangladesh. as ili may be caused by many different pathogens, it is possible that shared latrines may expose users to a number of different pathogens that may cause ili symptoms. specifically, influenza viruses [ , ] and coronaviruses [ ] have been recovered from faeces of patients, suggesting that some respiratory viruses may be transmitted through faecal contact. interactions with people with influenza have been shown to be associated with risk of secondary influenza [ ] [ ] [ ] [ ] [ ] , and ili [ ] ; it is plausible that those who use shared sanitation may have increased interactivity due to a commonly used resource (latrine). we did not observe an association between multiple daily interactions with the index-case patient and shared latrine use. however, we were unable to thoroughly investigate this possibility due to limited data. it is also possible that the association between sharing a latrine and ili may be spurious or that latrine sharing represents a proxy for an unknown factor that is associated with ili, but the effect estimates did not change substantially when adjusted for measures of wealth, age or smoking making this a less likely explanation. nearly % of household contacts reported ili in this study. this proportion is similar to previous investigations of the burden of ili in the general population of bangladesh [ ] . although age of the index-case patient did not modify the effects of household-level risk factors on ili, in this analysis and our prior analysis, ili incidence was higher in susceptible contacts who were younger than years compared with those who were years or older [ ] . residing in the index-case patient's household was not associated with ili risk, indicating that all members of a compound in a densely populated area are at risk of contracting infectious diseases from their compound members or the community at large. important limitations of this study include lack of detail regarding intravs. extra-household transmission pathways. we do not know whether pathogens were transmitted between members of the same household compound, whether they were acquired outside of the compound or whether the index-case patient we identified is in fact the primary ili case in each compound. it is possible that control households had contact with intervention households and subsequently modified handwashing behaviour. however, our main study results do not suggest an association between handwashing and secondary ili, so contact with the intervention arm is unlikely to have affected our results. as few participants had influenza, we did not test for other pathogens, and our definition of ili for those under years was broad, our results may not be relevant to influenza transmission, but rather, transmission of respiratory pathogens more broadly. air pollution is a wellestablished household-level risk factor for respiratory illness [ ] [ ] [ ] , but reliable data on concentrations of household air pollutants are not available from this study. however, we did observe associations between indoor smoking, one proxy indicator of air pollution and secondary ili incidence. as this study recruited participants from selected healthcare facilities, our sample may not be representative of people who sought care elsewhere [ , ] . in addition, our sample may not be generalisable to urban bangladesh, where there may be more crowding and more accessible health care. smoking in the home and use of shared latrines are associated with an increased risk of secondary influenza-like illness in households in this study. our data highlight the possible benefit of efforts to reduce exposure to indoor air pollution from environmental tobacco smoke, including effective approaches to smoking cessation and clean air initiatives. interventions focused on improving access to private latrines may also be helpful in low-income countries. influenza a and b infection in children in urban slum influenza is a major contributor to childhood pneumonia in a tropical developing country incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in bangladesh household transmission of pandemic (h n ) influenza-associated mortality in in 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illness influenza a/h n virus infection in humans in cambodia detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis measured dynamic social contact patterns explain the spread of h n v influenza what types of contacts are important for the spread of infections?: using contact survey data to explore european mixing patterns social contacts and mixing patterns relevant to the spread of infectious diseases social mixing patterns in rural and urban areas of southern china using data on social contacts to estimate age-specific transmission parameters for respiratory-spread infectious agents university at buffalo, school of public health and health professions first, we would like to thank the participants of bistis for their time and patience. we would like to thank our field staff for all of their hard work. this research study was funded by us centers for disease control and prevention. icddr,b acknowledges with gratitude the commitment of cdc to its research efforts. icddr,b also gratefully acknowledges the following donors who provide unrestricted support: government of the people's republic of bangladesh; global affairs canada; swedish international development cooperation agency and the department for international development. the findings and conclusions in his report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. key: cord- - zbsbcdd authors: silverman, justin d.; hupert, nathaniel; washburne, alex d. title: using influenza surveillance networks to estimate state-specific prevalence of sars-cov- in the united states date: - - journal: sci transl med doi: . /scitranslmed.abc sha: doc_id: cord_uid: zbsbcdd detection of sars-cov- infections to date has relied heavily on rt-pcr testing. however, limited test availability, high false-negative rates, and the existence of asymptomatic or sub-clinical infections have resulted in an under-counting of the true prevalence of sars-cov- . here, we show how influenza-like illness (ili) outpatient surveillance data can be used to estimate the prevalence of sars-cov- . we found a surge of non-influenza ili above the seasonal average in march and showed that this surge correlated with covid- case counts across states. if / of patients infected with sars-cov- in the us sought care, this ili surge would have corresponded to more than . million new sars-cov- infections across the us during the three-week period from march to march , . combining excess ili counts with the date of onset of community transmission in the us, we also show that the early epidemic in the us was unlikely to have been doubling slower than every days. together these results suggest a conceptual model for the covid- epidemic in the us characterized by rapid spread across the us with over % infected patients remaining undetected. we emphasize the importance of testing these findings with seroprevalence data and discuss the broader potential to use syndromic surveillance for early detection and understanding of emerging infectious diseases. the ongoing severe acute respiratory syndrome-coronavirus (sars-cov- ) pandemic continues to cause substantial morbidity and mortality around the world [ , ] . regional preparation for the pandemic requires estimating the growth rate of the epidemic, the timing of the epidemic peak, the demand for hospital resources, and the degree to which current policies may curtail the epidemic, all of which benefit from accurate estimates of the true prevalence of the virus within a population [ ] . confirmed cases are thought to be underestimates of true prevalence due to some unknown combination of patients not reporting for testing, testing not being conducted, and false-negative test results. estimating the true prevalence of sars-cov- would inform the scale of upcoming surges in hospital demand, the proportion of individuals who remain susceptible to contracting the disease, and estimates of key epidemiological parameters such as the epidemic growth rate and the fraction of infections that are subclinical. the current literature suggests that the predominant symptoms associated with covid- are fever, cough, and sore throat; that is, patients often present with an influenza-like illness (ili) yet test negative for influenza [ , ] . as covid- often presents with similar symptoms to influenza, existing surveillance networks in place for tracking influenza could be used to help track covid- . outpatient ili surveillance has proven to be a useful tool for assessing the impact of influenza [ , ] . when combined with the number of providers and patients in a given region, ili surveillance allows estimation of influenza prevalence and severity [ , , , , , , ] . studies of outpatient ili have repeatedly demonstrated that confirmed influenza case rates underestimate disease burden, likely due to preferential testing of more severe cases [ , , , ] . together these features suggest that ili surveillance could provide a crucial tool for estimating covid- prevalence within the us. here, we quantified the baseline prevalence of non-influenza ili in the us over the past years and identified a recent surge of non-influenza ili starting the first week of march, . this surge of excess ili correlated with known patterns of sars-cov- spread across states within the us yet was orders of magnitude larger than the number of confirmed covid- cases reported by the end of march. using influenza surveillance networks to estimate statespecific prevalence of sars-cov- in the united states admitted to the hospital to decrease. however, although the daily number of ili visits to emergency departments across new york city increased in march , the proportion of those patients who went on to be admitted also increased by as much as -fold compared to the baseline rate prior to march ( fig. s a ). this observation suggests that patients with mild ili presented less often to hospital emergency departments. such a decrease in care-seeking behavior for mild ili, if similar across the us, could deflate the estimated size of the ili surge in the later weeks of march by a factor of approximately . if non-ili patients were less likely to seek medical care, then we would expect that the number of patients complaining of other symptoms not typically associated with covid- (for example vomiting) would also decrease compared to prior years. in the month of march, the daily number of patients presenting with vomiting decreased by as much as a factor of compared to the baseline rate in prior years (fig . s b ). assuming that all non-ili conditions were similarly decreased during march, this would suggest that our estimates of the ili surge could be inflated by as much as a factor of . this assumption is conservative as it assumes that even individuals with severe conditions (such as severe trauma) would avoid seeking health-care in response to covid- at the same rate as those with more mild conditions such as vomiting. however, the potential -fold decreased care-seeking behavior for non-ili conditions cancels out the potential -fold decreased care-seeking behavior of mild ili, suggesting that our estimates of prevalence based on the ili surge may be insensitive to recent changes in care-seeking behavior (fig. s c). overall these estimates suggest a conceptual model in which health care utilization for both mild ili and non-ili conditions declined at similar rates as covid- increased in the us. to estimate the proportion and magnitude of the march us ili surge attributable to sars-cov- infections, we made the following three assumptions: ( ) that the patient population reported by sentinel providers is representative of their state each week; ( ) that changes in care-seeking behavior of ili patients is occurring at a similar rate as that of other non-ili patients; and ( ) that the total number of patients in the us who require medical care over the course of a year has not substantially changed since . our first assumption is common and underlies prior studies which have used ili to estimate influenza prevalence [ , ] . our second assumption is supported by our new york city analysis which suggests that both mild ili and non-ili conditions have seen similar changes in healthcare seeking behavior. our third assumption is based on the observation that the increasing need for health-care between march and march , due to covid- is likely small compared to the approximately billion outpatient encounters that occur annually [ , ] . these assumptions together with surveys describing the average number of patients seen by providers [ ] , the number of providers in each state [ ] , and the total number of outpatient visits per year [ , ] , allowed us to estimate that, if outpatient clinics remained open during the covid- epidemic, we would expect that there would have been approximately . million patient encounters with ili due to covid- between march to march , ( % credible set . million to . million). not all patients infected with sars-cov- will present to a health-care provider with ili. although we cannot directly measure the rate of such sub-clinical cases, a number of prior studies on asymptomatic rates of covid- and the careseeking behavior of ili patients in the us suggest a lowerbound on the subclinical rate of patients with ili. a recent study of passengers on the diamond princess cruise-ship accounted for a right-censoring of patients sampled and estimated that % of patients infected with sars-cov- are asymptomatic for the course of their infection ( % credible set % to %). this estimate likely represents an underestimate given that the majority of passengers were over years old, a demographic thought to have a lower asymptomatic rate than younger individuals [ ] . beyond asymptomatic individuals, a large study of adult health-care seeking behavior in the united states found that, of a random sample of over , individuals with ili, % of those went on to seek health care [ ] . together these additional contributions from sub-clinical cases correspond to a mean clinical rate of % (the overall rate at which sars-cov- cases seek medical care) and a lower bound of . million sars-cov- infections between march th and march th ( % credible set . million to . million). prevalence estimates for each state within this time-period are shown in fig. s . we define the syndromic case detection rate as the number of confirmed covid- cases in a week divided by the size of the ili surge that week. the syndromic case detection rate varied by state and over time ( fig. s ). our estimated syndromic case detection rates increased over the month of march; this was expected given increases in testing capacity across the us since the february detection of community transmission in washington state. for the week ending march , covid- cases in the states with the highest estimated syndromic case detection rate (washington, nevada, and michigan) only captured approximately % of ili surges in those states. in the last week of the month ending on march , the syndromic case detection rate across the us increased to . % ( % credible interval . %- . %). the true prevalence of sars-cov- is unknown at the time of this writing. however, if we assume the excess noninfluenza ili is almost entirely due to sars-cov- , an assumption that becomes more valid as sars-cov- becomes more prevalent, we can use the excess non-influenza ili to define lower bounds on the exponential growth rate of the us sars-cov- epidemic. by estimating the number of patients visiting clinics for covid- in the us in march, we can also identify the mutual dependence of exponential growth rates, the rate of sub-clinical infections, and the time between the onset of infectiousness and a patient reporting as ili (fig. ) . using stochastic susceptible, exposed, infectious and recovered (seir) simulations of us covid- epidemics with a january start date [ ] , we find that an initial epidemic doubling time longer than days is unlikely to explain the ili surge. doubling times longer than days fail to produce enough infected individuals to match the observed excess ili. doubling time faster than days can explain the observed excess ili with a clinical rate that depends on the growth rate. here, we define the clinical rate as the proportion of infected individuals who present to a health care provider. in keeping with our sub- day doubling times, we found that across the entire us, new deaths due to covid- doubled every . days over the month of march (± . , p-value of test that doubling rate is less than days approximately ). if there was only a -day lag from onset of infectiousness to presentation with ili and the entirety of the first week of the us ili surge is comprised of patients with covid- , then an epidemic starting january th and growing at the rate of deaths in the us would imply a % clinical rate ( fig. a) . a four-day lag between the onset of infectiousness and presentation with ili yields a clinical rate of % among the % of simulations which could account for the ili surge. the % overall clinical rate estimated from a january start date and the doubling time of us covid- deaths is in close agreement with the % clinical rate we estimated independently based on a % asymptomatic rate and % symptomatic clinical rate. although our epidemic model suggests the first week of the ili surge is consistent with the us epidemic start date and growth rate, the ili surge across the us peaked the week ending march , much earlier than our epidemic models, suggesting the epidemic in the us differed from the seir model through some combination of factors. such factors could include successful interventions, even faster decreases in care-seeking than observed in new york, heterogeneity in susceptibility [ ] , or an early epidemic doubling faster than every days. faster growth rates require lower clinical rates to explain the ili surge. epidemic curves growing at the rate of deaths in italy, doubling every . days, could better match the curvature of the ili surge by peaking around mid to late march, but would imply a clinical rate of . % the second week of march with a -day lag between onset and recorded as ili ( fig. b and c). if the entirety of the ili surge was attributable to covid- , the slowest-possible doubling time for the us epidemic which can explain the ili surge would be a doubling time of of days. any evidence of significant secondary introductions, super-spreading, or rapid transmission events in early transmission chains will decrease these estimated clinical rates [ ] . evidence of slow initial spread would increase the estimated clinical rates. last, estimating the infection fatality rate from the ili surge requires knowing the clinical rate and the delay from clinical presentation with ili to death. if patients present with ili at the onset of their illness, exhibit a day median lag between onset and death [ ] , and have a % clinical rate as estimated from the % asymptomatic rate and % clinical rate of symptomatic covid- cases, then the observed ili surge corresponds to an infection fatality rate of . %. we stress that estimating the infection fatality rate from this ili surge is highly sensitive to both the lag from presentation with ili to death and the clinical rate ( fig. s ). consequently, the ili surge is compatible with fatality rates ranging from . % to . % depending on the unknown sub-clinical rate and lag from presentation with ili to death. under the cdc planning scenarios specifying a -day lag from onset of symptoms to presentation to the doctor with ili [ ] and a day lag from onset to death, the resulting -day lag from ili to death produces ifr estimates of . % ( . - . % % credible set) for the unadjusted ili surge and . % ( . - . % % credible set) for the ili surge adjusted to account for asymptomatic and subclinical cases. we use outpatient ili surveillance data from around the us to estimate the prevalence of sars-cov- . we found a clear, anomalous surge in ili outpatients during the covid- epidemic that correlated with the progression of the epidemic in multiple states across the us. the surge of non-influenza ili outpatients was much larger than the number of confirmed case in each state, providing evidence of large numbers of probable symptomatic covid- cases that remained undetected. this result is also consistent with ili excess observed in france in late-february/early-march [ ] . additionally, this finding predicts that the slowest epidemic doubling time that could explain the ili surge would be days, and that this rate could only be achieved with unusually fast early transmission or super-spreading events and a clinical rate near %. consistent with this prediction, we found that deaths due to covid- within the us doubled every . days and note that this empirical growth rate for the us epidemic can account for the ili surge with a % clinical rate assuming a day lag from the onset of infectiousness to presentation as an outpatient with ili. together, these results suggest that sars-cov- spread rapidly throughout the us since its january th start date and was likely accompanied by a large undiagnosed population of potential covid- outpatients with presumably milder distribution of clinical symptoms than estimated from prior studies of sars-cov- + inpatients. excess ili appears to have peaked during the week starting on march th, leading the observed ili dynamics to diverge from the overall epidemic dynamics implied by the growth rate of covid- deaths in the us. if the ili dynamics were proportional to the epidemic curve then the two could be related via a constant subclinical rate. however, the changing ratio between covid- prevalence estimated by the ili surge and the epidemic curves parameterized by the growth rate of us deaths suggests additional mechanisms may be behind the ili slowdown. mechanisms which can explain the difference between our simulated epidemic curves and the ili surge include effective social distancing, disproportionate reductions in ili care-seeking behavior relative to non-ili care-seeking behavior, or heterogeneity in susceptibility or contact structure not captured in our seir model [ ] . our empirical estimate of the size of the ili surge has several potential limitations. first, the observed ili surge may represent more than just sars-cov- infected patients. a second epidemic of a non-seasonal pathogen that presents with ili could confound our estimates of ili due to sars-cov- . however, this seems unlikely as additional viral surveillance through the us centers for disease control and prevention (cdc) suggests that between march to march other monitored respiratory viruses were at low prevalence [ ] . nonetheless, were our approach to be used during winter months, additional steps would be needed to account for concomitant non-influenza seasonal pathogens. additionally, our assumption that changes in health-care seeking behavior are similar between mild ili and non-ili condition may be incorrect. although this assumption was supported by new york city emergency department surveillance data, it is possible that differential health-care seeking would be present in other locations or in the outpatient setting. last, it is also possible that our use of ili data has underestimated the prevalence of sars-cov- within the us. although early clinical reports focused on cough and fever as the dominant features of covid- [ ] , other reports have documented digestive symptoms as the complaint affecting up to half of patients with laboratory-confirmed covid- [ ] , and alternative presentations, including asymptomatic or unnoticeable infections, could result in underestimation of sars-cov- prevalence. additionally, our models have several limitations. first, we assumed that ili prevalence within states can be scaled to case counts at the state level. this is based on the assumption that the average number of cases seen by sentinel providers in a given week is representative of the average number of patients seen by all providers within that state in a given week. errors in this assumption would cause proportional errors in our estimated case counts and syndromic case detection rate. second, our us-wide seir models vary by growth rate alone and as such may not capture important heterogeneity in susceptibility or transmission as well as regional variation, intervention-induced changes in transmission, or clustering of infection outbreaks. our models were used to illustrate that the ili surge is consistent with an estimated growth rate and start date for the us epidemic and to specify the mutual dependency of growth rate, the lag between the onset of infection and presentation to a doctor, and clinical rates. finer models with regional demographic and case-severity compartments are needed to translate our range of estimated prevalence, growth rate, and clinical rates into actionable models for public health managers. last, our method of calculating the infection fatality rate relied on assumptions about the clinical rate and the delay from patients recorded as ili to death. our clinical rate required using patterns of care-seeking for typical seasonal causes of ili as did our delay from ili to death; consequently, neither should be relied on as a definitive source for covdi- and estimating the clinical rate and delay from ili to death for covid- specifically will reduce the large uncertainty around our iliestimated infection fatality rates. despite these potential limitations, the ili surge identified in syndromic surveillance time-series allowed early estimates of covid- prevalence, estimates that were not possible from confirmed case data due to early logistical delays in sars-cov- testing in the us. our prevalence estimates are supported by a serosurvey conducted in new york state. we estimated that over . % of new york state residents were infected by sars-cov- by march ; on april , , new york state announced that % of residents had evidence of past infection by sars-cov- by march at which time the cumulative pcr-confirmed case counts totaled only . % of new york's population [ ] . although an ili surge tightly correlated with covid- case counts across the us and consistent with the new york state serology strongly suggests that sars-cov- has potentially infected millions in the us, further laboratory confirmation of our hypotheses are still needed to guide public health decisions. our findings make testable predictions that one would find relatively high seroprevalence in other states that have already seen an ili surge and that seroprevalence of individuals infected in march across states is proportional to relative sizes of the states' ili surges. a study of ili patients from mid-march who were never diagnosed with covid- could produce a focused test of our predictions about the number and regional prevalence of undetected covid- cases presenting with ili during that time. if seroprevalence estimates beyond new york state continue to corroborate our prevalence estimates from syndromic surveillance, this would strongly suggest lower case severity rates for covid- than were assumed in late march by comparing pcr-confirmed case counts to deaths. further corroboration of our estimates of the magnitude of the ili surge would suggest ili and other public time-series of outpatient illness allow early and reliable estimates of crucial epidemiological parameters for rapidly unfolding, novel pandemic diseases. as not all novel pandemic diseases are expected to present with influenza-like symptoms, surveillance of other illnesses that commonly present in the outpatient setting could provide a vital tool for rapidly understanding and responding to novel infectious diseases. the goal of our study was to use publicly available data to estimate the number of patients seeking care for non-influenza ili in excess of seasonal trends during the three weeks spanning march to march , and then use this ili surge to estimate covid- incidence in march and parameterize epidemiological model growth rates and clinical rates. the ili surge detection above produced an excess proportion of patients visiting outpatient providers for non-influenza ili in each week and each state. to scale up the proportion of patients to a national number of covid- cases, we estimated the number of patients per sentinel provider in the cdc dataset, normalized that number of patients per provider to a number of patients per doctor, and scaled that up by an estimated number of practicing doctors in the us. the result was an estimated number of covid- patients visiting doctors in each state for each week -we called this our "unadjusted" ili surge. the unadjusted ili surge is an under-estimate of covid- prevalence due to only clinical infections, those that seek medical care. we accounted for both asymptomatic infections and symptomatic but sub-clinical infections to produce an "adjusted" ili surge as our final estimate of covid- incidence in each state and each week. we then used the unadjusted and adjusted ili surges to estimate syndromic case detection and fatality rates. we also used the unadjusted ili surge as an empirical observation to evaluate epidemiological modelling of covid- growth rates and clinical rates in the us. throughout our methods, we use i to denote the index state i and let t index week t (with t= referring to october , ; the start of state-specific ilinet surveillance). since the cdc has maintained ilinet for weekly influenza surveillance. each week approximately , enrolled providers distributed throughout all states as well within the ilinet dataset, new york city and new york were summed into a combined new york variable representing both new york city and the surrounding state. due to incomplete data in one or more of the data-sources described above the virgin islands, puerto rico, the commonwealth of the northern mariana islands, and florida were excluded from subsequent analysis. in addition, to match the weekly reporting of ili from ilinet, daily cumulative confirmed covid- cases were converted to weekly counts of new cases by ( ) to subtract influenza signal from it y we assumed that the population of patients with ili within a state are the same population that are potentially tested for influenza. this assumption allows us to calculate the number of non-influenza ili cases as mean imputation based on neighboring states was used to address missing values in laboratory influenza quantification. to assess the impact of this model for extracting noninfluenza ili signal, we calculated covid- prevalence without first removing signal from influenza, we found little change in our prevalence estimates ( fig. s ). this likely reflects that influenza also demonstrates strong seasonal patterns that can be addressed as discussed below. we to account for variation in the number of total patients, we modeled it y as binomial distributed. to account for correlation in non-influenza ili over time, we use a gaussian process model which assumes that weeks that are closer together will have more similar levels of non-influenza ili. the following model reflects these modeling choices: where  refers to a gaussian process. we made the following prior specifications: we set the bandwidth parameter for the squared exponential kernel as ρ= representing a strong local correlation in time that died off sharply beyond weeks, α= representing a signal to noise ratio of approximately , ν= and ξ= representing weak prior knowledge regarding the overall scale of variation in the latent space. collected using the function basset from the r package stray [ ] ; a total of such samples were collected, for each state, in this analysis. we defined the prevalence of non-influenza ili in excess of normal seasonal variation as to investigate whether our results were sensitive to the above model specification, we alternatively used the sample mean and variance from years - as an estimate of typical seasonal non-influenza ili. despite not accounting for the binomial count structure of ili data or correlations in the proportion ili between weeks, this simpler model resulted in nearly identical prevalence estimates ( fig. s ). still, we used the gp-derived estimates throughout this paper due to their better accounting for the known binomial count and week-to-week correlation structure of ili-causing pathogen prevalence. to exclude variation attributable to unseasonably high rates of other ili causing viruses (such as the outbreak of rsv in washington state in november-december ) we only investigated * it y for weeks after march th as only these later weeks had high correlation to the covid- confirmed case rate ( fig. s ). as new covid- case counts it z  represent the number of confirmed cases in an entire state and ilinet data represents the number of cases seen by a select number of enrolled providers, we had to estimate scaling factors i w to enable comparison of ilinet data to confirmed case counts at the state level. let * it π denote the probability that a patient with ili in state i has covid- as estimated from ilinet data. let i p denote the population of state i and let i b denote the number of primary care providers per , people in state i. we translated the inferred proportion of individuals with ili due to covid- to the state level by considering the average number of patients seen across all providers in the state in a -day work-week. in addition, we added a discount factor λ= . to calibrate these estimates with prior reports regarding the total number of outpatient visits per year [ ] . this yielded our estimated number of covid- cases (excess ili at the state level) as where m= . is the mean number of patients seen by physicians per day [ ] . to account for the contribution of sub-clinical sars-cov- infections we used a recent analysis of cohort surveillance from the diamond princess [ ] . monte-carlo simulations were used to propagate error from our uncertainty regarding potential asymptomatic infections affecting the clinical rate assuming that the majority of sars-cov- testing within the us has been directed by patient symptoms [ ] , the pool of newly diagnosed sars-cov- + patients is a subset of the pool of sars-cov- + patients who are identified as having ili. therefore, we calculated the probability that a sars-cov- + patient with ili who seeks medical care will be iden- the exact lag from an outpatient being recorded as ili to death is unknown, but estimated lag times from onset to death and from hospitalization to death [ ] can be used to understand the range of implied infection fatality rates from the ili surge. we calculated the infection fatality rate implied by the ili surge as a function of the unknown lag from patients being recorded as ili and death, and we repeat this calculation for both the raw and subclinical rate adjusted ili estimates. for a lag of l days from ili reporting to death, the infection fatality rate was estimated by dividing the magnitude of the adjusted or raw ili surge by all new deaths occurring within the dates ( / / + l, ..., / / + l). a plot of the fatality rate by lag for raw and unadjusted ili surges revealed a large range of fatality rates compatible with the ili surge and highly sensitive to the estimate of lag and clinical rates. one study [ ] estimated a median . days from hospitalization to death and . days from symptom onset to death. for the raw ili surge estimate, day and day lag times would produce median infection fatality rate estimates of . % and . %, respectively, without adjusting for any subclinical infections; for the subclinical-adjusted ili surge estimate, these lag times would produce median infection fatality rate estimates of . % and . %, respectively. as of april , , deaths from sars-cov- epidemic were still growing nearly exponentially as evidenced by nearly linear growth on a log y axis. early in the epidemic, estimating exponential growth rates by poisson regression with a log link function produces accurate estimates of the true growth rate [ ] , and so we estimated growth rates for the us and italy by poisson generalized linear models predicting new deaths using date as a quantitative explanatory variable. us covid- deaths from march , to april , , were summed by date to calculate national-level statistics. initially, april - were included but were found to have anomalously high leverage and were hence excluded from our analysis. we applied the same procedure to covid- deaths in italy, focusing on deaths from february until march . we used the slope from poisson regression as the estimated exponential growth rate, which yielded a us growth rate of the following seir models [ , ] combined with persistence of high loads of sars-cov- that can be cultured up to days after symptom onset [ ] , resulting in our use of a . - day % credible interval for the infectious period. finally, we parameterized β to ensure i(t) grew with a specified exponential growth rate early in the epidemic. we ran a total of , simulations for each of the two growth rate distributions (us and italy) analyzed. growth rates were drawn at random from a normal distribution with standard deviation of . figure s . excess ili for each us state. figure s . excess ili correlates strongly with patterns of newly confirmed covid- cases. figure s . surveillance data from new york city emergency departments. figure s : prevalence of sars-cov- infections between march and march , . figure s . syndromic case detection rates by state. figure s : estimating the infection fatality rate (ifr) of covid- based on the unadjusted ili surge. figure s : investigating model sensitivity when ili is modeled without first removing signal from influenza. figure s . investigating model sensitivity when seasonal trends in non-influenza ili are identified using an alternative statistical model. . an early surge of ili visits across the us. the proportion of patients presenting with ili that could not be explained by influenza or typical seasonal variation (that is, excess ili) is shown for four states (blue line and ribbons represent the posterior median as well as % and % credible sets; results from all analyzed states are shown in fig. s ). ili that could not be attributed to influenza was calculated based on influenza laboratory surveillance data ( - flu season shown in red, prior seasons are shown in black). a time-series model was used to infer seasonal variation of non-influenza ili. excess ili was then calculated as the difference between non-influenza ili from - and the seasonal baseline of non-influenza ili. excess ili after march th is highlighted in darker blue as these data correlated strongly with observed covid- case counts ( fig. s ). . epidemiological models were either stochastic (simulated via tau-leaping) or deterministic (solved by numerical integration). in addition to our raw estimates of the ili surge size (unadjusted), we provide adjusted prevalence estimates accounting for sub-clinical cases by assuming an % asymptomatic rate and a % rate of health-care seeking of symptomatic ili patients (adjusted). epidemic trajectories were simulated using an seir model (black lines). the increasing gap between ili prevalence estimates and seir trajectories (orange) suggest the presence of additional factors such social distancing, changes in care-seeking behavior, or heterogeneity in susceptibility or transmission. (c) more generally, the size of the clinical population estimated from ili data imposes a dependence between epidemic doubling time, the clinical rate, and the lag between onset of infectiousness and ili reporting. combinations of these three variables that are consistent (black) or inconsistent (gray) are shown as well as a smoothed estimate of clinical rate as a function of doubling time. china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china world health organization fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the sars-cov- epidemic epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in surveillance for influenza-united states, - , - , and - seasons update: influenza activity in the united states during the - season and composition of the - influenza vaccine do family physicians make good sentinels for influenza? spanish influenza surveillance system, estimating the burden of seasonal influenza in spain from surveillance of mild and severe influenza disease estimating the burden of influenza-associated hospitalizations and deaths in chile during - burden of influenza-associated outpatient influenza-like illness consultations in china detection of excess influenza severity: associating respiratory hospitalization and mortality data with reports of influenza-like illness by primary care physicians influenza surveillance in new zealand in estimating influenza disease burden from population-based surveillance data in the united states national ambulatory medical care survey: national summary tables united health foundation, america's health rankings analysis of special data request for information on active state licensed physicians the u.s. health system in perspective: a comparison of twelve industrialized nations estimating clinical severity of covid- from the transmission dynamics in wuhan, china self-reported influenza-like illness during the h n influenza pandemic-united states washington state -ncov case investigation team, first case of novel coronavirus in the united states epidemic size and probability in populations with heterogeneous infectivity and susceptibility superspreading and the effect of individual variation on disease emergence high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus . emerg influenza-like illness, the time to seek healthcare, and influenza antiviral receipt during the - influenza season-united states excess cases of influenza-like illnesses synchronous with coronavirus disease (covid- ) epidemic, france clinical characteristics of covid- patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study cumulative incidence and diagnosis of sars-cov- infection in new york bayesian multinomial logistic normal models through marginally latent matrix-t processes estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship coronavirus test: what you need to know estimating initial epidemic growth rates incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application presymptomatic transmission of sars-cov- -singapore sars-cov- viral load in upper respiratory specimens of infected patients temporal dynamics in viral shedding and transmissibility of covid- virological assessment of hospitalized patients with covid- competing interests: nh and jds declare that they have no competing interests. adw owns selva analytics llc. data and materials availability: all data associated with this study can be found in the paper or supplementary materials key: cord- -fdosch authors: nuvey, francis sena; edu-quansah, elijah paa; kuma, george khumalo; eleeza, john; kenu, ernest; sackey, samuel; ameme, donne; abakar, mahamat fayiz; kreppel, katharina; ngandolo, richard bongo; afari, edwin; bonfoh, bassirou title: evaluation of the sentinel surveillance system for influenza-like illnesses in the greater accra region, ghana, date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: fdosch background: influenza-like illness (ili) is a medical diagnosis of possible influenza or another respiratory illness with a common set of symptoms. the deaths of four schoolchildren, during a pandemic influenza outbreak in december in ghana, raised doubts about the ili surveillance system’s performance. we evaluated the ili surveillance system in the greater accra region, ghana, to assess the system’s attributes and its performance on set objectives. methods: cdc guidelines were used to evaluate the data of the ili surveillance system between and . we interviewed the surveillance personnel on the system’s description and operation. additionally, routinely entered ili data from the national influenza center provided by the six sentinel sites in accra was extracted. we sampled and reviewed ili case-investigation forms from these sites. surveillance activities were examined on system’s performance indicators, each being scored on a scale of to (poorest to best performance). results: all population and age groups were under ili surveillance over the period evaluated. overall, suspected case-patients, including ( . %) children under-five were reported, with being positive for influenza virus (predictive value positive = . %). the predominant influenza subtype was h n , recorded in ( . %) of positive case-patients. the system only met two out of its four objectives. none of the six sentinel sites consistently met their annual suspected case-detection quota. samples reached the laboratory on average hours after collection and results were disseminated within days. of case-investigation forms sampled, ( . %) were completely filled in. conclusions: the ili surveillance system in the greater accra region is only partially meeting its objectives. while it is found to be sensitive, representative and timely, the data quality was sub-optimal. we recommend the determination of thresholds for alert and outbreak detection and ensuring that sentinel sites meet their weekly case-detection targets. introduction influenza-like illnesses (ili), often also called acute respiratory infection or flu-like syndrome, are acute viral infections of the respiratory tract with similar signs and symptoms to influenza. ili is a syndrome and affected persons may become infectious before, during or after the onset of symptoms. the pathogen can be transmitted both directly (by droplets) and indirectly through contact with contaminated fomites. children, the elderly and pregnant women, as well as persons with chronic illnesses or immunosuppression are at the highest risk for morbidity and mortality from ilis [ , ] . according to the world health organization (who), most influenzas in the global circulation are of zoonotic origin. sub-types implicated in epidemics include h n , h n , h n , h n and h n [ ] . influenza has a global annual attack rate of - % in adults and - % in children, causing between - million cases of severe illness and about , deaths yearly [ ] . pandemics of influenza have had high fatality rates in the past and robust surveillance systems are key to global efforts to prevent similar outbreaks [ ] . due to the epidemic-prone nature of influenza pathogens and their high propensity for mutations, the world health organization (who) recommends strict adherence to infection control and prevention measures including increased handwashing during peak flu seasons [ ] . in ghana, laboratory-based surveillance for respiratory tract infections (rtis) only tests for influenza in suspected cases. rtis remain a major cause of morbidity and mortality in ghana ranking second among the top diseases seen at outpatient departments (opds) in healthcare facilities across the country, in [ ] . respiratory diseases may occur because of an invasion of a susceptible host by microbes including bacteria and viruses. three main types of influenza viruses exist, namely; influenza a, b, and c but epidemics are often linked to the influenza a strain [ ] . influenza surveillance data between and in ghana indicated positive influenza cases out of a total respiratory samples tested, with different subtypes [influenza a (h , h n , h , h ) and influenza b (victoria, yamagata)] identified [ ] . ghana began influenza surveillance in to obtain data on strains in circulation. the national influenza center (nic) was formally recognized by the who in after the pandemic influenza [ ] . the nic is a member of the who global influenza surveillance and response system (gisrs) and is located in the department of virology of the noguchi memorial institute of medical research (nmimr). in collaboration with the ghana health service (ghs) and the ministry of defense (mod), it currently operates sentinel surveillance for influenza in sites across all regions in ghana with support from the u.s. naval medical research unit no. (namru- ), centers for disease control and prevention (cdc) and who [ ] . ili surveillance is conducted all year round across the sentinel sites. the ili surveillance system aims to detect early unusual events indicating a change in circulating influenza sub-types, identify and monitor vulnerable groups for influenza, determine influenza thresholds and detect antiviral resistance. in december , an outbreak of influenza in a school in the ashanti region of ghana was followed by the death of four children (case fatality rate = . %) [ ] . this raised concerns about the effectiveness of the influenza surveillance system, particularly that in the greater accra region of ghana, which recorded no alerts over the past five years. we evaluated the effectiveness of the ili sentinel surveillance system to determine if its objectives are being met and to assess its attributes and usefulness. the findings of this study are important in order to give useful recommendations to improve the current system. we described the attributes and effectiveness of the ili sentinel surveillance system in the greater accra region (gar) of ghana using a descriptive cross-sectional survey. we extracted and evaluated routinely recorded ili data between january and december . the gar is one of ten regional demarcations in ghana with a population of about million people [ ] . as the most densely populated region in ghana, it is mainly an urban settlement and lies in the southeastern part of the country along the coast of the gulf of guinea. it is the administrative capital of ghana with over public and private health care facilities. six of these facilities in the region conduct ili surveillance including four ghs facilities, one military and one quasi-government facility (see fig ) . they fall under the three main levels of healthcare delivery in ghana; primary, secondary and tertiary. the manhean health center provides primary health services, while secondary healthcare providers involved in ili surveillance are tema polyclinic, achimota hospital and university of ghana hospital, legon. lastly, the greater accra regional hospital (garh) and military hospital provide tertiary care. data on ili from all six sentinel facilities in gar was extracted and abstracted from the nic database into microsoft office excel format. additionally, we selected three sentinel facilities, based on the different levels of care they provided, and obtained permission for site visits. these sentinel sites; garh, achimota hospital and manhean health center, were visited for at least one week each and their records were collected and reviewed. the researchers interviewed all personnel directly involved in ili surveillance and also partook in surveillance activities while observing practices. the staff members were interviewed using a structured questionnaire and guided interviews on their knowledge on ili surveillance activities. forty case investigation forms were randomly sampled from each of the three facilities, using microsoft excel. the unique serial numbers on case investigation forms for each sentinel site were used to retrieve the randomized forms for examination. in addition, we collected and reviewed ili registers at the sites. the evaluation was conducted using the cdcs guidelines for evaluating public health surveillance systems [ ] . nine attributes: simplicity, flexibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness and stability, usefulness and the utility of the system to achieve its objectives were evaluated. the indicators for each attribute or characteristic measured were scored one point each. based on the evaluator's assessment, an indicator is scored zero ( ) if the key finding from evaluation do not support the indicator assessed in relation to the attribute. a score of one ( ) is given to each indicator that supports the attribute assessed. the assessment scores were then summed and divided by the total number of indicators used in evaluating each attribute. attributes with relative scores of more than twothirds of the total score, were considered as major strengths of the system and scored overall. those with less than one-third were major weaknesses (overall score = ). scores between one-third and two-thirds were relative strengths (overall score = ). scores were on a scale of to : poorest to best performance, respectively. the evaluation was done within the framework of integrated disease surveillance and response matrix implemented by the ghana health service and therefore did not have to receive formal review by ethical review committees. the field epidemiology and laboratory training programme and national influenza center in ghana approved the study. permission was sought and obtained from the public health directorate of ghana health service and authorities in the sentinel facilities before commencement of the evaluation. all respondents provided informed, written consent and were assured of confidentiality. the surveillance system was found to be utilizing the syndromic approach by screening suspected cases and thereafter conducting further laboratory confirmatory tests on collected nasopharyngeal or oropharyngeal specimen. a reverse transcriptase-polymerase chain reaction (rrt-pcr) is used to confirm real influenza cases from among those suspected using the influenza case definition and determine the influenza virus sub-type. we found that data on patients meeting the ili case definition (s table) from the sentinel sites are collected together with nasopharyngeal or oropharyngeal specimen. specimen are stored in sample bottles with virus transport media (vtm) in a cold chain system and submitted to nmimr for laboratory confirmation. each site is required to suspect five cases weekly using the case definition. thus, the expected annual total for each site is suspected cases. firstly, clinicians at the opds suspect cases and trained personnel (i.e. nurses, laboratory scientists and surveillance officers) collect the specimen for storage in designated refrigerators at the disease control units. case detection is, in most cases, directly done by ili surveillance team members at the opds. socio-demographic, epidemiologic and clinical data are collected on each case using case investigation forms. the nic supplies the sentinel sites with the tools including case investigation forms, vtm, and specimen bottles, for the system's operation. they contact the sentinel sites via phone calls and visit the sentinel sites in the gar at least twice every week to pick up specimen. the nic reports results from the polymerase chain reaction laboratory test to each sentinel site, the national surveillance department (nsd), and who via e-mail. data is entered into the flu-net system, a who (gisrs) global database specifically designed for influenza surveillance, weekly. sentinel sites receive printed laboratory results from the nic averagely every days. at the facility level, health information units (hiu) enter the data into the district health information management system (dhims ). ili data entry into dhims started in . the ghs, nic and other stakeholders periodically hold review meetings to discuss influenza activities and publish the data generated by the system in reports and journals. the information and data flow in the ili surveillance system is further illustrated in fig . of the suspected cases tested, ( . %) cases were tested positive for influenza viruses. s table) . utility of the ili sentinel surveillance system in attaining set objectives. the ili surveillance system has four objectives: early detect events that show change in severity or patterns of influenza infection or emergence of new strains, determine influenza thresholds, identify and monitor at risk groups and detect antiviral resistance in circulating strains. the system met two out of its four objectives over the period evaluated. it was able to detect and characterize influenza viruses in circulation, as from influenza a and b lineages, and established the groups of persons most at risk of influenza infection. it however did not set thresholds, which is the minimum number of suspected cases above which the system is alerted, and was not performing antiviral resistance testing. surveillance system attributes. usefulness: data generated by the system informed the choice of vaccines used in controlling influenza outbreaks in ghana. in addition, who and cdc use the system's information to monitor influenza activity globally, through sharing of confirmed influenza samples. tables and show the key findings on the indicators and scores for all the qualitative and quantitative attributes evaluated respectively. sensitivity: the ili system in gar has been able to confirm influenza cases in each of the years evaluated (s table) . evidence of aggregate data from ghana can also be found in the who flunet system, available free at http://apps.who.int/flumart/default?reportno= . predictive value positive (pvp): the predictive power of the ili case definition is tested using rrt-pcr. overall pvp for the period was . % (range . %- . %). see s table for yearly pvps over the evaluated period. simplicity: surveillance personnel interviewed identified case definition to be simple. only opds partake in ili surveillance and follow-up of confirmed cases done within days. however, specialized training is required for specimen collection and laboratory confirmation, data collected on each suspected case is comprehensive and laboratory testing is not done at sentinel sites. flexibility: modification of the ili system in , enabled detection of severe acute respiratory infections (sari) with no difficulty. furthermore, addition of other common respiratory symptoms to case definition at the sites did not disrupt the system. data quality: key surveillance information was completely provided in % ( / ) of case investigation forms sampled and data extracts were comparable at sentinel facilities and the nic. in spite of this, only two sites: garh and achimota hospital, had influenza data table entered in the dhims- platform and laboratory results were not entered in ili registers at the three sites. acceptability: on average, five out of the six sentinel sites detect influenza cases each year ( %) and about % of case investigation forms sampled were completely filled out by ili personnel. all sentinel staff interviewed reported satisfaction with feedback from nic and test results from collected specimen are on average tested within days. most of the sites ( / ) however, do not meet the case detection quota for each year with some recording as low as three suspected cases per year only (see fig ) . representativeness: age and sex distribution of total cases detected reflect the general distribution of ghana's population. the median age of cases was years (range: weeks to years). females constituted % ( / ) of cases. timeliness: it takes on average days between symptom onset and detection at facilities. the majority of specimens are tested within hours after collection and results are disseminated within days. this conforms with the who set standard timelines for influenza surveillance. stability: data flow in the system conforms to set standards; testing is routinely done within hours after case detection and results are released within days to the sites on average. the system is sustainable with donors mainly funding its operation. there was not a time influenza surveillance in the greater accra region, ghana during the period evaluated when no site recorded cases, but all sites detected cases only for % of the period. influenza causes considerable morbidity and about , deaths per year globally. the disease is highly infectious with high pandemic potential. therefore, worldwide surveillance systems to record influenza-like illnesses (ili) in real time and detect possible influenza outbreaks are essential to prevent and control epidemics. ghana's ili surveillance system since its inception in , aims to early detect changes in circulating influenza, identify vulnerable groups to influenza infection, determine influenza thresholds and detect antiviral resistance to influenza viruses. the late detection of a pandemic influenza outbreak in ghana raised doubts about the ili surveillance system's performance. our study provides evidence, that the ili sentinel surveillance system in the greater accra region (gar), ghana, is only partially meeting its objectives because it did not have thresholds for alerting the health system and does not perform antiviral resistance testing. it is sensitive and timely in detecting influenza cases but of low predictive value positive (pvp). it is representative of the population under surveillance and flexible to modifications. the system is fairly stable and acceptable to key stakeholders; the quality of data is relatively high. predominant influenza subtype in circulation is influenza a (h n ) virus. the sentinel sites consistently failed to meet their case detection quotas annually over the period evaluated. despite these apparent weaknesses in the ili system, the good performance of the laboratory component is commendable and key to detection of novel influenza viruses for prompt response. even though the who's standards for influenza surveillance alluded to the possibility of resource limitation hindering achievement of all influenza surveillance system objectives, it advocates for influenza surveillance systems capable of collecting the minimum amount of data needed for decision making [ ] . for the past two decades of influenza surveillance in ghana, the system proved its utility by its ability to detect and classify circulating strains as well as providing key information for public health action. similar findings were made in the south african [ ] and madagascar [ ] influenza surveillance systems. even though the pvp is low, it generally conforms to other syndromic surveillance systems with broad case definitions but specific for respiratory diseases in this case, aimed at maximizing influenza case detection [ ] . nevertheless, the unmet objectives, lack of thresholds and antiviral resistance testing, of the system require attention. the lack of antiviral resistance testing to detect the emergence of treatment resistant strains and the absence of thresholds preventing the issuing of alerts, are a major drawback. this situation is not specific to the gar alone but is found in all sites in ghana. in addition, the mainly conservative management of clinical signs and symptoms, of confirmed influenza cases in the health system, without the use of recommended antiviral agents, may partly explain the absence of antiviral susceptibility testing. these shortfalls may, however, be caused by a lack of capacity or resources (antiviral drugs and laboratory reagents) at the sentinel facilities to determine thresholds for influenza alerts and test for antiviral resistance, as was observed in evaluations done in other settings [ , ] . threshold establishment for disease surveillance is paramount to be able to alert the health system early when outbreaks occur for prompt public health action [ ] . a study to test the moving epidemic method in determining thresholds for ili and sari surveillance systems in europe, was able to detect epidemic periods with few or no false alarms in different countries [ ] . other methods employed in cambodia [ ] and australia [ ] had similar findings. the nic must take the lead using the who manual [ ] , to choose an epidemic threshold determination method and train focal persons in the facilities to use it, to ensure this key information is available. even though implementing an integrated (human and animal) surveillance could be costeffective and improve case detection and response [ ] , discussions with ili surveillance personnel at sentinel sites revealed the absence of a link between influenza surveillance in humans and animals. human infection with zoonotic influenza strains is possible and may cause mild to severe form of the disease. there have been recorded episodes of pandemics in humans over a century due to cross-species transmission of zoonotic respiratory viruses including influenza viruses, notably spanish flu ( ), severe acute respiratory syndrome ( ) and swine flu ( ), resulting in high morbidity and mortality globally [ , ] . thus, the importance of a one-health approach in the surveillance and response with control of pandemics in an increasingly globalized world is evident. research findings in africa, europe and the americas, have shown incremental gains derived when interventions are integrated between human and animal health systems [ ] . the nic and veterinary service department in ghana should collaborate to formalize protocols for engaging each other to integrate influenza surveillance systems in humans and animals, using a one health approach, as this can provide additional key information on possible cross-species transmission in the country and enhance savings. the usage of one laboratory to test for both human and animal pathogens in canada was shown to cost about % less than the combined original operational costs of testing individually in both laboratories [ ] . in ghana, the timeliness of the system in case detection, laboratory confirmation and result dissemination is commendable. this strength may be as result of the weekly visits nic makes to sentinel sites to collect specimen, supply virus transport media in specimen bottles and transfer results. the nic should further take advantage of these visits and collaborative meetings to ensure each site meets weekly case detection quotas. focal persons at the various sites must take the lead. in spite of the usefulness and fair performance of ili surveillance in gar on indicators evaluated, it is only partially meeting its set objectives. it is sensitive in detecting circulating influenza types, representative of the population under surveillance and timely. however, sentinel sites do not consistently meet annual case detection quotas. there is the need to address shortfalls in the system's objectives as well as improving case detection at sentinel facilities. this would ensure that the successes chocked are not undermined, thereby preventing increasing morbidity and mortality related to influenza infections. failure of the system to address these shortfalls would also affect ghana's contribution to the who global influenza surveillance and response system. considering the zoonotic character of most influenza viruses, it is important that a one health approach is adopted with influenza surveillance in ghana. we propose strict adherence to case detection targets by individual sentinel sites and determination of alert thresholds for the system to allow for a more effective monitoring of influenza activity in the region for prompt public health actions. supporting information s table. ili case definitions used for screening and enrolment by the ili sentinel surveillance technical guidelines for integrated disease surveillance and response in the african region world health organization. community case management during an influenza outbreak: participant's handbook influenza at the human-animal interface; sumary and assessment influenza (seasonal) fact sheet [internet]. who. world health organization world health organization. global epidemiological surveillance standards for influenza virological surveillance of influenza-like illness among children in ghana ghana health service/ministry of health. interim report: surveillance response to influenza type a (h n ) outbreak in kumasi academy, asokore -mampong municipality population projections by districts updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group evaluation of two influenza surveillance systems in south africa evaluation of the influenza sentinel surveillance system in madagascar overview of syndromic surveillance: what is syndromic surveillance? morb mortal wkly rep strategy to enhance influenza surveillance worldwide a summary of influenza surveillance systems in australia influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method establishing seasonal and alert influenza thresholds in cambodia using the who method: implications for effective utilization of influenza surveillance in the tropics and subtropics exploring a proposed who method to determine thresholds for seasonal influenza surveillance world health organization. who interim global epidemiological surveillance standards for influenza creating a framework towards integrated health syndromic surveillance and response in africa global burden of influenza: contributions from resource limited and low-income settings sars, the first pandemic of the st century one health: the theory and practice of integrated health approaches. croydon: cab international the world bank. people, pathogens and our planet: towards a one health approach for controlling zoonotic diseases we acknowledge support received from the national influenza center and noguchi memorial institute of medical research, and afrique one alliance. in addition, we would like to thank the physicians, nurses, surveillance officers, laboratory scientists and health information officers who contribute to the influenza-like illnesses sentinel surveillance system in the greater accra region of ghana, as well as the staff and cohort residents of the ghana field epidemiology and laboratory program. key: cord- -wln og authors: alqahtani, amani salem; tashani, mohamed; heywood, anita elizabeth; almohammed, abdulrahman bader s.; booy, robert; wiley, kerrie elizabeth; rashid, harunor title: tracking australian hajj pilgrims’ health behavior before, during and after hajj, and the effective use of preventive measures in reducing hajj-related illness: a cohort study date: - - journal: pharmacy (basel) doi: . /pharmacy sha: doc_id: cord_uid: wln og this study assessed australian hajj pilgrims’ knowledge, attitude and practices throughout their hajj journey to understand their health behaviors, use of preventative measures and development of illness symptoms. a prospective cohort study with data collection at three phases (before, during and after hajj) was conducted among australian pilgrims between august and december . baseline data were collected from pilgrims before hajj, with providing follow-up data during hajj and after their home return. most participants ( % [ / ]) received one or more recommended vaccines; travel agents’ advice was the main factor affecting vaccination uptake. most participants ( % [ / ]) practiced hand hygiene with soap and sanitizers frequently, followed by disposable handkerchief use ( % [ / ]) and washing hands with water only ( % [ / ]). during hajj % ( / ) of participants reported one or more illness symptoms, % ( / ) of these symptoms were respiratory. cough was less often reported among pilgrims who received vaccinations, cleaned their hands with soap or alcoholic hand rubs, while a runny nose was less common among those who frequently washed their hands with plain water but was more common among those who used facemasks. this study reveals that most australian hajj pilgrims complied with key preventative measures, and that tour group operators’ advice played an important role in compliance. pilgrims who were vaccinated and practiced hand hygiene were less likely to report infection symptoms. the hajj pilgrimage attracts over two million pilgrims every year to saudi arabia, making it one of the biggest annual human mass gatherings on the planet [ ] . consequently, with such large numbers of pilgrims from around the world in close proximity to one another, there is amplified risk of transmission of infectious diseases among those present [ ] . to minimize the risks, saudi ministry of health (moh) require a valid vaccination certificate against meningococcal disease for all pilgrims, plus yellow fever and polio vaccines for pilgrims coming from or transiting through endemic countries. additionally, vaccinations against influenza, diphtheria, pertussis, tetanus, mumps and measles are recommended, particularly for pilgrims susceptible to more severe disease [ ] . other relatively inexpensive preventative measures such as hand hygiene and facemask use are also recommended [ ] . studies have consistently shown that there is an extensive variation in vaccine uptake among pilgrims based on their country of origin, demographics and vaccines received. similarly, hand hygiene and other preventative practices also vary among pilgrims, making it more difficult for researchers to ascertain whether vaccine uptake and health behaviors overall have improved in comparison to previous years or studies [ ] [ ] [ ] . although there is some research linking knowledge, attitudes, and beliefs of hajj pilgrims to their use of preventive measures, no studies have explored their knowledge, preparedness and preventive practices over the course of their travel (i.e., following the same cohort before, during and after hajj) [ , [ ] [ ] [ ] [ ] . to address these research gaps, we conducted a cohort study to explore australian hajj pilgrims' knowledge about the risk of diseases during hajj, assess their preparedness and use of preventive measures at three times points (before, during and after hajj) , investigate the factors affecting their preventive health behavior, and determine the number of reported infections during and after hajj. this was a prospective cohort study undertaken between august and december among australian hajj travelers aged years or older. the participants were residents of greater sydney, new south wales, australia. this region was chosen because it contains the largest muslim population in new south wales, australia. the participants were assessed before, during and after returning from their pilgrimage to makkah, saudi arabia. this study was approved by the university of sydney human research ethics committee (hrec) (project no: / ). all overseas pilgrims travelling to saudi arabia require a visa which can only be acquired through approved hajj travel agents [ ] . these tour operators play a pivotal role in pilgrims' preparation leading up to hajj, relaying important travel instructions prior to departure through 'pre-hajj seminars' which almost all prospective pilgrims attend [ ] . this made these seminars an ideal recruitment points for a representative sample. the list of accredited australian hajj tour operators and their addresses were collected from the saudi embassy in canberra, australia. a selection of participating travel agencies was decided on the basis of the number of hajj visas allocated for a given tour operator and the operators with the highest visa quotas were approached first. three questionnaires were used for data collection: ( ) a pre-hajj questionnaire completed prior to departure; ( ) a 'hajj diary' completed daily over six days during the peak hajj period; and ( ) a post-hajj questionnaire, completed upon return to australia. the surveys were mainly in english, with the availability of arabic, turkish and urdu translations for those who preferred it. we attended eleven seminars held by the hajj travel agents in sydney from august to september to explain our research. once pilgrims consented to participate in the study, their characteristics were obtained using a self-administered questionnaire documenting socio-demographic details, their travel itineraries, vaccination records, and presence of chronic medical conditions such as hypertension, diabetes, bronchial asthma and hyperlipidemia. barriers to, and facilitators of vaccine uptake and data on risk perception of diseases at hajj, such as influenza, pneumonia, blood borne infections and their preparedness for using preventive measures during hajj were also collected. we did not ask about the receipt of meningococcal vaccine because our previous survey showed that all australian pilgrims receive this as a mandatory visa requirement [ ] . we travelled to makkah, saudi arabia, during hajj period ( - september ) and identified and met the study participants upon their arrival in mina, greater makkah. the participants were given a diary for self-completion daily over the six consecutive days of hajj (the peak hajj days). they were followed from - september . each participant was asked to record the following in the diary for each day: actual use of preventative measures including, facemask use, hand sanitizer use, hand washing after touching an ill person and use of disposable handkerchiefs. as the data were collected electronically, no respondent could submit their responses with vitally important information missing. any respondent who used a preventive measure ≥ days during the peak hajj days considered to be 'frequently compliant', those who used the preventive measure < days were considered to be 'infrequently compliant' and those who did not use the preventive measure at all were considered 'non-compliant'. any development of symptoms suggestive of respiratory infection including cough, subjective fever sore throat, rhinitis and other symptoms including vomiting, diarrhea and nausea was also recorded. we considered those who complained of cough, subjective fever and sore throat to meet the criteria of influenza-like illness (ili). the respondents participated in a computer-aided telephone interview (cati) seven to days after their coming back to australia (until december ). they were asked about any development of symptoms suggestive of respiratory infection such as cough, sore throat, runny nose, fever, and also about some constitutional or gastrointestinal symptoms like vomiting, diarrhea and nausea after their home return from hajj. facilitators of and barriers to using preventive measures during hajj were also explored. for each participant we made three call attempts before being classified as lost to follow-up. a consecutive sampling strategy was used to ensure a representative sample of hajj pilgrims living in nsw. based on results from previous research, and considering an error margin of % to be acceptable for this survey, a sample size of pilgrims was deemed to be sufficient. considering a loss to follow-up rate of %, participants were targeted, representing % of australian pilgrims attending hajj in . statistical analysis was performed using the spss v. . (spss, inc., chicago, il, usa). chi-squared tests and pearson correlation were used to assess variables and establish associations and correlations. factors with p values < . in univariate analysis were entered into multivariable regression models. binary logistic regression, using the backward wald method, controlling for factors was used to investigate variables related to health behavior. p values less than . were considered statistically significant in multivariable models. a three-point likert scale was used to measure the pilgrims' perception about the risk of diseases during hajj and the effectiveness of the preventive measures. a total of pilgrims were recruited in the first stage of the study (before hajj); out of those, ( %) were followed during hajj; and finally, ( %) were reached after their return to australia ( figure ). of , % were female and their mean age was . (standard deviation [sd] ± . ) years. regression models. binary logistic regression, using the backward wald method, controlling for factors was used to investigate variables related to health behavior. p values less than . were considered statistically significant in multivariable models. a three-point likert scale was used to measure the pilgrims' perception about the risk of diseases during hajj and the effectiveness of the preventive measures. a total of pilgrims were recruited in the first stage of the study (before hajj); out of those, ( %) were followed during hajj; and finally, ( %) were reached after their return to australia (figure ). of , % were female and their mean age was . (standard deviation [sd] ± . ) years. over a quarter ( %) reported having one or more pre-existing medical conditions. over one third of participants ( %) had a university degree or higher qualification and two thirds ( %) were employed. the pilgrims intended to stay in saudi arabia for a median of (range: - ) days, and the majority ( %) were attending hajj for the first time. the participants' further demographic details are presented in table . the majority of participants ( %, [ / ]) received one or more recommended vaccines (i.e., vaccines that are recommended but not compulsory); of those, % ( / ) received only the influenza vaccine, % ( / ) received influenza plus other recommended vaccines while the remaining % ( / ) received only "other than influenza" vaccines. overall, the coverage of influenza, pharmacy , , of pneumococcal and pertussis vaccine was, respectively, % ( / ), % ( / ) and % ( / ) ( table ) . on the other hand, % ( ) did not receive any recommended vaccine. * some pilgrims cited more than one reason. thirty-one percent ( / ) were in an at-risk category; of those, % ( / ) were aged ≥ years and % ( / ) aged < but had one or more chronic medical condition (e.g., diabetes). the influenza vaccination rate among at-risk group was % ( / ), while the uptake of pneumococcal vaccine was % ( / ) . no significant differences of influenza and pneumococcal vaccine uptake were noted between 'at risk' and not 'at risk' groups. participants reported different sources of vaccination advice including, hajj (table ). multivariate analysis showed that being aged between and years was significantly associated with the receipt of recommended vaccines compared to being aged ≤ years or ≥ years (adjusted odds ratio [aor] = . , % confidence interval [ci] = . - . , p < . ). moreover, those who had a university qualification or higher education were more likely to receive vaccine than those who had lower level of education (aor = . , % ci = . - . , p < . ). . those who were very concerned about blood borne diseases (aor = . , % ci = . - . , p = . ) and pneumonia (aor = . , % ci = . - . , p < . ) were twice as likely to receive hepatitis b and pneumococcal vaccines respectively. yet, no association was found between the level of concern about influenza and the receipt of influenza vaccine. (table ). in addition, % ( / ) of pilgrims used antibiotics during hajj. there was no significant association between pilgrims' intended use of non-pharmacological measures and their actual use during hajj (table ) . in multivariate analysis, no demographic factors were associated with using hand hygiene with soap and sanitizers, but those who were concerned about developing pneumonia during hajj (aor = . , % ci = . - . , p = . ) were more likely to practice hand hygiene with soap and sanitizers during hajj compared to those who were not concerned. those aged ≥ years (aor = . , % ci = . - . , p = . ) were more likely to practice hand hygiene with alcoholic hand rubs than those aged < years. moreover, males were more likely to wash their hands with water only (aor = . , % ci = . - . , p < . ) and wash hands after touching an ill person (aor = . , % ci = . - . , p < . ) but were less likely to use disposable handkerchiefs (or = . , % ci = . - . , p < . ) compared to females. on the first day of hajj, some pilgrims were symptomatic; % had a cough, % had a sore throat, and % had a runny nose. however, on the th day up to % became symptomatic. the reported onset of daily symptoms among pilgrims during the peak days of hajj is presented in figure . on the first day of hajj, some pilgrims were symptomatic; % had a cough, % had a sore throat, and % had a runny nose. however, on the th day up to % became symptomatic. the reported onset of daily symptoms among pilgrims during the peak days of hajj is presented in figure . overall, % ( / ) of participants reported one or more illness symptoms throughout the hajj journey. eighty-six percent ( / ) of these symptoms were respiratory, including cough, % ( / ); sore throat, % ( / ); runny nose, % ( / ); and fever, % ( / ). ili was only reported among % ( / ) of participants. additionally, % ( / ) reported diarrhea, % ( / ) nausea and another % ( / ) reported vomiting. twenty six per cent ( / ) of respondents did not report any symptom during hajj. over half ( % [ / ]) reported symptoms after returning from hajj; of those, % ( / ) reported cough ( %, [ / ] of these also had cough during hajj), % ( / ) sore throat ( %, [ / ] of them reported sore throat during hajj), %, ( / ) runny nose ( %, [ / ] reported runny nose during hajj) and % ( / ) had fever ( %, [ / ] had fever during hajj). ili was reported in % ( / ) of participants; of those, % ( / ) also reported ili during hajj. moreover, % ( / ) reported diarrhea, % ( / ) nausea and another % ( / ) reported vomiting. as shown in table , cough was less likely to occur among those who were vaccinated against influenza and those who used alcoholic hand rubs. runny nose was less likely to occur who frequently washed their hands with plain water but was more common among those who used facemasks. vomiting was less likely to be reported among those who washed their hands frequently with soap and sanitizers during hajj, but more common in those who used disposable handkerchiefs. no association was found between use of any of the preventive measures and reduction in fever and sore throat. overall, % ( / ) of participants reported one or more illness symptoms throughout the hajj journey. eighty-six percent ( / ) of these symptoms were respiratory, including cough, % ( / ); sore throat, % ( / ); runny nose, % ( / ); and fever, % ( / ). ili was only reported among % ( / ) of participants. additionally, % ( / ) reported diarrhea, % ( / ) nausea and another % ( / ) reported vomiting. twenty six per cent ( / ) of respondents did not report any symptom during hajj. over half ( % [ / ]) reported symptoms after returning from hajj; of those, % ( / ) reported cough ( %, [ / ] of these also had cough during hajj), % ( / ) sore throat ( %, [ / ] of them reported sore throat during hajj), %, ( / ) runny nose ( %, [ / ] reported runny nose during hajj) and % ( / ) had fever ( %, [ / ] had fever during hajj). ili was reported in % ( / ) of participants; of those, % ( / ) also reported ili during hajj. moreover, % ( / ) reported diarrhea, % ( / ) nausea and another % ( / ) reported vomiting. as shown in table , cough was less likely to occur among those who were vaccinated against influenza and those who used alcoholic hand rubs. runny nose was less likely to occur who frequently washed their hands with plain water but was more common among those who used facemasks. vomiting was less likely to be reported among those who washed their hands frequently with soap and sanitizers during hajj, but more common in those who used disposable handkerchiefs. no association was found between use of any of the preventive measures and reduction in fever and sore throat. this cohort study captured and compared the health behavior, knowledge, attitudes and practices of australian hajj pilgrims regarding preventative measures against communicable diseases throughout the course of hajj travel (before, during and after the journey). vaccinated pilgrims and those who washed their hands and used alcohol rubs were less likely to develop respiratory symptoms during hajj. influenza vaccination coverage was relatively higher ( %) among australian pilgrims, compared to that reported in studies from other countries, but was lower compared to that in australian pilgrims in ( %) and in ( %) [ , ] . in contrast, coverage of other recommended vaccines, such as the pneumococcal vaccine, was suboptimal ( %), as has been reported in previous studies involving australian hajj pilgrims and other international pilgrims [ , ] . the coverage of influenza vaccine among pilgrims with high-risk conditions was %, while that of pneumococcal vaccine was only %; compared to the influenza vaccination coverage among pilgrims with high-risk conditions from other countries, australian hajj pilgrims had a higher vaccination rate [ ] . the low pneumococcal vaccine uptake is a concern because pneumococcal diseases, including invasive pneumococcal disease, are major causes of morbidity and mortality in the extremes of age and in individuals with chronic medical conditions worldwide, including hajj pilgrims [ , ] . of note, while the influenza vaccine is highly recommended by the saudi moh for hajj attendance, particularly for pilgrims aged ≥ years [ ] , there is no formal recommendation regarding the pneumococcal vaccine. this study found that a large number of participants were willing to use non-pharmacological preventative measures to reduce the prevalence of illnesses during hajj. however, there was no significant correlation between their intention to use measures and their actual use during hajj. although the level of concern for pneumonia and diarrhea was higher among first time hajj goers and pilgrims < years of age, these factors were not shown to be significant in their actual use of preventive measures during hajj. pilgrims who were concerned about catching diseases and those who joined hajj for the first time were more likely to accept the use of non-pharmacological measures before hajj. however, these intentions were not associated with actual use of preventive measures. previous studies have not explored if demographic factors were associated with an intention to use non-pharmacological protective measures before travel and their actual use during hajj. nonetheless, earlier studies did conclude that health education prior to departure was significantly associated with greater compliance with preventative practices, particularly the use of facemasks and hand sanitizers [ ] [ ] [ ] [ ] . demographic factors had minimal association with pilgrims' health behavior such as vaccine uptake, and the factors associated with pilgrims' willingness to use preventive measures were not associated with their actual use during hajj. consequently, these findings pose questions of other possible causes that might affect pilgrims' behaviors during hajj. in this study, several factors were identified as influencing pilgrims' health behavior, including disease risk perception, awareness of recommendations, the influence of people around them, age and medical history, the source of their travel health advice, and the lived experience of using preventive measures during hajj. these factors can be stratified into five categories: individual, interpersonal, organizational, community-associated and policy-related. relying on a single factor does not fully explain the interplay and dynamics between pilgrims' health behaviors and their influencers; rather, consideration of multiple factors at different levels may help to understand these cross-cutting relationships. these dimensions fit within the 'social-ecological model' which emphasizes the connectivity and relationship among multiple factors affecting health behavior, as shown in figure . the overlapping circles in the model illustrate how factors at one level influence factors at another level. this framework is based on evidence that no single factor can explain why only some people comply with preventive measures while remain influenced by multiple inter-related factors. the core of the model is at the individual level, surrounded by the outer four bands representing the interpersonal, organizational, community and policy levels ( figure ). it is important to understand and find the most common or influential reasons and the link between these levels to enhance pilgrims' health behavior uptake in regards to each measure. in addition, while some interventions such as vaccines are known to be clinically effective, their acceptability to pilgrims is a vital part of their overall effectiveness as a public health intervention. effective behavioral change is only made when changes interconnect between the individual, community, organizational and policy levels. this includes addressing underlying and related factors such as individual beliefs, culture, and miscommunication between organizations as well as government policies. this can help promote lasting changes in practices; on an individual level, cultural and religious beliefs; in attitudes and perception; in communication between the health care providers such as gps and the community; providing the service suppliers such as the travel agent with up-to-date health recommendations and also improving the communication between the international and local health policies (figure ) [ ] . future studies could be grounded in 'social ecological' theory, in order to further articulate the inter-level relationships between health behavior factors identified in this study, encouraging new insights to promote the health interventions among hajj pilgrims. care providers such as gps and the community; providing the service suppliers such as the travel agent with up-to-date health recommendations and also improving the communication between the international and local health policies (figure ) [ ] . future studies could be grounded in 'social ecological' theory, in order to further articulate the inter-level relationships between health behavior factors identified in this study, encouraging new insights to promote the health interventions among hajj pilgrims. during hajj, about % of participants reported developing one or more respiratory symptoms; a cough and sore throat were the commonest symptoms similar to what was found in earlier studies involving australian pilgrims who attended hajj in [ ] , and among french, iranian and malaysian pilgrims who attended hajj between and [ ] [ ] [ ] . during hajj, ili was reported by % of pilgrims in this study, a similar rate was reported previously among australian hajj pilgrims in [ ] . in other studies involving hajj pilgrims the reported incidence of ili varied (from % to %) [ ] . the risk of disease was equally high among returning pilgrims, with % to % of them reporting cough, sore throat and runny nose, and % suffering from ili as found in other studies [ ] [ ] [ ] . countries should continue to monitor the pilgrims and their contacts after the pilgrims' return from hajj to estimate the risk of post-hajj infections. influenza vaccine effectiveness was studied in several studies. in two studies conducted in and , a reduction in ili was observed, while in a few other earlier studies no significant reduction was noted [ ] . a synthesis of published and raw data from eleven hajj years between and showed that the rate of ili decreased among hajj pilgrims as the vaccination rate increased (relative risk . , p < . ) [ ] . subsequently, a 'test-negative' case-control study using data from individual hajj years involving participants from multiple countries shows trivalent influenza has an effectiveness of . % ( % ci . % to . %, p = . ) against laboratory-confirmed influenza [ ] . the varying results may due to heterogeneity in defining ili or may be because of ili symptoms representing non-influenza infections or even due to confounding factors such as use of facemasks or hygienic interventions [ , , ] . there are some limitations; % respondents failed to complete the study. this is a little higher than the expected loss to follow up ( %), however as this study was conducted over three months during hajj, about % of participants reported developing one or more respiratory symptoms; a cough and sore throat were the commonest symptoms similar to what was found in earlier studies involving australian pilgrims who attended hajj in [ ] , and among french, iranian and malaysian pilgrims who attended hajj between and [ ] [ ] [ ] . during hajj, ili was reported by % of pilgrims in this study, a similar rate was reported previously among australian hajj pilgrims in [ ] . in other studies involving hajj pilgrims the reported incidence of ili varied (from % to %) [ ] . the risk of disease was equally high among returning pilgrims, with % to % of them reporting cough, sore throat and runny nose, and % suffering from ili as found in other studies [ ] [ ] [ ] . countries should continue to monitor the pilgrims and their contacts after the pilgrims' return from hajj to estimate the risk of post-hajj infections. influenza vaccine effectiveness was studied in several studies. in two studies conducted in and , a reduction in ili was observed, while in a few other earlier studies no significant reduction was noted [ ] . a synthesis of published and raw data from eleven hajj years between and showed that the rate of ili decreased among hajj pilgrims as the vaccination rate increased (relative risk . , p < . ) [ ] . subsequently, a 'test-negative' case-control study using data from individual hajj years involving participants from multiple countries shows trivalent influenza has an effectiveness of . % ( % ci . % to . %, p = . ) against laboratory-confirmed influenza [ ] . the varying results may due to heterogeneity in defining ili or may be because of ili symptoms representing non-influenza infections or even due to confounding factors such as use of facemasks or hygienic interventions [ , , ] . there are some limitations; % respondents failed to complete the study. this is a little higher than the expected loss to follow up ( %), however as this study was conducted over three months in three different settings (before, during and after hajj) in two countries (australia and saudi arabia), it was very challenging to follow all participants, some of whom may have taken a side trip to other countries at the end of the hajj. a french study revealed that over a quarter of the pilgrims intended to delay their return to france after hajj, a situation that makes difficult to ensure maximum follow up [ ] . information and recall bias may have occurred due to the anecdotal nature of some survey questions, especially those of the post-hajj survey. although our results cannot be generalized to all pilgrims, this study is the first of its kind to assess pilgrims' kap continuously throughout the hajj journey to understand their health behaviors, experience of using preventative measures and the development of acute respiratory infections, and other symptoms of infections. in the meantime, studies conducted in france and malaysia have shown that compliance to some preventive measures such as hand hygiene and face mask use has increased, while the uptake of recommended vaccines including influenza and pneumococcal vaccines still remains low [ ] [ ] [ ] ; therefore, awareness campaigns should be continued to tackle respiratory infections including the ongoing covid- pandemic that, thus far, has taken a toll of over , people across the world (as of april ) and affected many muslim countries including saudi arabia [ ] . to mitigate the epidemic, the saudi arabian authority has already temporarily cancelled umrah (minor pilgrimage) visit to makkah [ ] ; the decision on whether this year's hajj pilgrimage (late july to early august) should be cancelled or not remains to be decided and may depend on the progress of the pandemic [ ] . our findings mean that preventive measures like hand washing and use of alcoholic hand rubs could be implemented readily during hajj, and tour operators may play important roles in improving compliance. in conclusion, this study reveals that most australian hajj pilgrims complied with key preventative measures, and that tour group operators' advice played an important role in compliance. pilgrims who complied with preventive measures were less likely to suffer from infection symptoms. researchers and policy makers should work together to explore ways to educate the pilgrims and their tour operators about the importance of vaccination and using simple but inexpensive preventive measures, such as hand hygiene, that may halt the spread of highly contagious infectious diseases, e.g., covid- , pandemic influenza and drug-resistant pathogens in mass gatherings. transmission of respiratory tract infections at mass gathering events preparing australian pilgrims for the hajj health conditions for travellers to saudi arabia for the pilgrimage to mecca (hajj)- vaccinations against respiratory tract infections at hajj vaccination in hajj: an overview of the recent findings non-pharmaceutical interventions for the prevention of respiratory tract infections during hajj pilgrimage protective measures against acute respiratory symptoms in french pilgrims participating in the hajj of : table predictors of protective behaviors among american travelers to the hajj pilgrims' knowledge about acute respiratory infections health risks at the hajj exploring australian hajj tour operators' knowledge and practices regarding pilgrims' health risks: a qualitative study hajj research team. influenza vaccination among australian hajj pilgrims: uptake, attitudes, and barriers pneumococcal vaccine uptake among australian hajj pilgrims in - attitude and practice (kap) survey concerning antimicrobial use among australian hajj pilgrims burden of clinical infections due to s. pneumoniae during hajj: a systematic review the potential for pneumococcal vaccination in hajj pilgrims: expert opinion using health educators to improve knowledge of healthy behaviour among hajj ( ) pilgrims hand hygiene compliance and effectiveness against respiratory infections among hajj pilgrims: a systematic review effectiveness of an education health programme about middle east respiratory syndrome coronavirus tested during travel consultations uptake and effectiveness of facemask against respiratory infections at mass gatherings: a systematic review an ecological perspective on health promotion programs pilot use of a novel smartphone application to track traveller health behaviour and collect infectious disease data during a mass gathering: hajj pilgrimage influenza viral infections among the iranian hajj pilgrims returning to shiraz, fars province circulation of respiratory viruses among pilgrims during the hajj pilgrimage the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims acute febrile respiratory infection symptoms in australian hajjis at risk of exposure to middle east respiratory syndrome coronavirus prevalence of influenza at hajj: is it correlated with vaccine uptake? health related experiences among international pilgrims departing through king abdul aziz international airport the prevalence and preventive measures of the respiratory illness among malaysian pilgrims in hajj season changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a -year retrospective analysis of data influenza vaccine effectiveness among hajj pilgrims: a test-negative case-control analysis of data from different hajj years pilot randomised controlled trial to test effectiveness of facemasks in preventing influenza-like illness transmission among australian hajj pilgrims in travel reported by pilgrims from marseille, france before and after the hajj: table hajj in the time of covid- respiratory tract infections among french hajj pilgrims from abubakar baaba, a. assessment of knowledge, attitude and practice towards prevention of respiratory tract infections among hajj and umrah pilgrims from malaysia uptake of recommended vaccines and its associated factors among malaysian pilgrims during hajj and umrah saudi arabias measures to curb the covid- outbreak: temporary suspension of the umrah pilgrimage the cancellation of mass gatherings (mgs)? decision making in the time of covid- this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - mnsjbib authors: maman, issaka; badziklou, kossi; landoh, essoya d; halatoko, afiwa w; nzussouo, talla n; defang, gabriel n; tamekloe, tsidi a; kennedy, pamela j; thelma, williams; kossi, komlan; issa, zoulkarneiri; kere, abiba b title: implementation of influenza-like illness sentinel surveillance in togo date: - - journal: bmc public health doi: . / - - - sha: doc_id: cord_uid: mnsjbib background: the emergence of avian influenza a/h n in as well as the pandemic influenza a (h n ) pdm highlighted the need to establish influenza sentinel surveillance in togo. the ministry of health decided to introduce influenza to the list of diseases with epidemic potential. by april , togo was actively involved in influenza surveillance. this study aims to describe the implementation of ili surveillance and results obtained from april to december . methods: two sites were selected based on their accessibility and affordability to patients, their adequate specimen storage capacity and transportation system. patients with ili presenting at sentinel sites were enrolled by trained medical staff based on the world health organization (who) case definitions. oropharyngeal and nasopharyngeal samples were collected and they were tested at the national influenza reference laboratory using a u.s. centers for disease control and prevention (cdc) validated real time rt-pcr protocol. laboratory results and epidemiological data were reported weekly and shared with all sentinel sites, ministry of health, division of epidemiology, who and cdc/namru- . results: from april to december , a total of samples were collected with % of the study population aged between and years. of the samples, ( . %) tested positive for influenza viruses; with ( . %) positive for influenza a and ( . %) positive for influenza b. the highest influenza positive percentage ( %) was observed in – years old and patients aged – and > years had the lowest percentage ( %). clinical symptoms such as cough and rhinorrhea were associated more with ili patients who were positive for influenza type a than influenza type b. influenza viruses circulated throughout the year with the positivity rate peaking around the months of january, may and again in october; corresponding respectively to the dry-dusty harmattan season and the long and then the short raining season. the pandemic a (h n ) pdm was the predominantly circulating strain in while influenza b was the predominantly circulating strain in . the seasonal a/h n was observed throughout year. conclusions: this study provides information on influenza epidemiology in the capital city of togo. influenza-like-illnesses (ili) is a significant source of morbidity and mortality worldwide [ ] . the world health organization (who) estimates that globally influenza accounts for between and million severe cases and . to . deaths annually, with most deaths occurring among elderly populations [ ] . in temperate regions, ili is reported throughout the year with a marked increase in cases recorded during winter periods [ ] . however, in tropical and subtropical regions where viral transmission occurs throughout the year, the data on the burden of influenza-like-illness are limited. nevertheless there is some evidence of a slight increase in cases during the rainy season [ , ] . the emergence of new highly pathogenic influenza a/ h n viruses in [ ] , their wide circulation in wild and domestic birds and its association with human infections which involves high mortality, has raised global concern about the risk of another influenza pandemic. the emergence of novel human pandemic influenza a (h n ) in april [ , ] and its rapid worldwide spread has motivated the monitoring of influenza and has enhanced preparedness to counter a possible emerging pandemic. in the african region, countries in collaboration with international partners (e.g. who, cdc, namru- , etc.) put efforts together to establish influenza surveillance capacities as part of the broader strategy for integrated disease surveillance and response (idsr) [ , ] . while most countries in asia, north america and europe have wellestablished influenza surveillance, few such systems have been established in sub-saharan africa [ , ] . influenza surveillance helps in understanding the epidemiology and impact of the disease; therefore providing information about seasonality and the groups at high risk of influenza infection. furthermore, the identification and characterization of circulating viruses will help to provide influenza isolates for monitoring changes in viral antigens and the development of vaccines. thus influenza surveillance provides data for pandemic influenza monitoring and planning as well as for decision-making [ ] [ ] [ ] . in togo, the first suspected cases of human avian influenza a/h n were reported between and in the maritime region, a few kilometers from the capital city lomé, which has a population of more than millions. between april and december , cases of ili were observed and were suspected to be pandemic influenza given the emergence of the novel human pandemic influenza a (h n ) pdm . with no ongoing influenza surveillance, our country was not yet ready to confirm and effectively monitor the severity of the disease. the lack of molecular laboratory technology to detect influenza viruses significantly reduced our ability to manage and control the pandemic. therefore, the ministry of health (moh) in collaboration with the institut national d'hygiène (inh) decided to add influenza to the list of diseases with epidemic potential to be monitored and reported through the idsr program. by april , togo was actively involved in ili surveillance with the support of united state government through the centers for disease control (cdc) and the naval medical research unit- (namru- ). this study aims to describe the implementation of ili surveillance and results obtained from april to december . the ili surveillance system constitutes a collaborative partnership between several togolese institutions within the ministry of health (moh). the departments involved in this surveillance are the division of epidemiology, the national influenza reference laboratory (nil) hosted by the institut national d'hygiène (inh), and the sentinel sites located at the hôpital de bè and military health services in the capital city lomé (figure ) . a protocol for influenza surveillance was written with the technical support of cdc and namru- experts. the ili sentinel surveillance sites were selected based on their accessibility and affordability to patients with low socioeconomic status, the qualifications of medical staff, adequate specimen storage capacity, and an established transportation system to the national influenza reference laboratory (nil). the first site was hôpital de bè, established in april and located in district n° . this site was chosen for its geographical location in an area of high population density and high consultation rate. this hospital hosts a pediatric unit and a general medicine ward. the second site established in december , is under the management of the military health services and located in district n° ; its selection was based on the essential role of the armed forces in case of a pandemic and their ability to serve both military and civilian populations. this military site is composed of three units and is attended by military personnel, their families as well as civilians. the two sentinel sites are located in the capital city of togo where approximately % of the country's population lives. lomé has two rainy seasons and two dry seasons: the long rainy season (april to june) and the short rainy season (mid-september to october). the long dry season extends from december through march, while the short dry season lasts for two months (july to august). lomé is a coastal city that borders the atlantic ocean to the south, ghana to the west, benin to the east ( figure ) and is at the crossroad with considerable commercial exchange of goods and movements of population. the who case definition [ ] that was used, defined ili as "any person with a sudden onset of fever (≥ °c) and cough or sore throat accompanied or not by general symptoms such as myalgia, prostration, headache or malaise". this definition was used during - period. in , the definition was changed to "any person with a sudden onset of fever (≥ °c) or history of fever and cough or sore throat accompanied or not by general symptoms such as myalgia, prostration, headache or malaise". at both sentinel sites, from monday to friday physicians enrolled the first two outpatients who met the case definition and samples were collected during consultation. the study population included every outpatient, between april to december , presenting at any of the sentinel sites and meeting the ili case definitions regardless of age or sex and who consented to participate in the surveillance. this population represents a wide cross-section of ethnic and socioeconomic groups. samples collected were nasopharyngeal and oropharyngeal swabs and were placed in the same tube containing a viral transport medium (vtm). they were stored between to °c at the bacteriology laboratory of the sentinel site prior to delivery to the nil within hours. before samples were transported, laboratory personnel at the sentinel site conducted quality control checks of information on patients' case report forms. the nil provides the sentinel sites with logistical and material support such as swabs, viral transportation media, cryovials, cool boxes, and ice packs. a quota of samples was targeted from each sites and transported twice a week (tuesday and thursday) to the nil. review meetings with all stakeholders were organized two or three times per year as part of a strategy to in at sigbéhoué (district des lacs), adétikope (district du golfe) and agodekê (district de zio). in at agbata (district des lacs). all theses foci were located at few kilometers from the capital city, lomé. improve the surveillance system by identifying strengths and areas of concern during these meetings. socio-demographic (age, sex, date of birth, residential area, travel history) and clinical (date of onset, date of consultation, previous treatment, vaccination status, co-morbidities) data were collected from all patients using a case report form (crf) during consultation. epidemiological data were stored in a single database with laboratory data using a single identification number for each patient. each week, nil provided reports on the distribution of total samples collected, as well as on the number of confirmed influenza cases to the moh, to the sentinel sites, who flunet, cdc, and namru- . samples collected were analyzed at the national reference influenza laboratory at inh. from every sample, three aliquots were made, two of which were stored at − °c for external quality control and further analysis (if not subtyped) at namru- in cairo, egypt. the other one was kept between to °c for rna extraction followed by influenza virus detection by real time rt-pcr within hours after sample reception. for the testing of influenza viruses, rna extraction was performed from μl of naso and/or oro-pharyngeal cells contained in the vtm by using a qiamp viral rna mini kit (qiagen) following the manufacturer's protocol. for detection and typing, it was run on an abi machine, the real time rt-pcr using the ambion enzyme agpath one-step (ambion, applied biosystems) that amplifies influenza a and b. the u.s. cdc provided the protocol used to detect influenza viruses [ ] . in order to determine the quality of the sample, the presence of human ribo-nucleoprotein (rnp) was assessed for each specimen tested. socio-demographic and clinical epidemiological data were entered into a database created using epi-info software version . . data analysis was conducted using spss software version . (spss inc., chicago, il). student t-test was used for comparison of mean age and the pearson chi-square or fisher exact test to compare laboratory results by age groups and clinical symptoms. the protocol was approved by the moh as part of the monitoring of diseases with epidemic potential and therefore did not require ethical review. verbal consent was obtained from all patients. a total of patients were enrolled in this study. seven hundred and twenty seven ( %) patients were enrolled from the hôpital de bè and ( %) from the military health service site (table ). there was no significant difference in the proportion of females compared to males enrolled in this study ( . % vs. . %; p = . ), and the gender distribution at the two sites was similar. most of patients ( %) were under years of age, while less than % were years or older. patients who presented at the hôpital de bè were significantly older (mean age = . years) compared to those who were seen at the military health service (mean age = . years with % of patients aged less than years) (p = . ). approximately % of the patients reported having received influenza vaccination within the last year. of the samples collected for ili surveillance, ( . %) tested positive for influenza viruses. of these, ( . %) tested positive for influenza a virus and ( . %) for influenza b virus ( table table ). the proportion of influenza positive cases varied between different age groups with a higher proportion of influenza a detected in the - year-old group ( %) than other age groups (p = . ; table ). significantly, the pandemic influenza a (h n ) pdm was more often detected in patients aged - (p = . ) and - (p = . ) years than in other age groups. seasonal a/h n was predominant in patients aged - years ( %; p = . ) and was the only influenza a subtype detected among patients who were years or older. ili was observed throughout every year with irregular peak activity occurring twice annually during the months of; may and november in ; may and october in ; april/august, october in ( figure ) . however, the number of patients/samples enrolled was not consistent. influenza a virus was detected predominantly in and correlated with the ili peak. the first cases of pandemic influenza a (h n ) pdm were only confirmed in may. during the ili peak, the influenza positivity rate was % in may and % in november with the pandemic influenza strain, the most subtype detected. the pandemic virus remained predominant between october and april (figure ). during the ili peaks in , the influenza positive rate ranged from % to % with the predominance of influenza b virus activity in may representing % of all virus detected ( / ). the second peak was correlated to the seasonal influenza a/h n activity in october with % of viruses detected ( / ). from october , there was a co-circulation of influenza type a and type b with low activity of pandemic strain until september while the seasonal influenza a/h n was detected throughout the year . fever ( %), cough ( %), and rhinorrhea ( %) were the major symptoms for all age groups although sore throat ( %) and headaches ( %) were also recorded ( table ). ili patients who tested positive for influenza were more likely to present with cough (p = . ) and headaches (p = . ) but were less likely to present with difficulty breathing (p = . ) compared to those who tested negative for the influenza virus. among symptoms, only cough was more common in patients testing positive for influenza a than those who tested positive for influenza b (p = . ). rhinorrhea was more common in patients with seasonal a/h n than in those with pandemic influenza a (h n ) pdm (p = . ). due to the lack of ili surveillance in togo, there was no information about the epidemiology of ili or influenza viruses until . the first samples collected were processed in may and the presence of pandemic influenza a(h n ) pdm virus was confirmed in togo one year after the novel pandemic influenza occurred in mexico (april ). this is the first report that describes the epidemiology of influenza in togo using data from the ili sentinel surveillance system. during the two and half year period of ili sentinel surveillance, influenza viruses were detected in ( %) of samples. the average percentage positive in this study was higher than the positivity rate observed in other african countries between and [ ] . however, during the same timeframe, other countries in the temperate climate region: madagascar ( %), morocco [ ] . there are several reasons to explain the difference in the percentage positive observed between countries including the temporal distribution of these viruses, the sample collection method, the number of samples collected and the geographical distribution of sentinel sites. most of our data includes post pandemic influenza a (h n ) pdm ; this is a different picture compared to other african countries ( ) ( ) ( ) ( ) ( ) and to the south american region. the sample collection method was different from one country to another. in our study, we used two swabs (one oro-pharyngeal and one nasopharyngeal) for each enrolled patient and put both swabs in the same cryotube, thereby increasing the viral load and enhancing rt-pcr detection while in some other countries samples were collected either with nasopharyngeal [ ] or oro-pharyngeal swab only [ , ] . in addition, the number of samples tested in most of the other countries was quite high compared to our sample numbers and they were collected from many sites ranging from only to as many as . we only used two sentinel sites as our catchment area. the influenza positivity rate varied by year with the highest rate obtained in . since the number of samples collected during this year was very low ( samples) than the two subsequent years, the rate could be influenced. nevertheless, our percentage positive was similar to that of ghana and rwanda in africa [ ] and that of taiwan [ ] , but at different periods of time. table distribution of influenza viruses confirmed and ili patients influenza a was predominant in with pandemic influenza a (h n ) pdm in our study; this observation was similar to that of other countries in west africa [ ] . however, in , the situation was different in other subregions with predominance of influenza b in central/south and north africa [ , ] and seasonal influenza a/h n in east africa [ ] . this difference could be explained by the fact that circulation of pandemic influenza a (h n ) pdm was delayed in west africa and occurred one year after it was predominantly circulating in other african subregions [ , ] . while two years is not sufficient time for an adequate description of the seasonality of influenza virus transmission, we did observe trends in the lomé commune region. the influenza b virus showed a peak activity during the rainy seasons (may and october) and the pandemic influenza a (h n ) pdm was more frequent during the long dry season while the seasonal a/h n was detected across both seasons. although the seasonality of influenza viruses in african countries is not yet clear, we observed that our trends were similar with the influenza peaks, which have often been associated with the rainy season activity in other tropical countries [ ] [ ] [ ] . in our study, influenza cases were highest ( %) in the - year age group but also high among other age groups, with lowest percent positive ( . %) among - and > years ( . %). this distribution is consistent with the observation in the study conducted in countries of africa during to and in peru [ , , ] in which young children and adults were shown to have the highest influenza viral disease. contrary to our study, a study from venezuela [ ] showed higher detection rates in - year olds. the percentage positive of influenza a was significantly higher in ili patients in the - age groups. therefore, we found that pandemic influenza a (h n ) pdm was detected significantly among - years old. this finding is consistent with other studies in the african region [ , , ] that have found that pandemic influenza a (h n ) pdm is most commonly identified in school-age children and young adults. while pandemic influenza a (h n ) pdm appeared more often in older children, seasonal influenza a/h n appeared more likely to infect adults in the - year-age category. our finding was similar to the observation from a study conducted in peru [ ] , where the author found that the seasonal influenza a/h n virus was detected with adults of to years. in conclusion, our results are consistent with studies from africa and south american regions which observed that seasonal influenza a/h n affected a wide range of age groups with predominantly to years old while the pandemic influenza a (h n ) pdm and influenza b virus infections occurred more frequently among older children and young adults. we observed that clinical symptoms were associated with influenza viruses. the influenza type a was more frequently detected than type b in patients presenting with cough and rhinorrhea. this result is consistent with the observation of a study from venezuela [ ] . in contrast with this study were pandemic influenza a (h n ) pdm was associated with ili patients with cough, our study showed that ili patients with rhinorrhea were associated with seasonal a/h n . our study had some limitations. our data were collected only from sites in an urban area in the capital city of togo and could not be generalized to the population. the percent influenza positivity and age distribution of positive cases were influenced by the low number of samples collected which may be attributable to the non availability of a physician to collect nasal and orpharyngeal swabs. the low number of samples may have also contributed to the high positivity rate. in addition, this low proportion may not be representative to better describe the distribution of influenza cases in the age groups. physician time limitations were due to the time consumption and their workload (number of patients viewed in consultation at the outpatients' department). children were over-represented in this study thus introducing a bias, as the number of adults was not comparable to children under years old. some possible reasons to explain this bias include the fact that the military health service has three units but only the family health care center was functional when added as a site in december . because this unit is a pediatric health center, the high number of children enrolled from this site can account for the observed figures. at the hôpital de bè site, we observed that many patients, mostly adults were not enrolled as ili patients due to the lack of recorded fever (≥ °c), suggesting that we should be considering history of fever as one of the enrollment criteria for ili. this study focused exclusively on outpatients thus limiting our ability to examine the severity of the influenza viruses in hospitalized cases. since our influenza surveillance system had challenges in collecting samples of severe acute respiratory infection (sari) and the lack of data on hospitalizations with patient follow-up we excluded discussions on sari from this study. to improve our influenza surveillance system, it will be necessary to expand the system in other regions by including sari surveillance for severe disease to give a complete picture of influenza burden and epidemiology in our country. these data provided information on the epidemiology of influenza in the lomé commune region in the capital city of togo. some efforts are needed to allow better understanding of influenza burden and epidemiology by expanding sentinel sites in other regions and including sari surveillance. future studies will also be focused on identifying the etiologic agents for the % of ili cases that were negative for influenza viruses. retrospective analyses of these stored samples will be necessary to identify other respiratory viruses 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wave from the amazon to the subtropics sentinel surveillance of influenza-like illness in two hospitals in influenza viruses in nigeria, results from the first months of a national influenza sentinel surveillance system implementation of influenza-like illness sentinel surveillance in togo we would like to thank all sentinel staff at the hôpital de bè and the military health services for their essential role in the ili sentinel surveillance. we are grateful to the ministry of health and the division of epidemiology for their support and coordination. we would like also express our sincere thanks to the team of the national influenza reference laboratory for their efforts in collecting samples, clinical data and detection by rt-pcr of influenza viruses. we wish to thank mr koffi akolly, field epidemiologist for designing the map of the figure . the influenza sentinel surveillance was successful established with the technical and financial support of cdc and namru- . we are grateful also to cdc reviewers for their precious analyses and revision of this paper prior it's submission for publication. the authors have declared that no competing interests exist.authors' contributions im contributed to the study design, statistical analyses of data and wrote the paper. kb and edl contributed to the study design, interpreted analysis and review the manuscript. awh and tat contributed in the study design and review the manuscript. tnn, gnd, wt and pjk provide technical advice for study protocol, methodology, revised critically the manuscript for important scientific content and have given final approval for the version to be published. zi and kk were involved in literature review and revising the manuscript. abk contributed to the facilitation of the project, participated in its design, coordination and review the paper. all authors read and approved the final manuscript. key: cord- -r nprlne authors: chughtai, a. a.; wang, q.; dung, t. c.; macintyre, c. r. title: the presence of fever in adults with influenza and other viral respiratory infections date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: r nprlne we compared the rates of fever in adult subjects with laboratory-confirmed influenza and other respiratory viruses and examined the factors that predict fever in adults. symptom data on healthcare workers (hcws) with a laboratory-confirmed respiratory virus infection were collected using standardized data collection forms from three separate studies. overall, the rate of fever in confirmed viral respiratory infections in adult hcws was · % ( / ). rates varied by virus: human rhinovirus ( · %, / ), influenza a virus ( %, / ), coronavirus ( · %, / ), human metapneumovirus ( · %, / ), respiratory syncytial virus ( · %, / ) and parainfluenza virus ( · %, / ). smoking [relative risk (rr) · , % confidence interval (ci) · – · ] and co-infection with two or more viruses (rr · , % ci · – · ) were significant predictors of fever. fever is less common in adults with confirmed viral respiratory infections, including influenza, than described in children. more than % of adults with a viral respiratory infection do not have fever, which is an important finding for clinical triage of adult patients with respiratory infections. the accepted definition of ‘influenza-like illness’ includes fever and may be insensitive for surveillance when high case-finding is required. a more sensitive case definition could be used to identify adult cases, particularly in event of an emerging viral infection. respiratory infections are common and one of the leading causes of morbidity and mortality, particularly in the extremes of age [ ] [ ] [ ] . influenza a and b, human rhinoviruses (hrv), respiratory syncytial virus (rsv), adenoviruses (adv) and parainfluenza virus (piv) are common respiratory viruses in adults and children [ ] [ ] [ ] [ ] [ ] . of respiratory infections, influenza is the most well studied viral infection, and is commonly reported (around %) as the cause of epidemics of respiratory infection, including nosocomial outbreaks [ ] . influenza virus is commonly isolated from febrile paediatric and elderly patients presenting with influenza-like illness (ili) and acute respiratory illness (ari) symptoms [ ] . the accepted clinical case definition of ili includes fever, which may be suitable for identifying paediatric cases, but less so for adults. fever is thought of as the most common presenting symptom of influenza in hospital emergency departments; however, the presence of fever depends on the age of person and the type of virus [ ] [ ] [ ] [ ] . it is known that fever is less common in adults than children with influenza, and that adults may have atypical presentations [ , , ] . in a matched case-control study in finland, laboratoryconfirmed influenza cases and controls with respiratory symptoms were recruited in children aged years. fever was present in · % ( / ) and · % ( / ) of cases and controls, respectively [ ] . in contrast to this, fever is not a common presentation in adults with laboratory-confirmed influenza. monto et al. [ ] examined clinical trial data of adult ili cases (defined as body temperature · °c or patients subjective feeling of feverishness) and of those ( %) had laboratory-confirmed influenza. fever ( · °c) was reported in % of laboratory-confirmed influenza cases, compared to % other ili cases [ ] . during a randomized clinical trial (rct) around the efficacy of facemask and hand hygiene in the household setting, % ( / ) of secondary cases with influenza a and % ( / ) of cases with influenza b had fever history [ ] . the rate of fever was % ( / ) in hospitalized influenza cases in a us study [ ] . another us study showed that less than half ( · %) of healthcare workers (hcws) with laboratory-confirmed influenza presented with fever [ ] . fever is a less common presenting symptom in elderly people which may lead to diagnostic and treatment delays [ ] . in patients admitted with myocardial infarction, % had unrecognized and undiagnosed influenza on testing at admission, highlighting the low level of clinical suspicion of influenza [ ] . the rate of fever also varies between influenza strains, being more common in influenza a strains than b, and higher in h n [ ] [ ] [ ] [ ] ] . fever is a commonly reported symptom during influenza outbreaks and pandemics due to novel and more virulent nature of strains. in china · % of the patients infected by influenza a(h n )pdm had fever [ ] . in another study in beijing suspected ili cases were tested and of those ( %) were positive for influenza virus (pandemic h n - and seasonal influenza h n - ) and all had history of fever [ ] . the aim of this study was to compare the rates of fever in adult subjects with confirmed influenza and other respiratory virus infections and examine predictors of fever. we analysed a dataset of laboratory-confirmed viral respiratory infections collected from three clinical trials of hcws where active surveillance for respiratory viral illness was conducted in prospective follow up [ ] [ ] [ ] . the same methods, data collection forms and outcome measures, were used across the three studies, allowing the data to be pooled [ ] [ ] [ ] . two studies were conducted in beijing china: trial ( / ) and trial ( / ) and another study (trial ) was conducted in hanoi, vietnam in / [ ] [ ] [ ] . in all clinical trials, participants were asked to complete diary cards on a daily basis to collect information on number of working hours, patients seen, mask use hours, high-risk procedures performed and appearance of respiratory symptoms. thermometers were given the participants to measure their temperature daily and at symptom onset. symptomatic cases were asked to complete sick patient follow-up forms and detailed information was collected on the following symptoms: chill or fever, cough, congestion, runny nose, sore throat, sneezes, lethargy, loss of appetite, abdominal pain, muscle or joint aches. swabs of both tonsils and the posterior pharyngeal wall were collected on the day of reporting. in all rcts, fever was defined as having body temperature °c. clinical respiratory illness (cri) and ili were in the primary outcomes in three clinical trials. cri was defined as two or more respiratory symptoms or one respiratory symptom and a systemic symptom and ili was defined as fever °c plus one respiratory symptom [ ] [ ] [ ] . we analysed data from all subjects with a positive isolation of a respiratory virus by multiplex polymerase chain reaction (pcr). descriptive analysis was conducted for rates of fever by virus type. a logistic regression analysis was used to determine the predictors of fever. a multivariable log binomial model was fitted, using a generalized linear model to estimate relative risk (rr). all variables were included in initial model. in the final model, we included only those variables that were significant (p < · ) in initial analysis. a backward elimination method was used to remove the variables that did not have any confounding effect, i.e. could not make meaningful change (± %) in the rr of the comparison arm. finally we estimated the rates of cri and ili in the laboratory-confirmed viral respiratory infections and laboratory-confirmed influenza infections. the data was analysed using sas v. . (sas institute inc., usa). the demographic characteristics of cases with laboratory-confirmed viral infections are presented in table . ninety ( %) cases were from china and ( %) were from vietnam. the mean age of hcws was · years and most participants were nurses ( %) and female ( %). most cases were non-smokers ( %) and had not received influenza vaccine ( %). viruses isolated included rhinovirus (n = , %), rsv (n = , %), influenza (n = , %), piv (n = , %), human metapneumovirus (hmpv; n = , %), coronavirus (n = , %) and adv (n = , %). more than one virus was isolated in cases ( · %), including nine cases with influenza co-infection. fever was documented in · % cases ( / ) with a positive laboratory viral diagnosis. table details rates of fever ( °c) associated with individual viral respiratory infections. hrv was the most common infection and · % ( / ) of these had a fever. in cases of rsv, four ( · %) had fever; · % ( / ) of piv and % ( / ) of influenza a cases had fever. seven cases of coronavirus and hmpv each were confirmed and of those two ( · %) had fever. when cases with influenza and a co-infection were included, · % ( / ) had fever. in univariate analysis, country, gender and smoking were significant predictors of fever. country and smoking remained significant predictors in multivariate analysis while gender became non-significant. fever rate was significantly higher in hcws in vietnam compared to hcws in china [rr · , % confidence interval (ci) · - · ]. smokers were around five times more likely to have fever compared to non-smokers (rr · , % ci · - · ). virus type was not associated with fever in univariate analysis; however, after adjusting for other variables, rates of fever were significantly higher in hcws co-infected with more than one virus compared to all other viruses excluding influenza (rr · , % ci · - · ) ( table ) . cri symptoms were present in · % ( / ) of hcws with laboratory-confirmed viral infections and · % ( / ) laboratory-confirmed influenza infections. the corresponding rates of ili in the two groups were · % ( / ) and · % ( / ), respectively. we have shown, using prospectively collected data, that the rate of fever in adults with confirmed viral respiratory infections is much lower than described in children [ , ] . the standard clinical case definition of ili requires fever to be presentthe majority of influenza cases in this series would have been missed using the ili definition. this has implications for effective triage, early antiviral treatment and preventive measures for adults with influenza, particularly during outbreaks and pandemic situations. for other respiratory infections, clinical case definitions need to be more sensitive, or > % of cases will be missed. the main implication for future surveillance, measurements and research studies is that the ili case definition in adults may be highly insensitive. for some types of surveillance systems, this may not be an issue, but for diagnostic screening in event of an emerging viral infection (such as for triage and implementation of infection control protocols) [ , ] , a more sensitive case definition is needed. rates of fever in influenza and other viral respiratory infections in this study were lower compared to other studies which report fever in around - % adult cases [ , , , ] . however, this variation may be due to different study base, case definition and viral strains, as well as the prospective measurement of incident infections. many research studies use fever as an inclusion criterion for laboratory testing [ , ] . while this may be suitable for studies in children, it is not adequately sensitive for studies of adults, as we have shown the majority of confirmed cases will be missed. the cut-off point for fever could be another factor in sensitivity. some studies have set lower cut-off points for fever, and report higher rates of fever in laboratory-confirmed influenza cases [ , ] . carrat et al. collected data of cases presented in general practices in france and collected nasal swabs from suspected influenza cases and defined fever as · °c. they found fever in influenza a(h n ), influenza a(h n ) and influenza negative cases in · %, · % and · %, respectively. applying a cut-off of · °c, the corresponding rates are · %, · % and · % [ ] . symptoms of feverishness (subjective feeling of fever) are included in ili definitions in some cases [ ] . previous studies report high rates of fever in children compared to the adults [ ] . low rates of fever in adults may also be due to protection via cross-reactive antibodies due to age-dependent differences in the immunity [ , ] . continued exposure to influenza throughout life may result in a broader protection with age. infection may provoke a stronger immune response in children with minimal to no exposure history compared to adults. therefore influenza infection history might help explain potential differences in clinical symptom severity (and presence of fever) between children and adults. a recent study reported high rates of influenza and other respiratory virus in afebrile hcws with only respiratory symptoms [ ] . of laboratory-confirmed influenza cases in this study, only three ( · %) had ili symptoms, which is very low compared to other studies. in a prospective influenza surveillance study, ili symptoms were present in % of adults and % of children with laboratory-confirmed influenza virus [ ] . cri symptoms were present in · % ( / ) of laboratory-confirmed influenza cases in this study. a highly sensitive definition of influenza may be required to diagnose most of adult influenza cases in the clinical setting to ensure rapid treatment and isolation, and prevention of nosocomial transmission. inclusion of ili cases may overestimate the proportion of febrile cases in influenza surveillance given fever is included in the definition. pre-symptomatic and asymptomatic influenza cases will also be missed, although infectivity and transmissibility of these cases is yet to be proven [ ] . longitudinal studies, where all participants are tested, provide similar estimates around rates of fever as in our study [ ] . we propose a more sensitive clinical case definition without fever as a requisite criterion. clinical signs and symptoms are less studied for other viral respiratory infections, but available evidence suggests that other respiratory viruses are associated with a lower rate of fever compared to influenza [ , [ ] [ ] [ ] [ ] . putto and colleagues [ ] examined the clinical records of children (> months) in a large hospital in finland, including adv ( cases), influenza a and b ( cases), piv ( cases) and rsv ( cases). fever ( · °c) was recorded in % cases with adv, % influenza a virus, % influenza b, % piv- , % piv- , % piv- , and % rsv. van den hoogen and colleagues estimated the prevalence and clinical symptoms of hmpv infection, in the netherlands and fever was reported in % of the hmpv-positive cases [ ] . in hong kong, hmpv was found in · % ( / ) of children admitted in hospitals and all had fever [ ] . manoha et al. examined nasal wash specimens from hospitalized children and found hmpv ( %), rsv ( · %), rhinoviruses ( · %), influenza a ( %), piv- ( · %) and piv- ( · %). fever was reported in · % cases with hmpv, · % cases with rsv and · % with rhinovirus [ ] . of the elderly patients with influenza and with rsv, fever was reported in % and %, respectively [ ] . a us study also reported low rates of fever in hcws infected with coronavirus e ( · %), coronavirus hku ( · %), coronavirus nl ( · %) and rsv ( · %) and all cases of hmpv were without fever [ ] . rate of fever for all other viruses (excluding influenza) was · % ( / ) in this study. co-infection with more than one virus was the strongest predictor of fever for adults with confirmed viral respiratory infections in the present study. previous studies also show high rates of fever in cases with dual respiratory viral infections compared to single viral infection [ , ] . rates of hospitalization and icu admission are also reported to be higher in cases with dual respiratory viral infections [ ] [ ] [ ] . increased severity of symptoms in co-infection cases might be due to an altered immune response [ ] . around % ( / ) of cases in our dataset were infected with more than one virus. drews et al. reviewed the data of eight prospective epidemiological studies and reported the rate of co-infection was % [ ] . studies in children generally report higher rates of co-infection cases ( - %) [ , , , ] . clinicians should consider the possibly of co-infection if a patient presents with fever; however, further epidemiological and clinical studies are required. smoking was also a significant predictor of fever in this study. smoking increases the risk of viral and bacterial infections through changes in respiratory epithelial and altered immune response [ ] [ ] [ ] [ ] .the risk of influenza also increases several times in smokers, compared to non-smokers [ ] . atypical clinical presentation of influenza and other respiratory infections in adults could be due to altered structural and immune response associated with active/passive smoking and other environmental hazards. the mechanism by which smoking increases the risk of fever is not clear. high rates of fever in smokers may also be due changes in immunoglobulin levels which could increase viral load. the severity of symptoms generally increases when high viral load is detected in the blood [ ] . the difference in fever rates between china and vietnam may be due to prevalence of viruses and co-infection. rsv was the most commonly isolated pathogen from china ( %), followed by rhinovirus ( %) and influenza virus ( %). in contrast to this hrv was the most commonly isolated pathogen from vietnam ( · %). the number of cases with co-infection were also different in the two countries - ( %) in china and two ( %) in vietnam. in multivariate analysis, we adjusted for country and type of virus. limited data are available regarding the prevalent viruses circulating in china during the study period. for the trial period, all influenza was influenza a(h n )pdm. for the trial period, · % were h n pdm, · % were h n , · % were influenza b victoria, · % were influenza b yamagata, · % were influenza a unsubtyped (y. zhang, beijing centre for disease prevention and control, personal communication). we could not obtain data on the viruses circulating in vietnam during the study period. there are some limitations to this study. we did not subtype the influenza strains, and studies show that the rate of fever also varies between influenza strains [ ] [ ] [ ] [ ] ] . fever data was self-reported but self-measured in three trials using a traditional glass and mercury thermometer. lower fever rates in chinese hcws in this study might be due to due to differences in circulating viruses (and their pyrogenicity) between the two countries when the studies were conducted. a japanese study of children with influenza reported a tendency towards shorter duration of fever with increasing age in children [ ] ; however, age and other demographic characteristics were not significant in that study. compared to children, this study shows that adults are less likely to have fever with a respiratory viral infection, even influenza. the implication of this finding is that for rapid treatment and reducing the risk of transmission of infection, clinicians should be aware that a diagnosis of viral respiratory infection, even influenza, is possible in the absence of fever. many of these infections are transmissible even when infected persons are asymptomatic or presymptomatic, and greater vigilance for respiratory symptoms in hcws could reduce nosocomial transmission of respiratory viral infections. the absence of fever should not preclude a differential diagnosis of influenza or other respiratory viruses in adults. influenza 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and interferon-gamma response correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection single, dual and multiple respiratory virus infections and risk of hospitalization and mortality dual respiratory virus infections multiple simultaneous viral infections in infants with acute respiratory tract infections in spain multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children cigarette smoking and infection smoking and the outcome of infection cigarette smoke extract suppresses human dendritic cell function leading to preferential induction of th- priming the associations of race, cigarette smoking, and smoking cessation to measures of the immune system in middle-aged men correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients the authors thank the staff at the beijing centre for disease control and national institute of hygiene and epidemiology. thanks are also due to the staff from the hospitals in china and vietnam which participated. m helped in fit testing during the first rct in china, no financial support was provided. the second rct in china was funded through the australian national health & medical research council of australia (grant no. ). funding to conduct the vietnam trial study was received from the australian research council (arc) (grant no. lp ). dr abrar chughtai had testing of filtration of masks by m for his phd. professor c. raina macintyre has held an australian research council linkage grant with m as the industry partner, for investigator-driven research. m have also contributed supplies of masks and respirators for investigator-driven clinical trials. she has received research grants and laboratory testing as in-kind support from pfizer, gsk and bio-csl for investigator-driven research. the remaining authors have no competing interests to declare. key: cord- - f q e authors: schnepf, nathalie; resche-rigon, matthieu; chaillon, antoine; scemla, anne; gras, guillaume; semoun, oren; taboulet, pierre; molina, jean-michel; simon, françois; goudeau, alain; legoff, jérôme title: high burden of non-influenza viruses in influenza-like illness in the early weeks of h n v epidemic in france date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: f q e background: influenza-like illness (ili) may be caused by a variety of pathogens. clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. the limited use of molecular tools underestimates the role of other common pathogens. objectives: during the early weeks of the – flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ili referred to two french university hospitals in paris and tours. aims were to investigate the different pathogens involved in ili and describe the associated symptoms. methods: h n v pandemic influenza diagnosis was performed with real time rt-pcr assay. other viral aetiologies were investigated by the molecular multiplex assay respifinder ®. clinical data were collected prospectively by physicians using a standard questionnaire. results: from week to , endonasal swabs were collected in patients. overall, samples ( . %) were positive for h n v. in of them, other respiratory pathogens were also detected. among h n v negative samples, ( . %) were positive for various respiratory agents, in single infections and in mixed infections. the most prevalent viruses in h n v negative single infections were rhinovirus ( . %), followed by parainfluenza viruses ( . %) and adenovirus ( . %). . % of h n v cases were identified in patients under years and none after years. there was no difference between clinical symptoms observed in patients infected with h n v or with other pathogens. conclusion: our results highlight the high frequency of non-influenza viruses involved in ili during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. in order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. in france, a network of general practitioners, ''réseau sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [ , ] . the criteria used by this network to define clinical influenza-like illness (ili) are the occurrence of a sudden fever above uc with myalgia and respiratory signs. in general no formal viral diagnosis is carried out. the groupes régionaux d'observation de la grippe (grog) is a second french network that surveys the emergence and the spread of the influenza viruses [ , ] . this network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. according to the sentinel network's criteria, french health authorities proclaimed that flu epidemic level was reached during the second week of september (week ) [ , ] . on the contrary, data provided by the grog showed only sporadic h n v activity until the last week of october (week ) [ , ] . thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [ , , , ] . as a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. in france, data on viral aetiologies associated with ili were at best sporadic and correlations with clinical symptoms were often lacking. extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. the aim of the present study was to investigate respiratory pathogens involved in ili during the early weeks of the - h n v diffusion in france (weeks through ) and describe the associated symptoms in paediatric and adult populations. this study was a non-interventional study with no addition to usual proceedures. biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). data analyses were carried out using an anonymized database. according to the french health public law (csp art l - . ), such protocol does not require approval of an ethics committee and is exempted from informed consent application. in the two academic hospitals, saint-louis hospital (sls) in paris and tours hospital (trs), influenza-like illness (ili) was defined as a patient suffering from at least one general symptom (fever above uc, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the french institut de veille sanitaire (invs), a governmental institution responsible for surveillance and alert in all domains of public health [ ] . criteria for severe clinical presentation were temperature below uc or above uc despite antipyretic, cardiac frequency above /min, respiratory frequency above /min, respiratory distress, systolic arterial pressure below mmhg or altered consciousness. predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with hiv infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [ , ] . a cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [ ] . in the two institutions, the prescription of h n v molecular testing was recommended for patients with ili and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. investigation of grouped suspected cases was also recommended. from week (last week of august) to (last week of october), endonasal swabs were collected in ml of universal transport medium (copan diagnostics inc, murrieta, ca) from adults and children seen in emergency rooms for suspected ili (table ) and sent to sls and trs laboratories for h n v detection. the two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza h n v. clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by invs [ , ] . this questionnaire included the presence or absence of the main general and respiratory symptoms associated with ili (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [ ] . total nucleic acid was extracted from ml of universal transport medium using the easymag system (biomérieux, marcy l'etoile, france) in sls or the ez advanced xl (qiagen, courtaboeuf, france) in trs, according to the manufacturers' instructions (elution volume: ml in sls or ml in trs). before extraction, ml of an internal amplification control (iac) which contained an encephalomyocarditis virus (emc) rna transcript was added into the sample. pandemic h n v infection was diagnosed by real-time reverse transcription-pcr (rt-pcr) assay on a real time pcr system (applied biosystems, foster city, ca) according to the protocol of the centers for disease control (cdc) [ ] . other respiratory infections were investigated by a multiplex molecular assay based on the multiplex ligation-dependent probe-amplification (mlpa) technology (respifinder h, pathofinder, maastricht, the netherlands) that allows the detection and differentiation of respiratory viruses, including influenza virus a (infa), influenza virus b (infb), rhinovirus (rhv), parainfluenza viruses to (piv- to piv- ), human metapneumovirus (hmpv), adenovirus (adv), respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb) and human coronaviruses e, oc and nl (cor- e, cor-oc , cor-nl ) [ ] . the test allows also the detection of h n influenza a virus and of four bacteria: chlamydophila pneumoniae (cp), mycoplasma pneumoniae (mp), legionella pneumophila (lp) and bordetella pertussis (bp). the amplified mlpa products were analyzed on an abi genetic analyzer (applied biosystems, foster city, ca). fragment sizing analysis was performed with the genemarker software (softgenetics, llc, state college, pa). further testing for h n v was carried out with simplexa tm influenza a h n ( ) (focus diagnostics, cypress, california) when the cdc real time rt-pcr assay was negative for h n and the respifinder h assay was positive for influenza a. if this latter assay was negative, h n typing was performed as previously described [ ] . data from our study are summarized as frequencies and percentages for categorical variables. quantitative variables are presented as medians, th and th percentiles. to compare those variables according to the viral infection status, fisher tests by using cdc reference assay, h n v was detected in samples out of ( . %), more frequently in sls ( samples) than in trs ( samples) (p, ). overall, weekly percentage of h n v positive endonasal swabs remained under % until week and increase significantly after (p trend , . ) ( figure ). rate of h n v detection reached % in sls at week and in trs at week . overall, this rate was in agreement with results provided by the grog network, showing an earlier start of h n v epidemic in paris area [ , ] . all nucleic acid extracts were analyzed using the respifinder h assay ( figure ). sixty six patients tested h n v positive with cdc real time rt-pcr assay were confirmed with the multiplex assay. thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( figure ). three of the h n v negative samples could not be studied with the multiplex assay because they contained rt-pcr inhibitors (no amplification of the internal control). two hundred and fifteen ( . %) of the remaining h n v negative samples were found positive for at least one respiratory pathogen ( figure ). two hundred and twelve were positive for non influenza pathogens ( single infections and mixed infections with two, three or four viruses) and three additional single infections by influenza a were identified in sls, including two by pandemic h n v and one by seasonal h n , as determined after molecular typing (data not shown). overall, patients ( . %) were then positive for h n v, one for h n and for non influenza pathogens. there were single infections ( with h n v and with other respiratory pathogens) and mixed infections ( with h n v and without h n v) ( figure ). among h n v negative single infections, the most prevalent viruses were rhinovirus ( . %, patients), followed by parainfluenza viruses to ( . %, patients), adenovirus ( . %, patients), human coronavirus e, oc and nl ( . %, patients) and respiratory syncytial virus a and b ( . %, patients) (figure ). in addition, respifinder h assay identified three patients with bacterial infection, two with mycoplasma pneumoniae (one years old female in sls and one years old female in trs) and one with bordetella pertussis (one years old male in sls). no single infection by influenza b, hmpv, chlamydophila pneumoniae or legionella pneumophila was identified ( figure to analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for adv (p = . ). non-influenza respiratory viruses presented a different epidemic profile compared to h n v. overall, in both hospitals, weekly rate of non-h n v respiratory viruses whether alone or involved in co-infection increased between week and (from . % to . %) and then consistently decreased ( figure ). rhv infections that represented nearly half of non-h n v viral infections ( out of , . %) were a significant contributing factor. in both hospitals, emergence of h n v cases was associated with a rapid decline of rhv rate of infection from - % down to less than % with a one to two weeks gap between sls and trs. data on age ( in both institutions, . % ( / ) children younger than years of age were infected by at least one respiratory pathogen ( table ). h n v infected patients were not significantly younger than h n v non infected patients ( years old vs. years old, p = . ) (figure ) . however, . % ( / ) of h n v cases were identified in patients under years old ( in sls and in trs) and no case was observed in patients older than years ( table ) . piv infection occurred in very young patients (median (figure ) . consequently, piv and adv were more frequently detected in the younger population of trs versus sls (p, and p, respectively). in contrast, although individuals with rhv infection were slightly younger than individuals without (median age = vs. for patients without rhv, p = . ) (figure ) , influenza-like illness associated with rhv was more frequent in sls than in trs (p = . ). finally, patients with viral multiple infection were significantly younger than those with single infection (median, idr: , - . vs. , - ) and rates of mixed infection at the time of medical attention, ( . %) standardized clinical questionnaires were collected out of patients. four of them could not be exploited because they were too incomplete. a review of the workable questionnaires showed that . % ( / ) of the patients included in this study fulfilled the criteria of ili as defined above, and . % had either a severe clinical presentation or an underlying risk factor of complications ( . %, / ), or were in a suspected cluster of grouped cases ( . %, / ). overall, most patients have fever ( . %) and cough ( . %) ( table ) . other classical clinical signs associated with ili such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. a sudden onset was also described in . % of cases. only . % of the patients had a temperature above uc; the age of these patients ranged from zero to years, with a median age of years and a mean age of years (data not shown). in h n v infected patients (including single and multiple infections), the main symptoms were also fever ( . %) and cough ( . %) ( we then compared clinical characteristics between patients positive for h n v, patients positive for other respiratory pathogens and negative for h n v and patients without any detection of respiratory pathogens (as detected with respifin-der h) ( table ). there was no difference between the three groups except for fever, cough, pharyngitis. however for these latter symptoms, the comparison between patients positive for h n v and those positive for other respiratory pathogens or between patients positive for h n v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for h n v than in patients positive for other respiratory pathogens ( table ) . as rhv was the most frequent aetiology in ili, we also compared clinical symptoms observed in patients with a single infection by rhv or by h n v (data not shown). there was no difference except that rhinitis and pharyngitis were significantly more frequent in rhv infection ( . % vs. . % [p = . ] and . % vs. . % [p = . ], respectively). viral multiple infection (including samples with h n v) was not associated with a different clinical presentation. fever and cough were observed in over % of the patients ( . % and . %, respectively), but only . % of these patients had a temperature above uc, which was not different from patients with single viral infection ( . %). our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the réseau sentinelles according to ili definition (a sudden fever above uc accompanied by myalgia and respiratory signs). these data extent previous observations in europe reporting high prevalence of rhv infections before seasonal influenza [ , ] or in , before h n v pandemic influenza [ , , , , ] . we confirm that rhv represent the most frequent aetiology of acute respiratory table . age of patients with respiratory samples positive for h n v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than % of cases. we show that other viral infections than influenza and rhv may represent up to % of aetiologies. we observed differences between the two hospitals, with a higher frequency of parainfluenza and adv infections in tours in contrast with a higher frequency of rhv in paris, likely explained by the higher proportion of paediatric samples collected in tours. however, despite the distance between the two institutions (about km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. in the two cities, high frequencies of rhv were seen at the same level with a likely different evolution speed, with sudden increase and decrease in sls and more progressive variation in trs. in both institutions, there was a decrease in the proportion and number of rhv diagnoses roughly in parallel with the increase of influenza diagnoses. indeed, h n v exceeds % of positive detection's rate only when rhv dropped under %. these data are thus consistent with negative interaction of the two epidemics at the population level. it was previously hypothesised that rhv epidemic could interfere with the spread of pandemic influenza [ , , ] . few in vitro data support this hypothesis. it has been reported that interferon and other cytokines production by rhv infected cells induced a refractory state to virus infection these data include the three patients whose respiratory samples could not be studied with the multiplex assay because of rt-pcr inhibitors. of neighbouring cells [ ] . further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. very few rsv infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks - [ ] . it has been confirmed by other laboratories and the french invs that the - rsv epidemic was delayed and had a lower impact compared with the previous winter season [ , ] . delayed and reduced rsv spread may be due to viral interference between rsv and influenza. another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [ ] . influenza infections were mainly detected in patient under years old and no case was found in patients older than . these results corroborate previous data suggesting that past seasonal h n infections or vaccination may give partial crossed protection [ , , ] . we have previously shown that the neutralizing titers against pandemic h n v virus correlate significantly with neutralizing titers against a seasonal h n virus, and that the h n v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [ ] . viral co-infections were predominantly seen in paediatric patients, as previously described [ , , , ] , both in influenza and non-influenza cases at a similar rate. no evidence of more pronounced respiratory impact was seen in these patients. our results showed the lack of specific clinical signs associated with proven h n v infections. clinical characteristics did not differ between influenza infections or other viral infections. in particular, the proportion of patients with fever above uc was not higher in h n v positive patients. in addition, the patients without any evidence of respiratory viral infections did not have different symptoms. these patients may have been infected with other virus not included in the multiplex assay (human bocavirus, coronavirus hku ) [ , , ] or were seen too late at the time of viral shedding was cleared [ ] . however, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (h n v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in sls and trs with respiratory syndromes, but not tested for h n v. in addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the h n v virus were acute and self-limited [ , ] . the higher proportion of non influenza viruses reported in ili in was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. the general lack of difference in symptoms in the particular context of h n v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. our data confirm that it may be virtually impossible to recognize symptoms heralding h n v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [ ] . molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [ , , ] . these data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. our results confirm the performance of respifinder h assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ili [ ] . respifinder h confirmed all h n infections detected by the cdc reference assay and was able to identify two additional h n cases suggesting a high sensitivity of this multiplex assay to detect influenza a infections. in conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for 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using a pseudotype neutralization assay multiplex real-time pcr for detection of respiratory tract infections frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate improving the clinical diagnosis of influenza-a comparative analysis of new influenza a (h n ) cases molecular diagnosis of respiratory viruses detection of respiratory viruses by molecular methods comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h n : what else is out there? we gratefully acknowledge the contribution of the members of the two virology laboratories and sandrine picco for their excellent technical assistance in the detection of h n v pandemic virus and other respiratory viruses, and catherine scieux for her help in epidemiological data analysis. key: cord- -mbiojk authors: benkouiten, samir; al-tawfiq, jaffar a.; memish, ziad a.; albarrak, ali; gautret, philippe title: clinical respiratory infections and pneumonia during the hajj pilgrimage: a systematic review date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: mbiojk background: the islamic hajj pilgrimage to mecca is one of the world's largest annual mass gatherings. inevitable overcrowding during the pilgrims' stay greatly increases the risk of acquiring and spreading infectious diseases, especially respiratory diseases. method: the medline/pubmed and scopus databases were searched for all relevant papers published prior to february that evaluated the prevalence of clinical symptoms of respiratory infections, including pneumonia, among hajj pilgrims, as well as their influenza and pneumococcal vaccination status. results: a total of papers were included in the review. both cohort- and hospital-based studies provide complementary data, and both are therefore necessary to provide a complete picture of the total burden of respiratory diseases during the hajj. respiratory symptoms have been common among hajj pilgrims over the last years. in cohorts of pilgrims, cough ranged from . % to . %. however, the prevalence rates of the most common symptoms (cough, sore throat, and subjective fever) of influenza-like illness (ili) varied widely across the included studies. these studies have shown variable results, with overall rates of ili ranging from % to . %. these differences might result from differences in study design, study period, and rates of vaccination against seasonal influenza that ranged from . % to % among study participants. moreover, the definition of ili was inconsistent across studies. in hospitalized hajj pilgrims, the prevalence of pneumonia, that remains a major concern in critically ill patients, ranged from . % to . %. conclusions: large multinational follow-up studies are recommended for clinic-based syndromic surveillance, in conjunction with microbiological surveillance. matched cohorts ensure better comparability across studies. however, study design and data collection procedures should be standardized to facilitate reporting and to achieve comparability between studies. furthermore, the definition of ili, and of most common symptoms used to define respiratory infections (e.g., upper respiratory tract infection), need to be precisely defined and consistently used. future studies need to address potential effect of influenza and pneumococcal vaccine in the context of the hajj pilgrimage. ksa for several weeks throughout the month-long hajj season, presenting a major public health and infection control concern, and a challenge both for the saudi authorities, as well as for the national authorities of the countries of origin of the pilgrims. in addition to physical exhaustion, sleep deprivation [ ] , and heat stress [ ] , inevitable overcrowding, both in housing and ritual sites, especially in mina encampment (this is approximately a -kilometer square area where pilgrims are accommodated in air-conditioned semi-permanent tents, some with up to - people) and inside the sacred mosque in mecca (with up to six pilgrims per square meter) [ ] , greatly increases the risk of acquiring and spreading infectious diseases [ ] [ ] [ ] , especially respiratory diseases [ , ] . to minimize the spread of infections during the pilgrimage or in the pilgrims' home countries upon their return, vaccination and non-pharmaceutical interventions are thus recommended by national and international public health agencies [ , ] . we carried out a systematic review of cohort and hospital studies that reported the prevalence of clinical symptoms of respiratory infections and pneumonia among pilgrims during the hajj, and both their influenza and pneumococcal vaccination status, with the aim to provide data allowing the investigation of the impact of this large mass-gathering event on public health policies and services and to identify potential targets for preventive measures. this review was performed according to preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines (http://www.prismastatement.org). the medline/pubmed and scopus databases were searched for all relevant papers published prior to february , using the terms: in addition, the saudi epidemiology bulletin (http://seb. drupalgardens.com/) was hand searched for additional papers for inclusion. finally, the reference lists of reviewed articles were searched for additional relevant papers. for inclusion, the article had to meet the following criteria: ( ) original study involving hajj pilgrims; ( ) detailed description of the study population, including influenza and pneumococcal vaccination status when available; ( ) clinical or self-reported respiratory symptoms and diseases. only articles published in english were included for review. we excluded cohort studies with less than participants and case reports. we also excluded studies conducted among selected groups of individuals suffering from respiratory tract infections, due to lacking denominator data. the two authors independently performed the searches, screened titles/abstracts for eligibility, selected papers that appeared to be relevant according to the review's inclusion criteria, and reviewed each of the selected manuscripts in full. the data were extracted from the included papers by one reviewer (sb) and collected in the summary table that was included in the review. the extracted data were checked by the two authors (sb and pg) for accuracy. minor discrepancies were resolved by the authors' discussion. the search strategy initially yielded records, of which were duplicates. twenty-nine additional papers were identified through manual searches. of the papers identified records were excluded after screening the title and abstract. of the full text articles reviewed, were deemed suitable for inclusion in this review influenza-like illness (ili) was defined according to the presence of the triad of cough, subjective fever and sore throat. c ili was defined as subjective (or proven) fever plus one respiratory symptom (e.g. dry or productive cough, runny nose, sore throat, shortness of breath). d ili was defined as subjective (or proven) fever and at least one respiratory symptom such as cough, sore throat and rhinorrhea. e ili was defined as symptoms and signs such as: sudden headache, dry cough, high grade fever, myalgia, coryza, malaise and loss of appetite with an abnormal general appearance. f upper respiratory tract infections (urti) was defined as any person who reported having developed at least one of the constitutional symptoms (fever, headache, myalgia) and one of the local symptoms (running nose, sneezing, throat pain, cough with/or without sputum) after reaching mecca for the hajj or within weeks from return to riyadh. g acute febrile respiratory infection (afri) was defined as the presence of subjective fever plus at least one respiratory symptom (cough, sore throat, runny nose or breathlessness). h two travelers who reported ''bronchitis'' as a symptom were also included. i ili was defined as fever plus sore throat and/or coughing. j common cold was defined as sore throat with coryzal symptoms, and low grade fever. k ili was defined as fever > . °c, myalgia, low back pain, coryzal symptoms and cough. l acute respiratory infection (ari) was defined as one of the constitutional symptoms (fever, headache, myalgia) along with one of the local symptoms (running nose, sneezing, throat pain, cough with/without sputum, difficulty breathing). m ari was defined as any person suffering from at least one of the constitutional symptoms (fever, headache, myalgia) along with one of the local symptoms (runny nose, sneezing, throat pain, cough with/without sputum, difficulty in breathing) developing after reaching makkah for the hajj. n ili was defined as cough and fever > °c with or without the coryzal symptoms and myalgia. o ari was defined as any person suffering from at least one of the constitutional symptoms (fever, headache, myalgia) along with one of the local symptoms (runny nose, sneezing, throat pain, cough with/without sputum, difficulty in breathing) developing after reaching mecca for the hajj. p ili was defined as sore throat with either temperature ≥ . °c or cough. q cough or sore throat or rhinorrhea or muscle ache or headache. according to the inclusion/exclusion criteria. the results of the search strategy are shown in fig. . a total of publications were identified. these studies were conducted among cohorts of pilgrims from the through the hajj seasons. the results of these studies are presented in table . various study designs were used, including cross-sectional studies, case-control studies, and prospective cohort studies with follow-up of pilgrims, before, during and after the hajj. participants were from different countries and continents (africa, north america, asia, europe, as well as from australia), with the majority from iran, and they were recruited from different settings, including travel medicine clinics, vaccination centers, hajj travel agencies, international airports and transit zones, mecca's city and mina encampments. their numbers varied widely in these studies, ranging from to , . respiratory symptoms were common during the hajj. overall, the prevalence of cough ranged from . % in domestic and international pilgrims in [ ] to . % in malaysian pilgrims in [ , ] ( table ) . more recent studies, conducted in different populations of pilgrims during the - hajj seasons, reported prevalence of cough ranging from . % to . % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these studies also reported a comparable prevalence of sore throat ranging from . % to % among pilgrims [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, many of these studies have investigated the epidemiology of respiratory tract infections among pilgrims by estimating the common prevalence of upper respiratory tract infection (urti), acute respiratory infection (ari) or influenza-like illness (ili), which were inconsistently defined across studies by a combination of general symptoms (e.g. cough, sore throat and fever). overall prevalence of ili varied in these studies from % to . % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( [ , ] . however, the ili syndromic case definition used in the - study (ili was defined as cough and fever of more than °c with or without the coryzal symptoms and myalgia) [ , ] was different with that used in the - study (ili was defined as symptoms and signs such as sudden headache, dry cough, high grade fever, myalgia, coryza, malaise and loss of appetite with an abnormal general appearance) [ , ] . also, it is unclear from the study [ ] if the definition used was consistent with those used in the two previous studies [ , , , ] . in a recent large study, conducted among egyptian pilgrims between and , the prevalence of ili was . % (ili was defined according to the world health organization definition as the presence of measured fever of ≥ c°, and cough; with onset within the last days) [ ] . other studies of different sizes (from to ) and design were conducted from through among different populations of pilgrims using a common ili definition (the association of cough, sore throat, and subjective fever). these studies have shown variable results, with overall rates of ili ranging from % to . % [ ] [ ] [ ] , , [ ] [ ] [ ] , , , , ] . thus, during the hajj season, while the highest prevalence of ili was observed among malaysian pilgrims, with a prevalence estimated at . % [ ] , a lower prevalence was observed among french pilgrims ( . %) [ , ] . coverage of seasonal influenza vaccination among pilgrims was evaluated in many studies, which have yielded varying results, with reported rates of influenza vaccination ranged from . % to % [ , , , [ ] [ ] [ ] , , , , [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a variation over time in influenza vaccination coverage was observed, as exemplified by a rate of . % observed in a survey of pilgrims from riyadh in [ ] , but . % in a similar survey in [ , , ] . during the hajj season, influenza vaccination rates also varied according to pilgrims' country of origin [ ] , with % observed among saudi pilgrims, % among qatari pilgrims, and % among australian pilgrims, while a study involving french pilgrims interestingly reported that none of them had received the influenza vaccine before departing for the hajj because the vaccine was not available at this time [ , ] . the majority of the studies reported influenza vaccination coverage among pilgrims, but only [ , , [ ] [ ] [ ] , , , , [ ] [ ] [ ] ] reported their pneumococcal vaccination status, with rates ranging from . % among a multinational cohort of pilgrims from countries (from africa, asia, usa and europe) in [ ] to . % among a small study of french pilgrims in [ , ] . of the publications that were included in this review, specifically addressed ill hajj pilgrims at health care facilities from through hajj seasons. medical facilities included primary health care centers (phccs) and different specialized wards in tertiary care hospitals, including ear, nose and throat (ent) departments, intensive care units, emergency units, infectious disease units and unspecified medical units. pilgrim participants were included either as inpatients or outpatients. the results of these studies are summarized in table . overall, the prevalence of upper respiratory tract infections (urti) ranged from . % to . % (table ). this prevalence was . % among pakistani pilgrims who attended the king abdul aziz hospital in medina during the hajj [ ] and . % among saudi and non-saudi patients ( . % of them were pilgrims) who attended the ent clinic at al-noor specialist hospital in mecca during the hajj [ ] . pharyngitis was also frequently reported among ill pilgrims. thus, in this study of pilgrims during the hajj, the overall prevalence of pharyngitis was . % [ ] . more recently, in , the prevalence of pharyngitis in a large cohort of outpatients patients from nationalities who attended randomly selected mina phccs ( . % of whom were pilgrims) was found to be . % [ , ] , and % in a study of saudi and non-saudi patients ( . % of them were inpatients) [ ] . however, in this second study of patients, only . % were pilgrims. on the contrary, lower prevalence rates of bronchitis were reported during the hajj ( . %- . %) [ ] [ ] [ ] [ ] [ ] . a recent retrospective cross-sectional multicenter study of turkish inpatients ( . % were pilgrims) who returned to turkey from the arabian peninsula countries between and reported a slightly higher prevalence of acute tracheobronchitis ( . %) [ ] . in addition, in this study, pneumonia was among the most common clinical diagnosis among the hospitalized hajj patients and represented about half of diagnoses [ ] . as pneumonia remains a major concern in critically ill patients, most of them reported the prevalence of pneumonia among pilgrims [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , with reported rates ranging from . % in in randomly selected mina primary health care centers [ , ] to . % in in two icu in mecca [ ] (table ). the prevalence of pneumonia was not reported in papers [ , , ] . pneumonia was the second most common admitting diagnosis ( %) in a study of patients admitted to the icus in four hospitals in mina during the hajj [ ] . this result is further confirmed by a recent study of critically ill hajj patients, of over nationalities, admitted to hospitals in and . in this study, pneumonia was defined as the primary cause of critical illness ( . %) of all icus admissions during the hajj [ ] . also, in another prospective study of pilgrims admitted in two major icus in mecca for the hajj season, community acquired pneumonia (cap) was the commonest source of sepsis, . % [ ] . [ ] acute bronchitis: . % a upper respiratory tract infection (urti) was defined as an acute infection that includes tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, and the common cold. b acute tracheobronchitis was defined as a patient with dry cough and/or low-grade of fever (< °c), sub-sternal pain, and fatigue in the absence of opacities on chest x-ray. c acute exacerbation of chronic obstructive pulmonary disease (copd) was defined as an association with increased frequency and severity of coughing and/or shortness of breath and wheezing, increased amount of sputum production, and/or a change in appearance of sputum in a patient with copd. d was not defined. the purpose of this review was to provide syndromic surveillance data that may be useful, in conjunction with microbiological data that will be presented in further papers, for the surveillance of respiratory infections and pneumonia during the hajj. despite the fact that some of the included studies in our review were performed among small numbers of pilgrims and cannot be extrapolated, it is clear from this work that respiratory symptoms have been common among hajj pilgrims over the last years, as evidenced by the high prevalence of cough (over %) among malaysian pilgrims during the hajj [ ] . cough is a common symptom among pilgrims [ , ] and likely results from crowded conditions during the hajj. this close contact among such individuals may increase the risk of the transmission of respiratory pathogens, and therefore may contribute to respiratory disease outbreaks. climatic conditions and air pollution in mecca and surrounding holy sites during the hajj [ ] may also play a role. recent follow-up studies thus evidenced a significant acquisition of respiratory viruses, particularly rhinovirus, influenza virus, and coronaviruses other than middle east respiratory syndrome coronavirus (mers-cov), and of bacteria, including streptococcus pneumonia, hemophilus influenza, staphylococcus aureus and klesiella pneumonia by hajj pilgrims upon their return from the hajj [ , ] . respiratory diseases are the most common diseases observed among pilgrims attending mina primary health care centers [ ] and a major cause of hospital admission during the hajj [ ] , with pneumonia a leading cause of admission to intensive care units [ , ] , where they are responsible for about half of the cases of sepsis [ ] . unfortunately, while numerous articles on hajj pilgrims were retrieved from our literature search, relatively few recent articles specifically addressed ill pilgrims in the context of hospital settings. the use of cohort studies allows investigators to evaluate the actual incidence of clinical events in hajj pilgrims since it provides a denominator, but may not identify and capture the prevalence of some underlying conditions and of severe forms of respiratory tract infections, which are more likely to be evidenced in hospital patient populations. conversely, hospital studies use data that may be biased, frequently lacking denominator values, and so probably overestimating the occurrence of severe illness. moreover, a hospital-based study will, by definition, not capture some minor illness cases that do not require hospitalization. the prevalence rates of cough, sore throat and subjective fever varied widely across the included studies. these differences may result from differences in study design that may lead to potential biases (for example bias related to the method of data collection, using either selfreport questionnaires or telephone interview), study period (with regards to the seasonality of respiratory viral infections), and rates of vaccination against seasonal influenza among study participants which may widely vary from one study to another, as described in this review. thus, all data regarding the pilgrims, including demographic data, medical history, clinical data and information on vaccination status and compliance with non-pharmaceutical preventive measures, should be carefully collected by using standardized questionnaires. in addition, in the context of syndromic surveillance for respiratory pathogens, data regarding the pilgrim's symptoms should be collected prospectively during face-to-face interviews by trained medical investigators who travel with the pilgrims. one important result of this review is the finding of a lack of consistency ili syndromic case definitions across included studies. thus, in a study (that did not fulfill the inclusion criteria for this review) [ ] , of malaysian pilgrims who had a clinic visit for upper respiratory tract symptoms at five clinics during the hajj, with the aim of determining influenza vaccine effectiveness against clinically defined ili, % had ili (defined as sore throat in combination with either temperature ≥ °c or cough) and % had influenza by the cdc definition (defined as measured fever [≥ °f ( . °c)] and a cough and/or a sore throat). only one of the studies reported here used the cdc definition of ili or the who definition (an acute respiratory infection with measured fever of ≥ c°a nd cough, with onset within the last days) [ ] . in his paper, rashid et al. demonstrated the low sensitivity of the cdc criteria and proposed therefore the use of the triad of 'cough, sore throat and subjective fever' to clinically define ili at the hajj or other mass gatherings, since this new simple clinical case definition is more specific and sensitive than the cdc definition [ ] . this definition was used over the last years by french [ , , , [ ] [ ] [ ] , ] , malaysian [ , , ] , indian [ ] and afghan [ ] investigators leading cohort studies among hajj pilgrims, thus allowing more reliable comparisons of findings between studies (table ) . respiratory diseases are a major concern during the hajj. nonpharmaceutical interventions (e.g., hand hygiene, wearing face masks, social distancing) are known to reduce the spread of respiratory viruses from person to person and are therefore recommended to pilgrims by public health agencies. although hand hygiene compliance is high among pilgrims, face mask use and social distancing remain difficult challenges. data about the effectiveness of these measures for preventing acute respiratory infections at the hajj are limited, and results are contradictory, highlighting the need for future large-scale studies [ ] . in addition to non-pharmaceutical interventions, vaccination against influenza is recommended for all hajj pilgrims by the ministry of health of saudi arabia [ , ] . differences in study design and heterogeneity in the ili definition across studies make it difficult to compare findings from different studies and inhibits the drawing of conclusions regarding the potential effects of this vaccination on related clinical symptoms of influenza disease. however, recent papers by alqahtani et al. and alfelali et al. found the influenza vaccine to be effective, respectively, against both laboratory-confirmed influenza [ ] and clinical influenza [ ] . as influenza vaccination is generally considered effective in reducing influenza-related infections, the scientific committee for influenza and pneumococcal vaccination guidelines (scipv) thus recommends, in its recent guidelines, an influenza vaccination for all people, especially those at high risk, at least weeks before the hajj [ ] . it also recommends, for the next hajj seasons that will take place from june to september, the administration (prior to the hajj) of the southern hemisphere influenza vaccine for pilgrims from the southern hemisphere (where influenza positivity rates are higher during this period). furthermore, as the influenza vaccine is not expected to be available for pilgrims from the northern hemisphere before these next hajj seasons, the scipv also recommends the administration of the southern hemisphere influenza vaccine for those pilgrims from the opposite hemisphere before the hajj [ ] . because of the mismatching between circulating and vaccine strains that has frequently occurred since [ ] , alfelali et al. recommends, when the composition of influenza vaccines differs and whenever logistically feasible, taking into consideration the dual vaccination of hajj pilgrims with both the southern and northern hemispheres' vaccines. however, such strategy is impaired by the frequent unavailability of the southern hemisphere influenza vaccine in the northern hemisphere. the issue of influenza vaccine availability to match southern and northern hemispheres was discussed by the saudi ministry of health in consultation with the who and it was recommended to use the available hemisphere strain as long as there is a match in circulating strains [ ] . despite the risk of acquisition of s. pneumoniae during the hajj, there is currently no consistent guideline on the use of pneumococcal vaccine for hajj pilgrims across pilgrim countries of origin [ , ] . thus, and because many of the hajj pilgrims are elderly and have chronic illnesses and underlying risk conditions for which pneumococcal vaccination is recommended [ ] , the scipv also recommended, in its pneumococcal vaccination guidelines, pneumococcal vaccination of the atrisk population at the appropriate time before the hajj, using the types of pneumococcal vaccines that are currently available: the valent polysaccharide pneumococcal vaccine (ppsv ) and the -valent conjugate vaccine (pcv ) [ ] . however, it did not recommend providing a pneumococcal vaccine routinely to healthy persons aged less than years, because of lack of evidence. in addition, it has been well demonstrated that the conjugate vaccine against s. pneumoniae targets the most virulent serotypes associated with invasive pneumococcal diseases (ipd) that are also associated with antibiotic resistance [ ] . these arguments reinforce the need for compliance with current recommendations for vaccinating at-risk hajj pilgrims against ipd and influenza [ ] . respiratory tract infections, including influenza, continue to be a major concern during the hajj. both cohort-and hospital-based studies provide complementary data and potentially useful information, and both are therefore necessary to provide a complete picture of the total burden of respiratory diseases during this mass gathering. large multinational follow-up studies are thus recommended for clinic-based syndromic surveillance, in conjunction with microbiological surveillance. matched cohorts ensure better comparability across studies, particularly in terms of origin of pilgrims and possible travelling conditions. however, the study design and data collection procedures should be standardized, to facilitate reporting and to achieve comparability between studies. furthermore, the definition of ili, and of most common symptoms used to define respiratory infections (e.g., urti), needs to be precisely defined and consistently used. future studies need to address the potential effects of influenza and pneumococcal vaccine in the context of the hajj pilgrimage. moreover, because of the mismatching between circulating and vaccine strains that has frequently occurred since [ ] , alfelali et al. recommends, when the composition of influenza vaccines differs and whenever logistically feasible, taking into consideration the dual vaccination of hajj pilgrims with both the southern and northern hemispheres' vaccines. however, such strategy is impaired by the frequent unavailability of the southern hemisphere influenza vaccine in the northern hemisphere. despite the risk of acquisition of s. pneumoniae during the hajj, there is currently no consistent guideline on the use of pneumococcal vaccine for hajj pilgrims across pilgrim countries of origin [ , ] . thus, and because many of the hajj pilgrims are elderly and have chronic illnesses and underlying risk conditions for which pneumococcal vaccination is recommended [ ] , the scipv also recommended, in its pneumococcal vaccination guidelines, pneumococcal vaccination of the at-risk population at the appropriate time before the hajj, using the types of pneumococcal vaccines that are currently available: the -valent polysaccharide pneumococcal vaccine (ppsv ) and the -valent conjugate vaccine (pcv ) [ ] . also, it did not recommend providing a pneumococcal vaccine routinely to healthy persons aged less than years, because of lack of evidence. respiratory tract infections, including influenza, continue to be a major concern during the hajj. both cohort-and hospital-based studies provide complementary data and potentially useful information, and both are therefore necessary to provide a complete picture of the total burden of respiratory diseases during this mass gathering. large multinational follow-up studies are thus recommended for clinic-based syndromic surveillance, in conjunction with microbiological surveillance. matched cohorts ensure better comparability across studies, particularly in terms of origin of pilgrims and possible travelling conditions. however, the study design and data collection procedures should be standardized, to facilitate reporting and to achieve comparability between studies. furthermore, the definition of ili, and of most common symptoms used to define respiratory infections (e.g., urti), needs to be precisely defined and consistently used. future studies need to address the potential effects of influenza and pneumococcal vaccine in the context of the hajj pilgrimage. none. the authors have no conflicts of interest to declare. the general authority for statistics in the kingdom of saudi arabia from hajj services to mass gathering medicine: saudi arabia formalizes a novel discipline hajj: journey of a lifetime social identification moderates the effect of crowd density on safety at the hajj hajj: infectious disease surveillance and control global perspectives for prevention of infectious diseases associated with mass gatherings health risks at the hajj 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research team. changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a -year retrospective analysis of data the saudi thoracic society guidelines for influenza vaccinations mismatching between circulating strains and vaccine strains of influenza: effect on hajj pilgrims from both hemispheres expected immunizations and health protection for hajj and umrah -an overview prevention of pneumococcal infections during mass gathering pneumococcal infections at hajj: current knowledge gaps the saudi thoracic society pneumococcal vaccination guidelines- emergence of drug resistant bacteria at the hajj: a systematic review none. supplementary data to this article can be found online at https:// doi.org/ . /j.tmaid. . . . key: cord- -tnnd s authors: yin, j kevin; lahra, monica m; iskander, mary; lambert, stephen b; heron, leon; nissen, michael d; rost, laura; murphy, jennifer; sloots, theo p; booy, robert title: pilot study of influenza vaccine effectiveness in urban australian children attending childcare date: - - journal: j paediatr child health doi: . /j. - . . .x sha: doc_id: cord_uid: tnnd s background: influenza outbreaks in the childcare setting are a significant cause of excess winter morbidity. this study explored methods of follow up and sample collection for a proposed randomised controlled trial of influenza vaccination in children attending childcare. methods: the study was conducted in four sydney childcare centres during . healthy children aged – months eligible for vaccination were recruited in two centres, with another two acting as controls. data on influenza‐like illness (ili: ≥ . °c plus at least one respiratory symptom) occurrence were collected weekly. in those children with an ili, parents were asked to collect nasal swabs and send via surface mail for viral polymerase chain reaction. vaccine efficacy (ve) for ili was estimated overall and for subgroups aged – and – months using the formula ve = − relative risk (rr). results: sixty‐three per cent ( / ) of eligible children had parents give consent. sixty‐three children received influenza vaccine and participated as controls. of specimens returned, a virus was detected in ( %); none with influenza. two symptomatic children had positive near‐patient influenza tests in general practice (one a vaccine failure). the rr with % confidence interval in all children and those aged – months were less than one, . ( . – . ) and . ( . – . ), respectively. conclusions: this study demonstrated the feasibility and utility of parent‐collected and mailed respiratory specimens for ve research in the childcare setting. two‐thirds of parent‐collected swabs proved positive for at least one virus. finding ways to reduce reluctance of parents to submit samples could improve the representativeness of samples collected and the power of the study. no evidence was found for influenza ve, but point estimates were in the direction of protection. influenza is a seasonal, vaccine-preventable disease which causes excess morbidity and mortality during winter in temper-ate climates. the health and economic costs associated with childhood influenza are substantial. for example, in australia during - , there were reports of hospitalisations and four deaths for influenza and pneumonia among children aged under years. the annual cost due to influenza-related diseases in australia is estimated to exceed $ million. the world health organization recommends annual influenza vaccination as the cornerstone for prevention and control. efficacious influenza vaccines have been available for over years, and yet, routine use in childhood remains the what is already known on this topic children in childcare are more likely to contract influenza and transmit infection to their siblings, parents, extended families and child-care workers. usa, canada and western australia currently have a routine influenza vaccine policy in place that includes children months of age and older. evidence for the effectiveness of influenza vaccine in children aged less than months is limited and high quality, appropriately powered, randomised controlled trials are needed. it is feasible to follow children weekly for months to obtain swabs for influenza-like illness. two-thirds of parent-collected swabs were positive for at least one virus demonstrating the utility of this approach for future studies. reluctance of parents to submit swabs for analysis may be a limitation of this approach. exception in most countries. the effectiveness of influenza vaccine for children in childcare has been demonstrated for children aged Ն months. [ ] [ ] [ ] [ ] [ ] although the us advisory committee on immunisation practices has recommended children aged - months to be vaccinated with influenza vaccine since , there is ongoing debate about vaccine effectiveness in this age group. three recent systematic reviews [ ] [ ] [ ] concluded that either influenza vaccine was not effective in children Յ months of age or that there were insufficient data to form a conclusion. influenza is transmitted from person to person through contact and respiratory droplets; however, the droplets do not remain suspended in the air for long nor do they travel far. transmission of influenza generally requires close contact with an infected person or contact with a contaminated surface or object. , the childcare setting provides enhanced opportunities for transmission of infections including influenza as there is prolonged close interaction between young children and the sharing of toys and other objects. further to this, young children are particularly susceptible to infection as they are immunologically naïve to many viruses. commercial childcare in australia is available in two broad categories: daycare centres (dcc) for children aged weeks until years and pre-school centres (psc) for children aged to years. commercial childcare usage in australia is increasing. the median attendance time for australian children who use childcare is h per week, but % attend h a week or more. children in childcare are known to be more likely to contract respiratory illnesses, including influenza, [ ] [ ] [ ] [ ] [ ] and are considered to be major transmitters of influenza to their siblings, parents, extended families and care workers. , , , the influenza season in australia ran from late may until october and notifications peaked during august. australia witnessed antigenically drifted influenza virus (a/brisbane/ / (h n )-like and a/brisbane/ / (h n )-like), and it was the most severe influenza season since a national influenza reporting system was established in . with this study, the primary process issues of conducting influenza vaccine research in the childcare environment were evaluated, including recruitment, retention, vaccination and specimen handling. while this pilot study was not powered to assess an efficacy end point, preliminary vaccine efficacy (ve) data were also examined. from july to august , children aged - months attending four childcare centres in new south wales were recruited for this study: two dcc caring for children aged - months and two psc caring for children aged - months. the four dcc were chosen by convenience (proximity to the children's hospital at westmead) with equal number of children between dcc and psc. one dcc and one psc were allocated to influenza vaccination, and one dcc and one psc were allocated to be controls. this study was approved by the royal alexandra hospital for children ethics committee, and informed parental consent for participation was obtained prior to study procedures. the par-ticipating children were evaluated in two age groups based on age at enrolment: - months and - months. the influenza vaccine administered was a southern hemisphere preparation, purified, inactivated, split vaccine (vaxi-grip junior, provided by sanofi pasteur, lyon, france), incorporating: children were administrated the vaccine according to the standard recommended dose and schedule for age. as all children at the centres randomised to receive vaccine were influenza vaccine naïve, each received two doses of vaccine month apart - . ml intramuscular dose for those less than months of age and . ml intramuscular dose for those aged - months at the time of their first dose. vaccines were administered between july and september . we defined influenza-like illness (ili) as an illness with fever > . °c and with one or more respiratory symptoms (cough, blocked nose or runny nose) to maximise sensitivity. as a protective level of antibody is usually detectable within weeks of the second dose of vaccine, , ili surveillance was commenced in vaccinated children at this time point. in control children, ili surveillance was arbitrarily commenced from the week ending august : at this time, just over half ( / ) of the children eventually fully vaccinated had received vaccine, and from that week, the ratio of child-weeks of follow up in vaccinated and unvaccinated children was similar (fig. ) . parent education for ili surveillance was provided at study entry. households received a weekly email or telephone call from july until october ( weeks) to monitor the study children for ili symptoms. parent training for the collection of nasal swabs was conducted by study nurses after the second immunisation. nasal swabs were collected using the virocult collection system (mw ) consisting of a rayon swab on a plastic shaft, with viral transport medium-soaked foam pad in the base of the transport tube (copan italia, brescia, italy), and were returned to the queensland paediatric infectious diseases laboratory using a pre-addressed, postage-paid envelope. returned specimens were tested using previously reported, real-time polymerase chain reaction assays with reverse transcription for rna viruses. a total of viruses were investigated: human rhinoviruses (hrv), influenza a, influenza b, rsv, adenoviruses, hmpv, parainfluenza viruses i, ii and iii, bocavirus hpyv-wu, hpyv-ki, and human coronaviruses: oc , e, nl and hku . while it was not part of the study protocol, some children had near-patient influenza tests performed by their general practitioners and these were reported by parents to study staff. ili incidence rates were calculated using child-weeks in the denominator. rate ratios (rr) and % confidence intervals were calculated comparing vaccinated and unvaccinated groups. these values were used to estimate ve using the formula ve = ( -rr) ¥ %. comparisons were performed in three age groups, - months (all children), - months and - months. the average ages of the children in the vaccine and control centre were . ( . - . ) and . ( . - . ) months, respectively, while the proportions who were males were . and . %. data on non-enrolled children were not recorded. there were children in total attending the four childcare centres, and children were enrolled giving a recruitment rate of %. complete information was available for analysis in children, with one vaccinated child lost to follow up during the study period (fig. ). there were ( children) and ( children) child-weeks of follow up in vaccine and control centres, respectively. a total of ilis were identified in all study children, and weekly ili incidence rates are provided (fig. ) . of the swabs received during the study period, ( %) had at least one virus identified, with bocavirus being the most common virus found in six swabs (table ) , followed by hrv in five swabs. one swab contained three viruses (hrv, adenovirus and bocavirus). thirteen of these swabs were from vaccinated children, with the other half from controls. two positive near-patient influenza tests (one a vaccine failure) were reported by parents to the study staff: one was from an unvaccinated child in a control centre; the other child was vaccinated with the test done days post-second vaccination ( table ) . there were a total of ilis identified with efficacy point estimates in the direction of protection for all age-groups but not significant ( table ). the key findings of this pilot study were that a high recruitment rate could be achieved, that recruited families were tolerant of regular weekly follow up over an extended period ( months) and that there was no evidence of protective efficacy, but point estimates of ve for the less-specific end point of ili were in the direction of protection. it is inevitable that ili would include non-influenza infections which cause respiratory signs and symptoms, especially as we used a sensitive definition (at the expense of specificity), so it is not surprising that a range of other viral pathogens were identified in our study. a population-based surveillance study showed that less than % of hospitalised children aged Յ months with ili had confirmed influenza infection. our study has some limitations. the childcare centres were not randomised. the commencement midway through an influenza season limited the number of influenza cases identified. less than half the episodes in children of ili ( out of , %) had a respiratory sample sent. this reduced sampling is probably due to the added burden on parents of sample collection (and posting) while a child is ill. in addition, as the childcare centres were in the suburbs with relatively lower socio-economic indexes for areas (and also involved larger families), this, too, may have limited parental cooperation. the participants were only followed from the nd half of august when the season was peaking, so some may have thought that sample collection in september or october was too late. given the limited data that were collected on symptomatology and the relatively small number of specimens, it was not possible to do an extensive analysis comparing symptoms by virus type to address if there are differences in symptoms among various viruses. furthermore, the demographic data (e.g. sex, age range) of those who did not participate in the study were not collected; therefore, it was not possible to identify if there was any recruitment bias. greater efforts are required for future studies in (i) improving the proportion of swabs collected and sent by initiating ili follow up before the influenza season starts and findings better ways to overcome parents' reluctance in submitting swabs; (ii) obtaining more detailed data on symptomatology of respiratory infection; and (iii) collecting de-identified demographic data on those who are not enrolled in the study. this study showed no evidence for influenza ve. there was only a suggestion of protection in that all the point estimates were in that direction. trivalent, live, cold-adapted influenza vaccine (caiv-t) may be a better option for young children and has been demonstrated to have significantly higher efficacy than inactivated vaccine among young children during moderate , and high attack-rate influenza seasons. caiv-t was also able to provide protection even when the circulating influenza virus was an antigenically distinct strain. there were two children with positive results for influenza a from the near-patient test. this type of test is known to have only moderate sensitivity but high specificity, so a positive test is unlikely to be false. week ending date ( older children ( - months) are more likely to have developed natural immunity through previous infection and are more likely to have developed better personal hygiene. for these reasons, older children are less susceptible to influenza as well as other non-influenza infections that may cause ili. this pilot study has shown the feasibility and value of parentcollected (and mailed) nasal sample for influenza ve research in childcare. two-third of parent-collected swabs proved positive for at least one virus. means of lessening reluctance of parents to submit respiratory samples from their children need to be found to improve the representativeness of samples collected and the power of the study. no evidence was found for influenza ve, but point estimates were all in the direction of protection. the cost of community-managed viral respiratory illnesses in a cohort of healthy preschool-aged children vaccine preventable diseases and vaccination coverage in australia influenza-related disease: the cost to the australian healthcare system influenza a vaccine decreases the incidence of otitis media in -to -month-old children in day care the effect of mass influenza immunization in children on the morbidity of the unvaccinated elderly effectiveness of influenza vaccination of day care children in reducing influenza-related morbidity among household contacts studies of the - inactivated influenza vaccine 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nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction detection of human bocavirus in respiratory, fecal, and blood samples by real-time pcr development and evaluation of real-time pcr assays for the detection of the newly identified ki and wu polyomaviruses real-time rt-pcr detection of respiratory viral infections in four triplex reactions human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens census of population and housing: socio-economic indexes for areas (seifa), australia live attenuated versus inactivated influenza vaccine in infants and young children various factors associated with the manifestation of influenza-like illness trivalent live attenuated intranasal influenza vaccine administered during the - influenza type a (h n ) outbreak provided immediate, direct, and indirect protection in children laboratory diagnosis of human seasonal and pandemic influenza virus infection key: cord- -jozrgcq authors: tan, xin quan; zhao, xiahong; lee, vernon j; loh, jin phang; tan, boon huan; koh, wee hong victor; ng, sock hoon; chen, mark i-cheng; cook, alex richard title: respiratory viral pathogens among singapore military servicemen – : epidemiology and clinical characteristics date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: jozrgcq background: few studies have comprehensively described tropical respiratory disease surveillance in military populations. there is also a lack of studies comparing clinical characteristics of the non-influenza pathogens with influenza and amongst themselves. methods: from may through october , consenting cases of febrile respiratory illness (fri) (temperature [greater than or equal to] . degreesc with cough or sorethroat) and controls in the singapore military had clinical data and nasal washes collected prospectively. nasal washes underwent multiplex pcr, and the analysis was limited to viral mono-infections. results: % of cases tested positive for at least one virus, of whom % had multiple infections. % of the fri cases fulfilled the definition of influenza-like illness (ili), of whom % were positive for at least one virus. the most frequent etiologies for mono-infections among fri cases were influenza a(h n )pdm ( %), influenza b ( %) and coxsackevirus ( %). the sensitivity, specificity, positive predictive value and negative predictive value of ili for influenza among fri cases were %, %, % and % respectively. on logistic regression, there were marked differences in the prevalence of different symptoms and signs between viruses with fever more prevalent amongst influenza and adenovirus infections than other viruses. conclusion: there are multiple viral etiologies for fri and ili with differing clinical symptoms in the singapore military. influenza and coxsackevirus were the most common etiology for fri, while influenza and adenoviruses displayed the most febrile symptoms. further studies should explore these differences and possible interventions. influenza-like illness (ili) is often used for influenza surveillance [ ] , as influenza is a disease of global interest with % of adults developing symptomatic disease annually and with case fatalities of . % in susceptible populations [ ] . while influenza surveillance remains a priority, ili can also be caused by a wide range of viral pathogens that present with a spectrum of respiratory symptoms [ ] [ ] [ ] [ ] . in the tropics, viral respiratory pathogens have been reported to exhibit different seasonality and transmission characteristics compared to temperate climates [ , [ ] [ ] [ ] . this necessitates a better understanding of their epidemiology to assess the utility and importance of surveillance in these settings. the year-round circulation of respiratory viruses in the tropics may also predispose patients to coinfection with multiple pathogens, with implications for severity of disease [ , ] and secondary bacteria infection [ , ] . while there have been studies comparing differences in clinical presentation between influenza and non-influenza cases [ ] , few describe the epidemiology and differences in clinical presentation among various non-influenza respiratory viruses. as influenza viruses have accounted for only between . % to . % of all ili cases [ ] [ ] [ ] , it is important to understand the contribution of other respiratory pathogens to overall morbidity and to determine their epidemiological distribution and clinical presentation. to address these issues, this study explores data obtained from a respiratory disease sentinel surveillance system in the singapore military to examine the etiologic viral agents of respiratory illnesses in a tropical environment, to determine the viruses that circulate post-influenza vaccination, and to compare the differences in clinical presentation. singapore is a city-state in tropical south-east asia with a population of . million people (mid-year ). the singapore military is based on national service in which all male citizens and liable permanent residents serve for two years after high school. servicemen typically live in barracks-style accommodation on weekdays and return home on weekends. the singapore military started a sentinel respiratory disease surveillance program in major camps (including a recruit training camp) on may , tracking febrile respiratory illness (fri) cases (temperature ≥ . °c with cough or sore throat). the definition of fri contrasts with influenza-like illness (ili, defined as fever ≥ . °c with cough or sore throat) to broaden the capture of other febrile cases that also result in absenteeism while limiting cases to those with fever as an indicator of severity. this allows for detection of a larger number of respiratory pathogens. patients who visited the primary healthcare clinics in the camps between may and october during regular consultation hours who met the fri criteria were recruited. healthcare workers obtained written informed consent, administered a questionnaire, obtained clinical specimens and performed a clinical examination on partcipants. repeat consultations were excluded if the healthcare worker determined that the patient had not recovered from the first illness episode. we also obtained samples from controls (those without respiratory symptoms or acute infections), who were recruited across the year at between to persons per week. informed consent, the baseline questionnaire, and clinical specimens were obtained. from december , all recruits were administered with the influenza a(h n )pdm (flu-a(h n )pdm ) vaccine. the trivalent seasonal influenza vaccination was first introduced to recruits in december , followed henceforth by all other personnel in november . nasal washes from each side of the nose were taken from consenting participants by trained medical staff, placed in viral transport media and refrigerated. the samples were transported to the laboratory on ice for etiological testing within hours. laboratory analysis was performed in an iso accreditated laboratory for molecular diagnostics which regularly takes part in external proficiency programs such as qcmd eqa programs. detailed laboratory methods were previously described [ ] . we used the multiplex pcr strategy based on the resplex assays described below, and performed additional singleplex pcr assays to determine the influenza subtype. total nucleic acids were extracted from each specimen using the dna minikit (qiagen, inc, valencia, ca, usa) according to manufacturer's instructions. a total of μl of extract were tested with resplex i and ii (version . , qiagen, inc., valencia, ca, usa) [ ] for respiratory micro-organisms on the liquichip workstation, according to manufacturer's instructions. the resplex i and ii (version . ) assays are multiplex pcr assays coupled with bead array detection technology and can simultaneously detect and subtype different pathogens including influenza a (flu-a) and influenza b (flu-b). specimens that were resplex ii positive for flu-a were further subtyped with real-time pcr for h or h (singapore ministry of health), or for flu-a(h n )pdm . briefly, μl of total genetic extracts were tested using an in-house developed assay based on the one-step superscriptiii/platinum taq kit (invitrogen, carlsbad, ca, usa) following manufacturer's instructions on the lightcycler machine from roche or the applied biosystems real-time pcr machine ( ). the analysis was limited to viral mono-infections amongst cases to discern clinical presentations and symptom complexes associated with each pathogen. we excluded viruses with fewer than cases ( . % of the total), as the number was too small to have a reasonable sample sizethese were coronavirus hku (cov-hku ), parainfluenza (hpiv- ), hpiv- , hpiv- , influenza a(h n ) (the prepandemic strain), respiratory syncytial virus a (rsv a), rsv b, cov and bocavirus (bv). this left viruses for the subsequent analyses. the main aim was to compare the differences in clinical expressions, including individual symptoms (or signs), pairs of symptoms, and overall symptom load between patients with different viral infections. we counted the clinical symptoms/signs and calculated the corresponding empirical proportions with % confidence intervals (cis) to evaluate the overall symptom load. logistic regression analysis was used to investigate the differences in symptom expressions for each pair. differences were identified at a significance level of . . to assess the presence of paired symptoms/signs for all viruses, we conducted binomial tests to compare the joint proportions of symptom pairs occurring together to the expected proportions assuming independence of symptoms. the ratio of the observed proportion of symptom pairs relative to the product of the marginal proportion of each symptom is defined as the excess probability ratio which measures effect size. multivariate logistic regression analysis was performed to compare the risk of having an individual symptom/sign among viral mono-infections by assigning a categorical variable for all viruses as the primary predictor. potential confounding was addressed by adjusting the model for age, smoking status, asthma and heart disease. non-significant variables were dropped at a significance level of . to obtain the final model. statistical analyses were performed using the r statistical software (version . . ) [ ] . ethics approval was given by the singapore military's joint medical committee for research, and the national university of singapore's ethics review committee. the basic demographic data are described in table . participants were mostly young male adults, with other characteristics largely similar. however, there were significantly less recruits amongst controls than amongst other groups. the temporal distribution of cases is described in figure . no obvious overall seasonal pattern can be observed. the peak in june and july corresponds to the flu-a(h n )pdm pandemic [ ] . as this peak tailed off, we observed an increase in flu-b cases (starting feb-mar ). subsequently, as the flu-b cases fell, adenovirus e (adv-e) cases started to increase. coxsackie/echovirus (cv) and rhinovirus (rv) infections were consistently present in the earlier periods but appeared to tail off by , corresponding to the rise in fri cases due to other viruses. the etiologies of selected infections are illustrated in table . at least one virus was detected in of the fri cases ( . %). in ( . %) of the cases, more than one virus was detected and these were excluded. ( . %) of the fri cases fulfilled the definition of ili; ( . %) of these ili cases were positive for at least virus. of the fri mono-infection cases, ( . %) were viral. ( . %) of these cases met the definition of ili. we examined the proportion of ili cases among those with viral mono-infections (table ) . influenza viruses accounted for only % of ili. among fri cases, more than % of patients with influenza and adenovirus infections presented with ili. however, several other viral infections led to high rates of ili, including cv, human metapneumovirus (hmpv) and covs. the sensivity, specifity, positive predictive value (ppv) and negative predictive value (npv) of ili for influenza was . %, . %, . % and . % respectively. on from the multivariate analysis ( figure ), compared to most other viruses, a flu-a(h n )pdm and flu-a (h n ) less commonly resulted in sorethroat. running nose was more common in enterovirus (ev) and rv cases and less common in adv. flu-b was more likely than a majority of the other viruses to cause dry cough while ev and adv-e were less likely to cause dry cough. cov-oc and hmpv were more likely to cause cough with phlegm than the most other viruses. influenza viruses and adenoviruses were more likely to cause fever ≥ . °c. in figure , we explored the associations (and dissociations) between different clinical symptoms and signs across all viruses. some are expected, such as association of fever ≥ . °c and fever ≥ . °c and dissociation of dry cough and cough with phlegm. fever ≥ . °c was also associated with systematic complaints, such as chills, bodyache, headache and eye pain. sorethroat was associated with an injected pharynx. our study shows the different viral etiologies of ili and compares the clinical characteristics of different viral etiologies in a tropical setting. this data series only spanned three years, and initial observations showed no clear seasonal variation compared to temperate regions, similar to previous reports of overall tropical respiratory disease patterns [ , , ] . the initial peak corresponded to the flu-a(h n )pdm pandemic [ ] , with the subsequent lower incidence in the recruit population likely due to vaccination with the pandemic vaccine a year before annual seasonal vaccination was started across all personnel [ ] . the number of fri cases remained fairly consistent throughout the study period (except the pandemic). however, prevalence of pathogens varied throughout, with some negative correlation observed between the virusese.g. a drop in flu-a followed by a rise in flu-b activity, and a drop in flu-b cases followed by a rise in adenovirus activity. correlation of viral activity have previously been reported -wang et al [ ] reported negative association between rv and adv rates, while bellei et al [ ] and razanajatovo et al [ ] described concomitant rise of influenza and rv, and influenza and adv activity respectively. in addition, kasper et al reported that ili rates remained constant despite varying prevalences of influenza [ ] . this supports our findings that multiple agents are capable of causing ili, and a decrease in the prevalence of one virus was replaced by an increase in prevalence of another. further studies across a longer time period are necessary, especially for vaccine effectiveness evaluation. . % of fri and . % of ili cases were positive for a virus, similar to the . % and . % reported by studies targeting similar panel of organisms [ , , , , , ] . the remaining fri cases may be due to non-viral agents, agents beyond the ability of the test, non-infectious causes, and possible sampling errors. viruses most commonly detected in ili cases were flu-a, flu-b, and cv in that order. influenza was also the top etiologic agent for ili in some studies [ , , , , ] although other pathogens have been identified to be most prevalent in different settings, such as rsv and hmpv in france [ ] , influenza and rsv in the usa [ ] , influenza and rv in central america [ ] and china [ ] and in italy, influenza and adv [ ] . the range of pathogens indicates a need to perform local continual surveillance since prevailing pathogens differ across different populations, geographic regions and climates. the high incidence of cv warrants further studyhand, foot and mouth disease is endemic to singapore and cv is frequently identified in pediatric samples [ ] , and it is possible that cv circulates at high levels in adults also. previous studies have identified a co-infection rate of . to . % [ , , , , ] for viruses, higher than the . % found in this study despite some studies using a less extensive diagnostic panel in a similar age group. possible reasons include our highly influenza-vaccinated population or a warmer climate with higher relative humidity resulting in lower virus circulation [ ] and a study population that did not include children (studies reported higher co-infection rates amongst pediatric patients [ , ] ). in both univariate and multivariate analysis, adv and influenza viruses were more likely to cause fever (≥ . °c). this finding has been demonstrated in other studies [ , , [ ] [ ] [ ] [ ] . fever also tends to be associated with other systemic complaints such as eye pain, bodyache and headache; this may be due to cytokine mediated systematic inflammatory response [ ] and could indicate more severe disease. bellei et al's [ ] study in brazil also found that ev, rv and cov were least likely to cause fever, and that rv and ev cases were the most likely to present with rhinorrhea. cough with sputum in flu-a(h n ) and flu-a were less prevalent than in other viruses, in contrast to reports in other settings [ , [ ] [ ] [ ] . we found that flu-b cases were more likely to report dry cough, similar to other studies [ , [ ] [ ] [ ] . the heterogeneity of results across different studies highlight the difficulty of using clinical symptoms in determining the etiology of ili. we also detected a small proportion of asymptomatic individuals who tested positive for the various viruses ( table ). these could represent carriage without infection or subclinical/asymptomatic infections during periods of virus circulation. although ili is widely used to identify influenza, the traditional definition would have picked up only % of influenza infections (except untyped flu-a) that were identified as fri. adv infections also frequently fulfilled the ili definition ( . % of adv-b and . % of adv-e), as did substantial fractions of cv, hmpv, and cov. only . % of viral mono-infections that met the ili definition were due to influenza. we found a fairly high sensitivity ( . %) of ili for influenza, but a low specificity ( . %) in keeping with sensitivities of . - . % and specifities of . - % reported elsewhere [ , , , ] . color cells represent variables that are significant at the % level, and the thickness of the cell wall represents the p-value (thin means . < p < . ; medium, . < p < . ; and thick, p < . ). the odds ratios are encoded by colors where a red cell indicates an odds ratio > ; and blue otherwise. for example, for a sore throat, flu-a(unknown), flu-b, cv, rv, adv-e, cov-oc and cov-nl have more of the sore throat than iflu-a(h n )pdm indicated by the red cells in the row for flu-a (h n )pdm and corresponding columns. ppv of ili was low ( . %) compared to other studies ( . - . %) likely because these studies were conducted during influenza seasons. this is supported by the higher npv ( . %) compared to other studies ( . - . %) [ , , , ] . two tropical studies [ , ] also report low ppv of . - % and high npv of - . %. adv are as likely as influenza viruses to present with fever and tend to be captured by the ili definition. however, they are more likely to present with cough and sorethroat than influenza. it may be possible to differentiate adv and influenza infection based on rhinorrhea and other symptoms. this could be useful in surveillance and clinical management, especially when deliberating whether to start antivirals. early etiologic diagnosis of influenza has been shown to be cost effective [ ] with reduced antibiotic use and may reduce complications with early antivirals. it may be possible to combine a clinical diagnostic model with rapid testing to achieve these goals. the analysis was limited to viral mono-infections and future studies should explore co-infections and bacterial infections. this study involved predominantly young adult males, and results may not be generalizable to the overall population, necessitating further studies among various age groups and gender. there were also less recruits amongst controls than amongst other groups, and this would be an important consideration when comparing the two groups in the future. finally, the actual clinical impact of differentiating between various viral etiological agents may be limited, and we could not determine the relative severity of symptoms other than fever. our study highlights the varied etiology for fri and ili in the tropical settinginfluenza and adv and cv were all common. influenza and advs tend to present with higher fever, and vaccination should be considered. the utility of ili for tropical surveillance of influenza needs to be reviewed given the low ppv and high npv compared to temperate regions. the surveillance system has enabled the singapore military to understand the etiologic agents affecting servicemen, hence implementing and evaluating controls measures such as vaccination. figure correlation of symptoms and signs across all viruses. clinical signs or symptoms are listed by average frequency from the most to the least. binomial test is used to assess the discrepancy between the observed proportion of symptom pairs and the expected proportion of symptom pairs which is the product of the two marginal distributions by assuming symptoms develop independently. color cells represent differences that are significant at the % level, and the thickness of the cell wall represents the p-value (thin means . < p < . ; medium, . < p < . ; and thick, p < . ). the excess probability encoded by colors measures the effect size. if the observed proportion is lower than the expected proportion, the cell will be shaded by blue color, and red color otherwise. mounts aw: strategy to enhance influenza surveillance worldwide acute respiratory illness in the community. frequency of illness and the agents involved viral agents responsible for febrile respiratory illnesses among military recruits training in tropical singapore acute respiratory symptoms in adults in general practice surveillance of respiratory viruses in patients with influenza-like illness in nanjing epidemiology and seasonality of respiratory tract virus infections in the tropics influenza in the tropics seasonal trends of viral respiratory tract infections in the tropics single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-[gamma] response epidemiology of multiple respiratory viruses in childcare attendees respiratory viral infections in infants: causes, clinical symptoms, virology, and 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key: cord- -r a orh authors: chu, yanhui; wu, zhenyu; ji, jiayi; sun, jingyi; sun, xiaoyu; qin, guoyou; qin, jingning; xiao, zheng; ren, jian; qin, di; zheng, xueying; wang, xi-ling title: effects of school breaks on influenza-like illness incidence in a temperate chinese region: an ecological study from to date: - - journal: bmj open doi: . /bmjopen- - sha: doc_id: cord_uid: r a orh objective: to assess the effects of winter/summer school breaks on occurrences of influenza-like illness (ili). methods: we jointly analysed ili surveillance data with the timing of school breaks in a temperate district in beijing, china from to . ili incidence rate ratios (irrs) of schoolchildren ( – and – years of age) to adults ( – and > years of age) were used to measure the age shift of ili incidence before, during and after the -week winter/ -week summer breaks. serfling-based poisson regression model with adjustment for unmeasured confounders was built to further assess the effect of winter school breaks. results: ili incidences were consistently lower during winter breaks than before winter breaks for all age groups. irrs of younger schoolchildren aged – to adults were higher during winter school breaks than before breaks, while the opposite was true for the irrs of older schoolchildren aged – to adults. schoolchildren-to-adults irrs during summer breaks were significantly lower than before or after school breaks (p< . ). conclusions: both winter and summer breaks were associated with reductions of ili incidences among schoolchildren and adults. our study contributes additional evidence on the effects of school breaks on ili incidence, suggesting school closure could be effective in controlling influenza transmission in developing countries. schoolchildren play a major role in the spread of influenza considering their high clinical attack rates, high social contact rates and increased viral shedding compared with adults. reactive school closure at the initial phase of a pandemic is considered as an effective non-pharmaceutical intervention to mitigate the spread of influenza. both schoolchildren and their caregivers are expected to have a lower risk of infection after school closure by altering social mixing patterns. several empirical studies have confirmed the effects of school closure on reducing influenza virus transmission after immediate closure of schools at an early stage of a pandemic, including studies from hong kong, france, germany, eight european countries, canada and usa. however, the assessment of effects of school closure on influenza transmission remains challenging. reactive school closure during an influenza pandemic was often accompanied by other control measures such as intensive screening, border control measures and improved sanitation, which may confound the assessment of effects of school closure on influenza incidence. to differentiate the effects of school closure from other accompanied interventions and changed health-seeking behaviour, some studies have evaluated the effects of school closure on influenza transmission by focusing on school breaks rather than reactive school closures. as school calendars are usually set at the beginning of each school term, observed changes of influenza incidence before, during and after school breaks are believed to be less confounded than school closures strengths and limitations of this study ▪ we assessed the impact of school breaks on influenza activity. ▪ age shift of influenza-like illness incidence from schoolchildren to adults during school breaks indicated a reduction of influenza transmission in schoolchildren. ▪ serfling-based poisson model was built to adjust for unmeasured confounders. ▪ residual confounding such as population mobility, change of health-seeking behaviour may exist. aimed to control a pandemic. significant associations between winter school breaks and temporary reductions of influenza-like illness (ili) incidence have been found in chile and argentina, as children were less likely to be gathered during school breaks thus reducing the chance of influenza transmission. we hypothesised that the -week winter/ -week summer breaks in china might also be relevant to ili incidence reductions among schoolchildren and adults. our study aims to assess the effects of winter/summer school breaks on ili incidence by estimating the ili incidence ratio of schoolchildren to adults before, during and after the breaks in xicheng district, beijing, china. beijing is located in northeastern china and has a temperate and continental climate with four distinct seasons. xicheng district is located in the centre of beijing. weekly district-wide ili time series during - was obtained from beijing medical institutions in communicable disease surveillance and early warning system. ili was defined as fever (temperature of °c or greater) and a cough and/or a sore throat in the absence of a known cause other than influenza. the ili reporting system recorded weekly ili cases into five age groups ( - , - , - , - and years or older). beijing children's hospital, affiliated with capital medical university is one of the sentinel hospitals for ili surveillance, and accounts for more than half of the total paediatric outpatient visits in beijing. to render our data more representative of ili rates across the entire beijing population, we age-standardised our ili visit data in xicheng district to the age distribution of beijing's population. age-specific population sizes in xicheng district were collected from china's population census. we assumed that the age-stratified population remained constant over the study period. we ignored the immunised population as the influenza vaccination coverage was below % in the general population. school calendars for winter/summer breaks of primary and secondary schools in beijing were obtained directly from beijing municipal education commission. school calendars were usually announced ahead of each academic year. winter breaks were often in january and february, which lasts for weeks and covered the -day statutory holidays for national chinese new year. summer breaks are in july and august, which last for weeks. the exact starting dates of winter/summer school breaks varied slightly across years and schools. on average, winter breaks in beijing started at the th week and ended at the th week, while summer breaks started at the th week and ended at the th week. data analysis ili incidence rate ratios (irrs) of schoolchildren ( - and - years of age) to adults (aged - and years or older) were used to measure the age shift of ili incidence before, during and after the -week winter/ -week summer breaks. in this study, we used years of age as the cut-off point for schoolchildren and adults, because the ili surveillance system in beijing reported weekly ili numbers by age groups of - , - , - , - and years or older, without specific age for each case. owing to data availability, we grouped individuals under years of age as schoolchildren, which was close to but might be a bit older than the true cut-off for schoolchildren. similar to previous literature, [ ] [ ] [ ] we compared schoolchildren-to-adults irrs before, during and after the winter/summer breaks using two-sided z tests. as the reduction in the ili incidence during the winter break was maintained on average for weeks after the end of the winter break, we allowed a -week window and defined before/after school breaks as weeks apart from the break. we considered a -week window before and after the winter/summer break as a sensitivity analysis. in order to control the effects of unmeasured confounders of long-term and seasonal trends, we built a serfling-specified poisson regression model to assess the effect of winter breaks. a vector of dummy variables for each year was considered and sinusoidal terms of weeks were added in the poisson model to adjust for long-term and seasonal trends, respectively. as weekly adjacent ili visits were temporally correlated, we further added an autocorrelated regression term to adjust for autocorrelation in the poisson model. details of the model can be found in online supplementary appendix and tables s -s . model fitting was evaluated by pseudo r values. irrs of during/after to before winter breaks were used to evaluate the change of ili incidence before, during and after winter breaks in each age group. irrs of during/after to before winter breaks smaller than indicated that ili visits during/after winter breaks were lower than those before winter breaks. we presented the estimates as weekly irrs, which was calculated by dividing the weekly ili incidence during/after winter breaks to an average of ili incidence weeks before winter breaks. data analyses were conducted in r v. . . using package nlme. from january to december , center for disease control and prevention of xicheng district in beijing recorded ili visits and a total of clinic attendances. percentage of number of ili visits to number of total clinic attendances (ili%) had been widely used as an indicator for influenza activities. figure a illustrates age-specific trends in weekly ili% throughout years. peaks of influenza activities appear from end of december to middle of january across different age groups in the past years. summer peaks were observed for groups of age - and - without a consistent annual pattern. during the winter school breaks, ili incidence decreased substantially in age groups of - and - , but modestly in age groups over (figure b). during the summer school breaks, only ili incidence in age group of - reduced dramatically, while levelled off for other age groups (figure b). a decline of schoolchildren-to-adults irr during school breaks implied an age shift in patients with ili towards adults, and further suggested a reduction of influenza transmission among schoolchildren. irrs of younger schoolchildren aged - to adults was higher during winter school breaks than before breaks, while the opposite was true for the irrs of older schoolchildren aged - to adults (table , figure c) . during summer breaks, schoolchildren-to-adults irrs were significantly lower than those of -week before or after school breaks ( p< . ). to be specific, irrs of age - to age above had declined by . % ( % ci . %, . %). the above conclusion was robust when we considered a -week window before and after winter/ summer break (see online supplementary table s ). since long-term trend and seasonal pattern of ili visits could be two confounders in the previous analysis of irrs, we used a poisson regression model to assess the effect of school breaks on ili incidence. the models generally well fitted the observed ilis with adjusted pseudo figure age-specific ili incidence rates, adjusted average ili incidence rates, adjusted average ili irrs of influenza surveillance data by week in beijing, - . (a) weekly ili incidence rates for five age groups. (b) adjusted average ili incidence per persons by week. (c) adjusted average ili incidence rate ratio of schoolchildren-to-adult by week. the upper plot is irrs of age - to adults; the lower plot is irrs of age - to adults. ili, influenza-like illness; irr, incidence rate ratio. r values for the five age groups ( - , - , - , - and years or older) ranging from . to . . after incorporating the autocorrelation within error terms, we estimated irrs of during/after to before winter break and their cis (table ) . during-to-before irrs were smaller than for all age groups. however, the starting time and duration of the ili reduction varied across different age groups. in age group of - , ili incidence dropped substantially by around % during the whole school break and lasted for weeks thereafter. declines of during-to-before irrs for age groups of - and - were also observable but not necessarily smaller than declines in adults (table ). for adults (age - and years or older), ili reductions were statistically significant in the second and third week of the break and lasted for about weeks (table ). among all age groups, incidence of ili visits returned to regular patterns with none of after-before irrs statistically significant weeks after the end of winter breaks (table ) . consistent with other studies in china and other temperate regions, we found peaks of influenza in beijing appeared synchronised across age groups in most winters and springs during - . for age groups of - and - , peaks of ili% had also been detected before summer breaks, which might be driven by final examination stress, poor classroom ventilation, incompletely developed immune system and other unfavourable factors related to young children. since ili visits could be caused by influenza, respiratory tract infection and other illness, ili% is a broad indicator of respiratory disease activity not entirely specific for influenza and could be affected by changes in health-seeking behaviour. therefore, laboratory surveillance data are necessary for further conclusions on age-specific seasonal influenza activities from a public health perspective. in this study, our findings generally support that winter/summer school breaks could achieve temporary reductions in ili incidence rates, especially among schoolchildren (age of - and - ). in consistency with previous empirical studies in chile, usa and european countries, the average reduction in schoolchildren-to-adult irrs in beijing lasted for up to weeks (table ) after school sessions resumed, and this allowed a few successive chains of transmission for influenza virus to reach full-scale transmission. the finding that ili incidence rates decreased substantially in schoolchildren yet modestly in adults during winter breaks is consistent with past work on age-specific influenza transmission. moreover, the reduction in ili during the school holidays went beyond the dips in ili ± weeks surrounding the holiday weeks (see online supplementary appendix), further supporting our suggestion of lower influenza transmission in schoolchildren during school breaks. the finding that irrs of schoolchildren aged - to adults increased significantly in winter breaks is inconsistent with findings of influenza activities in chile. although ili incidence rates of age group - had been reduced by . % ( % ci . % to . %) after entering winter breaks in - , reductions in ili incidence rates for adults' groups were even larger and thus resulted in increased irrs of age group - to adult. in addition to different strains of influenza circulating in a certain year, there could be many other human behaviour factors playing a role in regulating seasonal age-specific ili visits in china. for example, under pressure of final examinations before winter *small p values indicate that the irr for the period before the break is significantly higher than that for the period during the break (or the irr for the period during the break is significantly higher than that for the period after the break). ili, influenza-like illness; irr, incidence rate ratio. breaks, some schoolchildren are unwilling to spare any time to see a doctor when they had influenza-like symptoms. during chinese spring festival vacation (covered by winter breaks), most of migrant workers in beijing went back to their hometown and local people who stayed in the city were unwilling to seek medical advice for fear of missing family gatherings (see online supplementary figure s ). moreover, apparent drops of total outpatient visits were observed in every january (see online supplementary figure s ), suggesting the change of healthcare-seeking behaviour around the holidays. in fact, irrs of schoolchildren aged - to adults during spring festival were significantly higher than other weeks within winter breaks. consequently, we infer the enhanced irrs of schoolchildren aged - to adults in winter breaks are due to integrated effects of spring festival, population migration and changes in health-seeking behaviour. we established an autocorrelated poisson regression model to control the effect of potential confounders that may distort age-specific ili incidence rates, such as seasonal change of ili visits and year. we found a significant reduction in ili incidence rates for age group of - when entering winter breaks, compared with those of weeks before (irr . , % ci ( . to . )). however, the reduction is less significant when comparing with the rates throughout the entire break for the adults' groups. the phenomenon that reduction of ili incidence rates for age above was larger than those for age - and close to those for age [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ) could be due to age-specific health-seeking behaviour, immune system characteristics and residual confounders of seasonal ili visits. although a serfling model has been adopted, our model is still relatively conservative in controlling seasonal pattern of ili because timing of peaks of influenza activities had severe overlaps with timing of winter breaks, and the overfitting could underestimate the effect of winter breaks on decrease of irrs. therefore, additional analysis that considers high variability in the temporal relationship between winter breaks and weekly ili rates across influenza seasons are needed and incorporating environmental factors or human behaviour data may better clarify the relationship between age-specific influenza activity and winter breaks. we quantified the reduction in irrs, and also analysed age-specific timing of the decline in ili incidence. similar to the study in argentina, a stepwise trend of the effect on ili had been detected. age groups of - and - experienced the initial significant decrease in ili during the first week of winter breaks and lasted for weeks. the reduction effect was caught up by other age groups, and lasted for weeks for - age group and weeks for adults' groups. the spread of influenza likely varies according to population subgroup. overall, our study supports winter breaks as effective means to prevent the epidemic of winter-to-spring influenza activities. schoolchildren-to-adults irrs consistently decreased during summer breaks compared with -week before *irrs were used to estimate whether incidence of ili-associated visits in a particular week were lower, higher, or did not deviate from the expected seasonal ili patterns. each row represents a separate regression model. ili, influenza-like illness; irr, incidence rate ratio. summer breaks. according to our analysis, protective effect of summer breaks on schoolchildren of age - was more obvious than that on adults. we emphasise that results on effects of summer breaks on influenza activities should be interpreted with caution, because respiratory viruses other than influenza virus were active during summer that could make clinical surveillance less specific to influenza. although our study sustains the positive effect of school breaks in controlling influenza activities, insufficient evidence is provided on the effect of school breaks in order to support the decision on duration of the break and the timing with respect to the influenza season. furthermore, the economic cost of school breaks that is mainly due to absenteeism of working parents who have to stay home to take care of their children remains difficult to estimate. further analysis of the environmental or social factors influencing the transmission of seasonal and pandemic influenza is required in order to provide additional information for policymakers and public health officials to use when considering measures to control pandemic influenza. estimating household and community transmission parameters for influenza social contact networks for the spread of pandemic influenza in children and teenagers school closure and mitigation of pandemic (h n ) , hong kong closure of schools during an influenza pandemic social contacts of school children and the transmission of respiratory-spread 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differentiation of inter-provincial migration in china and characteristics of the flow field spatial pattern of severe acute respiratory syndrome in-out flow in in mainland china analysis on the multi-distribution and the major influencing factors on severe acute respiratory syndrome in beijing effects of school closures, winter influenza season, hong kong influenza-associated hospitalizations in the united states estimating the costs of school closure for mitigating an influenza pandemic competing interests none declared.provenance and peer review not commissioned; externally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. see: http:// creativecommons.org/licenses/by-nc/ . / key: cord- -miptf od authors: jeffery, diana d.; cohen, martin; brooks, arnold; linton, andrea; gromadzki, richard; hunter, christine title: impact of the influenza (h n ) pandemic on the united states military health care system date: - - journal: mil med doi: . /milmed-d- - sha: doc_id: cord_uid: miptf od background: during public health emergencies, the military health system experiences challenges similar to those across the u.s. public and private health systems. this study explored how such event, the / influenza (h n ) pandemic, impacted health care utilization and associated costs in the military health system. methods: data from the military data repository were used to examine diagnoses, claims data, and dates of services with respect to military or civilian care during – / influenza seasons. comparison analysis was conducted through two-tailed t-tests and regression models. results: there was a significant increase in inpatient and outpatient health care utilization during the / h n pandemic year, most markedly for emergency department visits. the / h n pandemic cost the department of defense $ million compared to influenza-related health care costs incurred in previous influenza seasons. highest health care utilization costs were found in children less than age . the greatest cost burden was attributed to immunizations for active duty personnel delivered at military facilities. conclusion: annual trend analysis of costs and health care utilization would be helpful to plan and resource emerging influenza pandemics and to identify subgroups at greatest risk for contracting influenza. public health emergencies have long been an area of concern for military health care systems because of the increased demand for health care services. over the last decade, there have been several instances where outbreaks of infectious disease, as public health emergencies, have repeatedly challenged military policies for preparedness. in addition to localized outbreaks, international pandemics have become a focal concern because of their impact on available resources in both civilian and military health care systems. since the influenza, the united states has helped mitigate the spread of disease during pandemics, but recent global epidemics have shifted the focus to strengthened preparedness for such public health emergencies. the growing number of emerging and re-emerging infectious diseases, like the severe acute respiratory syndromeassociated coronavirus in and the influenza a virus subtype h n pandemic in , have highlighted the rapid progression of localized epidemics into global pandemics. available treatment options with novel pathogens are typically limited during such global public health emergencies, thus necessitating greater reliance on emergency outpatient or inpatient supportive care. from a health systems management perspective, the large influx of patients being treated for either an unknown virulent etiology or supportive therapy can easily overburden health care facilities. the u.s. department of defense (dod) has long recognized potential threats posed by global pandemics to the readiness of combat forces, and the health and well-being of the nation they protect. the military health system (mhs), which maintains health care services for u.s. armed services personnel and their families, has extensive experience with influenza disease epidemics, such as the outbreak of h n that was detected after the death of an army recruit in fort dix, new jersey. this outbreak resulted in a mass vaccination program across the united states and initiated a collaborative approach between champus (now tricare, the system of health care plans for the u.s. armed forces and beneficiaries), the centers for disease control and prevention, and local/ regional health departments. twenty years after the fort dix incident, an outbreak of influenza a (h n ) on a navy ship led to the discovery of an antigenically distinct h n other types of communicable diseases have similarly challenged military preparedness policies such as outbreaks of pertussis, occupational exposures to blood-borne pathogens or enteroviruses, and food-borne infectious diarrhea. federal experiences identifying and controlling new strains of influenza are critical for military preparedness and the health care of dod beneficiaries. as part of national pandemic preparedness, dod policy now authorizes individual military base commanders to plan and coordinate response activities with local public health officials based on local declaration of public health emergencies. dod also contributes to national pandemic control by assisting in diagnostic testing of laboratory samples and reporting of influenza prevalence data through the dod electronic surveillance system for the early notification of community-based epidemics, published online at the force health protection pandemic influenza watch board. , during the h n pandemic, the world health organization reported , laboratory positive cases and , fatal cases from countries. in the united states, the centers for disease control and prevention laboratory confirmed , fatal cases and , reported hospitalizations. although laboratory-confirmed cases provide real-time case counts during a public health emergency, estimates are likely to be greater because of underreporting. the dod implemented a preparedness policy during this pandemic, including a stringent vaccination program and guidelines to providing health care during public health emergencies. antiviral therapy and pandemic (h n ) vaccination for active duty (ad) service members, including high risk beneficiaries, became a priority and this then served as the primary approach against this pandemic. tricare beneficiaries received the h n vaccine under another program, one with the same vaccine accessibility provided to civilian populations but with prioritization for vulnerable beneficiary populations. with the implementation of the vaccination program during the peak of the pandemic, military treatment facilities (mtfs) had to cope with the influx of ad personnel and beneficiaries who were seen in emergency departments, inpatient wards, or outpatient clinics during the pandemic. here, we report on the results of a retrospective study to assess the impact of the h n pandemic on the mhs with the overall goal of assessing and improving the effectiveness of pandemic preparedness in the dod. this study exam-ines the burden of disease caused by the h n pandemic strain on the mhs with respect to health care utilization and associated costs and provides insight into dod resource management of pandemics compared to previous nonpandemic influenza seasons. we used the mhs data repository (mdr) to assess health care utilization, prescription claims, and estimated costs of h n and influenza-like illnesses (ilis). the mdr captured immunizations rendered to patients in a military health care setting only. influenza immunization rendered in civilian settings were not tracked as non-ad beneficiaries may obtain immunization through multiple sources that may not be entered as mdr claims data (e.g., employment, free public health programs). our analytical dataset consisted of outpatient encounters, inpatient stays, and medication records rendered through mtfs and civilian care, october through april . we included encounter records if they had specific and nonspecific codes associated with ili, influenza immunization, and associated treatments. individuals with multiple influenza-related encounters were only counted once per season. each record contained the month/year of the service encounter, the patient pseudoidentifier, sex, age, beneficiary category, branch of service, state of residence, and type of tricare enrollment plan (mtf prime, managed care support contractor prime, and non-prime). the final dataset was aggregated to person-level records to enable characterization of the h n influenza patient. age was grouped by - , - , - , - , - , - , and years or older. beneficiary categories were ad, family members of ad, military retirees and family members of retirees age , and military retirees and family members age or older. cost data included costs associated with outpatient and emergency department visits, inpatient stays, influenza vaccine administration, and antiviral prescriptions rendered in the military and civilian settings from october to january (the last month when full cost data were available at the time of analysis). costs also included $ . million spent by dod to purchase h n vaccine although this expenditure could not be allocated to the patient level. for each ili service delivered from october through january , we used the full cost associated with services delivered in mtfs (direct care); for services delivered by civilian providers (care purchased in the tricare network or from non-network providers), we tabulated the actual amounts paid by dod using claim-level data. the study was reviewed by the tricare management activities human research protection program oversight office and determined to be exempt from full review by the dod institutional review board. demographics (number of cases by age group, gender, beneficiary category, branch of service, and tricare enrollment plan) for / influenza season and / pandemic season were examined using -tailed correlation analyses to identify differences between the two seasons. cost data for the / h n pandemic was derived by comparing the total ili-related costs for the / influenza season to the ilirelated costs associated with the five preceding seasons. the expected costs were then compared with the actual costs for the / season to isolate the incremental cost of h n . the ili-related costs excluded costs associated with services rendered outside a health care facility; costs borne by dhhs for h n immunizations administered in civilian facilities; costs associated with lost productivity because of ili; and costs of h n surveillance, reporting, and response activities. a series of linear regression models were then used to estimate the expected ili costs during the / influenza season as if it had been a typical influenza season, and to ferret out those factors that contributed to costs. eight regression models were specified, with separate models for military (direct) care and civilian (purchased) care as well as for four types of services: inpatient, outpatient nonpharmacy, outpatient pharmacy, and immunizations. the models controlled for the number of beneficiaries, age mix, beneficiary category, seasonality, and the underlying time trend. the time trend was modeled based on an explicit, independent variable that increased by one unit per month from october (month ) through january (month ). the model implicitly assumed that the underlying historical trend, controlling for the other independent variables, would continue. this underlying historical trend accounted for medical inflation and underlying trends in utilization per capita. the total and mean per-patient number of outpatient visits, emergency department visits, inpatient stays, and antiviral prescription fills by each type of ili service are shown in table i . relative to the preceding influenza season, utilization of - health care services for ili increased at both military and civilian-contracted facilities, with the largest increases observed for emergency department visits ( . % in military and . % in civilian facilities). ili office visits increased . % and . % at military and civilian facilities, respectively, whereas inpatient dispositions increased . % and . % at military and civilian facilities, respectively. in both tables ii and iii show the hi n cost impact by age group and type/source of care, respectively. for the estimated $ million h n impact that could be allocated to the patient level (table ii) , % of the cost increase was attributable to care rendered to beneficiaries aged to years, whereas % of the cost increase was due to care rendered to ad service members and their family members. table iii indicates where the total of $ . m was spent on ili from july to january . this total was approximately $ m more than projected for these months during a normal influenza season. table iii includes $ . m for dod-purchased h n vaccine that could not be allocated in the patient-level cost data. treatment costs accounted for % of the $ m overall cost impact, whereas immunization-related costs, including the dod purchase of vaccine, accounted for %. the regression models showed that there was a high cost burden for civilian inpatient hospitalization and civilian outpatient services across the period of analysis. the highest health care utilization costs were found for children ages to . the overall greatest cost burden, however, was incurred for immunizations delivered at mtfs. the estimated costs from the regression models and the actual costs for each influenza season by month between october and january are presented in figure . in / , the mhs realized an estimated $ million increase in ili-related costs compared to prior influenza seasons. the dod experience with the / h n pandemic is remarkably similar to experiences found in the nation as a whole, at least among populations with access to health care. our findings closely mirror those based on u.s. civilian populations, i.e., heavy reliance on emergency departments and high rates of ili among beneficiaries less than age . the increased rates of health care utilization and costs observed in the dod pediatric population is particularly concerning, given a new report that concludes that the nation's capacity to care for pediatric populations during the / influenza season was marginally adequate; had the / h n pandemic been more virulent, tertiary pediatric facilities would have been overwhelmed. further, in contrast to our findings, the u.s. civilian population saw a heavier burden of ili among young adults. most young adults included in our dod population were ad personnel, who, unlike their civilian counterparts, were required to be vaccinated for h n . based on our findings and those based on civilian reports, we recommend that dod, as well as the local communities where mtfs reside, develops specialized preparedness procedures for pediatric populations in anticipation of future influenza pandemics or epidemics. one strategy, based on estimated models when immunization resources are scarce and the influenza incidence is declining, is to consider immunizing school-age children as well as high-risk adults. although dod has clear requirements for vaccinating ad personnel, there are no such directives for vaccinating family members. another strategy is to consider closing elementary schools on dod installations, the length of closure dependent on pandemic severity as modeled elsewhere using civilian schools. in addition, to assist fiscal and resource planning, we recommend time trends that compare rates and costs to previous influenza seasons be conducted annually for each mtf. finally, we recommend that future study be directed to evaluating effective models of triaging individuals who present with ili symptoms, particularly for those presenting in emergency departments. our study has several limitations. our inclusion of nonspecific vaccine administration codes in the mdr may have captured vaccines unrelated to influenza. only vaccines administered in the health care setting are captured in the mdr, but some immunizations are administered to service members in the field; thus, total vaccine-related costs for dod are underestimated in our study. cost allocation data, particularly for care rendered in the military hospitals, were not specific to immunization clinics or different types of immunizations. immunizations or treatment for ili may have occurred as part of a broader health care visit, and ili costs may not have been identified. moreover, our cost estimation methodology may not have generated an accurate estimate for all events because we only had cost data through january . to some extent, comparison with prior influenza seasons mitigates the impact of this limitation, assuming that health care practices persisted across the - influenza seasons. yet another limitation, out of concern for patient privacy, is that we did not conduct analysis at the mtf level. therefore, we neither examined the prevalence of h n cases, health care utilization, or costs by individual mtfs nor consider mtf facility size, the number of available beds, and size of outpatient clinics in relation to the number of cases received at a particular facility. finally, while the cost of care provided in civilian settings is directly calculated from government payments/costs at a claim level, military care costs are reported in aggregate (by clinic) and then fitted and allocated to the detail "visit" level; hence, military care costs are inexact comparisons to civilian costs. the focus of this study was to examine the impact of the h n pandemic on the mhs and inform military commanders and policy makers about the burden of a public health emergency on health care systems used by dod beneficiaries. this information should assist in review of dod's response to this most recent pandemic and help strategize for preparedness response to similar events. in particular, review and evaluation of strategies that address our capacity to care for the dod pediatric population during an influenza pandemic is warranted, as is the need to manage increased demand on emergency departments during such events. pandemic influenza preparedness and community resiliency a novel coronavirus associated with severe acute respiratory syndrome extent of spread and duration of the outbreak outbreak of influenza in highly vaccinated crew of u.s. navy ship the national strategy for pandemic influenza, implementation plan (nspiip) armed forces health surveillance center (afhsc): pertussis diagnoses among service members and other beneficiaries of the u.s. military health system armed forces health surveillance center (afhsc): viral meningitis, active and reserve components diarrhea outbreak during u.s. military training in el salvador effectiveness of seasonal influenza vaccines against influenza-associated illnesses among us military personnel in - : a case-control approach department of defense instruction. number . . public health emergency management within the department of defense available at pandemic influenza watchboard pandemic (h n ) -update h n flu u.s. situation update defense officials prepare for h n flu syndromic surveillance during pandemic (h n ) outbreak hospitalized patients with h n influenza in the united states inpatient capacity at children's hospitals during pandemic (h n ) outbreak, united states estimates of the prevalence of pandemic (h n ) , united states an hhs retrospective on the h n influenza pandemic to advance all hazards preparedness available at rsif .royalsocietypublishing.org simulating school closure policies for cost effective pandemic decision making key: cord- -vxsbv t authors: loubet, p.; lenzi, n.; valette, m.; foulongne, v.; krivine, a.; houhou, n.; lagathu, g.; rogez, s.; alain, s.; duval, x.; galtier, f.; postil, d.; tattevin, p.; vanhems, p.; carrat, f.; lina, b.; launay, o.; seddik, k.; lesieur, z.; bonmarin, i.; loulergue, p.; bodilis, h.; servera-miyalou, m.; sadler, i.; momcilovic, s.; kanaan, r.; coolent, n.; tan boun, k.; blanche, p.; charpentier, j.; daviaud, f.; mongardon, n.; bretagnol, a.; claessens, y. e.; rozenberg, f.; yazdanpanah, y.; burdet, c.; harent, s.; lachatre, m.; rioux, c.; bleibtreu, a.; casalino, e.; choquet, c.; leleu, a.; belghalem, k.; colosi, l.; ranaivoson, m.; verry, v.; pereira, l.; dupeyrat, e.; bernard, j.; emeyrat, n.; chavance, p.; debit, a.; aubier, m.; pradere, p.; justet, a.; mal, h.; brugiere, o.; papo, t.; goulenok, t.; boisseau, m.; jouenne, r.; alexandra, j. f.; raynaud-simon, a.; lilamand, m.; cloppet-fontaine, a.; becheur, k.; pelletier, a. l.; fidouh, n.; ralaimazava, p.; beaumale, f.; costa, y.; munier, e.; betend, f.; amour, s.; loeffert, s.; francourt, k.; merle, c.; letois, f.; géraud, p.; driss, v.; noslier, s.; ray, m.; sebbane, m.; konaté, a.; bourdin, a.; klouche, k.; léglise, m. s.; couve-deacon, e.; fruit, d.; fenerol, c.; vallejo, c.; jouneau, s.; lainé, f.; thébault, e.; fillatre, p.; le pape, c.; beuzit, l.; chau, f.; goderel, i. title: clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france date: - - journal: clinical microbiology and infection doi: . /j.cmi. . . sha: doc_id: cord_uid: vxsbv t abstract objectives the aim of this study was to analyse characteristics and outcome of respiratory syncytial virus (rsv) infection in adults hospitalized with influenza-like illness (ili). methods patients hospitalized with ili were included in this prospective, multicentre study carried out in six french hospitals during three consecutive influenza seasons ( – ). rsv and other respiratory viruses were detected by multiplex pcr in nasopharyngeal swabs. risk factors for rsv infection were identified by backward stepwise logistic regression analysis. results a total of patients hospitalized with ili were included, of whom % ( / ) were > years and % ( / ) had underlying chronic illnesses. rsv was detected in % ( / ), and influenza virus in % ( / ). risk factors for rsv infection were cancer (adjusted or . , % ci . – . , p . ), and immunosuppressive treatment (adjusted or . , % ci . – . , p . ). patients with rsv had a median length of stay of days ( – ), and % of them ( / ) had complications, including pneumonia ( / , %) and respiratory failure ( / , %). fifteen per cent ( / ) were admitted to an intensive care unit, and the in-hospital mortality rate was % ( / ). pneumonia was more likely to occur in patients with rsv than in patients with rsv-negative ili ( % ( / ) versus % ( / ), p . ) or with influenza virus infection ( % versus % ( / ), p . ). conclusion rsv is an infrequent cause of ili during periods of influenza virus circulation but can cause severe complications in hospitalized adults. risk factors for rsv detection in adults hospitalized with ili include cancer and immunosuppressive treatment. specific immunization and antiviral therapy might benefit patients at risk. respiratory syncytial virus pneumonia adults influenza-like illness influenza elderly a b s t r a c t objectives: the aim of this study was to analyse characteristics and outcome of respiratory syncytial virus (rsv) infection in adults hospitalized with influenza-like illness (ili). methods: patients hospitalized with ili were included in this prospective, multicentre study carried out in six french hospitals during three consecutive influenza seasons ( e ). rsv and other respiratory viruses were detected by multiplex pcr in nasopharyngeal swabs. risk factors for rsv infection were identified by backward stepwise logistic regression analysis. results: a total of patients hospitalized with ili were included, of whom % ( / ) were > years and % ( / ) had underlying chronic illnesses. rsv was detected in % ( / ), and influenza virus in % ( / ). risk factors for rsv infection were cancer (adjusted or . , % ci . e . , p . ), and immunosuppressive treatment (adjusted or . , % ci . e . , p . ). patients with rsv had a median length of stay of days ( e ), and % of them ( / ) had complications, including pneumonia ( / , %) and respiratory failure ( / , %) . fifteen per cent ( / ) were admitted to an intensive care unit, and the in-hospital mortality rate was % ( / ). pneumonia was more likely to occur in patients with rsv than in patients with rsv-negative ili ( % ( / ) versus % ( / ), p . ) or with influenza virus infection ( % versus % ( / ), p . ). over the past decade, respiratory syncytial virus (rsv) has been increasingly recognized as an important pathogen in adults [ ] , and especially the elderly [ e ], immunocompromised patients [ , ] and individuals with underlying chronic respiratory diseases [ , ] . in the elderly population, rsv is one of the three most common causes of respiratory disease, along with influenza virus and rhinovirus [ , ] . respiratory virus detection has improved with the development of highly sensitive and specific molecular methods, which are providing interesting new epidemiological information. however, for reasons of cost and the lack of rsv-specific antiviral drugs, adult inpatients with influenza-like illness (ili) are frequently not tested for rsv but rather only tested for influenza virus. influenza surveillance systems based on ili and acute respiratory illness definitions are unsuitable for capturing cases of rsv in children and adults [ e ] . more studies are needed to determine the burden of rsv infection, and to characterize at-risk populations that could be targeted by vaccination and antiviral treatment. the aims of this study were to describe (a) the prevalence, (b) the clinical features, and (c) the outcome of rsv infection in adults hospitalized with ili. we analysed cases of laboratory-confirmed rsv infection during three consecutive influenza seasons ( / , / and / ), in a post hoc analysis of patients hospitalized with ili in the fluvac study. fluvac is a french prospective observational study of influenza vaccine efficacy conducted in six university hospitals (cochin hospital, paris; bichat hospital, paris; pontchaillou hospital, rennes; limoges hospital; university hospital, montpellier; edouard herriot hospital, lyon). the fluvac study design is further described in rondy et al. and loubet et al. [ , ] . each season, enrolments take place during periods of influenza circulation (from november to march). data on adults hospitalized for at least h for ili, with symptom onset < days before sampling, were collected. ili was defined as a combination of the following: (a) at least one of the following systemic symptoms: fever ( c), headache, myalgia or malaise, and (b) at least one of the following respiratory symptoms: cough, sore throat or dyspnoea [ ] . each participant was interviewed, and nasopharyngeal samples were obtained at enrolment. we collected demographic characteristics, chronic underlying diseases and their treatments, the characteristics of the current ili episode: clinical presentation, hospitalization ward, the length of hospital stay and outcome (occurrence of complication, intensive care unit admission and death). data were retrieved from the medical charts, interviews with the patients and families, and laboratory databases. all variables collected are detailed in the supplementary material (appendix s ). respiratory viruses were detected in nasopharyngeal swabs from all the patients by means of multiplex rt-pcr. any bronchoalveolar lavage fluid samples or tracheal aspirates ordered by the physician in charge were also tested. samples were first tested in the virology laboratory of each participating hospital by means of real-time influenza a and b pcr after manual nucleic acid extraction. all samples were then sent to the french national influenza reference centre (cnr-lyon) for influenza confirmation and screening for other respiratory viruses. rna and dna were extracted with the automated easymag system from biom erieux (marcy l'etoile, france), and influenza viruses were detected with an in-house real-time rt-pcr protocol [ ] . the samples were also screened for a panel of other respiratory viruses (adenoviruses, bocaviruses, coronaviruses, human metapneumovirus, parainfluenza viruses e , picornavirus and rsv) by real-time pcr using the respiratory multiwell system rgene ® on an abi analyser. we first described the characteristics of all the patients hospitalized with ili. then, univariate analysis was used to compare rsvpositive patients with (a) rsv-negative patients (including patients infected by other respiratory viruses and patients free of viral infection) and (b) influenza virus-positive patients. quantitative variables were expressed as mean and standard deviation (sd) or median and interquartile range (iqr), and qualitative variables as number and percentage. we used the wilcoxon rank sum test or fisher's exact test for univariable comparisons. missing data for each variable were excluded from the denominator. factors associated with rsv infection were identified by using rsv-negative individuals as the comparison group. individuals with influenza virus and rsv co-infection were excluded from the analysis. we used a backward stepwise logistic regression model, with rsv test results (positive/negative) as the dependent variable. all covariates with a p value < . in univariate analysis were tested in the multivariate model, namely age (considered as a binary variable (< and years)), dyspnoea, fever, myalgia, chronic lung disease, cancer (solid and haematological malignancies), diabetes, chronic renal failure, and immunosuppressive treatment. factors associated with pneumonia onset were analysed among all ili patients. individuals with influenza virus and rsv co-infection were excluded from the analysis. we used a backward stepwise logistic regression model in which pneumonia (positive/negative) was the dependent variable. covariates with a p value < . in univariate analysis were tested in the multivariate model, namely age (considered as a continuous variable), chronic heart disease, chronic respiratory disease, cancer (solid and haematological malignancies), diabetes, chronic renal failure, immunosuppressive treatment, rsv infection, influenza virus infection, and influenza vaccination. the final model was adjusted for chronic respiratory disease and age because of their known role in the onset of pneumonia. results from both regression models were expressed as odds ratios (or) and adjusted ors (aor) with their % ci. a p value of . was considered statistically significant. all analyses used stata software (v , © copyright e statacorp lpt, college station, tx, usa). the fluvac study (clinicaltrials.gov nct ) respected good epidemiological and clinical practices in clinical research, and the declaration of helsinki, and was approved by regional ethics committees. all the study participants gave their informed consent for respiratory virus testing. overall, patients hospitalized with ili were included during the three periods of influenza circulation (fig. fig. ). respiratory syncytial virus was the third most frequent virus, after influenza virus ( % ( / ) of patients with ili, % ( / ) of patients with at least one virus) and picornavirus ( % ( / ) of patients with ili, % ( / ) of patients with at least one virus). the other detected viruses were coronavirus ( . % ( / ) and % ( / ) respectively), human metapneumovirus ( % ( / ) and % ( / )), adenovirus ( % ( / ) and % ( / )) and bocavirus ( . % ( / ) and % ( / )). six patients ( / , . %) were diagnosed with both influenza virus and rsv infection and so were removed from further analyses. the median age of the patients with rsv infection alone was years (iqr, e ) ( table ) . chronic underlying diseases were present in cases ( / , %), and consisted mainly of chronic respiratory diseases ( / , %), chronic heart disease ( / , %) and cancer ( / , %; solid tumours, haematological malignancies). fifteen patients ( / , %) were on immunosuppressive therapy. twenty-six patients ( / , %) had been hospitalized in the previous year, an average of . times (sd . ). the median time from symptom onset to admission was days (iqr, e ). cough ( / , %), fever ( / , %) and dyspnoea ( / , %) were the main symptoms in patients with rsv infection. the median length of hospital stay was days (iqr e ). a total of medical complications occurred in patients ( / , %) during their hospital stay, including pneumonia ( episodes, % ( / ) of complications), respiratory failure ( / , %), heart failure ( / , %), and acute respiratory distress syndrome ( / , %). intensive care unit admission was necessary for eight patients ( / , %). four patients ( / , %) died during the hospital stay; all of them were men > years old with chronic respiratory diseases. furthermore, three patients had chronic heart disease. the mean time from admission to death was days (sd . ). patients with rsv were older than patients with influenza virus ( years (iqr e ) versus years (iqr e ), p . ) ( table ) . patients with rsv were more likely to have cancer or immunosuppressive treatment than patients without rsv (respectively % ( / ) versus % ( / ), p . ; % ( / ) versus % ( / ), p . ) and influenza virus-positive patients ( % ( / ) versus % ( / ), p . ; % ( / ) versus % ( / ), p . ). multivariate analysis of all patients with ili showed that cancer (or . ; % ci . e . , p . ) and immunosuppressive treatment (or . ; % ci . e . , p . ) were significantly associated with rsv detection ( table ). the patients with rsv and influenza virus infection did not differ in terms of the length of stay, icu admission or mortality. after adjustment for chronic respiratory disease and age, we found that rsv infection (or . ; % ci . e . , p . ), chronic renal failure (or . ; % ci . e . , p . ) and active smoking (or . ; % ci . e . , p . ) were significantly associated with the onset of pneumonia (table ). in this study conducted during three consecutive influenza seasons among adults hospitalized for ili in france, rsv was the third most common respiratory virus, being detected in % of patients, compared with % for influenza virus. this rate is lower than that found in the usa by sundaram [ ] . in the latter study, the rate was even higher ( / , . %) among hospitalized patients. in another study, falsey et al. reported a % ( / ) prevalence of rsv among adults aged years or with underlying cardiopulmonary diseases who were admitted with acute respiratory symptoms during four consecutive winters in rochester, ny [ ] . several reasons may explain the broad range of reported rsv detection rates. for example, our study was restricted to periods of influenza virus circulation, which may not have included the peak of rsv circulation, although enrolments in the two studies by falsey et al. started in mid-november. another factor is age: we included all adults > years, whereas sundaram et al. and falsey et al. included only older subjects, in whom rsv infection may be more frequent. furthermore, the laboratory methods used in the rochester study included viral culture and serological tests, and rt-pcr was the only positive test in only two-thirds of cases. finally, the clinical definition used to trigger swab collection also differed among the studies. the median age of the rsv-infected patients in our study was years but, contrary to other studies, we found no association between rsv infection and older age [ , , ] . most of the patients with rsv infection had chronic underlying conditions ( / , %), consisting mainly of chronic respiratory disease ( / , %) or chronic heart disease ( / , %). this is consistent with the report by walsh et al. that underlying pulmonary disease was a significant risk factor for severe rsv illness requiring hospitalization [ ] . we found that the two underlying conditions independently associated with rsv infection were cancer and immunosuppressive treatment. this association was statistically significant whether rsv-positive patients were compared with all rsv-negative patients or only with influenza virus-positive patients. although immunocompromised patients (especially patients with haematological malignancies) are known to be more susceptible to rsv infection [ ] , solid cancers and immunosuppressive therapy are not classical risk factors. respiratory syncytial virus was associated with significant morbidity: the median length of hospital stay was days; % ( / ) of rsv-infected patients were admitted to the intensive care unit, and % ( / ) died. these findings are consistent with the literature [ , ] . similar percentages were noted in the influenza group of our study. patients with rsv were significantly more likely than patients with influenza or without rsv to develop pneumonia ( % versus % and %, respectively). falsey et al. and lee et al. also found a high rate of pneumonia among rsv-infected patients > years admitted for ili in the usa ( / , %) and among rsv-infected adults admitted to three hospitals in hong kong ( / , %) [ , ] . jain et al. [ ] found at least one respiratory virus in % of american adults with communityacquired pneumonia requiring admission to one of five participating hospitals between and . rsv was found in % ( / ) of all patients. the strengths of this study include the large number of adults hospitalized with ili, the prospective multicentre design, standardized patient screening in the participating centres, centralized confirmation of respiratory viruses in an influenza reference centre and the lengthy study period spanning three consecutive influenza seasons. several limitations must, however, be acknowledged. first, as all the participating centres were teaching hospitals, the proportion of patients with underlying diseases may have been higher than in the general ili population, owing to a referral bias. second, although the sample was large, the study was probably underpowered to identify a possible impact of rsv on morbidity and mortality in multivariate analysis. third, the ili definition used here captures only a subset of rsv infections [ ] . our study does not reflect the real burden of severe rsv infection, which may include other clinical manifestations. fourth, we report post hoc results of the fluvac study, which was not designed to answer this research question. indeed, as the fluvac study was designed to assess influenza vaccine efficacy, patients were enrolled during periods of influenza virus circulation, which differ slightly from periods of rsv circulation. this means that our data reflect the prevalence of rsv among patients hospitalized for ili during periods of influenza virus circulation and not during peak rsv circulation, which usually occurs earlier, owing to epidemiological interference [ ] . however, french surveillance data (renal system) show that the peak of the rsv epidemic overlapped with the beginning of influenza virus circulation during the three seasons of interest, and that the two viruses co-circulated for at least e weeks (fig. ) . in conclusion, this prospective observational study conducted in france during three influenza seasons reveals that % of adults hospitalized for ili had rsv infection, of whom % developed cardiopulmonary complications and % died. it also shows that elderly individuals and patients with cancer and/or immunosuppressive treatment are more likely to have rsv isolated when hospitalized for ili. potential benefits of enhanced rsv testing, antiviral treatment, and vaccine development in these groups should be considered. the outcome analysed was rsv infection. the authors declare no competing interest related to the study. o launay is an investigator for clinical trials sponsored by janssen and other companies and received travel support to attend scientific meetings from pharmaceutical companies. the current work received no funding. however, the study sites received funding from sanofi pasteur and sanofi pasteur msd for the fluvac study. vaccine producers had no role in the study design, data 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virus associated hospitalizations among children in a rural community of northern india the diagnosis of viral respiratory disease in older adults progress in the surveillance of respiratory syncytial virus (rsv) in europe seasonal influenza vaccines effectiveness against confirmed a(h n ) influenza hospitalisation: pooled analysis from a european network of hospitals. a pilot study factors associated with poor outcomes among adults hospitalized for influenza in france: a three-year prospective multicenter study available at pandemic a(h n ) influenza virus detection by real time rt-pcr : is viral quantification useful? rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults hospitalizations associated with influenza and respiratory syncytial virus in the united states risk factors for severe respiratory syncytial virus infection in elderly persons respiratory syncytial virus infection in adults high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections respiratory syncytial virus and influenza a infections in the hospitalized elderly community-acquired pneumonia requiring hospitalization among u impact of the influenza a (h n ) pandemic wave on the pattern of hibernal respiratory virus epidemics we thank all the renal network laboratories and the cnr laboratory for agreeing to share their data on influenza and rsv epidemics. we specifically thank vincent enouf (cnr influenza, paris) for creation of fig. .we are very grateful to the following persons and institutions who made significant contributions to the fluvac study: r eseau the authors thank h el ene bricout, laurence pagnon and christine sadorge from sanofi pasteur msd for their inputs during the study design phase and for critical review of the results. additional supporting information may be found in the online version of this article at http://dx.doi.org/ . /j.cmi. . . . key: cord- -lvlbwcl authors: rogers, kimberly b.; roohi, shahrokh; uyeki, timothy m.; montgomery, david; parker, jayme; fowler, nisha h.; xu, xiyan; ingram, deandra j.; fearey, donna; williams, steve m.; tarling, grant; brown, clive m.; cohen, nicole j. title: laboratory-based respiratory virus surveillance pilot project on select cruise ships in alaska, – () date: - - journal: journal of travel medicine doi: . /jtm/tax sha: doc_id: cord_uid: lvlbwcl background: influenza outbreaks can occur among passengers and crews during the alaska summertime cruise season. ill travellers represent a potential source for introduction of novel or antigenically drifted influenza virus strains to the united states. from may to september – , the alaska division of public health, the centers for disease control and prevention (cdc), and two cruise lines implemented a laboratory-based public health surveillance project to detect influenza and other respiratory viruses among ill crew members and passengers on select cruise ships in alaska. methods: cruise ship medical staff collected – nasopharyngeal swab specimens per week from passengers and crew members presenting to the ship infirmary with acute respiratory illness (ari). specimens were tested for respiratory viruses at the alaska state virology laboratory (asvl); a subset of specimens positive for influenza virus were sent to cdc for further antigenic characterization. results: of nasopharyngeal specimens, % tested positive for at least one respiratory virus; % tested positive for influenza a or b virus. antigenic characterization of pilot project specimens identified strains matching predominant circulating seasonal influenza virus strains, which were included in the northern or southern hemisphere influenza vaccines during those years. results were relatively consistent across age groups, recent travel history, and influenza vaccination status. onset dates of illness relative to date of boarding differed between northbound (occurring later in the voyage) and southbound (occurring within the first days of the voyage) cruises. conclusions: the high yield of positive results indicated that influenza was common among passengers and crews sampled with ari. this finding reinforces the need to bolster influenza prevention and control activities on cruise ships. laboratory-based influenza surveillance on cruise ships may augment inland influenza surveillance and inform control activities. however, these benefits should be weighed against the costs and operational limitations of instituting laboratory-based surveillance programs on ships. approximately million cruise ship passengers visit the us state of alaska each summer. the inbound, summertime tourist population is . times alaska's wintertime resident population. some cruise ships in the region carry upward of passengers and crew members originating from many countries. these factors, along with close quarters and prolonged contact among travellers on ships and during land-based tours before embarkation, increase the risk of communicable disease transmission. influenza outbreaks among cruise ship passengers and crew members are relatively common, and may be prolonged and challenging to control as new, susceptible passengers embark at frequent intervals. , cruise ships destined for us ports of entry are required by federal regulations to report to the centers for disease control and prevention (cdc) all deaths on board, and certain signs and symptoms suggestive of infectious disease in passengers and crew members. cruise ships are required to report gastrointestinal illness to cdc's vessel sanitation program at least h before a ship's arrival at a us port. in contrast, cruise ships are not required to conduct respiratory illness surveillance. however, cdc's division of global migration and quarantine asks cruise ships to report outbreaks of influenza-like illness (ili) and to complete end-of-voyage cumulative ili reports. ships voluntarily report the total number of crew members and passengers with ili either electronically or by phone to the cdc quarantine station with jurisdiction over the port of call. if a ship's ili incidence rate exceeds the cdc-defined outbreak threshold of . cases per traveller days, cdc requests enhanced data collection and, in coordination with the state health department, provides consultation on influenza testing. if influenza viruses are detected in crew members or passengers, recommendations are made regarding control measures, including antiviral treatment and chemoprophylaxis, isolation of ill individuals, monitoring of exposed individuals, and vaccination of crew members. during low influenza activity periods such as summer, influenza outbreaks in the usa and canada have been linked to influenza virus transmission among tourists travelling on combined land-sea tours or cruise ship voyages, particularly in alaska. , cruise ship passengers who are at increased risk for more severe disease, such as the elderly or immunocompromised, may develop severe complications from influenza such as pneumonia, which may result in disembarkation for hospitalization. the value of cruise ship influenza surveillance is highlighted by outbreaks of seasonal and pandemic influenza among passengers and crews, , - and the potential for introduction of antigenically drifted seasonal influenza virus strains. for example, in , an antigenically drifted h n virus strain identified in ill australian cruise ship passengers became the predominant virus during the subsequent - influenza season in north america. cruise ship ili reporting to cdc is aggregate and does not include a laboratory component. integration of syndromic and virologic surveillance could improve the ability to detect and characterize influenza virus strains and other respiratory viruses circulating among cruise ship travellers. from may through september, - , the alaska department of health and social services, the alaska state virology laboratory (asvl), cdc influenza division, cdc anchorage quarantine station and two cruise lines initiated a pilot laboratory-based surveillance project to detect influenza and other respiratory viruses among ill crew members and passengers on a few cruise ships in alaska. the pilot project aimed to characterize respiratory viruses circulating among cruise ship travellers in the state, to correlate the timing of cruise ship ili incidence peaks with identification of respiratory viruses in alaska, and to compare influenza viruses identified in ill passengers and crew members on ships with influenza viruses identified at land-based influenza surveillance sites. specimens were collected from passengers and crew members reporting to the ship infirmary on participating cruise ships in alaska from may-september, - , and tested for respiratory viruses. four ships from two cruise lines participated in and , and two additional ships from the same cruise lines were added for a total of six ships in . the ships were relatively representative of those sailing in the region during the time period of the pilot project;~ - passengers, and - crew were on board each ship. participating ships had voyage lengths varying from to days. throughout the season, asvl provided specimen-collection kits to cruise ships, including nasopharyngeal swabs, universal transport medium, laboratory submission slips and packaging for shipping specimens. the case definition for inclusion in year ( ) was ili (temperature of ≥ °f and either cough or sore throat in the absence of a known cause other than influenza); in years and ( and ), the definition was expanded to include signs of an acute respiratory illness (ari) without fever, if the clinician suspected an infectious aetiology. ship staff obtained verbal consent from the individual (if an adult) or permission of a parent or guardian (for those under years old). each participating ship was asked to collect - specimens per week during the course of the pilot project. cdc approved the surveillance project with a determination that it did not meet the definition of human subjects research under code of federal regulations part . nasopharyngeal specimens were collected using flocked swabs (puritan ® ), placed in transport medium (puritan ® ), and refrigerated. specimens were kept on ice packs during their transit from the cruise ship to asvl. upon receipt at the laboratory, μl of each specimen was extracted (biomerieux nuclisens ® easymag ® ) to produce a final elution of μl of total nucleic acid. the genmark esensor ® -target respiratory virus panel was used per manufacturer guidelines to determine the presence or absence of influenza and other respiratory viruses. a random subset (~ of every ) of specimens testing positive for influenza viruses was shipped to the cdc influenza division laboratory for cell culture and antigenic characterization by using hemagglutination inhibition (hi) methods. for viruses that did not yield sufficient titre for hi testing, genetic characterization (sequencing) was conducted to determine the influenza type, subtype or genetic group. in addition to personal identifiers collected on the asvl requisition form per standard protocol for clinical specimens, ship medical staff provided asvl demographic, clinical and epidemiological information for each specimen. this information included whether the individual was a crew member or passenger, age, sex, country of residence, recent travel history outside the usa or canada, date and port of embarkation, influenza vaccination history (self-report), illness onset date and symptoms, receipt of antiviral treatment prior to specimen collection, and whether other travelers on board had respiratory illness. asvl coded and de-identified the data and laboratory results and shared that information with the cdc anchorage quarantine station. each month throughout the project period, the quarantine station compiled and issued reports to each cruise line. descriptive analyses were conducted on laboratory, demographic and epidemiological data from the -year pilot. while symptom history was analyzed, results were unremarkable and are not reported. in order to compare the pilot data to existing alaska state influenza surveillance data, asvl provided cdc with inland state surveillance data representing the same time period of the pilot project (may-september, - ). of the nasopharyngeal specimens tested from , and , ( %) were from passengers and ( %) from crew members with ili or ari. three hundred and forty ( %) specimens tested positive for at least one virus on the asvl target panel; ( %) tested positive for influenza a or b (table ) , representing % of sampled passengers and % of crew members (tables and ) . throughout the -year pilot period, respiratory viruses were identified (table ) . fifteen passengers and crew members tested positive for viruses; different co-infections were identified from these individuals. asvl sent a subset of samples ( , %) positive for influenza a or b viruses to cdc for further antigenic characterization; results are shown in table . beyond this subset, additional influenza a(h n ) viruses were genetically characterized at cdc; these viruses did not yield a sufficient titre for the hi test and thus are not presented in table . however, all belonged to the same genetic group as the predominant a(h n ) virus strains circulating during the - influenza season. of the samples collected by instate providers for alaska's statewide inland influenza surveillance program from may through september in - , ( %) tested positive for influenza a or b virus. while both the state inland surveillance program and the alaska cruise ship respiratory surveillance pilot project primarily identified influenza a(h ) among all individuals testing positive for influenza viruses, state inland surveillance results yielded a higher percentage of influenza b virus but fewer influenza a(h n )pdm viruses (table ). participating passengers and crew members on all ships represented countries. seventy-one ( %) of crew members submitting specimens during the pilot project period were permanent residents of the philippines or india, whereas most passengers were from the united states ( , or %). the median age of passengers submitting specimens was years (range: - ); the median age of crew members submitting specimens was years (range: - ). results were relatively consistent across age groups, although most ( , or %) influenza h n pdm positive specimens throughout the pilot came from individuals younger than . six percent ( ) of crew members and passengers submitting specimens had been outside the usa or canada in the days ( days in , days in and ) before symptom onset; countries included australia ( ), japan ( ), india ( ), taiwan ( ), south africa ( ), china ( ), israel ( ), singapore ( ) and trinidad and tobago ( ). aggregate virologic results from these individuals did not deviate markedly from those with no recent travel outside the us or canada. self-reported influenza vaccination coverage for passengers and crews varied greatly between participating ships and voyages. more than half of the passengers ( , or %) submitting specimens reported having been vaccinated for influenza during the previous year. in contrast, fewer than half ( , or %) of the crew members reported the same. one of the cruise lines reported that of those crew vaccinated, all had received the northern hemisphere influenza vaccine, regardless of ship's origin or the crew member's origin. aggregate data for all years showed that positive test results for influenza a or b were similar for passengers and crew members who reported recent influenza vaccination ( and % respectively) and those who did not ( and % respectively). no participants received antivirals before specimen collection. analysis of the timing of participants' illness onset relative to embarkation revealed a peak for northbound ships (originating in vancouver, canada or seattle, usa, en route to alaska) on day of the voyage (range: day pre-embarkation to days post-embarkation) and for southbound ships (which originate in alaska and sail toward the us pacific northwest) on day of the voyage (range: days pre-embarkation to days post-embarkation) (figure ). although onset dates differed from north vs. southbound cruises, no marked differences in influenza subtypes between individuals on north vs. southbound cruises were identified. the alaska cruise ship respiratory surveillance pilot project represents the first civilian sentinel respiratory virus surveillance program conducted on cruise ships in north america. although the number of specimens collected and participating ships were limited, the high yield of positive results ( % for at least one respiratory virus and % for influenza viruses) indicates that influenza was common among sampled passengers and crew members with ili or ari. this finding highlights the value of conducting such surveillance among cruise ship passengers and crews and reinforces the need to have robust influenza prevention and control activities on ships. influenza virus strains and respiratory viruses identified by the pilot were similar to findings of the state surveillance. the latter is conducted year-round in alaska, including in healthcare facilities in port cities where passengers disembark. data collected in the pilot project may aid in determining specific voyages with higher potential for influenza virus transmission. as southbound voyages originating in alaska had peak onset dates on the day before the start of the voyage, most influenza virus transmission may have occurred before embarkation. southbound voyages are generally associated with land-based tours immediately preceding the voyage involving buses and trains, which are additional semi-closed and close-contact environments that can potentially facilitate the transmission of influenza viruses. conversely, northbound voyages (which often have their land-based tours post-voyage) had peak onset dates occurring mid-voyage, suggesting that most influenza virus transmission occurred on board. additional surveillance is needed to better understand the role of land-based tours and potential differences in influenza virus transmission by voyage direction and route. adding a laboratory component to routine cruise ship respiratory surveillance in alaska, such as sending specimens from port to state laboratories, may better inform resource allocations and anticipate needs for cruise ship populations, which are typically disproportionately older. the average age of an alaska cruise ship passenger is years, and influenza in these passengers may be of more concern due to comorbidities in contrast to crew members, who are on average or younger and generally healthy. crew members may seek healthcare for no cost on board ship, but passengers without international or travel healthcare insurance coverage may incur medical costs for infirmary care on international waters. thus, cost could be a disincentive for passengers to seek care until they are severely ill or need to be medically evacuated to a hospital. this disincentive, in addition to participating cruise ship physicians' accuracy in targeting ill individuals for pilot project inclusion, may have contributed to the pilot's relatively high percent positive yield on respiratory pathogen and influenza testing. ili outbreaks can have economic and human resource costs to both cruise ships and public health agencies. additional staff are required to implement active and passive surveillance and coordinate testing; crew time is lost during illness or isolation; and antiviral agents for treatment and chemoprophylaxis of influenza can be costly. in some circumstances, land-based medical assistance may be required for follow-up and treatment of complications. influenza vaccination of crews may help reduce the impact of influenza virus transmission on cruise ships and serve as a cost-saving measure. both participating cruise lines have a target goal of vaccinating % of crew members, particularly in guest-facing departments. the findings of ship-based influenza surveillance can augment or supplement land-based surveillance. in alaska, influenza virus transmission during summertime is driven to a degree by the annual influx of tourists. states that experience off-season (summer) influenza virus transmission may benefit from extended summertime surveillance in order to guide recommendations on the timing of influenza vaccination and clinical decisions regarding testing and antiviral treatment, as well as to evaluate the match between circulating influenza virus strains and vaccine strains. these potential benefits should be weighed against the operational limitations in instituting such a surveillance program. while~ - cruise ships dock in alaska ports during the summer tourist season, only four to six participated in the alaska cruise ship respiratory surveillance pilot project each year. the small number, in addition to the relatively small number of specimens collected, does not allow generalization of results to all cruise ships in alaska or beyond. the results are also not generalizable to the entire cruise ship, as only ill individuals visited the ship medical centre for testing. as such, no true attack rate for influenza can be established. inclusion of only individuals reporting to the infirmary with respiratory illness may have biased selection of passengers and crew members to those with more severe symptoms or those more likely to seek healthcare. clinicians on participating ships were limited to collecting - specimens per week on average; thus, they may have chosen more overtly ill individuals to test. additionally, the case definition for participant inclusion changed between years ( ) and ( ). influenza vaccination history was obtained by self-report and may have been subject to recall bias, and the length of time between selfreported vaccination and illness onset was largely unknown. operational issues with specimen storage and shipping resulted iñ % ( / , table ) being lost in transport or deemed unsuitable for testing at the laboratory, and delays in shipping some specimens may have caused some falsely negative results. the alaska cruise ship respiratory surveillance pilot project served as a feasibility test for laboratory-based cruise ship surveillance in alaska. the pilot proved such surveillance is possible and thus may be helpful in informing public health officials and the cruise ship industry of the utility of surveillance to prevent and control outbreaks. in order to apply results to the whole alaska cruise ship population, additional ships should be included and sampling should be more representative of all ill persons. the program was operationally resource-intensive for all stakeholders, particularly cruise ship medical departments in collecting information on the ill individual, and the cdc anchorage quarantine station in equipment shipping and data management. subsequent similar laboratory-based surveillance projects may increase efficiency and lower the resource cost per sample. program evaluation is needed to weigh the time and operational limitations against the utility of this added layer of lab-based respiratory surveillance. despite its limitations, the pilot project's findings highlighted the usefulness of augmenting routine disease reporting and cumulative ili surveillance on ships in alaska. strengthening cumulative ili reporting on cruise ships to the level of required gastrointestinal illness reporting-an illness that generally is less severe than influenza-may be useful for improving the health of the travelling public. cruise ships should participate in requested cumulative ili reporting, and ship healthcare providers could collect recent travel history along with medical history during patient intake. cdc should be notified immediately of ili outbreaks on board, and its published outbreak management guidance should be followed, including specimen collection for respiratory virus identification and implementation of prevention and control measures. conducting limited laboratorybased surveillance during times of respiratory disease outbreaks or at the beginning and end of the cruise ship season, when detection of antigenic variants from ships repositioning to alaska is a higher possibility, should also be considered. cdc provided support to the alaska section of epidemiology through its existing epidemiology and laboratory capacity for infectious diseases (elc) grant for this pilot project. state of alaska department of commerce, community, and economic development influenza outbreaks among passengers and crew on two cruise ships: a recent account of preparedness and response to an ever-present challenge alaska/yukon territory respiratory outbreak investigation team. large summertime influenza a outbreak among tourists in alaska and the yukon territory outbreak updates for international cruise ships how to calculate the influenza or influenza-like illness (ili) case outbreak threshold for cumulative reports outbreak of influenza-like illness in a tour group-alaska outbreaks of pandemic (h n ) and seasonal influenza a (h n ) on cruise ship shipsan trainet project. the decision making process on public health measures related to passenger ships: the example of influenza pandemic cruise ships: high-risk passengers and the global spread of new influenza viruses world health organization. manual for the laboratory diagnosis and virological surveillance of influenza single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses guidance for cruise ships on influenza-like illness (ili) management we are grateful to the participating cruise ship clinicians, alaska maritime shipping agents, and dr sue gerber and dr dean erdman from the cdc division of viral diseases for their support during this project. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. key: cord- -p ofapbd authors: hellferscee, orienka; tempia, stefano; walaza, sibongile; variava, ebrahim; dawood, halima; wolter, nicole; madhi, shabir a.; du plessis, mignon; cohen, cheryl; treurnicht, florette k. title: enterovirus genotypes among patients with severe acute respiratory illness, influenza‐like illness, and asymptomatic individuals in south africa, ‐ date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: p ofapbd enteroviruses can cause outbreaks of severe acute respiratory illness (sari) and ev‐a, ‐b, ‐c, and ‐d species have different pathogenic profiles and circulation patterns. we aimed to characterize and determine the prevalence of enterovirus genotypes among south african patients with respiratory illness and controls during june to july . syndromic sari and influenza‐like illness (ili) surveillance was performed at two sentinel sites. at each site nasopharyngeal/oropharyngeal specimens were collected from sari and ili patients as well as controls. specimens were tested for enterovirus by real‐time pcr. positive specimens were further genotyped by sequencing a region of the vp gene. the prevalence of enterovirus was . % ( / ), . % ( / ), and . % ( / ) among sari, ili, and controls, respectively (sari/controls, p = . and ili/control, p = . ). among the / ( . %) enterovirus‐positive specimens that could be genotyped, we observed a high diversity of circulating enterovirus genotypes (a total of genotypes) from all four human enterovirus species with high prevalence of enterovirus‐b ( . %; / ) and enterovirus‐a ( . %; / ) compared to enterovirus‐c ( . %; / ) and enterovirus‐d ( . %; / ) (p = . ). of the enterovirus genotypes identified, echovirus ( . %, / ), coxsackie virus b ( . %, / ) and enterovirus‐d ( . %, / ) were most prevalent. there was no difference in disease severity (sari or ili compared to controls) between the different enterovirus species (p = . ). we observed a high number of enterovirus genotypes in patients with respiratory illness and in controls from south africa with no disease association of ev species with disease severity. pneumonia in children also include enteroviruses, rhinovirus, human bocavirus, and human coronaviruses ( e, nl , oc , hku ), although this remains controversial. , although influenza and respiratory syncytial viruses have been well described as causes of pneumonia in south africa, little is known about enterovirus prevalence and circulating genotypes. , enteroviruses are members of the enterovirus genus in the family picornaviridae. the capsid protein vp , the most variable protein containing the majority of neutralization epitopes, is commonly used to characterize enteroviruses. [ ] [ ] [ ] more than enterovirus genotypes are currently classified into four species, hev-a, hev-b, hev-c, hev-d, which have different pathogenic profiles and circulation patterns. , enteroviruses (ev) can cause symptoms similar to a mild cold, but have also been associated with severe respiratory infection requiring hospitalisation and may be fatal. an outbreak of a re-emerging ev-d lineage causing severe acute respiratory illness (sari) occurred in the usa and canada in , and these lineages were also detected in europe, asia, and south america from to samples. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] though evs have been associated with sari and influenza-like illness (ili), these viruses have been less well characterized in south africa. we identified high diversity among evs circulating in hospitalized south african patients, however it is unknown whether different ev genotypes are associated with mild or severe respiratory illness in south africa. we aimed to characterize the ev genotypes circulating among south african patients with sari, ili, and asymptomatic individuals from june to july . in addition we assessed the association of different ev species with mild (ili) or severe (sari) illness compared to asymptomatic individuals. an asymptomatic individual or control was defined as a person presenting at the same outpatient clinics with no history of fever, respiratory, or gastrointestinal symptoms during the days preceding the visit. these individuals commonly presented to the clinics for visits such as dental procedures, family planning, well baby visits, voluntary hiv counseling and testing, or acute care for non-febrile illnesses. we aimed to enroll one hiv-infected and one hiv-uninfected control every week in each clinic within each of the following age categories: - , - , - , - , and ≥ years. individuals were not followed-up to ensure they remained asymptomatic after enrollment. study staff completed case report forms for all enrolled sari and ili cases as well as controls. in addition, for sari cases, hospital records were reviewed to assess disease progression and outcome (ie, discharge, transfer, or in-hospital death). referral to hospital was recorded for all enrolled ili cases. ili cases that were referred to hospital were excluded from the analysis, as these patients did not adhere to the case definition of ili anymore. hiv results were obtained from a combination of two sources: (i) patient clinical records when available and (ii) for consenting patients, an anonymized linked dried blood spot was tested at the national institute for communicable diseases (nicd). when both results were available, the nicd result was used. nasopharyngeal aspirates for children < years of age and nasopharyngeal and oropharyngeal swabs from persons ≥ years of age were collected from all enrolled participants and placed in universal transport medium (copan, murruieta, ca). all respiratory tract specimens were tested for the presence of respiratory viruses (influenza virus a and b, respiratory syncytial virus, parainfluenza virus types , , and , adenovirus, human metapneumovirus, and rhinovirus) including ev, using a multiplex real-time reverse transcriptase polymerase chain reaction assay. the real-time reverse transpriptase polymerase chain reaction used for ev testing, is a sensitive method for the routine detection of all members of the enterovirus genus. this method has a weak cross-reactivity to hightitre rhinovirus stocks, but not with rhinovirus-positive clinical samples. all specimens testing positive for enterovirus were selected for molecular characterization. a base pair region of the vp gene was amplified and sequenced as previously described using primer set / during the first round of amplification and primer set an /an for the nested amplification. this assay was originally designed to detect and identify ev, but also detects and identifies a subset of rhinoviruses. amplicons were purified using the exosap enzyme system the genetic diversity of each vp sequence was first determined by comparison with the reference strains in genbank (us national center for biotechnology information, ncbi, http://www.ncbi.nlm.nih.gov/, accessed july ) using blast (basic local alignment search tool) and confirmed by phylogenetic analysis of the partial vp sequences. multiple sequence alignments were generated in the mafft (multiple alignment using fast fourier transform) multiple sequence alignment program and analyzed in the bioedit sequence alignment editor software. phylogenetic trees were generated using the neighbor-joining method and genetic distances were calculated with the kimura- -parameter model using mega (molecular evolutionary genetics analysis) software. the statistical significance of the phylogenies was estimated by bootstrap analysis using pseudo replicates. sub analysis was similarly done for the three dominant ev species in this study. sequences of enterovirus partial vp genes generated in this study have been deposited in genbank with the following accession numbers: kx -kx . the chi-squared or fisher's exact test were used for comparison of categorical variables. unconditional exact logistic regression was used to assess the association of ev species with disease severity among patients with mild (ili) or severe illness (sari) using asymptomatic individuals as control group. exact logistic regression was used to account for the fact that no ev-d species was detected among controls. ev-b was used as the comparison group as it was the species most frequently detected. significance was assessed for p < . . all models were adjusted for age, hiv serostatus and underlying medical conditions. the analysis was performed using stata on multivariable logistic regression analysis using ev species b as the reference group and controlling for age, hiv status and underlying illness, no association with disease severity (sari or ili compared to controls) was identified for any of the ev species (supplementary table s ). enterovirus genotypes could be identified in % ( / ), % ( / ), and % ( / ) of enterovirus-positive samples among sari and ili cases and controls, respectively. we identified a total of genotypes, distributed among all four ev species. the most prevalent genotype was e ( . %, / ), followed by cvb ( . %, / ) and ev-d ( . %, / ). all other genotypes identified in the study were detected in < % of genotyped samples (supplementary fig. s ). all e strains identified in this study displayed > % nucleotide (nt) homology and formed a distinct bi-phyletic cluster with % bootstrap support (fig. ) . in the bi-phyletic cluster, strains from edendale sari cases and klerksdorp sari and ili cases are distinctly located in each of the sub clusters with % bootstrap support, respectively. the e strains in our study differ from other e genotypes by mean nucleotide pairwise distance of . % ( . - . ) and mean amino acid sequence distance of % ( . - . ) in amino acid sequence. all cvb strains identified in our study (six sari samples and two ili samples from edendale) were categorized as genogroup c and formed a distinct sub cluster with a bootstrap value of % (fig. ) . the nucleotide the majority ( . %, / ) of ev-d strains identified in / distinctly clustered in lineage b ( % bootstrap support) of which three were ili cases and two were sari cases, together with strains from one of the two co-circulating ev-d lineages that caused the large usa outbreak (fig. ) . the nucleotide sequences for evd- strains were downloaded from genbank and sequences depicted in fig. represent all known lineages. the lineage b ev-d strains in our study differed from other lineage b ev-d strains by . % nucleotide and . % amino acid sequence. these included two sari cases (one from klerksdorp and one from edendale) and three ili cases (one from klerksdorp and two from edendale). we describe the ev species circulating among sari and ili cases and asymptomatic controls in south africa. we observed a high diversity of circulating ev genotypes (a total of genotypes) from ev species a-d with high circulation rates for ev-b and ev-a compared to ev-c and ev-d. from our systematic surveillance across all age groups, the majority of patients testing positive for ev were < years of age. we did not observe any difference in disease association due to different ev species since ev disease association cannot be conclusively ascribed since the analysis did not include coinfections with other pathogens. epidemics of human ev disease display a seasonal pattern, with infections more common in summer and early autumn in geographical regions with a temperate climate. , most clusters of ev-d showed an atypical late seasonality compared to other evs, with a peak in autumn, instead of summer. a common feature of e molecular epidemiology is the progression of circulating lineages within one prevalent genotype. , the e strains identified in this study clustered together (designated genotype k) although no differences were observed between viruses from sari compared to ili cases. there is no report of a concurrent e aseptic meningitis outbreak at the same time the e np/op positives were detected. it has been reported that genotypes of e can become dominant for - -year periods before they disappear from circulation, and they are therefore known as epidemic strains. in our study, it seems that e genotype k became dominant and circulated in south africa during - . phylogenetic studies with vp sequences indicate that e variants are continuously emerging and replacing the circulating e strains, largely due to the error prone ev rna polymerase . cvb has been detected for over years and, similar to e , sporadic cases of aseptic meningitis, as well as outbreaks, have been reported, remaining one of the most predominant reported ev genotypes in a number of countries. all of the cvb strains identified in this study formed a sub cluster in genogroup c. phylogenetic clustering by year of study enrolment was observed for the south african strains. different co-circulating strains of ev are often observed in ev disease outbreaks as ev genomes vary with respect to time but may also vary by geographical distribution. in , the largest outbreak to date of severe respiratory illness associated with ev-d occurred in the usa, and almost all of the case patients were children. coinciding with the usa outbreak the mechanisms for emergence of ev-associated outbreaks are not known; however, a combination of virus-specific, population-level, and other external factors are likely to be involved. our study has some limitations. previous studies observed a difference in the predominant ev genotypes among neonates and older children but we were not powered for this analysis. some rhinovirus sequences were detected in samples that tested positive for ev on our in-house assay indicating that our ev detection assay is not % specific to detect only evs. this study was not designed to describe the role of co-infections with other respiratory pathogens. in addition the association with ev detection with mild and severe illness was not investigated. in conclusion, we showed that there was a high diversity in the vp sequences of the ev species circulating in south africa during - and most of these genotypes are associated with meningitis worldwide. ev was detected in outpatient and hospitalized patients with sari but was also detected in controls. we determined no disease association of ev species with disease severity; however, some genotypes (e , cvb , ev-d ) were more prevalent in symptomatic cases. further studies are needed to determine if other factors such as viral load or host interactions play a role in ev-associated disease as well as a robust spatiotemporal phylogenetic analysis based on complete vp sequences. we thank all members involved in sari and ili surveillance for the collection of specimens and data management. this work was supported by the national health laboratory service, south africa, and the united states centers for disease control and prevention, atlanta, georgia, usa (co-operative agreement number: u ip ). the findings and conclusions in this paper are those of the authors and do not necessarily represent the views of their affiliated institutions or the agencies funding the study. childhood pneumonia-progress and challenges who pneumonia fact sheet no development and implementation of a molecular diagnostic platform for daily rapid detection of 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reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa rapid and sensitive routine detection of all members of the genus enterovirus in different clinical specimens by real-time pcr mafft multiple sequence alignment software version : improvements in performance and usability bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt mega : molecular evolutionary genetics analysis (mega) software version . phylogeography of circulating populations of human echovirus over years: nucleotide polymorphism and signature of purifying selection in the vp capsid protein gene worldwide emergence of multiple clades of enterovirus phylogenetic patterns of human coxsackievirus b arise from population dynamics between two genogroups and reveal evolutionary factors of molecular adaptation and transmission molecular epidemiology of echoviruses and in russia: different properties of genotypes within an enterovirus serotype an outbreak of aseptic meningitis caused by a distinct lineage of coxsackievirus b in china genetic variation of coxsackie virus b strains associated with aseptic meningitis in greece severe respiratory illness associated with enterovirus d -missouri and illinois enterovirus d- infection, prophylaxis, and vaccination in a novel permissive animal model, the cotton rat (sigmodon hispidus) presence of human non-polio enterovirus and parechovirus genotypes in an amsterdam hospital in to compared to national and international published surveillance data: a comprehensive review key: cord- -vwye pk authors: comach, guillermo; teneza-mora, nimfa; kochel, tadeusz j.; espino, carlos; sierra, gloria; camacho, daria e.; laguna-torres, v. alberto; garcia, josefina; chauca, gloria; gamero, maria e.; sovero, merly; bordones, slave; villalobos, iris; melchor, angel; halsey, eric s. title: sentinel surveillance of influenza-like illness in two hospitals in maracay, venezuela: – date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vwye pk background: limited information exists on the epidemiology of acute febrile respiratory illnesses in tropical south american countries such as venezuela. the objective of the present study was to examine the epidemiology of influenza-like illness (ili) in two hospitals in maracay, venezuela. methodology/principal findings: we performed a prospective surveillance study of persons with ili who presented for care at two hospitals in maracay, venezuela, from october to december . a respiratory specimen and clinical information were obtained from each participant. viral isolation and identification with immunofluorescent antibodies and molecular methods were employed to detect respiratory viruses such as adenovirus, influenza a and b, parainfluenza, and respiratory sincytial virus, among others. there were participants in the study (median age: years; range: month – years). viruses were identified in ( . %) subjects, and one participant was found to have a co-infection with more than one virus. influenza viruses, including pandemic h n , were the most frequently detected pathogens, accounting for . % ( / ) of the viruses detected. adenovirus ( / ), parainfluenza virus ( / ), and respiratory syncytial virus ( / ) were also important causes of ili in this study. pandemic h n virus became the most commonly isolated influenza virus during its initial appearance in . two waves of the pandemic were observed: the first which peaked in august and the second - higher than the preceding - that peaked in october . in , influenza a/h n re-emerged as the most predominant respiratory virus detected. conclusions/significance: influenza viruses were the most commonly detected viral organisms among patients with acute febrile respiratory illnesses presenting at two hospitals in maracay, venezuela. pandemic h n influenza virus did not completely replace other circulating influenza viruses during its initial appearance in . seasonal influenza a/h n was the most common influenza virus in the post-pandemic phase. acute respiratory infection (ari) remains a leading cause of global burden of disease, and is the second most common cause of illness worldwide, with an annual global incidence exceeding million [ ] [ ] [ ] . a prerequisite of public health planning to reduce global disease burden from ari is to examine data on its epidemiology in order to better define environmental factors as well as target populations for preventive interventions [ ] . respiratory viruses are predominant causes of aris, and the epidemiology of acute viral respiratory illnesses in developed countries with temperate climates has been well-characterized [ ] [ ] [ ] . in countries such as the united states, children have been shown to carry a large burden of viral respiratory diseases [ ] . recent prospective studies, which utilized more sensitive methods for detecting respiratory viruses such as multiplex polymerase chain reaction (pcr), have similarly demonstrated that the highest rates of viral respiratory infection occur among children and the frequency of infection tends to decrease with age due to increasing acquired immunity [ ] . respiratory syncytial virus (rsv), influenza virus, parainfluenza virus, and rhinovirus have long been identified as common causes of ari [ ] . recent improvements in molecular detection techniques have allowed the identification of multiple new respiratory viruses such as human metapneumovirus (hmpv), human bocavirus (hbov) and human coronavirus nl [ ] . while the body of literature describing the epidemiology of acute viral respiratory diseases in developed countries has rapidly expanded, knowledge of the distribution of these diseases in regions such as tropical south america remains limited. influenza viruses are among the most impactful acute respiratory pathogens in terms of morbidity and mortality. despite developed public health intervention programs, the estimated annual average number of influenza-related hospitalizations in the united states exceeds , , and , deaths are attributable to influenza infections yearly [ , ] . information on the contribution of influenza viruses to the global burden of disease due to acute respiratory illness is incomplete. data on the epidemiology of influenza viruses in developed countries are derived from multiple sources to include laboratory-based surveillance, sentinel surveillance, as well as hospitalization and outpatient records. in developing countries, where resources are sparse, sentinel surveillance methods may be more readily accessible and more cost-effective than laboratory-based or population-based surveillance for determining the viral etiology of influenza-like illness (ili) in these regions. better identification of the viral causes of ili will enable clinicians in resource-limited settings to appropriately treat and manage patients; more importantly, it will allow public health officials to formulate more effective prevention and control strategies, including monitoring of influenza vaccine efficacy in their communities [ ] . studies on the epidemiology of ili in the tropical south and central american countries of peru, brazil, ecuador, nicaragua, honduras, and el salvador have been published [ ] [ ] [ ] [ ] . a prospective study of adults with ili in sao paulo, brazil, revealed that while influenza viruses were the predominant cause of ili, rhinoviruses and other respiratory viruses were detected in . % and . % of subjects, respectively [ ] . this observation illustrated that a significant proportion of patients who are clinically diagnosed with influenza virus infection may have symptoms indistinguishable from other respiratory viruses. in a prospective study of ili in ecuador, the regional distribution of influenza virus infections varied; a higher detection rate of influenza a occurred in quito, located in the highlands where the level of absolute humidity is lower, whereas influenza a detection rate was lower in the coastal city of guayaquil, which has a more humid tropical climate [ ] . additionally, an expanded sentinel surveillance of ili conducted at health centers and hospitals located in peruvian cities showed that the distribution of ili-causing viruses varied by region [ ] . these studies illustrate the importance of conducting baseline and continuing ili surveillance in other countries of central and south america because ili can be caused by pathogens other than influenza viruses. the distribution of respiratory infections may vary within the region or within a particular country, depending on climate and topography. in venezuela, ari is the primary cause of weekly notifiable diseases registered by the national epidemiological surveillance system (ness) [ ] . nonetheless, very low numbers of respiratory samples were collected ( , from to ) and even lower numbers ( , ) were confirmed by the national system for virological surveillance of ari [ ] [ ] [ ] [ ] [ ] . furthermore, the epidemiology, clinical characteristics, and viral causes of ili have been poorly characterized in venezuela; to our knowledge, only one longitudinal study, carried out during a limited period of time (february -july ) and with a low number of patients (n = ) with ari, has been published [ ] . the objective of this paper is to describe the epidemiology of ili using data from a sentinel surveillance system at two major hospitals in maracay, venezuela. a total of subjects participated in this study. six hundred and thirty seven ( . %) were recruited at the hospital central de maracay (hcm). there was a slightly larger proportion of females compared with males who enrolled in the study ( . % vs . %), and the gender distributions at the two sites were similar. subject ages ranged from month to years with a mean age of . years (s.d. = . years) and a median of years. a significant percentage of the subjects ( . %) were under years of age, while less than . % were years or older. the - year old group was the most predominant group comprising . % of the study population, followed by the - year old age group ( . %). the subjects who presented to hcm were significantly older (mean age = . years; s.d. = . years) compared with those who were seen at the hospital del instituto venezolano de los seguros sociales jose maria carabano tosta (ivss-jmct) (mean age = . years; s.d. = . years). a large proportion ( . %) of the subjects at hcm belonged to the - age group, while nearly half of the patients ( . %) seen at ivss-jmct were younger than years old. approximately % of the subjects reported having received influenza vaccination within the last year. a small percentage of patients ( . %) received antibiotic treatment for their acute respiratory illness prior to enrollment in the study. no hospitalized cases with ili were recruited in this study; only outpatient subjects participated. demographic information is summarized in table . among the patient samples obtained from the sentinel surveillance, at least one viral organism was detected by viral isolation and/or reverse transcriptase-polymerase chain reaction (rt-pcr) in ( . %) subjects ( table ). only one participant with a co-infection was observed. influenza viruses were the most frequently detected organisms in this passive surveillance, consisting of . % ( / ) of all the viruses detected by viral isolation and/or rt-pcr. of the samples collected, ( . %) were positive for influenza a viruses and ( . %) for influenza b viruses by viral isolation and/or rt-pcr. in addition, noninfluenza viruses were detected only by virus isolation; they were: adenovirus ( . %), parainfluenza virus ( . %), rsv ( . %), and other viruses ( . % ), which included hbov ( . %), enterovirus ( . %), hmpv ( . %), rhinovirus ( . %), and herpes simplex virus (hsv, . %). the only co-infection was observed in a month old toddler in whom adenovirus and hsv were isolated. the virus etiology and detection rates varied with age ( table ). the most commonly detected viral pathogens in the - year old group were influenza a ( . %) and adenoviruses ( . %). influenza a and b were the most commonly detected viruses in the school-age group ( - year old: . % and . %, respectively) and the late adolescent and young adult group ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) year-old: . % and . %, respectively). influenza a was the only detectable virus among patients who were years or older. of the influenza a cases, h subtype was detected in , and was observed in all the age groups but in subjects aged - year old ( table ) . six cases had the h subtype (non-pandemic), and influenza a viruses were isolated but not subtyped by one-step rt-pcr; of the latter, can be classified as seasonal because they were isolated from patients samples collected before the start of the h n pandemic outbreak of . twenty-one cases of pandemic h n influenza virus (ph n ) infections were identified in this sentinel surveillance; thirteen occurred in the - year old group. there were no cases of ph n infections among adults years old or higher. genetic analysis based on partial hemagglutinin gene sequences (approximately bp) of influenza isolates is shown in figure (see material and methods for genbank accession numbers). this analysis shows that the circulating seasonal a/h n strains in venezuela ( figure a ) were similar to the ones previously described in central and south america [ , ] and all of these samples can be grouped with the a/ brisbane/ / like - genotype. the ph n samples were part of only one cluster similar to previously reported strains in latin america [ ] . influenza a/h n samples possessed more genetic variability ( figure b) ; isolates from revealed two genotypes, a/brisbane/ / -like and a/ california/ / -like, while the more recent isolates were closer to the a/perth/ / -like genotype. finally, in figure c , the genetic analysis for influenza b isolates disclosed the presence of two different genotypes, b/florida / -like and b/malaysia/ / -like, in agreement to what was previously found in the region [ , ] . adenovirus was detected more frequently from december to april. rsv infection occurred more frequently from june to november. parainfluenza viruses were detectable throughout the year but without distinct seasonality. the passive surveillance illustrated the impact of ph n on the distribution of respiratory viruses associated with ili in ( figure ). during the ili peaks from - , the percentage of ili cases attributable to influenza viruses ranged from % to %. the first cases of ph n in this surveillance system were detected in july , three months after the swine origin influenza outbreak began in mexico [ ] . during the first wave in july , the monthly virus detection rate rose to %, and % ( / ) of all the viruses detected were of the pandemic strain. the first wave simulated a typical ili peak activity similar to those observed in other years. however, during the larger second wave in october , the virus positivity rate exceeded %, and the pandemic strain was identified in greater than % ( / ) of the viruses detected. figure demonstrates that ph n did not completely replace seasonal influenza a viruses. the peak pandemic activity in october was followed by a rapid decline in the rate of pandemic strain detection one month later. meanwhile, seasonal influenza a viruses remained in circulation throughout the pandemic period comprising % of all the influenza a viruses detected in october . in , % of influenza a specimens obtained via oropharyngeal swabs were randomly selected and tested for the pandemic strain via rt-pcr, and no cases of ph n were detected ( figure ). however, seasonal influenza a/h n continued to be detected by our surveillance system with monthly positivity rates ranging from % to %. the clinical features of subjects in our surveillance are summarized in table . compared with those whose respiratory secretions tested negative, subjects in whom virus was identified were more likely to have sore throat, headache, pharyngeal congestion, and ear pain. there were no significant differences in the symptoms of individuals who had seasonal influenza a when compared with those who suffered from ph n influenza, except that a higher proportion of the latter subjects had expectoration, myalgias, and lymphadenopathy. the symptoms of influenza a (either seasonal or pandemic) and influenza b were clinically indistinguishable. when compared with patients who had ili due to other viruses, a higher percentage of those with confirmed influenza virus infection experienced sore throat, myalgias, and headache. there is scarce information about the epidemiology of acute febrile respiratory illness in venezuela and, to our knowledge, only one longitudinal study has been published [ ] . this investigation, however, was limited to few number of subjects (n = ) recruited during a short period of time ( months) and did not describe the transmission seasonality of the viral infections. thus, our study is the first to fully describe the epidemiology and viral etiology of ili in venezuela and provides baseline levels of ili activity in a typical highly-populated urban city. our study demonstrated that influenza viruses are a main cause of ili at hcm and ivss in maracay in agreement with findings reported by venezuelas ness [ ] [ ] [ ] [ ] [ ] . nevertheless, the rate of confirmed influenza virus infections found in our surveillance ( . %, / ; tables and ) during the study period was lower than the one ( . %) reported by the ness for the same period [ ] [ ] [ ] [ ] [ ] . these differences in detection rates may be attributable to different strategies for capturing ari patients, especially those with influenza, used by the ness and by this study protocol (flores e, director of epidemiology, corposalud , personal communication). the ness randomly selected a sample of ari cases (including those with influenza) with emphasis on severe hospitalized cases, whereas in our protocol we recruited ili subjects in an outpatient setting where the majority had symptoms that were not severe. on the other hand, the percentage of influenza viruses (not including ph n ) detected in our study during a similar period of time, but in different years accounted for the significant differences found in both studies: a) the collection, preservation and further processing of respiratory samples, and b) the type of cells and ifa reagents used for virus isolation and identification. the proportion of influenza cases was significantly different when comparing non-pandemic and pandemic periods. before the h n pandemic, the ness [ ] detected influenza virus in . % of subjects in whom a virus was isolated; a similar proportion to the % (data not shown) found in our study. during the height of the pandemic (from july through dec ), the ness confirmed % of all ari cases with a virus as having either influenza a or b virus compared with % observed in our study. the predominance of influenza viruses as etiological agents of ili in maracay, venezuela, is consistent with observations of surveillance studies in other tropical central and south american countries [ , , ] . while the overall virus detection rate ( / , . %; table ) was lower compared with other studies, the positive rates for influenza a ( . %) and influenza b ( . %) in this surveillance were comparable to those observed in a similar study in el salvador, honduras, and nicaragua [ ] . our findings were also consistent with a study in indonesia, which identified influenza a or b in % of all respiratory samples using viral isolation and rt-pcr [ ] . our findings were consistent with those from a prospective study of outpatient children in northern taiwan, in which influenza a or b were isolated in . % of subjects with ili, using madin-darby canine kidney (mdck) cell cultures and hemagglutinin inhibition assay for antigen detection [ ] . however, our detection rate for all respiratory viruses, as well as influenza a and b viruses, was generally lower compared to other studies conducted in south america. in an expanded sentinel surveillance study in peru, which utilized a similar methodology for viral detection as our study, the virus positivity rate was . %, and influenza a and b rates were . % and . %, respectively [ ] . in a prospective surveillance study of ili in two ecuadorian cities using similar methods, at least one virus was detected in % of the participants; influenza a was detected by pcr in . % while . % tested positive for influenza b [ ] . the lower virus detection rates found in our study is unclear because we used the same operative protocol described in the mentioned studies [ , ] . nevertheless, the different skills to collect respiratory tract specimens from ili patients, by the health personnel employed in their studies and ours may have accounted for the lower virus detection rates found in our study. while influenza viruses were observed to be the most prevalent viral pathogen, adenovirus, parainfluenza virus, and rsv were important causes of ili at the two hospitals (table ). in venezuela, the ness reported rsv as the most frequently detected non-influenza respiratory virus followed by parainfluenza virus and rhinovirus [ ] [ ] [ ] [ ] [ ] . the study performed in zulia, venezuela, also reported rsv as the most common detected virus, followed by adenovirus, parainfluenza virus and influenza viruses [ ] . as previously mentioned, the differences in the detection rates may be attributed to the different procedures used by the ness, other venezuelan researchers. [ ] and us. these viral pathogens were also frequently detected in other surveillance studies in central and south america [ ] [ ] [ ] . the highest detection rate of respiratory viruses was observed in the - year old group ( . %; table ). the rate of viral positivity generally decreased with age as acquired immunity increased in older subjects. a slight increase can be seen between the - year old group ( . %) and years or older group ( . %). subjects who were years old or older comprised less than % of the study population, and thus, the prevalence in this age group may be unreliable. contrary to our study, the other study from. venezuela had higher detection rates in both the - and $ year old groups ( . % and . %, respectively) [ ] . on the other hand, our findings are consistent with observations in the tecumseh study, a community-based surveillance in michigan, in which children were shown to have the highest rate of viral respiratory diseases [ , ] . the tecumseh study further illustrated the variable impact of influenza viruses among the different age groups depending on the influenza virus subtype. for example, influenza a/h n affected a wide range of age groups while influenza a/h n and influenza b virus infections occurred more frequently among older children and young adults [ , ] . our findings were similar being seasonal influenza a/h n viruses and influenza b detected primarily in children and young adults, and seasonal influenza a/h n found in all age groups. as expected, ph n was the most common influenza a subtype identified among the subjects with ili in ( figure ). in , . % (figure ) of ili cases were due to ph n ; this detection rate was lower than the . % detection rate reported by the venezuela's ness [ ] . nevertheless, this finding was similarly demonstrated in respiratory illness surveillance networks in other tropical and temperate south american countries such as guatemala [ ] , peru [ ] , argentina [ ] , and brazil [ ] . in , non-pandemic influenza viruses continued to circulate in venezuela, and ph n was not detected in our surveillance study, suggesting that ph n did not displace seasonal influenza a viruses. during the same year, . % of the ari cases reported to venezuela's ness were due to ph n [ ] . infection with ph n may have stimulated immunity among the residents of this community during its initial arrival in . this observation suggests that the circulating ph n in may not have significantly mutated relative to the strain in , so that antibodies stimulated by the natural infection during its initial arrival may have still been highly efficient in protecting the influenza-like illness in maracay, venezuela plos one | www.plosone.org community from another wave of ph n outbreak. in , the infection rate of influenza a/h was higher than the rates observed during previous years, further illustrating the lack of cross protection between ph n and influenza a/h . our study shows that hmpv and hbov were not commonly associated with ili, based on the low detection rates observed in our surveillance. in a study from ecuador which utilized methods similar to those employed in our study, a low detection rate for hbov ( . %) was reported [ ] . the latter study, as well as another from central america which used methods similar to ours, reported rare detection of hmpv (, . %) [ , ] . in contrast, a prospective study of ili among brazilian adults, which utilized viral isolation and rt-pcr testing on respiratory samples, detected rhinoviruses in . % of patients [ ] . although rhinoviruses are typically associated with milder illness, they can contribute to the misdiagnosis of influenza based on clinical case definition alone. a cohort study of vietnamese children hospitalized for acute febrile respiratory illness, which applied multiplex-pcr assays on respiratory samples, revealed slightly higher prevalence rates of hbov ( %) and hmpv ( %) infections [ ] . it is important to note that our method of identification (culture on three cell lines), compared to molecular diagnostic methods, substantially lacked sensitivity for detecting rhinoviruses, hmpvs, and hbovs. our study shows that patients with ph n infections were more likely to have myalgias, productive cough with expectoration, and lymphadenopathy than with those infected with seasonal influenza a virus (table ). in contrast, clinical manifestations in guatemalan subjects hospitalized for ph n and seasonal influenza a infections did not significantly differ [ ] . our study had limitations worth noting. data collection at only two hospitals in an urban area limits our ability to generalize our findings to the population. the low virus detection rates may be attributable to variations in the skills of the health staff employed to collect the respiratory specimens. the sampling method may have a significant effect on the proportion of respiratory viruses identified, and nasopharyngeal washes may yield higher sensitivity over nasopharyngeal or oropharyngeal swabs [ ] . study participants were exclusively seen in the outpatient setting, thus limiting our ability to examine the impact of respiratory viruses in hospitalized cases. a large proportion of ili cases were not associated with any pathogen, and the impact of bacteria on this clinical syndrome cannot be determined from this study, since the respiratory samples were not cultured for bacteria. two methods of detection were used for identification of influenza (pcr and culture) whereas only one method of detection was used for the other viruses (culture). twenty-two percent ( / ) of the respiratory samples which were positive for influenza viruses by rt-pcr were negative by viral isolation illustrating that viral detection by culture underestimated the true prevalence. viral culture may not be the ideal way of isolating organisms such as rsv, hmpv, hbov and rhinoviruses leading to significant underestimation of their detection rates [ ] . despite these limitations, our study contributes information on the distribution and etiology of ili at two hospitals in maracay, venezuela. this knowledge can serve as a baseline for future, more expansive population-based surveillance studies of influenza and other respiratory viruses in this region. maracay is located in the central northern region of venezuela ( u n, u w) , approximately miles from the caribbean coast (figure ) . the climate is tropical with two seasons: dry (december-april) and rainy (may-november). the monthly averages for temperature, relative humidity and rainfall are . uc (range = . uc- . uc), % (range = %- %) and mm (range: mm- mm), respectively. maracay is comprised of two main urban municipalities, named girardot and mario briceñ o iragorry, and had an estimated total population of , in . the hcm and the ivss-jmct are located approximately miles apart. both are major county hospitals and referral health centers with adult and pediatric departments including emergency services and (ivss-jmct) to (hcm) hospitalization beds. they provide services to people in maracay as well as those from the adjacent city of aragua and three neighboring states. the study population included every outpatient with ili, regardless of age, who sought attention at the two study sites between october and december , and agreed to participate in the study. at each site, trained medical personnel were responsible for properly identifying and classifying patients with ili. each person with ili was asked to enroll in the study. a person was defined as having ili if he or she had a sudden onset of fever ($ uc) and either cough or sore throat for less than five days in duration, with or without general symptoms such as myalgias, prostration, headache, or malaise [ ] . data on gender, age, previous treatments, medical attention before enrollment, influenza vaccination status, and days of work/ school lost at the time of acute illness were collected utilizing a case report form (crf) from all participants who met the case definition criteria. temporal distribution of the results were recorded by month during the study period, taking into account the number of ili cases identified and the number of confirmed cases of influenza a and b in each study site. monthly reports of enrolled ili participants and laboratory results were sent to the venezuelan ministry of health. regular personnel training in protocol procedures were conducted as part of the strategy to improve sampling, storage,and shipping procedures. this protocol was approved as less than minimal risk research by the naval medical research center (nmrc), silver spring, maryland. institutional review board (irb; protocol nmrcd. . ) authorization was given to perform the study using an information sheet approved and stamped by the irb. as this was part of clinical care and routine surveillance benefiting the ministry of health, verbal consent was obtained from all participants. this method of consent was accepted by the nmrc irb as well as the venezuelan institutions involved. a verbal consent was approved by both irbs following cioms and cfr (the common rule), ) it was a minimal risk study, and ) local health installations would not require a written consent for the procedures required in this study. additionally, this document included all the information that a written consent would require; a copy was provided to each study subject; and study personnel responsible for administering the process of informed consent were trained at each site on human research protection issues and were certified through the collaborative institutional training initiative (citi). finally, the frequent monitoring visits conducted by the study team found no problems in the process nor complaints from study participants. sample collection. nasal ( of , . %) or oropharyngeal ( of , . %) swabs were obtained from each subject for viral isolation and identification. the swabs were placed in viral transport media and stored at - uc until they were delivered on dry ice to namru- in lima, perú, for laboratory analysis. duplicate swabs were processed and analyzed at the laboratorio regional de diagnostico e investigacion del dengue y otras enfermedades virales/instituto de investigaciones biomedicas de la universidad de carabobo (lardidev/ biomed-uc) in maracay, venezuela, following the same operative protocols used in namru- . virus isolation and identification. nine-hundred and sixteen nasal or oropharyngeal swabs were processed for viral isolation and identification following the procedure described by laguna-torres et al. [ ] . briefly, patient specimens were inoculated onto four cell lines: madin-darby canine kidney (mdck; atcch number ccl- ), african green monkey kidney (vero ; atcch numbercrl- ) and veroe atcch number crl- ), and rhesus monkey kidney (llc-mk atcch number ccl- ). upon the appearance of cytopathic effect or after ten days of culture (or thirteen days in the case of vero cells), the cells were spotted onto microscope slides. cell suspensions were dried and fixed in chilled acetone for minutes. virus isolates were identified using direct fluorescence antibody (dfa) assays. the respiratory virus screening and identification kit (d dfa respiratory virus diagnostic hybrids; athens, oh) was utilized for the identification of adenoviruses, influenza a virus, influenza b virus, parainfluenza viruses (types , , and ), and rsv. the d dfa herpes simplex virus (hsv) identification kit and the d ifa enterovirus id kit (diagnostic hybrids; athens, oh) were utilized for the identification of hsv (both hsv- and hsv- ) and enteroviruses, respectively. for isolation of hmpv, we used vero e and llc-mk cell lines. for detection of hmpv antigens by direct fluorescence assay, we used an anti-hmpv mouse monoclonal antibody from diagnostic hybrid (athens, oh). all assays were performed following the manufacturers' instructions. hbov was identified using the methods described by salmon-mulanovich, et al [ ] . since duplicate swab samples were analyzed in different laboratories (lardidev/biomed-uc and namru- ) with the same diagnostic kit and standard operating procedures, a virus isolation result was considered positive if the specific virus was isolated and identified at either site. a subset of specimens ( nasals and oropharyngeals) was analyzed by one-step rt-pcr and/or real time rt-pcr in order to sub-type influenza a/h n (including ph n ), influenza a/h n , and influenza b viruses. one-step rt-pcr and/or real time rt-pcr were not used to identify noninfluenza viruses because the specific primers and protocols were available only for sub-typing influenza viruses; thus, only virus isolation was used to detect and identify non-influenza viruses. of the specimens, ( . %) were randomly selected from repository samples collected before the pandemic and tested for influenza a sub-typing by one-step rt-pcr and/or real time rt-pcr. at the onset of the pandemic, of ( . %) respiratory samples were tested for ph n by real time rt-pcr at the request of the venezuelan ministry of health. after the peak of the second wave of the pandemic, the percentage of respiratory samples tested by real time rt-pcr was reduced to % ( of ). one-step rt-pcr was performed according the procedure and influenza primers described below. real time rt-pcr was these protocols are available from cdc upon request. for the purpose of this study, an ili case with a confirmed viral respiratory infection was one in which viral isolation and/or rt-pcr identified a virus. rna extraction and one-step rt-pcr. viral rna extraction was performed from the supernatant of infected mdck cells using a qiaamp viral rna kit (qiagen; valencia, ca) following the manufacturer's protocol. the onestep rt-pcr was performed following a procedure described previously [ ] with primers that amplified the hemagglutinin (ha) gene of influenza a and influenza b viruses using the superscript iii one-step rt-pcr system kit (invitrogen; san diego, ca). the following primers were used for the amplification of h influenza a viruses: h f- ( -aagcaggggaaaa-taaaa- ) and h r- ( -gtaatcccgttaatggca- ); for h influenza a viruses: h f- ( -actat-cattgctttgagc- ) and h r- ( -atggctgctt-gagtgctt- ); for influenza b viruses: bhaf- ( -gaagg-caataattgtact- ) and bhar- ( -accagcaatagctccgaa- ). five ml of the extracted rna was added to ml of master mix containing the enzyme mixture (superscript iii rt/platinum taq), x reaction mixture (containing . mm of each dntp and . mm of mg so ) and mm of each primer. cycling conditions included a reverse transcription step at uc for minutes and a denaturation step at uc for minutes. cycling conditions of the pcr were cycles of uc for seconds, uc for seconds, and uc for seconds, followed by a final incubation step at uc for minutes. dna sequencing and phylogenetic analysis. for confirmation of serotype and genotype of the circulating influenza viruses, samples were sequenced. as part of the respiratory surveillance protocol in venezuela, these viruses were randomly selected from approximately % of the positive samples. only the ha gene region of influenza viruses was analyzed routinely for genotyping the one-rt-pcr products amplified with the primers described before were purified using centri-sep columns (princeton separation; englishtown, nj) and sequenced using the bigdye terminator v. . cycle sequencing kit (applied biosystems; foster city, ca) following the manufacturers' instructions. sequences were analyzed and edited using the sequencer . software (applied biosystems; foster city, ca). phylogenetic trees were constructed by the neighbor-joining method and bootstrap analysis to determine the best-fitting tree for the gene using mega software (version ). the statistical significance of the tree topology was tested by bootstrapping ( , replicas). pairwise distances between and within the genotypes at the nucleotide level were calculated with kimura parameters and with poisson correction at the amino acid level with mega software. genbank accession numbers are listed in table s . information on the crfs was entered into a database created in microsoft office access . the chi square and fisher exact tests were used to compare means and associations using spss software version . (spss inc.; chicago, il) and r version . . (r development core team; vienna, austria). projections of global mortality and burden of disease from to acute respiratory infections are the leading cause of death in children in developing countries the global burden of disease occurrence of respiratory virus: time, place and person viral respiratory infections in the community: epidemiology, agents, and interventions the tecumseh study of respiratory illness epidemiology of viral respiratory infections community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens viral pneumonia influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states cdc ( ) prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices influenza-like illness sentinel surveillance in peru acute respiratory infection and influenza-like illness viral etiologies in brazilian adults sentinel surveillance of influenza-like illness in two cities of the tropical country of ecuador influenza and other respiratory viruses in three central american countries boletin epidemiologico semanal semana epidemiologica available boletin epidemiologico semanal semana epidemiologica boletin epidemiologico semanal semana epidemiologica boletin epidemiológico semanal semana epidemiologica boletin epidemiologico semanal semana epidemiologica etilogía viral de las infecciones respiratorias agudas genetic analysis of influenza a/h n of swine origin virus (soiv) circulating in central and south america outbreak of swine origin influenza a (h n ) virus infection -mexico influenza surveillance in indonesia surveillance of respiratory viral infections among pediatric outpatients in northern taiwan a comparison of the epidemiology and clinical presentation of seasonal influenza a and pandemic influenza a(h n ) in guatemala pandemic influenza in a southern hemisphere setting: the experience in peru from pandemic (h n ) cases epidemiology of human infection with the novel virus influenza a(h n ) in the hospital das clinicas, sao paolo, brazil viral pathogens associated with acute respiratory infections in central vietnamese children respiratory viruses in adults with community-acquired pneumonia role of cell culture for virus detection in the age of technology frequency of human bocavirus (hbov) infection among children with febrile respiratory symptoms in argentina we would like to express our gratitude to all personnel working at the two sentinel health centers (hcm and ivss-hjmct) in venezuela for supporting this surveillance study. we thank mr. eduardo guerra, the informatics superior technician of lardidev/biomed-uc, for his irreplaceable technical work in venezuela. we also thank the professional staff of the virology department of namru- for invaluable laboratory and technical support in the execution of the study.disclaimer: the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the department of the navy, department of defense, nor the u.s. government. the corresponding author had full access to all data in the study and final responsibility for the decision to submit this publication.additional key: cord- -f m wuyj authors: hoogeveen, martijn j.; van gorp, eric c.m.; hoogeveen, ellen k. title: can pollen explain the seasonality of flu-like illnesses in the netherlands? date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: f m wuyj current models for flu-like epidemics insufficiently explain multi-cycle seasonality. meteorological factors alone, including the associated behavior, do not predict seasonality, given substantial climate differences between countries that are subject to flu-like epidemics or covid- . pollen is documented to be allergenic, it plays a role in immuno-activation and defense against respiratory viruses, and seems to create a bio-aerosol that lowers the reproduction number of flu-like viruses. therefore, we hypothesize that pollen may explain the seasonality of flu-like epidemics, including covid- , in combination with meteorological variables. we have tested the pollen-flu seasonality theory for - flu-like seasons, including covid- , in the netherlands, with its . million inhabitants. we combined changes in flu-like incidence per k/dutch residents (code: ili) with pollen concentrations and meteorological data. finally, a predictive model was tested using pollen and meteorological threshold values, inversely correlated to flu-like incidence. we found a highly significant inverse correlation of r( )= - . (p < . ) between pollen and changes in flu-like incidence, corrected for the incubation period. the correlation was stronger after taking into account the incubation time. we found that our predictive model has the highest inverse correlation with changes in flu-like incidence of r( ) = - . (p < . ) when average thresholds of total pollen grains/m , allergenic pollen grains/m , and a solar radiation of j/cm are passed. the passing of at least the pollen thresholds, preludes the beginning and end of flu-like seasons. solar radiation is a co-inhibitor of flu-like incidence, while temperature makes no difference. however, higher relative humidity increases with flu-like incidence. we conclude that pollen is a predictor of the inverse seasonality of flu-like epidemics, including covid- , and that solar radiation is a co-inhibitor, in the netherlands. makes pollen less airborne, and cools the bio-aerosol down. very high humidity levels (rh %) are even detrimental to pollen (guarnieri, ) . an rh % effect on pollen could thus provide an alternative explanation as to why flu-like incidence in tropical countries is higher during the rainy season, and reduced during the rest of the year. we hypothesize that pollen bio-aerosol has an inverse effect on flu-like incidence, including covid- (see figure ), whereby pollen is known to be triggered and influenced by meteorological variables, which can then jointly explain the seasonality of flu-like incidence. this indirect explanation of the pollen effect is based on the fact that pollen bio-aerosol and uv light exposure lead to immuno-activation, and sometimes allergic symptoms, which seem to protect against flu-like viruses, or at least severe outcomes from them. the indirect pollen effect is explained by the spread of pollen bio-aerosol under sunny and dry conditions. further, it is unknown how viral bio-aerosol and pollen bio-aerosol interact with each other in the air, and whether anti-viral phytochemicals in pollen could then play a role in an alternative explanation. to further understand the impact of pollen as an environmental factor influencing the life cycle of flulike epidemics, the objective of this study is to determine the correlations of pollen and meteorological variables with (changes in) flu-like incidence and develop and test a discrete predictive model that combines pollen and meteorological co-inhibitors. our main hypothesis, therefore, is that pollen is the missing link, jointly explaining with certain meteorological variables, flu-like seasonality, and that a compound threshold based factorcombining detected flu-inhibitorsis a good unified predictor of such seasonality. regarding covid- , we have limited ourselves to observing whether or not covid- at the tail-end of the / flu-like season is able to break with the flu-like seasonality pattern. to study the relationship between pollen and flu-like incidence in the netherlands, we used the public datasets of elkerliek hospital (elkerliek.nl) about the weekly allergenic, low-level allergenic and total pollen concentrations in the netherlands in grains/m , whereby for types of pollen particles the j o u r n a l p r e -p r o o f journal pre-proof numbers are counted and averaged per day per m of air. the common burkard spore trap was used, through which a controlled amount of air was ingested. the applied classification and analysis method conforms to the eaaci (european academy of allergology and clinical immunology) and the ean (european allergy network) standards. allergenic pollen includes nine types of particles that are classified as moderate (corylus, alnus, rumex, plantago and cedrus libani) , strong (betula and artemisia), or very strong allergenic (poaceae and ambrosia). additionally, we included low-level allergenic pollen concentrations in addition to the allergenic ones because we assume that they may also have effects. low-level allergenic pollen includes the other particles that are classified as nonallergenic to low-level allergenic (cupressaceae, ulmus, populus, fraxinus, salix, carpinus, hippophae, fagus, quercus, aesculus, juglans, acer, platanus, pinus, ilex, sambucus, tilia, ligustrum, juncaceae, cyperaceae, ericaceae, rosaceae, asteraceae, ranunculaceae, apiaceae, brassicaceae, urtica, chenopodiaceae, fabaceae, humulus, filipendula, and indet) . total pollen concentration is the sum of the average allergenic and low-level allergenic pollen concentrations. advantages of using the total pollen metric are that there are hardly any values (only out of ), and we did not need to limit ourselves to just parts of the seasonal cycle, which might introduce subseasonal bias into our research. we also assumed that long-distance pollen transport is accounted for, as foreign pollen will also be counted by a pollen measuring station that works all year round. furthermore, we used the data from the dutch state institute for public health (rivm.nl) gathered by nivel (nivel. nl) about weekly flu-like incidence (who code "ili" -influenza like illnesses) reports at primary medical care level, per , citizens in the netherlands. primary medical care is the using the number of influenza-like reports per primary care unit divided by the number of patients registered at that unit. this is then averaged for all primary care units and then extrapolated to the complete population. the datasets run from week of up to week of (n = data points) to include the recent covid- pandemic at the tail-end of the / flu-like season. to underpin the relative importance of covid- : sars-cov- has been detected in the netherlands since week , . according to the figures of nivel.nl ( , see figure ), from week onward sars-cov- is the outcome of the (vast) majority of positive tests for patients at primary care level with flu-like complaints, and by week % of positive tests indicate sars-cov- (other tested viruses are five influenza a and b subtypes, rsv, rhinovirus and enterovirus). furthermore, we also included meteorological datasets from the royal dutch meteorological institute (knmi.nl), including average relative humidity/day, average temperature/day and global solar radiation in j/cm per day as an indicator of uv radiation. these datasets were obtained from the knmi's centrally located de bilt weather station. next, we calculated the weekly averages for the same periods that featured in the other datasets. de bilt is traditionally chosen as it provides an approximation of modal meteorological parameters in the netherlands, which is a small country. furthermore, all major population centers in the netherlands, which account for around % of the total dutch population, are within a radius of only kilometers from de bilt. we therefore assumed in this study that the measurements from de bilt are sufficiently representative for the meteorological conditions typically experienced by the dutch population. to test allergenic versus low-level allergenic pollen assumptions, against hay fever and pre-covid-prevalence of allergic rhinitis that is more or less similar to that in western europe, being around %, and frequently undiagnosed (bauchau & durham, ) . furthermore, it can be noted that the prevalence of allergic diseases in general in the netherlands is around % (van de ven et al, ) . datasets were complete, except that three weekly pollen concentration measurements were missing ( . %). this was due to a malfunctioning monitoring station during week of , week of and week of . these missing measurements appeared to be completely random. we imputed missing values to avoid bias and maintain power. we used a four-week surrounding average to estimate the three missing data points and thus avoid breaking lines in visuals. we checked that the missing data has no material impact on the results by comparing these averages with the data of previous years for similar periods, and by observing whether removal from statistical tests had any effect on outcomes and conclusions. regarding the incidence of flu-like symptoms, we calculated the weekly change compared to the previous week (Δili=ili t -ili t- ). this was to obtain an indication of the flu-like epidemic life cycle progression, whereby a decline is interpreted as ro< and an increase as ro> (ro is the reproduction number of flu-like viruses). furthermore, to cater, in one time-series metric, for changes in flu-like incidence as well as for an incubation period of up to two weeks, we calculated a three-week moving average ( wma) of changes in flu-like incidence, of which two weeks are forward looking: (Δili wma = (Δili t + Δili t+ + Δili t+ )/ ) thus, Δili wma has on average a one week lag. a general advantage of a moving average is that it reduces statistical noise. it should be noted that whenever we use the term incubation time, we also mean to include reporting delay (estimated to be around . days). we have not assumed delay effects for meteorological variables or pollen concentrations, so we have not calculated moving averages for other time series. compared with our previous study (hoogeveen, ) , there is an overlap in datasets of less than %. the datasets are extended by the extension in time, the addition of meteorological datasets and non-allergenic pollen, and the introduction of newly calculated variables, such as total pollen j o u r n a l p r e -p r o o f journal pre-proof concentration, Δili wma , the compound predictor and the log transformations on pollen, ili and the hay fever index. we formulated the following statistical null hypotheses for falsification. h : there are no inverse correlations for total pollen concentrations with flu-like incidence (corrected for incubation period). h : there are no inverse correlations between pollen and changes in flu-like incidence (Δili or corrected for incubation time: Δili wma ). h : there is no predictive significance of a discrete model's compound value, based on thresholds for pollen and meteorological co-inhibitors, related to changes in flu-like incidence (Δili wma ). to understand the role of meteorological variables, to check whetherin our datasetsmeteorological variables show their well-established effects on pollen as assumed, and to select coinhibitors: h : meteorological variablessolar radiation, temperature and relative humidityhave no effect on pollen and/or flu-like incidence change (Δili wma ). low-level allergenic pollen is sometimes known to have a slight allergenic effect. to understand how to interpret adding none-to-low-level allergenic pollen to the total pollen metric, we wanted to verify their effects on the hay fever index: h : low-level allergenic pollen has no effect on hay fever and (changes in) flu-like incidence. note that with the exception of h , all hypotheses are related to potential causality: the temporal sequentiality (temporality) of the respective independent variables, and flu-like incidence corrected for incubation period. whenever we refer to temporality, we mean to indicate that the datasets behave as if there is causality, on the understanding that statistics alone cannot prove causality in uncontrolled settings. variables are presented with their means (m) and standard deviations (sd). we calculated correlation coefficients to test the hypotheses and to assess the strength and direction of relationships. as a sensitivity analysis, we also calculated the bootstrapped correlation coefficients. we used the full datasets, to avoid sub-seasonal bias, and by extending the number of years the distortions by incidental and uncontrolled events are supposed to be minimized. however, as a second sensitivity analysis, we removed from the datasets the autumn weeks between and , which typically show low pollen concentrations of up to grains/m , which are applied to analyze the main outcome (h ). further, as a third sensitivity analysis we calculate correlations per individual time lag included in Δili wma in relation to h . next, linear regression (f-test) on identified inhibitors and interactions was used descriptively to determine whether the relationship can (statistically) be described as linear, and to determine the equation using estimates and intercept values, and produce probability, significance level, f-value, and the multiple r squared correlation to understand the predictive power of the respective inhibitor. standard deviations and errors, and degrees of freedom (df) were used as input for calculating the % probability interval. we have reported in the text the outcome of statistical tests in apa style, adapted to journal requirements. for relationships that appear non-linearlogarithmic or exponential we have used the log function to transform the data if that makes the relationship appear linear, before re-applying linear regression. we have also used the log transformed datasets for the calculation of correlation coefficients, to correct for skewness. finally, we created a simple, discrete model resulting in one compound value, using selected flu-like inhibitors. this was to determine the optimal average threshold values for these inhibitors, which have between total pollen and flu-like incidence, including the first cycle of the covid- pandemic. furthermore, we can reject h in favor of our assumption that it makes sense to also include low-level allergenic pollen concentrations in our study. low-level allergenic pollen is inversely correlated to flulike incidence (r( ) = - . , p < . ), especially when corrected for the weeks incubation time (r( ) = - . , p < . ). the fact that the correlations become stronger when taking into account incubation time, implies temporality. furthermore, we can also observe from figure that flu-like incidence starts to decline after the first pollen bursts. moreover, flu-like incidence starts to increase sharply after pollen concentrations become very low or close to zero. this is a qualitative indication of temporality. furthermore, we can notice that the first covid- cycle behaved according to pollen-flu seasonality, at least does not break with it. when testing the impact on Δili, the weekly changes in medical flu-like incidence, the extended dataset till , including covid- , shows a strong and highly significant inverse correlation with total pollen (r( ) = - . , p = . ). therefore, we can falsify the null-hypothesis (h ) that there is no inverse correlation between the weekly pollen concentrations and weekly changes in flulike incidence (Δili), including the period covering the first cycle of the covid- pandemic. this inverse correlation therefore provides further support for the alternative hypothesis that the presence of an elevated level of pollen has an inhibiting effect on flu-like incidence, and starts to immediately influence the direction and course of the epidemic life cycle. also, during the covid- dominated period of the last weeks, it appears that flu-like incidence behaves according to the expected pollenflu seasonality. this strengthens the idea that covid- might itself be seasonal, like all other flu-like pandemics since the end of the th century. also when studying other data from rivm.nl about covid- hospitalizations, we cannot conclude that covid- breaks through the seasonal barrier. for example, new covid- hospitalizations decreased from a peak of on march to just on may , the last day of week . using the three-week moving average (Δili wma ) of changes in flu-like incidence, the correlation coefficients become stronger and are again highly significant for total pollen concentration (r( ) = -j o u r n a l p r e -p r o o f journal pre-proof . , p < . ). the bootstrapped correlation coefficient calculation gives a comparable outcome (r( ) = - . , p < . ). as a second sensitivity analysis, we used the reduced dataset (minus the weeks of low pollen activity) and again found similar correlations (r( ) = - . , p < . ; bootstrapped r( ) = - . , p < . , ci % - . to - . )). finally, as a third sensitivity analysis, we analyze each time lag included in the Δili wma calculation separately. per individual time lag there are as well highly significant inverse correlations: as given before r( ) = - . , p = . in case of no time lag (Δili t ); r( ) = - . , p = . in case of a time lag of one week (Δili t+ ); r( ) = - . , p = . in case of a time lag of two weeks (Δili t+ ); and the bootstrapped correlations for these are similar. we can thus also reject the null-hypothesis (h ) that there is no inverse relationship between pollen and changes in flu-like incidence including incubation time (Δili wma or Δili t+ or Δili t+ ). these correlations (see also figure ) are a further indication of temporality and does not contradict the idea that covid- is subject to pollen induced fluseasonality. the fact that the correlation with Δili wma is stronger than those for each of the included time lags might be an indication of the noise reduction effect of this moving average, and makes thus the compound effect of the three covered time lags more visible. linear regression analysis shows that pollen has a highly significant inhibitory effect on flu-like incidence change (Δili wma ) of f( , ) = . , p < . (see table , line ), as a further basis for using total pollen concentration as a predictor. a log transformation of pollen to compensate for visual non-linearity leads to a similar outcome: f( , ) = . , p < . (see table , line ). at least visually, it is a good fit (see figure ). of the meteorological variables, only solar radiation has a highly significant inverse correlation with changes in flu-like incidence (Δili wma ): (r( ) = - . , p = . ). thus, of the meteorological variables, when it comes to solar radiation and relative humidity the nullhypothesis (h ) can also be rejected, as they seem to effect the flu-like epidemic lifecycle. of these two, only solar radiation is a flu-like inhibitor in line with its positive effect on pollen concentration, its association with immune-activation and the effect that uv has on viruses. a univariate linear regression also shows the highly significant negative correlation for solar radiation on flu-like incidence change (Δili wma ) (f( , ) = . , p < . (see table , line ). as the correlation is weak (multiple r-squared = . ), we have interpreted solar radiation as a co-inhibitor in relation to pollen; as a stand-alone independent variable its effect is too weak to explain flu-like seasonality. taking into account all these findings, we developed a discrete, compound model in which we included the changes in flu-like incidence (Δili wma ), a threshold value for solar radiation (k r ), and both pollen threshold values for allergenic (k ap ) and total pollen (k p ). we found that the compound model has the highest inverse correlation (r( ) = - . , p < . ) for the following threshold values: k r : j/cm , k ap : allergenic pollen grains/m , and k p : total pollen grains/m . the bootstrapped correlation coefficient calculation gives a comparable outcome (r( ) = - . , p < . ). in line with the previous outcomes, the inclusion of relative humidity, low-level allergenic pollen or temperature did not improve the correlation strength of this model. furthermore, given that they showed no significant interaction effects with pollen, it was not necessary to take such interactions into consideration in the model. in each of the observed years, the now (re)defined pollen thresholds are passed in week (± weeks), depending on meteorological conditions controlling the pollen calendar and coinciding with reaching flu-like peaks, and again in week (± weeks), marking the start of the new flu-like season. there is a highly significant inverse relationship between our compound threshold-based predictor value with flu-like incidence change (Δili wma ) of f( , ) = . , p < . and a multiple r-j o u r n a l p r e -p r o o f squared correlation of . (see table , line ). this confirms the usefulness of a discrete, pollen and solar radiation threshold-based model as a predictor of switches in flu-like seasonality, whereby the effect of pollen is stronger than that of solar radiation. as a consequence, we can reject the nullhypothesis (h ) that this compound pollen/solar radiation value has no predictive significance for flulike seasonality. first of all we will discuss the possible implications of the results for our theoretic model and alternative explanations. next, we will discuss our methods. we found highly significant inverse relationships between pollen and solar radiation and (changes in) flu-like incidence: a higher pollen concentration or an increase in solar radiation in the netherlands is related to a decline in flu-like incidence. this inverse correlation with pollen becomes stronger when the / period is included, which has been increasingly dominated by covid- during the last weeks. given that more time will be needed to draw conclusions about whether the spread of covid- is seasonal or not, from the data in this study it can only be observed that covid- is not breaking with the flu-like seasonality pattern. alternatively, social distancing may have contributed to flattening both the flu-like epidemic and covid- pandemic curves at the tail-end of the / flu-like season. the dutch government imposed hygiene measures from march , onward and a mild form of a lockdown, that included social distancing, from march . such behavioral policies the highly significant inverse correlation between hay fever and flu-like incidence confirms that allergic rhinitis makes it more difficult for flu-like viruses to propagate. solar radiation, the only meteorological variable that has a co-inhibitive effect on changes in flu-like incidence, has a stimulating effect on aerosol pollen formation and is responsible for melatonininduced immuno-activation. relative humidity reduces pollen aerosol formation, and correlates positively with flu-like incidence. we did not specifically look at precipitation, but it might make sense to explicitly consider this independent variable, given that it reduces pollen dissemination. in our study we showed that temperature, aside from the fact that it influences pollen, has no predictive value for changes in flu-like incidence. therefore, its inverse correlation with flu-like incidence might be interpreted in a number of ways: a) as spurious: the common causal factor is solar radiation, or b) as a stressor that has immediate effects on the functioning of the immune system of already infected persons. when discussing the influence of meteorological variables, we assume that the associated behavioral aspects are covered. these are sometimes summarized as seasonal behavior, but this independent variable might have a cultural dimension that needs to be better understood. we showed that a compound value, based on threshold values for pollen and solar radiation, results in a stronger correlation with the flu-like lifecycle than the individual inhibitors. this model could form an empirical basis for understanding flu-like seasonality, its ro and reliably predicting the start and end of each flu-like cycle. given that behavior, in the form of hygiene and social distancing, is also widely seen as an inhibitor, it might be worthwhile to also include this factor in our compound value. this will probably lead to an even stronger predictor for the evolution of the reproduction number ro of flu-like epidemics, although this might be beyond explaining the seasonality effect itself. for as long as the level of herd immunity (fine et al., ) for covid- is still below required thresholds for ending pandemics (plans-rubio, ) , it might make sense to also include indications of herd immunity levels in the theoretic model. finally, despite air pollution not been seen as an inhibitor of flu-like incidence (coccia, ), it still might interact with pollen. a more complete theoretic model, controlling for the (interactions with) air pollution, could give more insight in how to interpret the findings of this or similar studies. in general, statistical research cannot prove causal relationships in uncontrolled environments, even if datasets seem to behave as if there is causality. such statistics, however, can provide indications and identify reliable predictors, help filter out bad ideas, and be the inspiration for testable hypotheses that can be verified in laboratory and other fully controlled experiments. with a predictor we mean that a reliable temporal relationship between two variables is identified, without yet having validated causality, i.e., a bellwether factor. although the datasets seem to be sufficiently representative, there appears to be room for improvement. for example, including the data of more weather stations might help to improve the approximation of the weather conditions the dutch population experiences on average, and help to distinguish patterns per province. furthermore, it might be useful to include wind speeds, given that these constitute a vector for the dispersal of pollen in the netherlands, which has a maritime and temperate climate. additionally, the effects of climate change on pollen maturation (frei & gassner, ) might also be an important factor. another example of improving the representativeness would be by including more pollen types in the particle counts than are currently covered by the current methodology of the european allergy network. further, reclassification or recalibration of pollen types on a rational scale in terms of allergenicity, let's say - %, would be very useful. for example, if pollen types are identified with a reliable score of %, these could be used to differentiate impact of pollen eleven faces of coronavirus disease herd immunity": a rough guide seasonality in risk of pandemic influenza emergence towards a data-driven characterization of behavioral changes induced by the seasonal flu ripe pollen carbohydrate changes in trachycarpus fortunei: the effect of relative humidity climate change and its impact on birch pollen quantities and the start of the pollen season an example from switzerland for the period - pollen exposure weakens innate defense against respiratory viruses nivel zorgregistraties eerste lijn -surveillance -wekelijks bulletin over symptomen en aandoeningen op basis van gegevens van huisartsen why lungs keep time: circadian rhythms and lung immunity the vaccination coverage required to establish herd immunity against influenza viruses dynamical prediction of flu seasonality driven by ambient temperature: influenza vs. common cold current understanding of the pathophysiology of allergic rhinitis covid- transmission in mainland china is associated with temperature and humidity: a time-series analysis humidity and latitude analysis to predict potential spread and seasonality for covid absolute humidity and pandemic versus epidemic influenza the role of temperature and humidity on seasonal influenza in tropical areas: guatemala, el salvador and panama global influenza seasonality: reconciling patterns across temperate and tropical regions potential utility of melatonin in deadly infectious diseases related to the overreaction of innate immune response and destructive inflammation: focus on covid- & asfiang p. correlation between weather and covid- pandemic in jakarta atopic diseases and related risk factors among dutch adolescents receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus association between ambient temperature and covid- infection in cities from china no association of covid- transmission with temperature or uv radiation in chinese cities clinical characteristics of patients infected with sars-cov- in wuhan key: cord- -y bdjnhg authors: dai, yaoyao; wang, jianming title: identifying the outbreak signal of covid- before the response of the traditional disease monitoring system date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: y bdjnhg new coronavirus cases and related deaths are continuing to occur worldwide. early identification of the emergence of novel outbreaks of infectious diseases is critical to the generation of timely responses. we performed a comparative study to determine the feasibility of the early detection of the covid- outbreak in china based on influenza surveillance data and the internet-based baidu search index to evaluate the timelines of the alert signals compared with the traditional case reporting and response systems. an abnormal increase in the number of influenza-like illnesses (ili) occurred at least one month earlier than the clinical reports of pneumonia with unknown causes and the conventional monitoring system. the peak of the search volume was days earlier than the issuance of the massive official warning about the epidemic. the findings from this study suggest that monitoring abnormal surges of ili and identifying peaks of online searches of key terms can provide early signals of novel disease outbreaks. we emphasize the importance of broadening the potential of syndromic surveillance, internet searches, and social media data together with the traditional disease surveillance system to enhance early detection and understanding of emerging infectious diseases. synopsis: early identification of the emergence of an outbreak of a novel infectious disease is critical to generating a timely response. the traditional monitoring system is adequate for detecting the outbreak of common diseases; however, it is insufficient for the discovery of novel infectious diseases. in this study, we used covid- as an example to compare the delay time of different tools for identifying disease outbreaks. the results showed that both the abnormal spike in influenza-like illnesses and the peak of online searches of key terms could provide early signals. we emphasize the importance of testing these findings and discussing the broader potential to use syndromic surveillance, internet searches, and social media data together with traditional disease surveillance systems for early detection and understanding of novel emerging infectious diseases. introduction new coronavirus cases and related deaths are continuing occur worldwide. [ ] the who, on march , , declared the coronavirus disease (covid- ) outbreak a global pandemic. this pandemic dates back to december , when a cluster of unexplained pneumonia cases was identified, which were linked to a seafood market in wuhan, china. [ ] subsequent investigations determined that a novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), was the causative agent now at the heart of the pandemic of an emerging infectious disease (eid). the virus jumped from the transportation hub to other areas during the peak seasonal travel periods of the winter holiday and the traditional spring festival. [ ] to control the spread and mitigate the risk of the virus, a series of strong, unprecedented measures were taken by the chinese government. these measures included the mandatory wearing of face masks in public, canceling of mass events, closing of scenic attractions, suspending of long-distance buses, and asking hundreds of millions of chinese citizens to stay indoors to break the transmission chain. [ , ] despite the rapid increase in the number of covid- cases in january, china has now passed the peak of the epidemic and has effectively controlled the disease. [ ] no new infections of the novel coronavirus were reported on march in wuhan, the epicenter of the epidemic in china, marking a notable first success in the months-long battle with the virus and showing hope of suppressing the pandemic. because this is an infectious disease caused by a new virus, it took approximately one month from the initial detection of unexplained pneumonia cases to the definite conclusion of "human-to-human transmission" and the inclusion of the disease in the management of statutory infectious diseases by the national health commission, china. the traditional disease monitoring system is useful for detecting the outbreak of common infectious diseases, but it is insufficient for the discovery of new diseases. [ ] how to build a comprehensive early warning system of public health emergencies from multiple sources has become the focus of attention of all countries. to compensate for the shortcomings of the traditional disease monitoring system, some scholars have tried to use digital data streams, [ ] network density, [ ] and google trends (gt) [ ] as early warning indicators; these attempts have achieved remarkable results; nevertheless, the roles of these indicators in covid- remain unclear. in this study, we performed a comparative study to discuss the early warning capability, timelines, and validity of alert signals for the first wave of the covid- outbreak in china based on the surveillance data of influenza-like illness (ili) and the baidu search index (bsi) compared with the traditional case reporting system. covid- data from china were obtained from the center for disease control and prevention of china and national health commission of china as well as the report of the who-china joint mission on coronavirus disease (https://www.who.int) and vital surveillances report on the epidemiological characteristics of an outbreak of covid- -china, . [ ] the data source of influenza-like illnesses we extracted data regarding ili reported from january to may from the national health commission of china. after the sars epidemic, the chinese government built the world's most extensive internet-based disease reporting system, called the china information system for disease control and prevention (cisdcp). [ ] cases of infectious diseases, categorized as class a, b, and c, are required to be reported through the cisdcp within a limited time. we compared the monthly morbidity of ili during the last five years and plotted a line chart to describe the long-term trend. we also compared the peak of ili with the onset of the covid- in the late in china. we used the baidu search engine (http://index.baidu.com/v /#/) to analyze the bsi for searches of the keywords of "pneumonia" and "sars" from november , , to february , . baidu is the world's largest chinese search engine and china's largest internet integrated service company. the bsi reflects active searches by internet users. we compared the timeline of peak searches for these key terms with the time of official response to the epidemic. data were entered into excel and analyzed using spss (ibm, ny, usa). the ili cases across several years were compared using the analysis of variance. the dunnet method was used for pairwise comparison. the test level for significance was set at . . data of this study were extracted from a public database. no individual information was published in this paper. therefore, this study is exempt from ethical approval. on december , , the department of health of hubei province and wuhan city received a report from a local hospital regarding patients with unexplained pneumonia, all of whom were employees of the south china seafood wholesale market. on december , the national health commission and cdc sent a team of experts to wuhan. the investigators excluded several suspected causes, including influenza, avian influenza, adenovirus, severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov). on january , , the local government closed this seafood market and disinfected the area. on january , , the chinese government informed the who of the outbreak of unexplained pneumonia. on january , , the pathogen was identified as a new type of coronavirus, and then, the full genome sequences of this new virus were shared. on january , an expert group and a who team were invited to visit wuhan for a field investigation. by january , novel coronavirus cases have been reported in wuhan. as of january , the risk of human-to-human transmission of this new virus had not been determined, and officials have not realized the potential global epidemic risk. on january , the novel coronavirus pneumonia was incorporated as a notifiable disease under the infectious disease law and health and quarantine law in china. on january , the whole city of wuhan was locked down, and all the residents were required to stay at home. two days later, the chinese government made the highest-level commitment to mobilize all forces to stop the epidemic. [ ] as of january , , there were more than confirmed cases and more than suspected cases of covid- across chinese provinces or municipalities. [ ] human-to-human transmission of the pathogen was also confirmed. [ ] huang et al. analyzed laboratory-confirmed covid- cases in wuhan and showed that the symptom onset date of the first patient was december , . [ ] it is estimated that the origin of covid- was most likely earlier than december . as shown in fig a, it took more than one and a half months for the traditional surveillance system to trigger the alert of the outbreak of this eid. as shown in fig b, there was a search peak for the terms of "pneumonia" ( times) and "sars" ( times) on december , , mainly in wuhan (pneumonia: times; sars: times), where the outbreak of covid- occurred. with the official announcement of the exclusion of sars and the absence of apparent human to human transmission, the number of searches decreased rapidly the following day. until around january , the bsi of these two terms began to rise again, resulting in a second search peak, which was consistent with the increase in confirmed covid- cases countrywide (fig b) . overall, there were differences in the number of ili in - (f = . , p< . ). as shown in fig a, the ili case numbers in were significantly higher than those reported in the previous years of - (p< . ). we observed an early spike in ili in winter of , with a fast-growing period from november to december (fig b) . this observation suggests that covid- cases may have occurred before december . the signal of the abnormally rapid increase in ili cases was earlier than the report of clinical cases of pneumonia with unknown causes through the official routine disease monitoring system. early identification of the emergence of an outbreak of a novel infectious disease is critical to generating a timely response. the traditional monitoring system is adequate for detecting the outbreak of common diseases; however, it is insufficient for the discovery of novel eids. in this study, we used covid- as an example to compare the delay time of different tools for identifying disease outbreaks. the results showed that both the abnormal spike in ili and the peak of online searches of key terms could provide early signals of novel eids. for centuries, infectious diseases have been among the leading causes of death and have presented growing challenges to human health. the threat is further increased by the continued emergence of new and unrecognized infectious disease epidemics. [ ] due to the lack of sensitive and specific diagnostic tools, infections are often undiagnosed and therefore untreated, or are diagnosed at late stages. early detection of infectious diseases plays a crucial role in all treatment and prevention strategies. a crucial goal of infectious disease surveillance is the early detection of epidemics, which is essential for disease control. in china, the current surveillance system is based on confirmed case reports. [ ] it is not practical for health units to perform laboratory tests to confirm a novel infectious disease. most infectious disease outbreaks start with clinicians noticing unusual patterns. patients may present with patterns of symptoms that are similar to those of more common diseases but which, after repeated observation and diagnostic testing, may deviate in scale, seasonality, or severity. [ ] the discovery of covid- is an example. in december , clinicians from wuhan city reported several patients with unexplained pneumonia, all of whom were employees of south china seafood wholesale market. bronchoalveolar lavage samples were collected and sequenced for the whole genome. bioinformatic analyses indicated that the pathogen was a novel coronavirus, showing the closest relationship with the bat sarslike coronavirus strain batcov ratg . on january , , the novel coronavirus was confirmed as the cause of unexplained pneumonia. however, at that time, people did not realize the potential risk of an epidemic (even less a pandemic) caused by this new pathogen. it was not until mid-to late-january that the risk of widespread transmission was taken seriously. in other words, clinical symptom monitoring and case reporting can help identify new diseases; however, these practices do not provide timely signals of an epidemic. internet-derived information has recently been recognized as a valuable tool for epidemiological investigation. [ ] timeliness and precision in the detection of infectious disease outbreaks from the information published on the web are crucial for prevention of their spread. arsevska et al. retrieved data from a corpus of relevant documents and compared them with african swine fever (asf) outbreaks from the google search engine and the pubmed database. [ ] the results showed that relevant documents could serve as a source of terms to detect infectious animal disease emergence on the web. walker et al. used google trends (gt) to investigate whether there was a surge in searches for information related to the covid- epidemic. these authors observed a strong correlation between the frequency of searches for smellrelated information and the onset of covid- infection in italy, spain, uk, usa, germany, france, iran and netherlands. [ ] li et al. demonstrated that the data obtained from gt, bsi and the sina weibo index on searches for the keywords 'coronavirus and 'pneumonia' correlated with the published daily incidence of covid- , with the maximum r > . . [ ] however, few studies explored the role of web-based search index in detecting the first occurrence of the covid- . in this study, we used the bsi to explore the correlation between the internet search index and the outbreak of covid- . the bsi is a public sampling database of search queries users entered into the predominant search engine (baidu) in china. unlike gt, the bsi reflects the absolute baidu search volume and is not displayed as normalized values. [ ] one important issue that emerges from web-based searches is that they tend to underestimate the real epidemiological burden when the general population has poor knowledge of the disease. [ ] additionally, the bsi can be influenced by media clamor. therefore, the real scientific usefulness of the so-called "digital epidemiology" remains questionable, at least when using gt or bsi. although the source of information cannot be taken for granted or even replace the "real life" epidemiological data, mining the web is an intriguing perspective for eids. when and where sars-cov- originated remains unclear. the similarity between covid- and influenza symptoms makes it possible that the excess ili cases were due to covid- cases. the presence of sars-cov- -positive swabs in the patients supports this possibility. [ ] the predominant symptoms associated with covid- are fever, cough, and sore throat; that is, patients often present with an ili. at the early stage of the epidemic, covid- cases may have been misdiagnosed as influenza or other respiratory diseases. thus, we hypothesized that ili surveillance data could be used as a tool for early detection of covid- . kong et al. analyzed throat swabs collected from patients with ili in wuhan from october , , to january , , and found that nine samples were positive for sars-cov- , suggesting community transmission of sars-cov- in wuhan in early january . [ ] the dramatic increase in ili in wuhan in early december further supported this hypothesis. [ ] spellberg et al. observed a seasonal spike in ili in los angeles, usa. [ ] among patients with mild ili, % were tested positive for sars-cov- . such transmission is consistent with the countywide unusual third ili spike that occurred late in the season and with declining rates of influenza positivity. [ ] however, seasonal influenza activity was lower in than in previous years in japan. [ ] it may have been affected by temperature or virulence and by measures taken to constrain the sars-cov- outbreak. [ ] the coinfection of covid- and influenza a reported in iran also highlighted the importance of considering sars-cov- pcr assay regardless of positive findings for other pathogens during the epidemic. [ ] silverman et al. explored how ili outpatient surveillance data could be used to estimate the prevalence of covid- , and they found a surge in noninfluenza ili above the seasonal average in march and showed that this surge correlated with covid- across states. [ ] in our study, several potential limitations should not be neglected. first, the web-based search for key terms or ili surge counts in relation to the emergence of covid- may be attributed to potential confounders. second, the observed ili surge may represent more than just sars-cov- -infected patients. whether ili surveillance data could be used for the signal of the eids without dominant features of covid- , such as cough and fever, is unclear. third, the web-based search can be affected by media coverage, the population's knowledge or the degree of information disclosure. in conclusion, monitoring abnormal surges in ili and identifying online search peaks of key terms can provide early signals of novel disease outbreaks. we emphasize the importance of testing these findings and discussing the broader potential to use syndromic surveillance, internet searches, and social media data together with traditional disease surveillance systems for early detection and understanding of 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data, china, . euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin correlations of online search engine trends with coronavirus disease (covid- ) incidence: infodemiology study pubmed central euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin sars-cov- detection in patients with influenza-like illness community prevalence of sars-cov- among patients with influenzalike illnesses presenting to a los angeles medical center pubmed central seasonal influenza activity during the sars-cov- outbreak in japan co-infection of coronavirus disease and influenza a: a report from iran using influenza surveillance networks to estimate state-specific prevalence of sars-cov- in the united states key: cord- -ssjl w l authors: chamberlain, samuel d; singh, inder; ariza, carlos a; daitch, amy l; philips, patrick b; dalziel, benjamin d title: real-time detection of covid- epicenters within the united states using a network of smart thermometers date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ssjl w l containing outbreaks of infectious disease requires rapid identification of transmission hotspots, as the covid- pandemic demonstrates. focusing limited public health resources on transmission hotspots can contain spread, thus reducing morbidity and mortality, but rapid data on community-level disease dynamics is often unavailable. here, we demonstrate an approach to identify anomalously elevated levels of influenza-like illness (ili) in real-time, at the scale of us counties. leveraging data from a geospatial network of thermometers encompassing more than one million users across the us, we identify anomalies by generating accurate, county-specific forecasts of seasonal ili from a point prior to a potential outbreak and comparing real-time data to these expectations. anomalies are strongly correlated with covid- case counts and may provide an early-warning system to locate outbreak epicenters. epidemic dynamics of the coronavirus disease (covid- ) have varied widely across the globe, since the outbreak emerged from wuhan, china in december ( ) . countries with proactive management and widespread testing have more effectively limited spread (i.e. 'flattening the curve'), whereas countries with limited response, such as the united states, have experienced steeper growth rates in cases ( ) . syndromic monitoring systems-collecting information about symptoms often prior to, or instead of, interaction with the formal healthcare system-have played a pivotal role in successfully mitigating covid- spread in countries such as taiwan and south korea ( ) . rapid syndromic detection also played a role in curtailing the [ ] [ ] [ ] ebola outbreak in west africa ( ) , where speed of detection was important in reducing outbreak intensity ( ) . networks of geolocated, user-generated physiological measurements hold the potential for improved tracking and prediction of outbreak epicenters ( , ) . data from these networks are typically less specific, but more sensitive, than formal surveillance, and are often available more rapidly, because formal surveillance is constrained by testing speed ( ) and/or time for record aggregation ( ). data from syndromic monitoring networks can be physiologically grounded in disease processes (e.g. fever), in contrast with internet-based proxies, such as search engine logs, that are often used as a substitute for real-time syndromic data ( ) . physiological data from connected devices, such as thermometer data, or elevated heart-rate data ( ) , are thus a potentially valuable resource for outbreak response, and, at present, may be able to detect covid- symptoms in the absence of widespread and rapid testing in the us. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint here, we outline a method to identify illness incidence anomalies using a geospatial network of smart thermometers, where county-scale anomalies are flagged in real-time. we then use the method to identify covid- outbreaks in the continental united states. our anomaly detection method follows three core steps: ) generate county-specific forecasts of influenza-like illness (ili) from a time point prior to a potential outbreak, ) compare real-time thermometer-derived ili to forecast expectations when new data is aggregated daily, and ) flag anomalous ili values by evaluating the probability that the current signal is driven by regular seasonal influenza. in this case, we flag ili values that exceed the . % percentile of expected influenza trajectories. we track real-time ili and create ili forecasts using data collected from a network of smartphone-connected personal thermometers managed by kinsa, inc. we track ili in the network by classifying users with elevated temperature readings over multiple days as having influenza-like illness (see methods). this sensor network records the temperature and approximate geo-location when a user takes their temperature (e.g. during an illness episode). these recordings store temperature readings, via kinsa's smartphone app, and locations are logged via gps location or ip address. readings are aggregated to county-scale ili and anonymized. the temperature readings are used to construct an ili signal that is highly correlated to center for disease control and prevention (cdc) ili nationally (r > . ) and across cdc regions (r range . - . ), and these signals have been demonstrated to improve regional ili surveillance and forecasting ( , ) . we construct the ili signal at the county-scale, allowing identification of anomalous ili incidence at the scale of individual cities. more information on constructing illness incidence from the thermometer network can be found in the supplemental methods. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint our forecast builds upon the findings that individual cities have predictable, distinct epidemic intensity patterns governed by both population size and climate ( , ) . as population size and climate change relatively slowly from year-to-year, this allows the use of the county-specific, daily network data to learn the typical seasonal flu transmission patterns of a region. from this we construct local ili forecasts that form the basis for real-time anomaly detection. following ( ), we estimate a county's daily reproductive number (r t ), the average number of secondary cases that each infected individual would infect if the conditions remained as they were, at time t by solving the following equation: where i t is kinsa-derived ili incidence, and w k is the infectivity profile for influenza, representing the probability that an individual who becomes infected on day t acquired the infection from an individual who became infected on day t-k. we approximate w k by a gamma distribution with a mean of . and variance of . days ( ) . the summation term in equation ( ) . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint we compare the real-time ili to these influenza trajectories to identify localized anomalies, daily. we flag anomalous ili when the daily, real-time data exceeds the . % percentile of the expected influenza forecast ensemble. we define anomaly incidence as the difference between the real-time thermometer ili and the . % percentile drawn from the influenza forecast ensemble. to validate this method, we apply our approach to detect covid- anomalies by forecasting expected influenza starting on march st , , before widespread outbreaks were underway ( ). we assess detection quality by comparing anomaly fever counts to state-and county-level covid- confirmed cases aggregated from a variety of government sources ( ) . anomaly fever counts are calculated from cumulative anomaly incidence multiplied by the estimated user base of a region. the skill of the ili forecast in the anomaly detection system compares favorably with the carnegie mellon (cmu) epicast and stat models ( ) at cdc region and national scales (fig. a ). to calculate these forecast error rates, we compare cmu forecasts to cdc ili, while comparing anomaly detection forecasts to thermometer-based ili, given that these models are trained on different data sources. in contrast to other ili forecasts, our models do not need to be initialized with now-cast predictions, (i.e. inferred initial conditions) as thermometer-based ili, upon which the model is trained, is available in real-time. additionally, our forecast errors do not appear to increase much past the -week forecast horizon, suggesting the ili forecasts in the anomaly detection system are stable at long-time horizons (fig. a) . forecast error rates display seasonality, where forecasting errors are higher in winter months during periods of increased illness incidence, and lower in the spring and summer (fig. s ). . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint our detected anomalies correlate strongly to positive covid- cases at both county (fig. b) and state scales (fig. c) , validating this method for the rapid detection of covid-related illness anomalies. specifically, we find significant correlations between total confirmed covid- cases and total fever anomalies at the county (fig. b: r = . , p < . ) and state scales ( fig. c : r = . , p < . ). we restrict this analysis to counties with at least one confirmed covid- case, given that a lack of confirmed cases could be due to either true covid- patterns or an absence of testing and reporting. we do not observe anomalous fevers in three states where covid- cases are confirmed; minnesota, wisconsin, and south dakota. countylevel total anomalous fevers also correspond to the spatial distribution of major known outbreak centers, with hotspots in the seattle area, san francisco bay area, new york metro area, and florida (fig. d ). we provide an example for how this method is applied in real-time for brooklyn, ny, usa (fig. e ). here, we forecast expected influenza trends from march st , before large-scale outbreaks were occurring in the new york metro area ( ), and we observe a strong divergence from expected influenza trajectories (fig. e) . however, we also observe an inflection back toward declining ili shortly after social distancing efforts were enacted (fig. e) , beginning with school closures on march th and a later 'stay-at-home' order on march th , . we observe even sharper declines in ili post-social distancing in santa clara, ca, where a 'shelterin-place' order was enacted on march th , (fig. f) . . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint an . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint our estimates of anomaly incidence are likely impacted by social distancing in these cases, as distancing should also reduce influenza transmission. we are thus likely underestimating anomaly incidence after social distancing is enacted, given that our model assumes typical seasonal influenza transmission patterns. we therefore explore the sensitivity of our method by reducing r t values used in the influenza forecast for all days after social distancing efforts were implemented. in brooklyn ny ( fig. a) , dropping future r t values by % reduces the trajectory of seasonal influenza, leading to an increased magnitude of anomaly incidence ( fig. a ). in santa clara county, without accounting for social distancing we observe only a short period of detected anomalies, where real-time ili falls below the expected range of seasonal influenza (fig. f) . here, reducing r t following the 'shelter-in-place' order leads to additional anomaly detections post-social distancing (fig. b) . similarly, following school closures in miami-dade county, real-time ili values fell below anomalous levels by late march (fig. s ) , though accounting for reduced influenza transmission leads to more anomaly detections (fig. c ). in these case examples, we assumed r reduced by % given social distancing directives. while the true value cannot be estimated at present, it likely varies with both policy implementation and adherence. these findings suggest social distancing plays an important role in the quality of anomaly detection, and future research should address the impact of social distancing on influenza transmission rates. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint of . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint working in synergy with the formal healthcare system, high-throughput signals from distributed syndromic monitoring networks, such as we describe here, could play a decisive role in managing local outbreaks, and alter the trajectories of pandemics. these networks may be of particular use for situations where rapid testing and isolation are key to pandemic control ( , ) , such as the current covid- outbreak, where the absence of widespread and rapid testing have led to a massive growth in cases worldwide. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint covid- ) -situation report - response to covid- in taiwan: big data analytics, new technology, and proactive testing ebola surveillance -guinea the role of rapid diagnositics in managing ebola epidemics a smartphone-driven thermometer application for real-time population-and individual-level influenza surveillance improving state-level influenza surveillance by incorporating real-time smartphone-connected thermometer readings across different geographic domains accurate estimation of influenza epidemics using google search data and argo harnessing wearable device data to improve state-level real-time surveillance of influenza-like illness in the usa: a population-based study. the lancet digital health urbanization and humidity shape the intensity of influenza epidemics in us cities human mobility patterns predict divergent epidemic dynamics among cities a new framework and software to estimate time-varying reproductive numbers during epidemics time lines of infection and disease in human influenza: a review of volunteer challenge studies author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint the anomaly detection and forecasting code can be accessed on github, and all examples here are reproducible (https://github.com/kinsahealth/therm_anomaly_detection). we thank c. jessica metcalf for her input on an initial draft of this manuscript. author contributions sdc conceived and implemented the anomaly detection approach with feedback from bdd and pp. is conceived of and designed the kinsa products, including its use as syndromic early warning and monitoring systems. pp, ca, ad, and sdc designed and managed the real-time illness signal and geospatial platform. sdc wrote the manuscript with feedback from all authors.competing interests sdc, is, pp, ad and ca are employees of and shareholders in kinsa, inc.is conceived of and designed kinsa products to track the spread of infectious disease. bdd has no competing financial interests. all authors have completed the icmje uniform disclosure form at www.icmje.org/coi_disclosure.pdf . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity.is the (which was not peer-reviewed) the copyright holder for this preprint this work is not a clinical trial and is instead a population-level observational study, where all user information is anonymized. more specifically, this study uses aggregated temperature readings to estimate the prevalence of influenza-like illness at the scale of united states counties.raw measures are temperature, time of reading, and approximate geolocation from smart thermometer readings, where user information is anonymized and aggregated to whole population influenza-like illness at the scale of us counties. it therefore does not use individual, private or personally identifiable information. given the nature of this data, it does not require irb review.. cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity.is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- - oac bg authors: silverman, justin d; hupert, nathaniel; washburne, alex d title: using ili surveillance to estimate state-specific case detection rates and forecast sars-cov- spread in the united states date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: oac bg detection of sars-cov- infections to date has relied on rt-pcr testing. however, a failure to identify early cases imported to a country, bottlenecks in rt-pcr testing, and the existence of infections which are asymptomatic, sub-clinical, or with an alternative presentation than the standard cough and fever have resulted in an under-counting of the true prevalence of sars-cov- . here, we show how publicly available cdc influenza-like illness (ili) outpatient surveillance data can be repurposed to estimate the detection rate of symptomatic sars-cov- infections. we find a surge of non-influenza ili above the seasonal average and show that this surge is correlated with covid case counts across states. by quantifying the number of excess ili patients in march relative to previous years and comparing excess ili to confirmed covid case counts, we estimate the syndromic case detection rate of sars-cov- in the us to be less than %. if only / of patients infected with sars-cov- sought care, the ili surge would correspond to more than . million new sars-cov- infections across the us during the three week period from march to march . combining excess ili counts with the date of onset of community transmission in the us, we also show that the early epidemic in the us was unlikely to be doubling slower than every days. together these results suggest a conceptual model for the covid epidemic in the us in which rapid spread across the us are combined with a large population of infected patients with presumably mild-to-moderate clinical symptoms. we emphasize the importance of testing these findings with seroprevalence data, and discuss the broader potential to use syndromic time series for early detection and understanding of emerging infectious diseases. the ongoing sars-cov- pandemic continues to cause tremendous morbidity and mortality around the world [ , ] . regional preparation for the pandemic requires forecasting the growth rate of the epidemic, the timing of the peak, the demand for hospital resources, and the degree to which current policies may curtail the epidemic, all of which benefit from accurate estimates of the true prevalence of the virus within a population [ ] . confirmed cases are thought to be underestimates of true prevalence due to some unknown combination of patients not reporting for testing, testing not being conducted, and false-negative test results. estimating the true prevalence informs the scale of upcoming hospital, icu and ventilator surges, the proportion of individuals who are susceptible to contracting the disease, and estimates of key epidemiological parameters such as the epidemic growth rate and the fraction of infections which are sub-clinical. the current literature suggests that the predominant symptoms associated with covid are fever, cough and sore-throat; that is, patients often present with an influenza-like illness (ili) yet test negative for influenza [ , ] . with many covid patients having a similar presentation as patients with influenza, existing surveillance networks in place for tracking influenza could be used to help track covid. ili correlates with known patterns of sars-cov- spread across states within the us, suggesting the surge is unlikely to be due to other endemic respiratory pathogens, yet is orders of magnitude larger than the number of confirmed covid cases reported. together this suggests that the true prevalence of sars-cov- within the us is much larger than currently appreciated and that even the highest symptomatic case detection rates are likely lower than % corresponding to approximately million new ili cases due to sars-cov- . our analysis provides empirical corroboration of previous hypotheses of substantial undocumented cases [ ] yet places the estimated undocumented case rate an order of magnitude higher than prior reports [ ] . moreover, these updated prevalence estimates predict that epidemic doubling times greater than . days [ , ] would be unable to account for the magnitude of the ili surge. we test our hypothesis of sub -day doubling times in the us by analyzing both state and national covid surveillance data, finding a broad agreement of doubling time less than . days in both confirmed covid case counts and documented covid deaths. our findings highlight probable trajectories of the us epidemic and provide evidence for a conceptual model for covid spread in the us in which more rapid spread than previously reported is coupled with a larger undiagnosed population to give rise to currently observed trends. we identified excess ili cases by first subtracting cases due to influenza and then subtracting the seasonal signal of non-influenza ili ( figure ). many states, including washington, new york, oregon, pennsylvania, maryland, colorado, new jersey, and louisiana, have had a recent surge in number of non-influenza ili cases far in excess of seasonal norms. for example, in the second week of march, , oregon saw % higher non-influenza ili than it had ever seen since the inception of the ilinet surveillance system within the us. we find that with % probability approximately % of all outpatient visits in oregon during this time were for ili that could not be explained by either influenza or the normal seasonal variation of respiratory pathogens. we find that as the seasonal surge of endemic non-influenza respiratory pathogens declines, this excess ili correlates more strongly with state-level patterns of newly confirmed covid cases suggesting that this surge is a reflection of ili due to sars-cov- (pearson ρ = . and p < − for the last two weeks; figure s ). to equate this surge to state-wide or national case counts, we assume that the average number of patients seen per week by sentinel providers is representative of their respective states that week. using this assumption, the total excess non-influenza ili across the us was approximately . million excess individuals in the week starting march th, compared to the same week in ( % credible interval of . - . million). the rate at which sars-cov- + patients with ili symptoms are identified as having covid varies by state and over time ( figure s ). our estimated symptomatic case detection rates have been increasing over the month of march, which can be expected given increases in testing capacity across the us since the february detection of community transmission in washington state. for the latest week ending march , covid cases in the states with the highest estimated symptomatic case detection rate (washington, nevada, and michigan) are only capturing approximately % of ili surges in those states ( figure ). across the entire us we find the average symptomatic case detection rate to be . % ( % credible interval . %- . %). the true prevalence of sars-cov- is unknown. however, if we assume the excess non-influenza ili is almost entirely due to sars-cov- , an assumption that becomes more valid as the virus becomes more prevalent, we can use the excess non-influenza ili to understand the constraints and mutual dependence of exponential growth rates, the rate of subclinical infections, and the time between the onset of infectiousness and a patient reporting as ili figure . with a january start date of the us epidemic [ ] , allowing early stochasticity from start-time to the onset of regular exponential growth, we find that it's impossible to explain the ili surge with an epidemic whose doubling time is longer than . -days, as such slow growth scenarios fail to reach the observed excess ili even when accounting for possible asymptomatic cases. we tested our hypothesized constraint that doubling times across the us would be shorter than . days by analyzing the doubling time confirmed covid cases and deaths due to covid. we find that both confirmed case data and covid fatality data support our estimated doubling-time bound derived from ili data. across the entire us, the doubling rate for deaths due to covid is . days (± . , p-value of test that doubling rate is less than . days approximately ) and the doubling time for confirmed cases is . days (± . , p-value of test that doubling rate is less than . days approximately ). under a -day lag from the onset of infectiousness to reporting as ili, these doubling times capture the ili surge with clinical rates of . % and . %, respectively. here we use outpatient ili surveillance data from around the us to estimate the prevalence of sars-cov- +. we find a clear, anomalous surge in ili outpatients during the covid epidemic that correlates with the progression of the epidemic across the us. the surge of non-influenza ili outpatients is much larger than the number of confirmed case in each state, providing evidence of large numbers of symptomatic probable covid cases that remain undetected. moreover, this excess ili surge allows us to develop a bound for the slowest epidemic doubling time, and the lowest clinical rate that would still be consistent with the observed surge. we test whether or not observed epidemic doubling times are shorter than our upper-bound and find that the observed doubling time for both confirmed cases and deaths is faster than days within the us. together, the surge in ili and analysis of doubling times suggest that sars-cov- has spread rapidly throughout the us since it's january th start date and is likely accompanied by a large undiagnosed population of potential covid outpatients with presumably milder clinical symptoms than would be thought based on prior studies of sars-cov- + inpatients. our study has several limitations. first, the observed ili surge may represent more than just sars-cov- infected patients. a second epidemic of a non-seasonal pathogen that presents with ili or changing patient behaviors causing higher rates of presentation for typical seasonal ili could confound our estimates of ili due to sars-cov- . alternatively, it is also possible that our use of ili data has underestimated the prevalence of sars-cov- within the us. while early clinical reports focused on cough and fever as the dominant features of covid [ ] , other reports have documented digestive symptoms as the complaint affecting up to half of patients with laboratory-confirmed covid [ ] , and alternative presentations, including asymptomatic or unnoticeable infections, could result in ili surges underestimating sars-cov- prevalence. additionally, our models have several limitations. first we assume that ili prevalence within states can be scaled to case counts at the state level. this is based on the assumption that the average number of cases seen by sentinel providers in a given week is representative of the average number of patients seen by all providers within that state in a given week. errors in this assumptions would cause proportional errors in our estimated case counts and symptomatic case detection rate. second, our epidemic models are crude, us-wide seir models varying by growth rate alone and as such do not capture regional variation or intervention-induced changes in transmission. our models were used to estimate growth rates from ili for testing with covid data and to estimate the mutual dependency of growth rate, the lag between the onset of infection and presentation to a doctor, and clinical rates; these models were not intended to be fine-grained forecasts for municipality hospital burden and other common goals for covid models. finer models with regional demographic, and case-severity compartments are needed to translate our range of estimated prevalence, growth rate, and clinical rates into actionable models for public health managers. while an ili surge tightly correlated with covid case counts across the us strongly suggests that sars-cov- has potentially infected millions in the us, laboratory confirmation of our hypotheses are needed to test our findings and guide public health decisions. our conceptual model for the epidemic with the us makes clear and testable predictions. our model would suggest rela- tively high rates of community seropositivity in states that have already seen an ili surge. a study of ili patients from mid-march who were never diagnosed with covid could test our model's predictions about the number and regional prevalence of undetected covid cases presenting with ili during that time. if seroprevalence estimates are consistent with our estimated prevalence from these ili analyses, it would strongly suggest lower case severity rates for covid and indicate the value of ili and other public time-series of outpatient illness in facilitating early estimates of crucial epidemiological parameters for rapidly unfolding, novel pandemic diseases. since not all novel pandemic diseases are expected to present with influenza-like symptoms, surveillance of other common presenting illnesses in the outpatient setting could provide a vital tool for rapidly understanding and responding to novel infectious diseases. in what follows, let i index state i and let t index week t (with t = referring to october , ; the start of ilinet surveilance). our analysis centers around decomposing the probability of testing positive for covid, δ into the product of the symptomatic case detection rate for ili patients, δ s , and the probability that a covid patients presents to the clinic with ili, δ c . since the cdc has maintained ilinet for weekly influenza surveillance. each week approximately , enrolled providers distributed throughout all states as well as puerto rico, the district of columbia and the us virgin islands, report the total number of patient encounters n it and the total number of which met criteria for influenza-like illness (ili -defined as a temperature for influenza y f lu it . therefore ilinet data can be thought of as a weekly state-level time-series representing the superimposed prevalence of various viruses which can cause ili. ilinet data was obtained through the cdc fluview interactive portal [ ] . in addition to ilinet data, us state population data for the year was downloaded from https://worldpopulationreview.com/states/. the number of primary care providers in each state per , residents b was obtained from becker's hospital review [ ] . covid confirmed case counts were obtained from the new york times' database maintained at https://github.com/nytimes/covid- -data. this dataset contains the daily cumulative confirmed case count for covid for each state z il for day l. within the ilinet dataset, new york city and new york were summed into a combined new york variable representing both new york city and the surrounding state. due to incomplete data in one or more of the data-sources described above the virgin islands, puerto rico, the commonwealth of the northern mariana islands, and florida were excluded from subsequent analysis. in addition, daily cumulative confirmed covid cases were converted to weekly counts of new cases bỹ to subtract influenza signal from y it we assume that the population of patients with ili within a state are the same population that are potentially tested for influenza. this assumption allows us to calculate the number of non-influenza ili cases as the resulting time-seriesỹ it are shown in figure s . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . we identified ili surges inỹ it by training a model onỹ it for all data prior to july , . we then used this model to predict the prevalence of non-influenza ili (π it ) for dates after and including july , . we calculated the ili surge as the difference between the observed proportion of non-influenza iliỹ it /n it andπ it . more specifically, to account for variation in the number of reporting providers, we trained the following binomial logistic-normal model we made the following prior specifications: we set the bandwidth parameter for the squared exponential kernel as ρ = representing a strong local correlation in time that died off sharply beyond weeks, α = representing a signal to noise ratio of of approximately , υ = and ξ = representing weak prior knowledge regarding the overall scale of variation in the the latent space. finally, we set θ = − . representing an off-season prevalance of . % non-influenza ili. samples from the posterior predictive density p(π it |y i , . . . ,ỹ it , n i , . . . , n it ) were collected using the function basset from the r package stray [ ]; a total of such samples were collected in this analysis. we define the prevalence of non-influenza ili in excess of normal seasonal variation to exclude variation attributable to unseasonably high rates of other ili causing viruses (such as the outbreak of rsv in washington state in november-december ) we only investigate y * it for sets of or more weeks (t, t + , . . . , t + k) such that the following criteria are met: . the nd order moving average of y * i(t+k) < y * i(t+k+ ) must remain positive for all k . at least one week in (t, t + , . . . , t + k) corresponds to a week after february , together these criteria for attributing excess non-influenza ili to covid reflect our assumption that we expect covid must be increasing within communities since febuary , . as covid new case countsz it represent the number of confirmed cases in an entire state and ilinet data represents the number of cases seen by a select number of enrolled providers, we must estimate scaling factors w i to enable comparison of ilinet data to confirmed case counts at the who state level. let π * it denote the probability that a patient with ili in state i has covid as estimated from ilinet data. let p i denote the population of state i and let b i denote the number of primary care providers per , people in state i. we simulated the number of covid cases (excess ili meeting criteria above) as that is we translate the inferred proportion of individuals with ili due to covid to the state level by considering the average number of patients seen by each provider in the study ( nit dit ) and the number of primary care providers in state i ( bipi ). notably to account for potential errors in these scaling factors, we add propogate uncertainty into our calculation by using monte-carlo simulation of the average number of patients seen by each provider in the study. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . assuming that the majority of sars-cov- testing within the us has been directed by patient symptoms [ ] , the pool of newly diagnosed sars-cov- + patients is a subset of the pool of sars-cov- + patients who are identified as having ili. therefore, we calculate the probability that a symptomatic sars-cov- + patient will be identified as having sars-cov- as δ s it =z ij /π * it ( figure s ). to account for the contribution of assymptomatic sars-cov- + patients we use a recent analysis of cohort surveillance from the diamond princess [ ] . monte-carlo simulations were used to propagate error from our uncertainty regarding potential asymptomatic infections affecting the clinical rate δ c into our calculation of posteriors for epidemic trajectories. to match posterior estimates from mizumoto et al. [ ] , we use quantile matching to parameterize δ c ∼ beta(α, β) to achieve a mean of . and a % probability set of (. , . ). we include this contribution to the overall detection rate δ it as δ it = δ s δ c it . for a given epidemic growth rate, the clinical rate was estimated by comparing the excess ili across the us y † t = i y † it to the number of infections one might expect under simulated epidemic parameterized by the growth rate. an seir model, was parameterized for the us to a timescale of units days by setting ζ = . × − corresponding to a crude birth rate of . per per year, a baseline mortality rate ω b = . × − corresponding to . per per year, an infectious mortality rate ω i = . × − , incubation period γ − of days, infectious period ν − of days, and β parameterized to ensure i(t) grew with a specified exponential growth rate early in the epidemic. a total of , simulations were run, each having a unique exponential growth rates, r, drawn uniformly from r ∈ [log( )/ , log( )/ . ], reflecting doubling times in the range of . ≤ t ≤ days. each simulation was initialized with (s, e, i, r, t) = ( . × , , , , ) where time was january . to simulate the stochastic time it took from the first case to the onset of regular exponential growth, a gillespie algorithm was used from the initial conditions until either t = (march , ) or e(t) + i(t) = . the output from gillespie simulations was input as an initial value into the system of differential equations and integrated until the august , . the number of infected individuals on a given day was the last observed i(t) for that day, and a weekly pool of infected patients was computed by a moving sum over the number of infected individuals every day for the past week, i w (t) = k= k= i t−k . defining y t = i y † it as the national excess ili, the clinical rate was for a given time delay t d it takes from the onset of infectiousness to a patient reporting to the doctor with ili. an estimate of clinical rate as a function of exponential growth rate was obtained by a generalized additive model log(δ c ) = s(log(r)))+ where s(.) denotes a piece-wise cubic spline. simulated epidemic trajectories were matched to posterior samples of y t through kernel density estimation. for t corresponding to the weeks starting on march and , , the density each simulated trajectory i(t) was evaluated against the density v t ∼ log-normal( , . ) . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint where noise v t was added to account for additional sources of error in our calculations of scaling factors, and p is a density estimated via kernel density estimation. the two weeks in march were chosen as the ili surge had the largest correlation with covid case counts during these two weeks. doubling times for confirmed cases and deaths due to covid were estimated using poisson regression with a log-link with an intercept and a covariate for the day of the epidemic. poisson regression parameters r were converted to doubling times d using d = log( )/r. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . non−influenza ili proportion figure : the excess non-influenza ili is extracted from all non-influenza ili by identifying the amount of non-influenza ili in excess of seasonal norms (blue point and error bars represent the posterior median and % credible set for ili not explained by non-covid endemic respiratory pathogens). a binomial logistic-normal non-linear regression model was fit to non-influenza ili data from - (grey lines). the model predicted the expected amount of non-influenza ili in the - season (grey ribbons represent the % and % credible sets; the black line represents the posterior median). observed non-influenza ili outside of the % credible set was attributed to covid based on the observed correlation across states with newly confirmed covid case rates ( figure s ). a number of regions are not represented due to insufficient laboratory influenza data to complete our analysis (see methods for full details). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . : assuming the non-influenza excess ili for the week starting march consists entirely of patients with covid, the probability that a symptomatic covid+ patient will be detected varies by state but even the highest symptomatic case detection rates are likely below %.in figure s we show how the symptomatic case detection rate varies over time across states. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . figure : (a) , random seir models predict a range of current number of covid cases in the us; less transparant trajectories are better agreements with the ili surge under an assumption that % of infections are subclinical [ ] . the inset plot shows covid forecasts comparable to weekly ili counts (red dots) by summing i(t) over the prior week. a t = . day doubling time (solid black lines) is the slowest possible growth rate which can account for the observed data with a -day lag between the onset of infectiousness and reporting as ili. faster doubling times, such as t = . (dashed black lines) can explain observed ili data but require a larger rate of subclinical infections to do so. (b) different growth rates imply different clinical rates in order to account for the excess ili surge, and the relationship between growth rate and clinical rate varies with the unknown time it takes from the onset of infectiousness to presentation an ili outpatient. the estimated . - . day doubling times across the us, for example, produce an estimated - % clinical rate. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . figure s . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . figure s : excess ili correlates strongly with patterns of newly confirmed covid cases. this correlation is strongest for the last two weeks of data, when other seasonal respiratory pathogens are at their lowest. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . figure s : since march , , the case-detection of symptomatic covid patients has increased by a factor of ≈ . this likely represents increased awareness of community transmission within the us combined with increased availability of testing. still, the symptomatic case detection rate remains below % for most states with many states with detection rates closer to . %. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . figure s : once the signal attributable to influenza is extracted, the proportion of patient encounters in which patient had non-influenza ili (ỹ it /n it ) displays strong seasonal trends. the most notable deviations from these trends occur around febuary to march of the - flu season and align with the onset of the covid epididemic within the us. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint a novel coronavirus from patients with pneumonia in china coronavirus disease (covid- ): situation report fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the sars-cov- epidemic epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study first case of novel coronavirus in the united states estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship, yokohama, japan key: cord- -spap b authors: silva, denise r; viana, vinícius p; müller, alice m; livi, fernando p; dalcin, paulo de tarso r title: respiratory viral infections and effects of meteorological parameters and air pollution in adults with respiratory symptoms admitted to the emergency room date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: spap b background: respiratory viral infections (rvis) are the most common causes of respiratory infections. the prevalence of respiratory viruses in adults is underestimated. meteorological variations and air pollution are likely to play a role in these infections. objectives: the objectives of this study were to determine the number of emergency visits for influenza-like illness (ili) and severe acute respiratory infection (sari) and to evaluate the association between ili/sari, rvi prevalence, and meteorological factors/air pollution, in the city of porto alegre, brazil, from november to october . methods: eleven thousand nine hundred and fifty-three hospitalizations (adults and children) for respiratory symptoms were correlated with meteorological parameters and air pollutants. in a subset of adults, nasopharyngeal aspirates were collected and analyzed through ifi test. the data were analyzed using time-series analysis. results: influenza-like illness and sari were diagnosed in ( · %) and ( · %) patients, respectively. thirty-seven ( · %) samples were positive by ifi and of ( · %) were ifi and/or pcr positive. in a multivariate logistic regression model, ifi positivity was statistically associated with absolute humidity, use of air conditioning, and presence of mold in home. sunshine duration was significantly associated with the frequency of ili cases. for sari cases, the variables mean temperature, sunshine duration, relative humidity, and mean concentration of pollutants were singnificant. conclusions: at least % of infections in adult patients admitted to er with respiratory complaints were caused by rvi. the correlations among meteorological variables, air pollution, ili/sari cases, and respiratory viruses demonstrated the relevance of climate factors as significant underlying contributors to the prevalence of rvi. respiratory tract infections are the most common causes of infection, and viruses account for the majority of these infections, leading to significant levels of morbidity and mortality. emergency rooms (ers) serve as the frontline for patients at highest risk for respiratory infection diseases, especially because of the acute nature of these illnesses. the prevalence of respiratory viral infection (rvi) in adults admitted to the er is largely unexplored, as most relevant data concern infants and children. , rvi can be severe in elderly patients, especially in those with underlying respiratory or cardiac disease. during winter months, rvi can account for many of the admissions to hospitals. , the etiology of respiratory infections in adults remains undetermined in more than % of cases. , in a study with adults hospitalized with pulmonary diseases, an overall prevalence of respiratory viruses (rvs) in the lower respiratory tract was of Á %, with rhinoviruses and influenza a virus being the most common. in adults with acute asthma admitted to the er, a prevalence of Á % of rvi was found. in another study, with adults admitted to hospital with respiratory symptoms, viruses accounted for % of hospital admissions for respiratory infections. seasonality of certain acute respiratory tract infection pathogens can be explained by meteorological variations. in a study, temperature was highly inversely correlated with respiratory syncytial virus (rsv), influenza a, and adenovirus frequency; rhinovirus was also associated with relative humidity (rh). climatic factors may influence the interaction among the host, pathogen, and environment, increasing the probability of exposure, susceptibility, and infection. in addition, experimental data have shown that air pollutants affect lung immune responses and inflammatory reactions and that these effects may underlie the increased risk for respiratory infections. , because of the large impact respiratory virus infections have on morbidity and even mortality, it is important to understand whether and how meteorological factors and exposure to air pollutants could influence respiratory virus infections. the aims of this study were to determine the number of emergency room visits for influenza-like illness (ili) and severe acute respiratory infection (sari) and to evaluate the association between ili/sari frequency, respiratory virus prevalence, and meteorological factors/air pollution, especially in adult population, in a humid subtropical climate. the present study was divided into two parts: in the first one, we characterized the symptomatic respiratory subjects attending the er at hospital de cl ınicas de porto alegre (hcpa), during year (november to october ). in the second part, we included patients with respiratory symptoms ≤ days to determine the prevalence of rvi; this part was conducted during years (november to october ). in the first year of the study, we also collected climate and air pollution data. the study was conducted at hcpa, in the city of porto alegre, southern brazil. the hcpa is a general, tertiary care, university-affiliated hospital with beds and approximately hospitalizations/year. with a population of inhabitants, porto alegre is surrounded by a metropolitan area that encompasses municipalities ( inhabitants). porto alegre has a humid subtropical climate, with the hallmark of the great variability (classification cfa in k€ oppen-geiger). the ethics committee at hcpa has approved access to patient records. all subjects selected for the study gave written informed consent to participate. in the first part of the study, the clinical records of all daily visits (adults ≥ years and children) to the er were reviewed by the research team. patients with respiratory symptoms (cough, coryza, nasal obstruction, odynophagia, dyspnea, chest pain, dysphonia, wheezing, and fever), regardless of the onset time, were included in the study. patients presenting only with fever were not included. demographic, clinical, and laboratorial characteristics were registered in a standardized questionnaire: sex, age, race, years of schooling, smoking status, symptoms at admission, duration of symptoms, presence of comorbidities, admission vital signs (temperature, heart rate, respiratory rate, and peripheral oxygen saturation measured by a digital oximeter), breath sounds, radiological findings, length of hospital stay, intensive care unit (icu) admission, hospitalization outcome (death or discharge). in the second part, adults ≥ years with respiratory symptoms ≤ days were included, and nasopharyngeal aspirates were collected. every day, a daily shift (morning, afternoon, or evening) was randomized for inclusion of patients. the patients were interviewed by a member of the research team, and the following data were registered in a standardized questionnaire (in addition to the data already registered in the first part): family income, influenza vaccine, previous antibiotic use, and occupational exposure, flu symptoms in the family, presence of smokers at home, family history of respiratory disease, air conditioning at home or at work, use of wood stoves, mold in home. the total cases of ili (fever > °c and cough or sore throat) and sari (fever > °c and cough or sore throat and shortness of breath or difficulty breathing) were registered. nasopharyngeal aspirates were obtained according to a standard protocol for the second part of study. an indirect immunofluorescence assay (ifi) was carried out using the respiratory daily meteorological parameters, like temperature (maximum, average, and minimum, in°c), relative (%) and absolute (g/kg) humidity, rainfall (mm), and sunshine duration (number of sunshine hours per day), were obtained from the climate laboratory at the federal university of rio grande do sul. also, the mean concentration of pollutants (lm/m ) was recorded. the methodology used by this laboratory informs automatically the pollutant that reached the highest concentration in the last hours. in the city of porto alegre, it is probable that the pollutant that is responsible for more than % of the days is ozone, and in the rest of days, particulate matter of < lm dynamic diameter (pm ). data were presented as number of cases, mean ae standard deviation (sd), or median with interquartile range (iqr). categorical comparisons were carried out by chi-square test using yates's correction if indicated or by fisher's exact test. continuous variables were compared using the t-test or wilcoxon test. odds ratios (ors) and nominal % confidence intervals (ci) were presented. a two-sided p value < Á was considered significant for all analyses. data analysis was performed using spss Á (statistical package for the social sciences, chicago, il, usa) and the free statistical software r (http://www.r-project.org). the data were analyzed using time-series analysis, through a generalized linear model (glm) to examine the association between ili or sari and air pollution/meteorological variables, using logistic regression. an autoregressive integrated moving average (arima) model was developed for ili or sari. pollution and meteorological parameters were inserted as explanatory variables into these arima models. for the second part of the study, further models were explored to include ifi and/or pcr results as outcome variables. volatility of the mean model variance error(s) was addressed using autoregressive conditional heteroscedasticity (arch) models within an arima modeling framework. an arima was developed for ili and sari. arima (p, d, q) are useful tools for analyzing time-series data containing non-stationary common trends, and these models were proposed by box and jenkins in . the arima (p, d, q) allow to make predictions from their parties ar (autoregressive) and ma (moving average). for both sg and sars, we made the identification of the order of the parts and autoregressive moving average following the box-jenkins approach. first we identified the need for differentiation, for ili and sari, the graphical analysis of time-series and autocorrelation functions, as well as for testing the unit root (dickey-fuller) who did not reject the hypothesis of non-stationarity for ili and sari. in a second moment, it was noted that there was no seasonality for ili and sari, by analyzing the periodograms and autocorrelation functions and partial autocorrelation. subsequently, variable selection models arima (p, d, q) for ili and sari followed the approach backward. the akaike information criterion (aic) which acts to penalize the number of parameters in the modeland the schwarz information criterion (sic) (or bayesian information criterion, bic) were also used to select models. sample size requirements were estimated from the literature review. , using a prevalence of rvi in adults of %, with a significance level of %, and total confidence interval amplitude of Á , we calculated that patients would be needed in the second part of the study. during the -month study period, there were admissions to the er ( adults and children), of which ( Á %) presented with respiratory symptoms. the most common symptoms were cough ( Á %), fever ( Á %), dyspnea ( Á %), chest pain ( Á %), and coryza ( Á %). the median duration of symptoms before admission was days (iqr: - days). a total of ( Á %) patients admitted to er needed to be hospitalized; of these patients, ( Á %) required icu admission. ili and sari were diagnosed in ( Á %) and ( Á %) patients, respectively. the overall mortality rate among all study participants was of ( Á %). demographic and clinical characteristics of the study population are shown in table . according to the selection criteria of the study, patients met the inclusion criteria and were invited to participate, and patients declined to participate. then, adults were enrolled for virological investigation, in the first year and in the second one. there were no differences in age, sex, race, years of schooling, and symptoms between selected sample and all adults admitted to the er in the same period. however, as expected, the median duration of symptoms was lower in the selected seven samples considered to be unsatisfactory for ifi were positive by pcr. another samples that were negative in ifi were positive by pcr. of the samples positive in ifi, were sent for pcr too, and in nine, the result was positive. in seven of these cases, the results of ifi and pcr were concordant. in one patient, adenovirus was identified on ifi and influenza a was found in pcr. in another case, ifi detected piv type , and pcr identified piv types and . the characteristics of patients with pcr positive and negative were shown in table . figure shows timesseries graph for virus percent positive by ifi and by pcr by month. table shows the descriptive statistics corresponding to the environmental variables considered in this study. figure shows the modeled and observed values for ili and sari cases. figures and show the daily number of patients with ili and sari and meteorological parameters. we checked the autocorrelation between the covariates of climate data and observations from previous days, and we considered the following lags (in days) for each variable: average temperature ( ), rainfall ( ), sunshine duration ( ), relative humidity ( ), mean concentration of pollutants ( ), and absolute humidity ( ) . the number of ili and sari cases tends to be higher between july , , and august , . in this period, the mean temperatures (tmin: Á ae Á °c and tmax: Á ae Á °c) and the sunshine duration ( Á ae Á hours of sun per day) were lower, as expected in winter. the rainfall tends to be higher than the median calculated for the entire year ( mm, iqr: - Á mm). in addition, the absolute humidity (ah; Á ae Á g/kg) and mean concentration of pollutants ( Á ae Á lm/m ) were lower in this period compared with the annual values. table shows multivariate logistic regression model for ifi and pcr. we included patients in logistic regression for ifi and patients in logistic regression for pcr, because we have climate data only for the first year of study. ifi positivity was statistically associated with ah (or: Á ; % ci Á - Á ), use of air conditioning (or: Á ; % ci Á - Á ), and presence of mold in home (or: Á ; % ci Á - Á ). on the other hand, pcr positivity was statistically associated with use of air conditioning (or: Á ; % ci Á - Á ), average temperature (or: Á ; % ci Á - Á ), and mean concentration of pollutants (or: Á ; % ci Á - Á ). the multivariate time-series models for ili and sari cases are summarized in table . sunshine duration was the only independent covariate that was significantly associated with the frequency of ili cases. the b-coefficient for this parameter was negative, indicating increasing ili frequency with decreasing sunshine duration. in the model for sari cases, the following variables proved to be significant: mean temperature (b = Á ; p = Á ), sunshine duration (b = À Á ; p = Á ), rh (b = À Á ; p = Á ), and mean concentration of pollutants (b = À Á ; p = Á ). acute rvis are responsible for causing significant levels of morbidity and mortality. the most common respiratory syndrome caused by these pathogens is ili. a more severe presentation, named sari, was also related to some rvs. , in this study, we have examined the relationship between ili and sari cases, meteorological variables, and air pollution using multivariate time-series analyses. we found that ili cases were inversely correlated with sunshine duration. in addition, sari cases were significantly associated with mean temperature, sunshine duration, rh, and concentration of pollutants. seasonal cycles of infectious diseases have been attributed to changes in atmospheric variables, the prevalence or virulence of the pathogen, or the behavior of the host. earlier investigations have demonstrated that lower temperatures and sunshine duration, conditions usually encountered in winter, were associated with admissions for rvi. , temperature was found to be highly inversely correlated with rsv, influenza a, and adenovirus frequency. interestingly, we found a positive correlation between temperature and sari cases. one possible explanation is that it was demonstrated that for every one degree celsius rise in temperature, the risk of premature death and acute morbidity especially among respiratory patients is up to six times higher than in the rest of the population. second, evidence is emerging that increasing temperature is associated with increases in air pollution, especially ground-level ozone, and can amplify the adverse effects of poor air quality. taking this evidence into account, we could expect that higher temperatures may have increased concentration of pollutants, leading to more sari cases. however, our data showed a decrease in air pollution during the months with a higher prevalence of sari. the third hypothesis to explain the relationship between higher temperatures and sari cases was related to el niño southern oscillation (enso) phenomenon. enso undergoes cycles between warm phases (el niño episodes) and reverse cold phases (la niña episodes). in the southern region of brazil, this phenomenon is associated with elevated temperatures and rainfall, especially in spring and in the period between may and july. previous reports have determined that el niño events were associated with increased hospitalizations and more severe influenza epidemics. , severe acute respiratory infection cases were found to be negatively related to rh in our study. previous studies have demonstrated that higher rh decreases the survival of lipidenveloped virus, like influenza a, influenza b, rsv, and piv. [ ] [ ] [ ] the use of indoor heating in winter lowers the rh; breathing dry air could cause desiccation of the nasal mucosa, epithelial damage, and reduced mucociliary clearance, increasing the host susceptibility to rvis. however, even in tropical regions with humid climate (rh > %), a higher activity of influenza can be found. this observation could be explained by the variation of viral stability in different rh levels. the stability of aerosolized influenza virions is maximal at lower rh ( - %), moderate at higher rh ( - %), and minimum at a mid-range rh ( %). in a multivariate logistic regression model for ifi-positive patients, we found that ah was a protect factor for rvi. a recent study suggested that ah may better correlate with influenza virus survival and transmission. unlike rh, ah measures the actual water vapor content of air irrespective of temperature and has a prominent wintertime low, both indoor and outdoor. such findings suggest that humidification measures could be helpful decreasing survival and transmissibility of influenza. air pollution has been associated with adverse health outcomes. studies have suggested acute effects causing respiratory symptoms, cardiovascular events, hospital admissions, and mortality. although the available evidences indicate associations between exposure to pollutants and increased risk of rvi, potential mechanisms mediating these effects are largely unexplored. , surprisingly, our results showed that sari cases were associated with a decrease in mean concentration of pollutants. in fact, this could be a reflection of higher rainfall in the same period, as rain acts washing out or scattering pollutants from atmosphere. on the other hand, we cannot exclude an effect of indoor pollution. in the last years, indoor pollution has been recognized as an emerging health problem, as about % of our time is spent indoors where we are exposed to chemical and biological contaminants. we estimated indoor pollution indirectly in our study, questioning patients about the use of wood stoves and air conditioning, and the presence of mold in home. our findings suggested that ifi-positive patients were more prone to live in a residence with mold growth. dampness and mold are two important sources of indoor pollution, consistently associated with respiratory symptoms. home dampness may be a marker for mold growth, dust mites, endotoxins, and reduced ventilation, which could increase concentrations of indoor pollutants. cough, wheezing, and upper respiratory symptoms were associated with dampness and mold in a metaanalysis. according to these results, the prevalence of cough and wheezing was higher in patients with mold in home and ifi positive. air conditioning was also positively related to ifi test in this study. air conditioning use was associated with fewer hospital admissions for cardiovascular diseases, chronic obstructive pulmonary disease, and pneumonia on days with high concentrations of pm , as individuals are less exposed to outdoor pollutants. nevertheless, the majority of virus transmission occurs within indoor, air-conditioned (i.e., cooler, lower humidity) environments that favor airborne virus survival and transmission. , in hot and humid conditions, indoor transmission in air conditioning environments may account for most of the transmission. we found a prevalence of % of rv, which is higher than that previous studies have demonstrated (between % and % in adults). , moreover, the length of stay was lower in our ifi-and/or pcr-positive patients. this finding is consistent with existing knowledge that virus identification allows the prompt initiation of therapy when indicated and avoids the unnecessary use of antibiotics, decreasing the length of hospital stay. the present study has some limitations. first, it was based on data collected from a single center, which may have potential biases because of the characteristics of the catchment population, like vaccination coverage. second, it is also important to note that this investigation was performed in a group of hospitalized patients, which is a bias toward the most severe disease cases. additionally, we do not have the concentrations of individual air pollutants, but it is implausible to reliably separate the effects of air pollutants because they frequently react with each other, sometimes potentiating individual effects. , the short study period should also be considered a limitation. finally, the use of molecular techniques (pcr) in all study patients could be useful, increasing the number of viruses detected, as limited sensitivity of ifi method is well known. , despite these limitations, this is the first study, to our knowledge, to analyze the relationship between rv, meteorological parameters, and air pollution in an adult population. in conclusion, we found that in adult patients admitted to er with respiratory complaints, at least % of infections were caused by rv. the correlations found among meteorological variables, air pollution, ili/sari cases, and rv demonstrated the relevance of climate factors as significant underlying contributors to the prevalence of rvi in a temperate region. there is still a need of additional investigations to clarify and confirm these data, perhaps using longer time-series observations. fundo de incentivo a pesquisa -sbpt; fipe-hcpa; fapergs. communicable respiratory threats in the ed: tuberculosis, influenza, sars, and other aerosolized infections role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy the contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter excess hospital admissions for pneumonia and influenza in persons > or = years associated with influenza epidemics in three 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seasonal variation in host susceptibility and cycles of certain infectious diseases influenza virus transmission is dependent on relative humidity and temperature climate change and respiratory disease: european respiratory society position statement association of normal weather periods and el nino events with hospitalization for viral pneumonia in females: california, - respiratory virus and the effects of climate ª the authors. influenza and other respiratory viruses published by survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids the effect of environmental parameters on the survival of airborne infectious agents aerosol transmission of influenza a virus: a review of new studies absolute humidity modulates influenza survival, transmission, and seasonality air pollution and health ambient air pollution and health climate change and human health the index project: executive summary of a european union project on indoor air pollutants variations of formaldehyde and voc levels during years in new and older homes meta-analyses of the associations of respiratory health effects with dampness and mold in homes air conditioning and source-specific particles as modifiers of the effect of pm( ) on hospital admissions for heart and lung disease incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the american heart association advances in the laboratory diagnosis of viral respiratory disease the authors have no competing interests to disclose. key: cord- -otx b x authors: conway, nicholas t.; wake, zoe v.; richmond, peter c.; smith, david w.; keil, anthony d.; williams, simon; kelly, heath; carcione, dale; effler, paul v.; blyth, christopher c. title: clinical predictors of influenza in young children: the limitations of “influenza-like illness” date: - - journal: j pediatric infect dis soc doi: . /jpids/pis sha: doc_id: cord_uid: otx b x background: influenza-like illness (ili) definitions have been infrequently studied in young children. despite this, clinical definitions of ili play an important role in influenza surveillance. this study aims to identify clinical predictors of influenza infection in children ≤ years old from which age-specific ili definitions are then constructed. methods: children aged – months with a history of fever and acute respiratory symptoms were recruited in the western australia influenza vaccine effectiveness (waive) study. clinical data and per-nasal specimens were obtained from all children. logistic regression identified significant predictors of influenza infection. different ili definitions were compared for diagnostic accuracy. results: children were recruited from winter influenza seasons ( – ; n = ). of eligible children, ( . %) had laboratory-confirmed influenza infection. predictors of infection included increasing age, lack of influenza vaccination, lower birth weight, fever, cough, and absence of wheeze. an ili definition comprising fever ≥ °c, cough, and no wheeze had % sensitivity ( % confidence interval [ci], – ), % specificity ( % ci, – ), % positive predictive value ( % ci, – ), and % negative predictive value ( % ci, – ). the addition of other symptoms or higher fever thresholds to ili definition had little impact. the centers for disease control and prevention definition of ili (presence of fever [≥ . °c] and cough and/or sore throat) was sensitive ( %; % ci, – ), yet lacked specificity ( %; % ci, – ) in this population. conclusions: influenza-like illness is a poor predictor of laboratory-confirmed influenza infection in young children but can be improved using age-specific data. incorporating age-specific ili definitions and/or diagnostic testing into influenza surveillance systems will improve the accuracy of epidemiological data. population surveillance is used to guide preventative strategies for influenza such as choosing strains for seasonal influenza vaccine constitution and early identification of pandemic or epidemic antigenic drift variants [ ] . early diagnosis of influenza disease also influences clinical decision making, especially when managing those at higher risk of severe disease [ ] . influenza disease surveillance usually includes a combination of community-and hospital-based syndromic surveillance and routinely collected data concerning morbidity and mortality, with only some including laboratory confirmation of influenza infection. consistent with other countries, australian children < years old experience the highest laboratoryconfirmed influenza notification rate ( . times the rest of the population in ), the highest rate of general practice consultations with influenza-like illness (ili) ( / population in ), and the greatest morbidity [ ] . in the united states, children < years comprised % of ili presentations in the centers for disease control and prevention (cdc) national network during the - influenza season [ ] . the value of these data using ili surveillance depends on a reliable and robust definition of ili as well as a clear understanding of how ili activity relates to influenza. the definition of ili varies between countries and surveillance systems, but it usually includes the presence of fever and symptoms of acute respiratory tract infection [ ] [ ] [ ] . influenza-like illness is a poor predictor of actual influenza infection in adults, despite attempts to improve the accuracy of the definition [ ] , but there is limited data on the reliability of ili in predicting influenza infection in children [ ] [ ] [ ] [ ] . in this study, we have used data collected from young children recruited as part of a community and hospital influenza surveillance program to assess the clinical predictors of influenza infection in children years old. we developed age-specific ili definitions and tested their diagnostic accuracy against existing definitions and parental opinion. the western australia influenza vaccine effectiveness (waive) study is an observational study designed to measure influenza vaccine effectiveness in children. the study design has been described elsewhere [ , ] . in brief, children - months of age were eligible for participation if they presented with symptoms suggestive of an acute respiratory infection to selected general practices ( only), emergency departments (eds; and ), and pediatric inpatient facilities ( and ). the recruitment period was for the duration of the winter influenza season as determined by local population surveillance. children were eligible for enrollment in whether they had a history of fever (by parental report) or a measured temperature > . °c on presentation in addition to any acute respiratory symptoms within hours before recruitment. to enhance recruitment in , children were enrolled whether they had a history of fever or a measured temperature of > . °c on presentation plus the presence of any acute respiratory symptoms within hours of recruitment. the study received approval from the relevant local human research ethics committees. on enrollment by trained research staff, parents were asked to complete a questionnaire detailing demographics, medical history, and presenting symptoms. temperature measured at enrollment was recorded by research staff. per-nasal swabs (copan diagnostics inc., murrieta, ca) placed in viral transport medium or per-nasal aspirates were collected. influenza testing on nasal swabs was performed by polymerase chain reaction (pcr) directed at hemagglutinin and matrix gene targets in multiplex real-time assay [ , ] , and by conventional cell cultures [ ] . in addition, samples underwent pcr directed at other common respiratory viruses including respiratory syncytial virus (rsv), parainfluenza viruses - , human metapneumovirus, rhinoviruses, adenoviruses, coronaviruses (other than severe acute respiratory syndrome coronavirus), and bocavirus [ ] . presence of influenza a or b detected by pcr or culture was collapsed into dichotomous dependent outcome: influenza present or absent. the predictor variables of interest fell into groups: ( ) demographic factors (age, sex, race [indigenous or other], deprivation quintile, influenza vaccination status, prematurity [< completed weeks gestation], birth weight, past medical history, child care usage, household composition, and household smokers); and ( ) symptomatology (recorded temperature and presence or absence of parentally reported: cough, coryza, wheeze, breathing difficulties, earache, sinusitis, sore throat, irritability, rash, diarrhoea, vomiting, lethargy, poor feeding, sleep disturbance, fever, and pallor). vaccination status was verified via the australian childhood immunisation register (acir) [ ] . where acir conflicted with parental report, the family doctor was contacted to establish the number of trivalent influenza vaccine (tiv) doses given previously. because they are not normally distributed, categorical variables were created for age, household composition, and duration of fever and respiratory symptoms. postal addresses were geo-coded before conversion to deprivation quintiles using data produced by the australian bureau of statistics [ ] . statistical analysis was performed using spss . . (spss inc., chicago, il). after initial univariate analysis, variables were analyzed simultaneously within both groups (demographic factors and symptoms) by forced entry into a multivariable logistical regression model. factors found to be significant (p < . ) at the group stage were then entered into a model encompassing both groups. various definitions of ili were then constructed based on the significant predictors of influenza infection. sensitivity, specificity, and positive predictive value (ppv) and negative predictive value (npv) and their respective binomial % confidence intervals (cis) were then calculated. in addition, positive and negative likelihood ratios (lr+ and lr-) were also calculated. ninety-five percent cis for likelihood ratios were calculated in the manner described by simel et al [ ] . new ili definitions were compared with definitions used by the cdc ( presence of fever ( o f [ . °c ]) and a cough and/or sore throat in the absence of a known cause other than influenza [ ] ) and parents' response to the questionnaire question: "do you think your child has influenza ('flu')?" nine hundred and forty-four subjects were recruited ( subjects in and subjects in ). the majority were recruited within the hospital setting (general practice, subjects; eds, subjects; inpatient wards, subjects). twenty-five subjects were withdrawn (specimens not processed, ; invalid consent, ; incorrect age, ; other, ), resulting in a total sample population of . altering the eligibility criteria in did not result in any additional recruits that were ineligible by criteria: subjects had a temperature recorded > . °c yet < . °c; however, all of these subjects also had a history of fever within the previous hours. respiratory viruses were identified in subjects ( . %). rhinovirus (n = ), rsv (n = ), and influenza virus (n = ) were most frequently detected. of those in whom influenza was detected, had influenza a (including subtyped as influenza a/h n ) and had influenza b. cultures proved positive for % ( of ) of those with influenza a and % ( of ) of those with influenza b. influenza was detected in . % ( of ) of recruits in , compared with . % ( of ) of eligible recruits in (p = . ). the median age was months and of ( %) were male. chronic comorbidities were uncommon: children ( %) had a comorbid condition, of which asthma was the most common ( of , %). premature births (< weeks gestation) accounted for of ( %) subjects. nearly one-half of all children enrolled ( of , %) had received the recommended schedule for seasonal influenza vaccination (ie, doses of tiv in the first year of vaccination followed by dose in subsequent years [ ] ) with a further of ( %) having received dose in total ( table ) . the most common symptoms reported by parents were cough ( of , %), coryza ( of , %), poor feeding ( of , %), sleep disturbance ( of , %), and irritability ( of , %), although wheeze and respiratory distress were less prevalent ( of , % and of , %, respectively) ( table ). for those that tested positive for influenza, the average temperature at enrollment was . °c (range, - . ; standard deviation . five hundred and ninety-eight parents recorded a response to the question, "do you think your child has influenza ('flu')?" seventy-eight of the ( %) answering "yes" proved positive for influenza, whereas of the ( %) answering "no" were positive for influenza (p < . ). age > years, lack of tiv, lower birth weight, sharing a home with or more other children, and being cared for by a single adult were significant demographic predictors of influenza infection on univariate analysis (table ) . with the exception of the number of adult household members, all of these variables were significant when entered simultaneously within this group, and so they were entered into the final model. the symptoms that were significant univariate predictors of influenza infection were as follows: raised temperature, fever for > days, presence of cough, absence of wheeze, respiratory distress, and rash. fever, presence of cough, and absence of wheeze remained significant when entered simultaneously within the symptom group, and so they were retained for the final model. with the exception of number of children in the house, all variables entered into the final model remained significant (see table ). based on the results of the regression equation, ili was defined in this population using various combinations of the following criteria: presence of cough, absence of wheeze, and incremental thresholds of fever (see figure and table ). a complete data set for each of these variables was available for cases, which were used for subsequent calculations of the performance of ili definition. the presence of cough alone was highly sensitive ( %; % ci, - ) yet lacked specificity in diagnosing influenza infection ( %; % ci, [ ] [ ] [ ] [ ] [ ] [ ] . if the definition of ili comprised cough and the absence of wheeze, sensitivity was reduced ( %; % ci, - ) but specificity improved ( %; % ci, - ). the addition of fever °c to this definition resulted in a small the above analyses were repeated with data stratified according to vaccination status (no tiv vs any tiv) and age group ( years vs > years) (see supplementary digital content). the observed ppvs in the unvaccinated group were significantly greater than the vaccinated group. subgroup analysis of each vaccination group stratum by year of illness was undertaken with no appreciable difference noted (data not shown). for those years, an ili definition that included fever °c, cough, and the absence of wheeze had a significantly lower sensitivity and ppv and a higher specificity compared with those > years. the reliability of parental opinion was unaffected by vaccination status or age group of child. young children and infants with seasonal influenza can present with a wide variety of symptoms and may not yet be developmentally capable of verbalizing symptoms to their caregivers [ ] . current definitions of ili are largely derived from adult studies and lack validation within the pediatric setting [ ] [ ] [ ] . fever is a common presenting symptom for young children with influenza. when all children with acute respiratory illness (irrespective of presence or absence of fever) are tested, % of those who test positive for influenza had a history of fever [ , ] . systematic review of the literature has failed to find any particular combination of additional symptoms that can reliably predict influenza infection [ ] . only studies in this systematic review enrolled preschool children: in study, of cases with fever and respiratory illness were < years old [ ] , whereas the other did not stratify by age [ ] . later, ohmit and monto [ ] reported a ppv of % in a subset of children < years old presenting with cough and fever (> . °c). the generalizability of these figures is limited, however, because the study design excluded children with rsv infection, which would have falsely elevated the incidence of influenza infection in the study population [ ] . a further prospective pediatric study assessing the predictive nature of an ili diagnosis in children with fever and symptoms suggestive of respiratory infection (n = ; age < years) did not stratify results by age, making it less generalizable to younger children [ ] . this study recruited young children presenting with acute respiratory symptoms during successive winter influenza seasons, which included the first wave of pandemic influenza a/h n . in this population, we have found that an existing ili definition in common usage (cdc definition) proved highly sensitive yet lacked specificity in identifying those with influenza infection. a surveillance system that uses this ili definition in isolation would therefore grossly overestimate influenza prevalence by virtue of the large number of false positives generated. in contrast, we found that an ili definition comprising fever °c, cough, and absence of wheeze achieved a greater balance between sensitivity and specificity in the study population. however, ili was a poor predictor of influenza infection regardless of which definition was tested. children with ili had a %- % probability of actually having influenza infection, whereas those without ili (regardless of definition tested) had a %- % probability of testing positive for influenza. each definition tested had positive and negative likelihood ratios approaching , indicative of little appreciable change in the odds of an individual having disease if they met ili definition criteria or not. furthermore, parental prediction of influenza infection in their children compared favorably with use of an ili definition, underlining the poor diagnostic accuracy of ili overall. decisions regarding the investigation and management of children suspected of having influenza take into account a number of factors, including the known incidence of influenza at that time, the likelihood of severe disease, and the availability of antiviral medication [ ] . however our findings would suggest that, due to the inaccuracy of syndromic definitions, clinicians should maintain a low threshold for influenza testing in children with possible influenza. this is especially important for children with moderate to severe illness and/or those requiring hospitalization. similarly, diagnostic testing is required to obtain accurate influenza surveillance in young children. ideally, this should include testing by highly sensitive and specific methods such as pcr, although immunofluorescent antigen detection tests are sufficiently sensitive for use on nasopharyngeal aspirates. the rapid antigen tests have been used in some studies [ , ] , but they are less sensitive than laboratorybased tests (especially for influenza a/h n ); their performance is influenced by specimen type, test brand used, and the virus type and subtype; and they do not identify influenza a subtypes. these tests have been successfully incorporated into public health influenza surveillance systems in the past [ ] ; however, they need to be reassessed now that influenza a/h n is circulating. the eligibility criteria for our study included a history of fever. this methodology is similar to a number of previous studies [ ] [ ] [ ] [ ] and highlights the need for external validation of any clinical predictor tool that is intended to be used in unselected populations. however, our findings remain relevant to the general pediatric population given that the vast majority of children with influenza present with fever [ , ] . the diagnostic accuracy of ili differed between those vaccinated and those unvaccinated (reflecting the lower prevalence of influenza in those who were vaccinated). the pediatric population studied had high vaccination rates with tiv as a result of a state-wide campaign introduced in that provides free seasonal influenza vaccination to this age group. the high prevalence of influenza vaccination in our study population contributes to the generalizability of our findings to other countries where influenza vaccination is readily available to children < years of age. the reliability of ili as defined by fever ( °c), cough, and absence of wheeze was age dependent, with less sensitivity and greater specificity in those years of age or younger. this is a reflection of proportionally higher numbers of children years old with wheeze irrespective of influenza status (data not shown), presumably as a result of a bronchiolitic-type illness. this age-dependent manifestation of influenza infection further highlights the problems inherent in applying adult-derived ili definitions to the pediatric population. because the presence of wheeze was determined by the parents, the calculated prevalence is likely to be different from one based on a clinical definition [ ] . however, because this bias is constant between those who tested positive for influenza and those who were negative, it is not expected to have affected the results. also, in practice, assessing children with an ili usually relies on parental history as well as clinical findings, making our findings relevant to real-life circumstances. to our knowledge, this is the first study attempting to construct a definition of ili for children aged years and under using prospectively gathered data from a general pediatric population presenting with symptoms suggestive of acute respiratory tract infection. we have demonstrated that when predicting influenza infection in younger children, an ili definition constructed using age-specific data and comprising presence of fever ( °c), cough, and absence of wheeze results in a greater balance between sensitivity and specificity compared with a definition of ili used by current surveillance systems. the diagnostic accuracy of influenza virus surveillance systems would be enhanced by developing age-specific ili definitions aimed at the pediatric age group and/or by incorporating diagnostic testing into the system. author contributions. n. t. c. and z. v. w. analyzed the data and revised the manuscript. p. c. r., d. w. s., a. d. k., s. w., h. k., d. c., and p. v. e. devised the waive study and revised the manuscript. c. c. b. supervised the present study, analyzed the data, and revised the manuscript. financial support. the waive study is funded by the western australia department of health. potential conflicts of interest. n. t. c., p. c. r., and c. c. b. are members of the vaccine trials group, telethon institute for child health research. the vaccine trials group has received funding from vaccine manufacturers for conducting clinical trials, although not in relation to this study. p. c. r. has served on a scientific advisory board regarding influenza vaccines for csl ltd., has received travel support from baxter and glaxosmithkline to present at scientific meetings, and received institutional funding for investigator-led who global influenza surveillance network - influenza antiviral medications: summary for clinicians annual report of the national influenza surveillance scheme - influenza season overview of influenza surveillance in the united states heterogeneous case definitions used for the surveillance of influenza in europe working towards a simple case definition for influenza surveillance does this patient have influenza? evaluation of clinical case definitions of influenza: detailed investigation of patients during the - epidemic in france diagnosing influenza: the value of clinical clues and laboratory tests symptomatic predictors of influenza virus positivity in children during the influenza season clinical predictors of influenza in children lessons from the first year of the waive study investigating the protective effect of influenza vaccine against laboratoryconfirmed influenza in hospitalised children aged - months vaccine effectiveness against laboratory-confirmed influenza in healthy young children. a case-control study duplex real-time reverse transcriptase pcr assays for rapid detection identification of pandemic (h n ) and seasonal influenza a/h , a/h , and b viruses australian childhood immunisation register seifa: socio-economic indexes for areas likelihood ratios with confidence: sample size estimation for diagnostic test studies the australian immunisation handbook th edition clinical presentation of influenza in unselected children treated as outpatients the underrecognized burden of influenza in young children performance of six influenza rapid tests in detecting human influenza in clinical specimens accuracy and interpretation of rapid influenza tests in children enhancing public health surveillance for influenza virus by incorporating newly available rapid diagnostic tests what do parents of wheezy children understand by "wheeze"? [see comment epidemiological research from glaxosmithkline and csl ltd. d. w. s. is a director of not-for-profit organizations (the influenza specialist group and the asia-pacific alliance for the control of influenza) that receive funding from vaccine manufacturers. all other authors report no potential conflicts.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- -wbhji g authors: tay, ee laine; grant, kristina; kirk, martyn; mounts, anthony; kelly, heath title: exploring a proposed who method to determine thresholds for seasonal influenza surveillance date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: wbhji g introduction: health authorities find thresholds useful to gauge the start and severity of influenza seasons. we explored a method for deriving thresholds proposed in an influenza surveillance manual published by the world health organization (who). methods: for - , we analysed two routine influenza-like-illness (ili) datasets, general practice sentinel surveillance and a locum medical service sentinel surveillance, plus laboratory data and hospital admissions for influenza. for each sentinel dataset, we created two composite variables from the product of weekly ili data and the relevant laboratory data, indicating the proportion of tested specimens that were positive. for all datasets, including the composite datasets, we aligned data on the median week of peak influenza or ili activity and assigned three threshold levels: seasonal threshold, determined by inspection; and two intensity thresholds termed average and alert thresholds, determined by calculations of means, medians, confidence intervals (ci) and percentiles. from the thresholds, we compared the seasonal onset, end and intensity across all datasets from - . correlation between datasets was assessed using the mean correlation coefficient. results: the median week of peak activity was week for all datasets, except hospital data (week ). means and medians were comparable and the % upper cis were similar to the (th) percentiles. comparison of thresholds revealed variations in defining the start of a season but good agreement in describing the end and intensity of influenza seasons, except in hospital admissions data after the pandemic year of . the composite variables improved the agreements between the ili and other datasets. datasets were well correlated, with mean correlation coefficients of > . for a range of combinations. conclusions: thresholds for influenza surveillance are easily derived from historical surveillance and laboratory data using the approach proposed by who. use of composite variables is helpful for describing influenza season characteristics. influenza infection remains a significant public health problem, resulting in considerable global morbidity and mortality [ ] [ ] [ ] . in temperate regions of australia, seasonal influenza outbreaks usually occur between late autumn and early spring and are associated with an increase in disease burden and utilisation of health service [ , ] . due to differences in circulating viruses, population immunity and environmental factors, the onset, duration and severity of a season may differ from year to year [ , ] . ongoing monitoring of influenza is therefore needed to determine the onset and severity of seasons and to monitor changes in disease trends. surveillance which involves laboratory testing can add to data on virus characteristics. influenza thresholds have been developed to indicate a level of disease activity that would signal the start or end of a season or provide an alert to an unusually severe or atypical season. the onset of a season may stimulate diagnosis, enhance case detection, promote awareness of the need for patient cohorting or isolation in hospitals, remind people about vaccination and encourage early prescription of anti-viral medication, especially in vulnerable populations [ , ] . in the setting of a particularly severe season or pandemic, thresholds may inform the appropriate allocation of resources [ ] . many influenza surveillance systems around the world have incorporated the use of thresholds. these include australia, new zealand, europe and united states (us) [ ] [ ] [ ] [ ] . methods using a variety of surveillance systems have been developed to establish thresholds for influenza activity. the methods vary in their complexity and can use either short-term or longer historical data to create time-varying or fixed thresholds. there is currently no gold standard or consensus for calculating thresholds. the simplest method uses visual inspection of historical data to create a fixed threshold used throughout the year [ ] . other methods include regression models [ ] [ ] [ ] [ ] , time series methods [ ] , calculation of means and medians [ ] [ ] [ ] and adaptation of industrial control processes such as shewhart charts [ ] , cumulative sum (cusum) [ , , ] and the exponentially weighted moving average [ ] . the us centre for disease control and prevention (cdc) calculates the baseline for their influenzalike-illness (ili) surveillance by adding two standard deviations to the mean percentage of ili visits during non-influenza weeks for the previous three seasons, with non-influenza weeks defined as periods with less than % of the year's total positive specimens for influenza for ≥ consecutive weeks [ ] . in victoria, the general practice sentinel surveillance (gpss) for ili has historically relied on thresholds determined by inspection [ ] . in a novel but simple method for defining thresholds was proposed by the world health organization (who) as part of the development of global standards for influenza surveillance [ ] . the proposed method aligns several years of historical data on the median week of peak activity and assigns thresholds based on means and standard deviations of aligned data. to our knowledge, this method has not yet been field tested. the aim of this study was to explore the feasibility of the who method for the calculation of influenza thresholds using a range of existing surveillance and laboratory data sources in one surveillance system. we used these data sources to compare the onset, duration and intensity of influenza seasons. victoria is a state with a population of over million people located in the south-eastern part of australia. it has a temperate climate with annual seasons of influenza occurring occur between late autumn (may) and early spring (october). it has a well-established influenza surveillance system that monitors influenza activity using syndromic surveillance of ili presentations to sentinel general practitioners (gp) and a medical locum service; laboratory-confirmed influenza; hospital admissions for influenza; and more recently google flu trends and the influenza complications alert network (flucan), which monitors hospitalised patients from sentinel australian hospitals, including four victorian hospitals [ ] . four independent surveillance data sources were used: (i) the victorian gpss, (ii) sentinel data from the melbourne medical deputising service (mmds), (iii) routine laboratoryconfirmed influenza (lab data) from the victorian infectious diseases reference laboratory (vidrl) and the (iv) victoria admitted episode dataset (vaed) for hospital admissions. the gpss is an annual surveillance system for ili and laboratory confirmed influenza that was established in , with laboratory support added in [ ] . surveillance extends from week to each year during the influenza season. the number of participating general practitioners (gp) has varied from to since the scheme's establishment. the ili definition used is based on the nationally agreed case definition of cough, fever (measured or reported) and fatigue [ ] . approximately % of ili patients seen by sentinel gps were swabbed and of these, influenza virus was detected from an average of % ( %- %) of the swabbed ili patients tested from to [ , ] . an alternative source of community sentinel ili surveillance is the mmds, an out-of-hours medical locum service that covers an approximate km radius from central melbourne. gps from the deputising service consult with patients in their own home or aged care facility. the diagnosis made by the attending doctor is recorded electronically and de-identified summary data are available on a password protected website within hours. ili data are extracted weekly based on a previously developed search algorithm [ ] . there is no laboratory support for the mmds surveillance which has nonetheless been shown to provide equivalent information to surveillance data from sentinel general practitioners [ ] . lab data from vidrl consist of laboratory detections of influenza viruses from all routine respiratory samples sent to vidrl, tested using an in-house respiratory multiplex reverse transcriptase polymerase chain reaction (rt-pcr) test that identifies influenza viruses, adenovirus, picornavirus, respiratory syncytial virus, parainfluenza virus, coronavirus and human metapneumovirus [ ] . many of the samples are referred from major adult teaching hospitals in victoria [ ] . the vaed is a hospitalisation dataset on all patients admitted to public and private acute care hospitals in the state of victoria. the clinical information coded for each episode of care is based on the international classification of diseases and related health problems, tenth revision, australian modification (icd- -am). we extracted records containing influenza codes j - in primary or secondary diagnostic fields. in addition, a further two composite variables were created from the product of ili and lab data. these were the gpss composite = proportion of ili cases in gpss x proportion of laboratory samples tests positive for influenza in gpss exploring a who method for influenza thresholds plos one | www.plosone.org mmds composite = proportion of ili cases in mmds x proportion of routine laboratory tests positive for influenza in lab data. while the gpss proportion and laboratory testing are part of the same system, the mmds is independent of routine clinical tests referred to vidrl. however, both mmds and routine vidrl clinical testing focus on older age groups and were matched based on age profiles [ , ] . the metrics used for threshold calculations were the weekly gpss ili proportion per ; the weekly mmds ili per ; the proportion of laboratory test positive for influenza using the total number of tests for influenza as the denominator (lab test positive influenza); the weekly proportion of influenza admissions in the population using the mid-year estimated resident population in victoria as the denominator and expressed per , population [ ] ; and the product of weekly ili and lab data per . we preferred the use of test positive influenza to count data to compensate for changes in testing behaviour over time [ ] . depending on availability, data were extracted from to for gpss and mmds, to for lab data and to for vaed. for mmds, lab data and vaed, data were complete for all weeks but only weeks to were available for gpss. for vaed, only aggregated data containing admissions of more than five counts were provided to protect privacy and confidentiality of individuals. we defined the three levels of influenza threshold based on the terminology used in the who manual and the existing victorian surveillance thresholds that had been adapted from the united kingdom: seasonal, average and alert [ , ] . seasonal threshold defines the start and end of an influenza season. the two intensity thresholds, termed average and alert thresholds, describe relative seasonal intensity. the who manual did not prescribe a specific method for determining the seasonal threshold, that is, the start of the season. we used the method of inspection of the complete data for the six datasets to determine the seasonal threshold. for each dataset this was done independently by four of the co-authors (et, kg, am, hk) and differences were resolved by discussion. for the four datasets that provided data for the whole year (mmds, lab data, vaed and mmds composite), we calculated the % confidence interval (ci) of the metrics used for each dataset for the period defined as out-of-season, that is, the values below the seasonal threshold, that had been determined by inspection. we also explored the seasonal threshold using the th percentile of out-of-season data without assuming data were normally distributed. we then compared the seasonal threshold set by inspection with the % ci and th percentiles of the average out-of-season values. average and alert thresholds were calculated for each dataset using a variation of the who protocol [ ] (figure ). we first determined the median week of peak occurrence using historical data, excluding the pandemic year of which was atypical from both surveillance and testing perspectives [ ] . we then aligned the transmission peaks around the median week of peak occurrence ( figure a ) and calculated the weekly mean and standard deviations for each week centred on the median week of peak occurrence ( figure b and c) . the who protocol suggests the use of the normal distribution to assign thresholds based on the mean and standard deviation of the aligned data for weekly counts. however, we believed data were unlikely to be normally distributed for all years and tested this by inspection and formally using the shapiro-wilks test for normality for gpss, mmds, lab data and the vaed for each year during season [ ] . in addition to the mean and standard deviations, we explored the thresholds using the median and th and th percentiles. the average threshold was determined by a comparison of the peak weekly mean and median, while the alert threshold was determined by a comparison of the peak weekly upper % and % ci upper limits with the th and th percentiles. we also performed log transformation of all datasets and calculated the corresponding geometric mean and / ci upper limit. once the seasonal thresholds were assigned, we determined the start and end of each season independently for all datasets, each defined as the two consecutive weeks where the seasonal threshold was crossed. we used the average and alert thresholds to categorise the influenza seasons, based on the threshold range of peak seasonal activity, specifically seasonal-average, average-alert or alert. comparisons were made for the onset, duration and intensity of a season across all datasets from - based on data availability. we also compared how all seasons compared to the average season created using aligned historical data described above. finally, to determine how correlated the six datasets were, we calculated the correlation coefficient for each year from to for a combination of datasets, from which a mean correlation coefficient and its corresponding % confidence limits was derived. data were analysed using microsoft® office excel and stata version . (stata corp., college station, tx, usa). the study was approved as a quality assurance project by the melbourne health office of research. gpss and mmds data in this study were collected, used and reported under the legislative authorization of the victorian public health and wellbeing act and public health and wellbeing regulations . during the study period, the highest number of ili or influenza cases annually ranged from to per week for the gpss; to per week for the mmds; to per week for test positive influenza; and to per week for influenza admissions. testing for normality of the weekly count data for each year suggested no seasonal surveillance data had a classical normal distribution graphically (data not shown). data were consistent with a normal distribution by formal testing for / seasons in the gpss, for / seasons in the mmds, for / seasons in lab data and for / seasons in the vaed. when data were log-transformed, the number of seasons with normal distribution increased ( / , / , / and / respectively). the threshold parameters from the adapted who method are summarised in table . the median week of peak occurrence for all datasets was week except for the mmds composite and vaed. the values assigned for seasonal thresholds by inspection were similar to the % ci upper limit and th percentile of the average out-of-season values for all datasets. for the average threshold, we found the peak mean values for the gpss, test positive influenza and vaed were similar to the median but the peak mean was higher for the mmds and mmds composite due to the high call out proportion in . we therefore use the peak mean to define (table ) . to set the alert thresholds, the peak % ci upper limit was used as we found the parameter to be similar to the peak th percentile across all six datasets (table and figure ). the geometric means and % ci upper limits from log-transformed data also produced similar parameters (data not shown). using the gpss dataset as an example, figure compares how an annual season compares against the average season calculated using ten years of historical data. onset, end and duration of influenza season across all datasets. for the seven years where data were available for all datasets, gpss assigned seasons tended to start much earlier for most years compared to other datasets. the use of the gpss composite suggested a later start to the season. season onset according to the vaed generally lagged behind other datasets for most years and was variable for the mmds ( table ) . for most pre-pandemic years, there was generally good agreement for defining the end of a season across datasets. however, a divergent trend, not reflected in other datasets, was noted in the vaed from onwards ( table ) . the vaed assigned seasons ended much later, resulting in a longer assigned seasonal duration. category of influenza season. there was agreement in describing the intensity of influenza seasons in / years prior to (table and figure ). during the pandemic year, the season intensity varied according to different data sources and from onwards, peak seasonal influenza activity was between the seasonal and average thresholds (or seasonalaverage) for all datasets except the vaed. datasets were found to be well correlated, with mean correlation coefficients of > . for a range of combinations (table ) . correlations between the vaed and other datasets improved once the vaed was aligned with other datasets to correspond to the one week lag in median week of peak occurrence. thresholds for influenza surveillance were easily derived using a simple method proposed by the who. the method was adapted to a non-parametric approach that produced similar findings to the suggested protocol based on the normal distribution. log transformation of the data produced comparable findings to both approaches. comparison of thresholds derived from different datasets revealed variations in defining the start of a season but relatively good agreement in describing the end and intensity of influenza seasons, except in the hospital data after the pandemic year. as the who protocol does not prescribe a method for defining the seasonal threshold, we used the simplest method of visual inspection but showed that the levels were consistent with variation in out-of-season virus circulation. numerous other approaches exist, based on more complex statistical techniques but many of these approaches usually require a pre-determined threshold to be nominated [ , , ] , again often by inspection. in the setting of the average and alert thresholds for our datasets, we used the peak mean values to set the intensity thresholds as per the who protocol after observing the data agreement between the peak weekly means and medians and % ci upper limit and percentiles. however, the median and or percentiles may be a more appropriate option when data are not normally distributed and no transformation has been performed. in practice, another point of consideration for the setting of the alert threshold may be a level of influenza activity that corresponds to an increased demand on the health care system [ ] . this would be dependent on local health care systems. we also incorporated a two week consecutive rule into the definition of the onset and intensity of a season to reduce the number of false positive signals. based on the seasonal thresholds, we found inconsistencies in defining the start and end of a season across the datasets. given the variations in timeliness of influenza reporting [ ] , we would expect the onset of ili surveillance to precede laboratory confirmed influenza and hospital admissions, and that both the ili surveillance systems might coincide with one another. by incorporating a laboratory component to the ili measure, the use of the composite variable appeared to improve the specificity and agreement between vaed and ili surveillance data. the finding is consistent with emerging literature that these composite variables may be a better proxy indicator of influenza incidence than either ili or lab data alone [ , ] . the use of composite variables in surveillance warrants further investigations. in comparing the intensities of an influenza season, there was good agreement across all datasets, except for the vaed after . the number of hospital admissions coded for influenza has increased both in and out of the influenza season, with the duration of the season prolonged due to the late end signal. these changes were not reflected in the ili or lab datasets. while there may be a number of possible explanations, such as changes in testing behaviours or disease coding, a recent study investigating the increase in out-ofseason influenza in australia suggests a genuine increase in influenza activity, combined with increased testing that occurred following the pandemic [ ] . this may reflect an increased awareness of influenza in hospitalised patients among health professionals after the pandemic. additionally, surveillance data at vidrl indicate that approximately % of patients with an influenza-like illness were swabbed prior to [ ] but this rose to % during the pandemic [ ] and has since remained at about this proportion [ ] . the measurement of the intensity a season was based on the peak of influenza activity and whether or not the thresholds were exceeded for two weeks. this reflects only a single dimension of measurement and does not take into account how long the influenza activity remained within a particular category or the rate of increase in the number of cases. for example, a short-term acute rise in influenza cases that marginally exceed the alert threshold does not correspond to a gradual or persistent elevated level of activity that may represent a higher disease burden. finally, when we compared the current parameters to the previous threshold based on seven years of historical ili data from - in victoria, the baseline threshold for the gpss was lower at per cases compared with the revised threshold of and the alert threshold was higher at per cases compared with the revised threshold of [ ] . these differences indicate the need of regular review of surveillance-derived thresholds. in conclusion, this study has shown that the proposed who threshold protocol is simple to implement and could be easily adapted for any influenza surveillance system with adequate historical data. however, the study was based in a region with a temperate climate, and its application in the tropics would require further work. further exploration of the proposed who method in another temperate region would be of interest. while thresholds are useful as a warning system, they should always be interpreted with other available information. influenza (seasonal) factsheet epidemiology of influenza the annual impact of seasonal influenza in the us: measuring disease burden and costs global influenza seasonality: reconciling patterns across temperate and tropical regions how to deal with influenza: worthwhile surveillance system is in action world health organization ( ) who global surveillance standards for influenza. geneva: global influenza programme, surveillance and monitoring team, world health organization resource allocation during an influenza pandemic virological surveillance: influenza weekly updates centers cdcfor disease control and prevention ( ) flu activity & surveillance: weekly us influenza surveillance report weekly influenza surveillance overview in: control establishing thresholds for influenza surveillance in victoria can syndromic thresholds provide early warning of national influenza outbreaks? methods for monitoring influenza surveillance data a routine tool for detection and assessment of epidemics of influenza-like syndromes in france a statistical algorithm for the early detection of outbreaks of infectious disease epidemic features affecting the performance of outbreak detection algorithms influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method modelling influenza epidemic -can we detect the beginning and predict the intensity and duration? detection of epidemics in their early stage through infectious disease surveillance do cusums have a role in routine communicable disease surveillance? detecting the start of an influenza outbreak using exponentially weighted moving average charts overview of influenza surveillance in the united states laboratory-supported influenza surveillance in victorian sentinel general practices estimation of influenza vaccine effectiveness from routine surveillance data a medical locum service as a site for sentinel influenza surveillance influenza-like illness surveillance using a deputising medical service corresponds to surveillance from sentinel general practices laboratory diagnosis and surveillance of human respiratory viruses by pcr in h n swine origin influenza infection in the united states and europe in may be similar to h n seasonal influenza infection in two australian states in a comparison of data sources for the surveillance of seasonal and pandemic influenza in victoria influenza surveillance in australia: we need to do more than count the use of thresholds to describe levels of influenza activity an analysis of variance test for normality (complete samples) improving the estimation of influenza-related mortality over a seasonal baseline predicting the epidemic sizes of influenza a/h n , a/h n , and b: a statistical method the significance of increased influenza notifications during spring and summer of - in australia pandemic influenza h n infection in victoria, australia: no evidence for harm or benefit following receipt of seasonal influenza vaccine in we gratefully acknowledge the ongoing support of general practitioners and their practice staff participating in the general practice sentinel surveillance system and the continued involvement of the melbourne medical deputising service in influenza surveillance in victoria. we thank the victorian government department of health for provision of hospital admissions data and staff at the viral identification laboratory at vidrl for provision of laboratory data. we acknowledge the world health organization global influenza program for the threshold methodology. sentinel surveillance in victoria is supported by the victorian government department of health. ee laine tay is a post-graduate scholar in the master of philosophy in applied epidemiology program, the field epidemiology training program in australia. conceived and designed the experiments: et kg hk am. analyzed the data: et kg mk hk. contributed reagents/ materials/analysis tools: am. wrote the manuscript: et hk. provided critical revisions to the article: et kg mk am hk. key: cord- -z ygy e authors: mccaw, james m; howard, peter f; richmond, peter c; nissen, michael; sloots, theo; lambert, stephen b; lai, michael; greenberg, michael; nolan, terry; mcvernon, jodie title: household transmission of respiratory viruses – assessment of viral, individual and household characteristics in a population study of healthy australian adults date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: z ygy e background: household transmission of influenza-like illness (ili) may vary with viral and demographic characteristics. we examined the effect of these factors in a population-based sample of adults with ili. methods: we conducted a prospective cohort study in community-dwelling australian adults nested within an influenza vaccine effectiveness trial. on presentation with ili, participants were swabbed for a range of respiratory viruses and asked to return a questionnaire collecting details of household members with or without similar symptoms. we used logistic and poisson regression to assess the key characteristics of household transmission. results: participants from multi-occupancy households experienced ili episodes and returned a questionnaire. of these, were the primary case in the household allowing assessment of factors associated with transmission. transmission was significantly associated in univariate analyses with female sex ( % vs. %, risk ratio (rr) = . ( . , . )) and the presence of a child in the house ( % vs. %, rr = . ( . , . )). the secondary household attack proportion (shap) was . , higher if influenza was isolated (rr = . ( . , . )). vaccinated participants who nonetheless became infected with influenza had a higher shap (incidence rr = . ( . , . )). conclusions: the increased shap in households of vaccinated participants who nonetheless had confirmed influenza infection supports the hypothesis that in years of vaccine mismatch, not only is influenza vaccine less protective for the vaccine recipient, but that the population’s immunity is also lower. improved characterisation of the determinants of household transmission of influenza-like illness (ili) remains an important public health priority, particularly in light of the past decade's events in which we have witnessed the emergence of severe-acute-respiratory-syndrome (sars) and the h n influenza pandemic. the evidence base for pandemic influenza public health interventions such as home-quarantine, provision of antiviral agents for post-exposure prophylaxis, school-closure and vaccination builds upon an appropriate understanding of the patterns and timing of infection within the household unit [ ] [ ] [ ] [ ] [ ] [ ] . while influenza viruses, rhinoviruses (hrvs), adenoviruses, respiratory syncytial virus (rsv) and parainfluenza viruses (pivs) are the most common aetiological agents in acute-respiratory-infection (ari) episodes [ , ] , in - % of all ari episodes no known respiratory virus can be identified [ , ] . this is despite discovery of a number of previously undescribed viruses since from clinical specimens from the human respiratory tract (human metapneumovirus [ ] , sars coronavirus [ ] , coronavirus nl [ ] , coronavirus hku [ ] , novel rhinoviruses [ ] , human bocaviruses [ ] and k and wu polyomaviruses [ , ] ). reflecting the need to improve our understanding of household transmission of ari, the literature examining factors associated with household transmission of influenza [ , [ ] [ ] [ ] [ ] has expanded significantly since the h n influenza pandemic [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , including a systematic review and meta-analysis [ ] . donnelly et al. estimated the serial interval for all ili (without laboratory confirmation) from case reports during the pandemic [ ] . only two studies of which we are aware explicitly consider the impact of virus type on infectiousness. principi et al. found less onwards transmission to household members from influenza-negative than influenza-positive children presenting to a hospital emergency department [ ] . similarly, in a cohort study of ari in young children, lambert et. al. observed significant heterogeneity in the proportion of participants' households in which one or more illness events were observed (ranging from % for isolation of hmpv from the child to % for isolation of influenza) [ ] . here we report on the household transmission of a range of viruses in a cohort study of healthy, community-dwelling adults reporting symptoms of influenza-like illness (ili). the study population was sourced from a large, industry sponsored placebocontrolled phase iv efficacy trial of a licensed seasonal trivalent influenza vaccine (fluvax w , csl ltd), conducted prior to the pandemic between march and november . in a previous article [ ] we have described the viral aetiology of ili in the cohort, and examined the influence of virus type, host and spatiotemporal factors on disease symptomatology. full details of subject recruitment and selection for the primary phase iv vaccine efficacy trial have been described previously [ ] . briefly, across study sites in australia and new zealand, healthy adults aged ≥ to < years were recruited for a placebocontrolled trial of a licensed trivalent influenza vaccine (fluvax w , csl ltd) in (clinicaltrials.gov #nct ). study participants were randomized to receive either placebo or vaccine in a : ratio prior to the southern hemisphere influenza season. from an available pool of participants from the primary study at study sites, we consider the persons (adults) who experienced at least one ili episodemeeting the case definition of at least one respiratory symptom (cough, sore throat, runny nose or nasal congestion) and at least one systemic symptom (fever (oral temperature ≥ . °c), feverishness, chills or myalgia) [ ] , and who provided written informed consent for participation in the nested cohort-study which required contribution of a valid biological sample (copan tm dry flocked swab). samples were tested for a range of respiratory viruses using a combination of multiplexed and uniplexed conventional and real-time polymerase chain reaction (pcr) assays [ ] . of those, were members of multi-occupancy households, allowing investigation of transmission within the household. using a non-specific and sensitive ili definition, they reported an episode of ili on occasions. for each episode they returned a study questionnaire (additional file ) detailing respiratory symptoms (see [ ] for details), health seeking behaviour (health care provider consultations, hospital admission, time off work), household characteristics (number of adults (≥ years) and children (< years)) and temporally associated symptoms of ili (if any) in other household members. from herein, we consider the illness episode as the primary unit of analysis. the virology results were classified into virus groups [ ] : influenza (influenza a, influenza b), coronaviruses (oc , e, nl , hku ), picornaviruses, other viruses (parainfluenza viruses ( , , ), adenoviruses, human metapneumovirus (hmpv), bocaviruses, rsv and ki and wu polyomaviruses) or none, where none indicates that no 'tested-for' virus was detected in the participant's sample, as opposed to a missing sample or inconclusive result. study participants' vaccination status (as determined by the primary phase iv trial intervention), physical location (i.e. study site), and socio-demographic characteristics were also recorded. the relevant outcome measure for this sub-analysis was evidence of transmission within households based on experience of symptomatic illness in at least one other member of the study participant's household. study participants were asked to complete the diary on the day following cessation of their own symptoms. they recorded the date of onset of symptomatic ilis in household members from between days prior to the study participant's illness through to the day of diary completion. note that the primary case in the household may or may not be the study participant. for household ili events in which the participant was the primary case ( of ), transmission may or may not have occurred in the household and so it is statistically valid to develop univariate and multivariate explanatory models. for participants who reported recurrent ili episodes during the study in which the same virus was isolated, we exclude all but the first episode. events with co-introduction, defined as onset of symptoms in the participant and one or more household members on the same day, were also excluded. following these exclusions, episodes remained for analysis. for household ili events in which the participant was not the primary case ( of ), while we do know who the introducer was for these events (via the questionnaire data), other household ili events initiated by that introducer that did not involve the participant are unobserved. that is, any events in which a child (or for that matter, any other adult member of the household) introduced an infection that did not infect the study participant are not captured by the study protocol. this observation necessitates the exclusion of all household ili events in which the participant was not the primary case from the analyses. in the one remaining household ili event, the status of the participant (primary or not) was unknown, so the episode was excluded from the analysis. for the episodes in which the participant was the primary case, logistic regression models were used to explore associations between host, demographic or virus variables with any observation of within-household transmission (outcome variable = transmission in household for each recorded ili episode in a study participant). the secondary household attack proportion (shap) was calculated as the proportion of potentially exposed household members (assumed susceptible) experiencing illness, averaged over all recorded episodes. we present descriptive statistics for the shap and its variation by virus, participant and demographic variables. poisson regression models were used to assess the influence of virus, participant and demographic variables on the number of secondary cases within a given household, offset against the number of potentially exposed household members (outcome variable = number of secondary cases in household for each recorded ili episode in a study participant). vaccination status of participants was not included in the primary logistic and poisson statistical analyses due to its known mitigating effect on the likelihood of influenza acquisition [ ] . investigation of the influence of prior immunisation on influenza transmission in 'breakthrough cases' was explored in a secondary analysis by inclusion of a statistical interaction term between vaccination and influenza-identification status. we make an empirical calculation of the mean time between the onset of symptoms in the primary case and the onset of symptoms in the household contacts (the serial interval), for all household ili events, events in which the participant was the primary case, and events in which influenza was isolated from the participant's virological sample. all statistical analyses were conducted in stata/ic . . figure reports characteristics of the multioccupancy households in which transmission did and did not occur. study participants reported two or more ili episodes during the course of the study. for two participants, who both experienced two episodes, picornavirus was isolated on both occasions. we only retain the first episode for each participant. the distribution of household size is dramatically different based on the absence or presence of children within the household ( figure ). in households without children, the distribution is left-skewed (mean household size = . , standard deviation (sd) = . , skewness = . ), while in households with children there is minimal skew (mean household size = . , sd = . , skewness = . ). table summarizes the descriptive statistics (and logistic model results) associated with presence or absence of transmission in the household for the household ili events in which the participant was the primary case. there is marked co-linearity between the variables 'presence of child in household' , 'age-category' and 'household size'. for example, respondents aged - years had significantly greater odds of having a child in the household than those aged - years (or . ( . , )), while no participant aged more than years lived with a child. the relationship between the household size distribution and presence or absence of children is depicted in figure . we retained 'presence of child in household' in the final multivariate model for transmission due to its strong predictive role, intuitive appeal, presumed causal role in our observed (univariate) association with age-category, and previous research indicating an association between transmission and children [ , , , , ] . in the multivariate model, the observed increased risk of transmission with female sex remains (or = . ( . , . ), p = . ). presence of children in the household is both the strongest and most statistically significant factor associated with transmission (or = . ( . , . ), p = . ). within multi-occupancy households, primaryparticipant introductions gave rise to secondary cases among potentially exposed individuals, a secondary household attack rate (shap) of . . of exposed children, in households in which the participant was female, secondary infections were reported among exposed household members (shap = . ), compared with secondary cases among contacts in households in which the participant was male (shap = . ), a riskratio of . ( . , . ), p = . ( -sided fisher's exact). in households with children, secondary infections were reported among exposed household members (shap = . ), compared with secondary cases among contacts in households without children (shap = . ), a risk-ratio of . ( . , . ), p = . . a multivariate poisson regression model ( table ) was used to consider the influence of virus group and demographic characteristics on the number of reported secondary cases within a given household, offset against the number of potentially exposed household members. in correspondence with the logistic regression model for transmission, we include presence of children in the in a secondary analysis, we considered the influence of prior vaccination on the reported number of secondary household cases among participants testing positive for influenza compared with all other participants. in a poisson model for secondary attacks including a statistical interaction between influenza detection (true/false) and vaccination status (placebo/vaccine), the irr for influenza positive cases in those receiving placebo was . ( . , . ), p = . . the factor increase (interaction term) for the irr for vaccinated participants was . ( . , . ), p = . , yielding a net irr for vaccinated influenza-positive participants relative to vaccinated influenza-negative participants of . ( . , . ), p < . ). under the simplifying assumption that the introducer of infection into the household is responsible for all subsequent infections, we may calculate an empiric serial interval, the time from symptom onset in one individual until symptom onset in another. we first consider infections to be related if symptoms are reported within days following onset in the primary case. across all virus-type isolations, we calculate a mean serial interval of . days (sd = . ) for all household ili events (where the study participant was the primary case or otherwise), and . days (sd = . ) for the events in which the participant was the primary case. for the five events in which the primary participant had virologically confirmed influenza and transmission occurred, the mean serial interval was . days (sd = . ). if we limited the maximum serial interval to seven days, the mean was reduced to . days (sd = . ) for all household ili events and . days (sd = . ) for events in which the in our main analysis, we made two assumptions that we now subject to a sensitivity analysis. of the events in which our participant was the primary case, were classified as co-introductions as (at least) one other household member recorded symptoms beginning on the same day. as the latent period for respiratory infections may vary from individual to individual, here we exclude a further episodes in which there was a day interval from onset of symptoms in the study participant to onset of symptoms in another household member. the resulting multivariate models (equivalent to tables and ) are materially unchanged, with the expected slight reduction in statistical power (data not shown). a second assumption made was that, for participants who reported multiple ili episodes during the study period, we only excluded the latter ili episode where the same respiratory pathogen was isolated on both occasions. however, if we conservatively exclude all ili episodes except for the first ( episodes excluded (by virus type: "none", "picornavirus", "influenza", "coronavirus")), again we find no material change in either the logistic or poisson analyses (data not shown). this study, notable in its consideration of a broad range of respiratory pathogens in addition to influenza, demonstrates that household transmission of ili is most strongly associated with host and demographic factors: female sex and the presence of children within the household (tables and ) . the observation that female sex may be associated with increased transmission in the absence of children (rr = . ( . , . ), p = . ) is novel, perhaps suggesting that females are fundamentally more infectious, and not simply more connected to children (in terms of both their susceptibility compared with males if a child introduces infection, and their infectiousness to children if they are the primary household case). behavioural differences whilst ill may drive such an observation. alternatively, mechanisms by which influenza pathogenesis is sex dependent have been investigated [ ] ; whether or not differences extend to infectiousness and susceptibility is not clear. barbara et al. have recently identified that the reporting of respiratory symptoms may be linked with risk perception [ ] and hence gender [ ] . clearly, we cannot exclude the possibility of gender difference in the reporting of within household transmission. the association between transmission and the presence of children within the household is consistent with many other studies [ , , , , ] . the logistic and poisson model findings (tables and ) are consistent with an increased susceptibility for children. this is further supported by the observed increased shap in children compared to adults ( . compared to . , a risk-ratio of . ( . , . ). the shap in adults did not differ by whether or not their household contained children, suggesting that other 'indirect' effects of children are less likely. as our study design limited the analysis to household events with an adult introducer, we were unable to assess the hypothesis that children may be more infectious than adults. our poisson regression analysis on the number of secondary cases given that the participant was the primary case ( table ) indicates that isolation of influenza in the introducer of infection to the household is associated with an increase in the number of secondary cases. we explored this finding more deeply using a statistical interaction model. while somewhat limited by sample size, we found that in placebo recipients identification of influenza was not significantly associated with an increase in the number of secondary cases (irr = . ( . , . ), p = . ), while in vaccine recipients the irr (relative to identification of any other virus, including 'none') was . ( . , . ) , p < . . note that our previous analysis confirms that vaccination is associated with a reduced probability of influenza virus identification [ ] . additionally, 'breakthrough' influenza cases have similar symptoms compared to unvaccinated individuals [ ] . we therefore suggest that our finding of increased transmission may be explained by infection with an influenza virus mismatched to the vaccine-strain (known to be in circulation during the year of study [ ] ), which furthermore may be relatively antigenically novel and to which household members may be expected to have heightened susceptibility. with no virological samples available from other household members and the small number of vaccinated participants who were infected with influenza we are unable to explore this hypothesis further. across all virus types isolated and all household ili events, and assuming that all secondary cases within the household are directly infected by the introducer, we calculate a serial interval of . days. restricting to events in which the participant was the primary case and in which influenza was isolated, we calculate a serial interval of . days. this simple approach, as taken by others [ , ] , cannot account for two important factors: community importation and infection of household members by other non-introducing members (i.e. tertiary cases). while others have partially accounted for these effects [ , , ] , a mechanistically-motivated statistical model is required to fully account for such possibilities, for example as introduced by cauchemez et al. [ ] who determined a serial interval for influenza of . days (sd = . ) compared to . days if calculated directly from empirical observations. with just events, application of these more advanced model-based techniques is not justified for our data. of primary interest for this sub-analysis focussed on transmission is the complication introduced by the monitoring and assessment of ili in an individual rather than a household. ideally, a protocol such as that suggested by klick et al. would have been employed [ ] . the lack of virological assessment of household secondary cases and the broad nature of the question used to establish the secondary case count in each house also contributes to uncertainty with regards to our assignment of temporally associated ili to within-household transmission. both of these limitations were an unavoidable consequence of the nesting of the data-collection protocol within a randomized placebo-controlled trial. furthermore, due to the requested timing for completion of the questionnaire, we cannot exclude the possibility that late onset of secondary (or tertiary etc.) cases may have been missed, particularly if a participant's experience of symptoms was of short duration. similarly, because the study protocol and analyses effectively assume that individuals are infectious until the end of their symptoms, any systematic differences (by virus type) in this relationship may influence the results. however the prompt to return the diary upon symptom cessation was in an effort to ensure timely reporting of questionnaire information to minimise recall bias. conversely, our poisson model implicitly assumes independence among household members, attributing all household infections to the primary case. more advanced model based methods that account for tertiary (and subsequent) cases and community introduction would be warranted with more complete data sources. as with all protocols based purely on symptomatic presentation (as opposed to active surveillance for nonclinical signs of infection such as virological or immunological measures [ , ] ), we are unable to account for potential sub-clinical infection routes, with potential impact for our assessment of whether or not transmission did occur, the primary case status of our participants and determination of the size of the susceptible pool within a given household. conversely, taking a nonsimulation approach to analysis, we are unable to discount our estimate for the shap due to the effects of community introduction into the household, or account for community introduction and tertiary cases in our estimate for the serial interval [ ] . our study sample had an over-representation of females ( of ( . %) individuals for the captured episodes; of ( . %) individuals for the primary-participant introductions). furthermore, it should be noted that the study population were originally volunteers in a randomized controlled trial and as such more likely to represent a group who were more concerned with their health than the general population. eligibility was restricted to healthy adults without recognized risk factors for severe influenza infection. in the context of a literature focussed on the transmission characteristics of laboratory confirmed influenza, our study is the only one that we know of to systematically explore the relationship between transmission and virus aetiology. the analyses suggest that influenza is more transmissible than other causative agents of ili, at least when introduced to the household by an adult. host and demographic factors are also of importance. further studies combining active surveillance of all household members with specimen collection and testing for a range of respiratory pathogens are warranted to elucidate these relationships. additional file : illness visit questionnaire. competing interests pcr has previously served on a scientific advisory board regarding influenza vaccines for csl ltd and has received a grant for an investigator initiated epidemiological study of otitis media from glaxosmithkline australia. he has also received travel support for himself and staff employed by the vaccine trial group to attend and present data at scientific meetings from baxter, glaxosmithkline, sanofi and pfizer. mdn has received travel grants from wyeth australia to present independent research at international meetings, and currently and previously has been the principal investigator for clinical trials sponsored by abbott, baxter, csl, gsk, medimmune, merck, novartis, sanofi-pasteur, wyeth, and pfizer. ml is an employee of csl limited and has an equity interest in the company. authors' contributions jmc, ph and jmv conducted the statistical analyses, provided the primary interpretation of the results and wrote the manuscript. pr was principal investigator on the vaccine efficacy trial within which the sub-study was conducted. tn, jmv, ts, mn, sl and pr conceived the sub-study and secured funding for its conduct, in partnership with csl limited represented by ml and mg. jmv coordinated conduct of the study at multiple sites and oversaw collation of the questionnaire data. ts, mn and sl oversaw conduct of and reporting of the virological testing at the queensland paediatric infectious diseases laboratory. ml was medical monitor for the main vaccine study and a partner investigator on the sub-study, as was mg. all authors contributed to critical revision of the manuscript and have seen and approved the final version of the manuscript. reducing the impact of the next influenza pandemic using household-based public health interventions the transmissibility and control of pandemic influenza a (h n ) virus effective, robust design of community mitigation for pandemic influenza: a systematic examination of proposed us guidance estimating antiviral effectiveness against pandemic influenza using household data modeling targeted layered containment of an influenza pandemic in the united states 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and medical records on signs and symptoms of respiratory illness gender differences in risk perception: theoretical and methodological perspectives annual report of the national influenza surveillance scheme estimation of the serial interval of influenza optimal design of studies of influenza transmission in households. i: case-ascertained studies submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank study staff at the sites for the recruitment of participants and collection of and processing of samples for this sub-study. we thank dale key: cord- -lir ht i authors: kinar, y.; lanyado, a.; shoshan, a.; yesharim, r.; domany, t.; shalev, v.; chodcik, g. title: predicting individual risk for covid complications using emr data date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: lir ht i background: the global pandemic of covid- has challenged healthcare organizations and caused numerous deaths and hospitalizations worldwide. the need for data-based decision support tools for many aspects of controlling and treating the disease is evident but has been hampered by the scarcity of real-world reliable data. here we describe two approaches: a. the use of an existing emr-based model for predicting complications due to influenza combined with available epidemiological data to create a model that identifies individuals at high risk to develop complications due to covid- and b. a preliminary model that is trained using existing real world covid- data. methods: we have utilized the computerized data of maccabi healthcare services a . million member state-mandated health organization in israel. the age and sex matched matrix used for training the xgboost ili-based model included, circa , rows and features. the available dataset for covid-based model included a total sars-cov- positive individuals who were either not hospitalized (n= ), or hospitalized and marked as mild (n= ), or as having moderate (n= ) or severe (n= ) complications. findings: the auc of our models and the priors on the covid- patients for predicting moderate and severe complications as cases and all other as controls, the auc for the ili-based model was . [ . - . ] for the covid -based model - . [ . - . ].. interpretation: these models can effectively identify patients at high-risk for complication, thus allowing optimization of resources and more focused follow up and early triage these patients if once symptoms worsen. we have search pubmed for coronavirus[mesh major topic] and the following mesh terms: risk score, predictive analytics, algorithm, predictive analytics. only few studies were found on predictive analytics for developing covid complications using real-world data. many of the relevant works were based on self-reported information and are therefore difficult to implement at large scale and without patient or physician participation. we have described two models for assessing risk of covid- complications and mortality, based on emr data. one model was derived by combining a machine-learning model for influenza-complications with epidemiological data for age and sex dependent mortality rates due to covid- . the other was directly derived from initial covid- complications data. the developed models may effectively identify patients at high-risk for developing covid complications. implementing such models into operational data systems may support covid- care workflows and assist in triaging patients. since january , the covid- pandemic has become a global emergency. healthcare organizations and governments, worldwide, are strained due to shortage of resources and the need to make timely decisions based on very little reliable data. these decisions includewho to test, how to treat positive cases, how to manage social distancing and reach-out to population at risk, contact tracing, and more. many of these decisions could benefit from decision support tools based on emr and additional data sources, such as geospatial information. unfortunately, accurate data-driven tools are still difficult to develop due to the limited availability of covid- patients' data with historical emr records. many of the relevant works - describe risk factors and the tools already developed , are based on self-reported information and are therefore difficult to implement at large scale and without patient or physician participation. here, we describe two approaches and tools to assess the individual risk of developing covid- complications based on medical records: a model developed by combining a machinelearning approach for influenza-like illness (ili) to be used as a proxy model for covid- and a second model using data on covid- patients. the models were trained using data from maccabi health service (mhs)a large israeli hmo with a central emr database containing longitudinal data for million active individuals each year between and . the data included full emr information -demographics (e.g. age and sex), behavioral info (smoking status), vital signs, lab test results, diagnoses and procedures (using the international classification of diseases th version ), medication prescriptions and purchases, and hospital admissions (dates and departments only). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . since the number of in mhs members who are positive for sars-cov- is relatively low, and the data available is biased due to the current limitations of tests and challenges of data collection and curation, we have therefore chosen to test two complimentary approaches. first, we use a proxy model that we derived for identifying patients with high risk of developing complications due to influenza and apply some required adjustments. although influenza and covid- are clearly very different diseases , it is already apparent that both diseases have common risk factors for developing complications. however, the initial epidemiological data for covid- [china cfr, nyc cfr] already show some major differences between the two diseasesprimarily in the effect of increased age on the risk of complications (which seems much stronger for covid- ) and the much higher risk among men for covid- complications and mortality, a trend less evident in influenza (another difference is seasonalitywhich is clear for influenza and less evident for covid- ). following these differences, we modified the ilibased model and forced it to ignore age and sex as risk factors, and then used bayesian correction to add these risk factors using external priors. for the training covid- ased model, we used information on sars-cov- positive individuals aged or above within the mhs population, as well as information regarding hospitalization and in-hospital complications. as an initial prior we used the information based on covid- mortality available from china [https://www.worldometers.info/coronavirus/coronavirus-age-sex-demographics/] as proxy for complications probabilities (appendix table ). fatality rate by sex is given in appendix table . due to the over-representation of women among the elderly, we had to replace the : . ratio of female-to-male risk with a higher : ratio per age group, as shown in appendix table . a detailed description of our approach to developing a model based on emr data is given elsewhere , . for training the ili-based model, a training set of all mhs members at september st of every calendar year who were not vaccinated during the following flu-season. we marked them as cases if they were diagnosed with ili followed by complications (death, hospitalization in internal ward, or severe illness, e.g. pneumoniasee appendix for list of icd- codes) within months, and controls if otherwise. bins were matched for age ( yegendar groups) and sex. in addition, we matched for calendar year to avoid biases due to change in collection, registration, or healthcare policy over the period. given the matched set, we generated a large matrix of features per each sample (a sample corresponds to an individual per each relevant year) and applied a process of univariant age-andsex conditioned feature selection on this matrix, and then trained and recalibrated xgboost model using isotonic regression. to combine the prediction of the calibrated model with age and sex priors for complications, we used the following formula - where odds is the overall odds of the model's predictions. see the appendix for derivation of the formula. we used the definitions of the israel ministry of health for covid complications: moderate (defined as pneumonia, with one of the following: respiratory rate above breaths per minute, all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint respiratory distress, or oxygen saturation below %) or severe (pneumonia accompanied by sepsis, shock, ards or death). we then created a vector that of features per each individual, including risk factors and underlying conditions (see appendix). we used xgboost on the features matrix to learn a covid- complications predictor based on these features. given that real world data on covid- are currently limited, it is difficult to evaluate the performance of our models. we report here several methods we have used to estimate the value of the models. . for the covid -based model, we report the performance (auc) in predicting influenza complications as an initial indication of the value of the models . we examined the excess risk of underlying health conditions, compared to information from the cdc [https://www.cdc.gov/mmwr/volumes/ /wr/mm e .htm#f _down]. for cdc information, we took the proportion of individuals admitted to icu given various comorbidities; for our models, we used the mean prediction over individuals with the corresponding icd- codes. evaluating performance of the model on initial covid- complications records. for the model directly derived on covid- data, we used cross-validation for performance evaluation. lift was evaluated by calculating the average prediction over the population with the underlying conditions, and comparing to the average prediction over a reference population all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the age and sex matched matrix used for training the xgboost model included, after feature selection, about , rows and features (compared to about , rows and features for the non-matched model). the top important features are given in an appendix. the available dataset included a total sars-cov- positive individuals who were either not hospitalized (n= ), or hospitalized and marked as mild (n= ), or as having moderate (n= ) or severe (n= ) complications. individuals who were hospitalized but not assigned severity level were excluded. all individuals were linked to their mhs medical record in order to generate the features matrix the auc of the full (non-matched) model for predicting influenza-complication was . , the matched model auc was . . after adjusting for the age and sex priors, the auc for predicting influenza-complications deteriorates to . . in comparing our results to cdc data (table ), we can see that, although the prevalence of various conditions is quite different between mhs and cdc data, lifts seem similar and correlated, both for the ili-based and the covid -based models, with the exception of pregnancy. we defined three groups according to the model's predictionhigh risk (top %), intermediate (next %) and low (bottom %). the confusion matrix of our grouping and the mhs status is given in table . the overall distribution of sars-cov- positives over the three groups is as expected for random infection (ili-based model: % for the low risk, % for the intermediate risk, and % for the high risk; all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint %, % and % respectively for the covid -based model). the hospitalized population is enriched in high and intermediate risk groups ( % of for the ili-based model, % for the covid -based model). this sensitivity is even higher for the moderate ( % of for both models) and severe cases ( % and % of ). we also compared the performance of our score to using only the priorsthe sensitivity of the severe and moderate cases in the top % (high all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint we have described two approaches for assessing risk of covid- complications and mortality, based on emr data. one model was derived by combining a machine-learning model for influenza-complications with epidemiological data for age and sex dependent mortality rates due to covid- . the other was directly derived from initial covid- complications data. such models have many potential applications during the covid- epidemics, prioritization of tests and antibody testing, follow-up on patients with the disease, decision on hospitalization, reach-out for population at risk when social-distancing and restrictions are gradually lifted, and in possible future outbreaks of the disease, and, hopefully in the near future, prioritization of vaccination. both approaches have many weaknesses, due to the speedy and urgent manner of their derivations. performance evaluation is indicative, at best, of the true performance of the models. a better model will surely be derived once more reliable covid- real world data will be available. however, we believe that currently such models can be of great use for health systems and public health entities coping with pandemic. although performance of the covid -based model seems better than the ili-based model, it is reasonable to suspect due to the small size of the dataset that the latter model is too specific to the mhs and less generalizable compared to the ili-based model. the auc of the ili-based model on the subset of sars-cov- positives is the same as the priors only. however, we note a couple of points -first, the significant difference in performance when considering all population, as well as some manual curation of the dataset suggest a possible bias toward older individuals in the definition of covid- complications and sars-cov positives. second, even though the auc is similar, the ili-based model can all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint identify younger populations at risk of complications, which the priors-only model, of course, cannot. in comparing the two models it is interesting to note that the effect of bmi on the risk for covid- complications seems much higher than the risk for influenza complications. this suggests future work of further adjusting the more robust ili-based -model by inserting exterior priors for bmi as well. in comparing our results to cdc data similar lifts both for the ili-based and the covid -based models, with the exception of pregnancy that was associated with low risk in our model, compared to slightly elevated risk in the cdc data (though based on very few cases). this might be due differences in age and sex distribution between the us and israel populations pregnancy. for all underlying conditions, the covid -based model showed lower lift compared to the ili-based model despite the model's inherent weaknesses, and due to the clear and urgent needs, the ili-based model was integrated at mhs to support two covid- care workflows. first use is triaging testing. with limited resources available for outpatient testing, there is a need to prioritize testing to those individuals at highest risk of complications and mortality from covid- . the second use is for outpatient virtual management and triage. mhs established a virtual covid- management center, occupied by primary care physicians and nurses. the medical staff are the first to contact confirmed covid- patients, question them and decide on the appropriate treatment facility based on their symptoms and overall medical assessment. patients can be hospitalized, sent to a special covid- care facility, or stay at home. nurses are then following-up on those patients at homecare to continuously assess their condition. a flag was added to patients estimated to be at high-risk, thus allowing optimization of resources and more all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint focused virtual follow up and also helping clinicians to triage these patients if their symptoms worsen. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . there was no funding for the study. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . we learn a model for flu complications on an age-matched training set, and evaluate the following probabilities -= ( = | + ) and = ( = | + ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ ); all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . and get the formula in the paper. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint clinical characteristics of coronavirus disease in china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease (covid- ): a meta-analysis prediction models for diagnosis and prognosis of covid- infection: systematic review and critical appraisal clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study prediction of progression from pre-diabetes to diabetes: development and validation of a machine learning model key: cord- -l f gp authors: nan title: oral and poster manuscripts date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: l f gp nan pandemic influenza h n (h n pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. , it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h n pdm and seasonal h n influenza virus in the human respiratory tract, however h n pdm virus differed from seasonal h n influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h n with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h n pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into - mm fragments within h of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. viruses investigated included h n pdm (a ⁄ hk ⁄ ⁄ ), swine h n virus (a ⁄ swine ⁄ hk ⁄ ⁄ ), which shares a common derivation for seven genes with h n pdm, a natural swine reassortant h n (a ⁄ swine ⁄ hk ⁄ ⁄ ), which has acquired the na gene from h n pdm and other swine influenza h n viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h n and pandemic h n swapped were also studied. lung fragments were cultured at °c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at and °c respectively. tissue fragments were infected for h and incubated for , , and h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h n viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus and a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus (with the na from h n pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h n pdm virus, but not seasonal h n virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h n pdm, seasonal viruses, and the swine related h n viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h n pdm, but not the polymerase genes, were required for the conjunctival tropism of h n pdm ( figure ). in contrast with highly pathogenic influenza h n virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) we studied were similar to h n pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h n pdm virus differs in modest but subtle ways from seasonal h n virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h n pdm virus. the pandemic reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus isolated from swine with the na from h n pdm shares with h n pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [ ] [ ] [ ] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, others support replication of a virus, but do not enable the formation of mature viral particles, whereas others show only a weak level of replication or are not permissive at all. caco- cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco- cells is as effective as in mdck, and sometimes is even better. the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. , influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h viruses provoke more strong apoptotic response than h viruses do ). nevertheless, it has been demonstrated that caco- cells do not follow the apoptotic pathway and die through necrosis. the sjpl cell line also dies through necrotic pathway and not apoptosis. the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h n a ⁄ kurgan ⁄ ⁄ , low pathogenic avian h n a ⁄ gull ⁄ kostanai ⁄ ⁄ , swine h n a ⁄ swine ⁄ ⁄ , human h n v a ⁄ california ⁄ ⁄ , human h n v a ⁄ saint-petersburg ⁄ ⁄ , human h n a ⁄ brisbane ⁄ ⁄ , and human h n a ⁄ brisbane ⁄ ⁄ . the viruses were propagated in -days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at ) °c for further use. to evaluate tcid for each virus on all cell cultures, -well plates were used. the cells were seeded ae ml per well (concentration of - ae · cells ⁄ ml). the confluent -h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then ae ml of tenfold viral dilutions from viral aliquots were added and left for min for contact at °c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin ( lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes ( ae %). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration - · ) were inoculated with viral dilutions (moi = - ). after min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in -well plates ( ml in each well). the results were fixed after h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst- staining. cells were grown in -well plates with seeding concentration · cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = - ) and left for h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h n and h n viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h n v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv- , l- , and rd lines. a- and a- were poorly infected, as well as all suspension cell lines tested. seasonal human h n , as well as h n viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t- g cell lines. strikingly, swine influenza virus and human pandemic h n v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv- , rd, and t- g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h n v. swine influenza virus differed because it infected weakly a- and girardi heart cell cultures, which was not the case for h n v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst- staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a- , ecv- , and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a- turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h n and h n viruses and h n viruses induced apoptosis at about h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h n v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table ). we've shown earlier thus, we've tested the ability of swine and pandemic h n v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h n virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h n v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv- endothelial line and t- g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. , also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, , and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table . replication, apoptosis induction, and np synthesis of influenza viruses in a- , ecv- and flech cell cultures. the numbers represent the log tcid ⁄ ae ml calculated by reed-muench method as described in. the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst- staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h n v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h n v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. , in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in - and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin u ⁄ ml, streptomycin mg ⁄ ml, and gentamicin lg ⁄ ml), incubated for minutes at room temperature, and centrifuged at g for minutes. the supernatant ( ae ml ⁄ egg) was inoculated into the allantoic cavity of four -day old embryonated hens' eggs as described in european union council directive ⁄ ⁄ eec. embryonic death within the first hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at °c for days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive ⁄ ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive ⁄ ⁄ eec. rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised ae ll of dmpc water, ll of · first strand buffer (invitrogen), ae ll of mm dntp mix (amersham biosciences), ll of mm uni primer, u of rnaguard (amersham biosciences), u of mmlv reverse transcriptase (invitrogen) and ll rna solution in total volume of ll. the reactions were incubated at °c for minutes followed by inactivation of the enzyme at °c for min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained · platinum taq buffer (invitrogen), lm dntp, ae mm mgcl , nm each of fw primer and rw primer, u platinum taq dna polymerase (invitrogen) and ll cdna. reactions were placed in a thermal cycler at °c for min, then cycled times between °c seconds, annealing at °c for seconds, and elongation at °c for seconds and were finally kept at °c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version ae chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version ae ae ae with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version ae ) software using neighbor joining tree inference analysis with the tamura-nei c-model, with bootstrap replications to assign confidence levels to branches. [ ] [ ] [ ] [ ] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [ ] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj , fj ae , fj , fj , gu -gu for ha and fj , fj ae , fj , fj , gu -gu for na. avian influenza prevalence in our study h , h , and h influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year to , and previously published in the genbank. phylogenetic analysis of the h ha gene showed that all viruses separated into the american and eurasian lineages ( figure ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h ha genes co circulate in a fashion similar to avian h ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade ae ) h n virus in the last years, while h n outbreaks in korea and japan may represent the longitudinally transmitting pathway. ha of subtype h so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. further we analyzed na genes of influenza viruses (figure ) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, and they show little amino acid changes. , the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h aivs have occurred frequently in chicken farms in the world. although vaccination is not permitted, h n aivs have circulated in taiwan for a time. the seroprevalence in chicken flocks reaches about % in the field. h n aivs invades internal organs, such as kidney and lung. thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h n aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized : in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at g for minutes. the supernatant was passed through a ae lm filter. the homogenate was examined for the presence of virus by inoculation into five -to -day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h n aivs were isolated in this laboratory during and from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of chicken h n aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g ⁄ , dq ; g ⁄ , dq ; ⁄ , dq ; na ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ns ⁄ , dq ; sp ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; a ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ch ⁄ , dq ; ⁄ , dq ; a ⁄ , dq and ⁄ , dq . the viruses isolated were propagated in the allantoic cavities of -day-old embryonated spf eggs for hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with lg of virion at week . five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp ⁄ -ag ). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with % fetal bovine serum at °c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding % polyethylene glycol- . the resulting pellet was plated into well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at ) °c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch -d , eb -b , eb -e , eb -f , ff -f , and ff -f , respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with % chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from % to %). the extent of drop in egg production depended on the chicken ages. for example, the age of case was weeks, a stage of increasing egg production. however, after h n aiv infection, the egg production decreased % instead of increasing and then stayed at % for a week. the infected chickens showed signs of decreasing activity, anorexia from g per bird to g per bird, and respiratory signs. case showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h n viruses, we found that amino acid changes in ha were higher than those in ha , showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h n viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a- s, i s, v i, n s, e k, l m, e d, q k, a v, or t, s n, s r, k n, y d, n t, s i, g d, l v, i v, g e, t n, g s, a v, k e, d n, i m, and m i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch -d , all other monoclonal antibodies elicited from v ⁄ showed different hi titers with the different h n viruses (table ). however, those mabs showed negative hi to and , the early h n strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h -h , n -n ) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the th century emerged from this region. the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h n , h n , and h n viruses, have become established and long-term endemic in different types of poultry in this region. [ ] [ ] [ ] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h n , h n , and h n virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august onwards. cloacal swabs and fresh fecal droppings were taken. about birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing ae ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in ) °c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during during - a total of cloacal swabs from migratory ducks and cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, influenza isolates were obtained from migra- tory ducks and from sentinel ducks; isolation rates of ae % and ae %, respectively (table ) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡ ) were identified from blood samples collected during november and december in . among these, samples were positive to h , were positive to h , were positive to h , and were positive to h . one serum sample was positive to both h and h , which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [ ] [ ] [ ] [ ] [ ] [ ] , the overall virus isolation rate from migratory ducks was less than %, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during years of surveillance. the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h , h , h were the predominant subtypes, while h , h , and h were the major subtypes in sentinel ducks. hpai h n was only detected from migratory ducks in early on two sampling occasions. from phylogenetic analyses the h n viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. therefore, it appears that h n viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h viruses were detected from sentinel ducks at poyang lake during this period. whether h n virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in did not match well with the epidemiological results during [ ] [ ] [ ] [ ] [ ] [ ] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h n influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ ⁄ (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g ⁄ (g -like) have become established and endemic in poultry in southern china. these established h n lineages continue evolving to generate many different reassortant variants (or genotypes) , and are causing sporadic cases of human infection. , studies of h n viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. , in addition, h n viruses isolated beyond the late s had preferential binding with a- , -neuacgal human-like receptors. these observations suggest that the h n influenza viruses still have pandemic potential. unlike highly pathogenic h n influenza viruses that have been rarely detected in the live-poultry markets in hong kong since , h n viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h n viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from to . a total of terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january and december . of those samples, were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in -to -day old embryonated eggs as described previously. virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. dna sequencing was performed using bigdye terminator v ae cycle sequencing kit on an abi dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene ae (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version ae . all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli ae . estimates of the phylogenies were calculated by performing neighbor-joining bootstrap replicates using paup* ae . systematic surveillance of live-poultry in hong kong from to resulted in h n isolates from samples (overall isolation rate, ae %) ( table ). there were strains isolated from chicken samples (isolation rate, ae %). of these viruses, four were isolated from tracheal swabs (isolation rate, ae %), while isolates were isolated from cloacal or fecal swabs (isolation rate, ae %). an additional isolates were collected from drinking water samples (isolation rate, ae %). there were strains of h n viruses isolated from minor poultry samples (isolation rate, ae %) ( table ) . of these viruses, only one was isolated from tracheal swabs (isolation rate, ae %), whereas strains of viruses were isolated from cloacal or fecal swabs (isolation rate, ae %). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with strains isolated from drinking water samples (isolation rate, %). taken together, these findings suggest that the h n viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h n viruses in the field. to better understand the evolutionary pathway of h n viruses in southern china, representative viruses, isolated from hong kong live-poultry markets from to , were sequenced and genetically characterized. phylogenetic analysis of the h ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g -like ha gene ( figure ). this is the first time the g like h ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. subgroup is represented by qa ⁄ st ⁄ ⁄ and subgroup is represented by dk ⁄ hk ⁄ y ⁄ . all h n viruses in this study belonged to subgroup of the ck ⁄ bei-like lineage except for the virus with the g -like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb , pa, np, ns and m genes clustered with those of h n lineage viruses previously prevailing in ter- restrial poultry in southern china. phylogenetic analysis of the pb gene revealed three different lineages; g -like (n = ), ck ⁄ sh ⁄ f ⁄ -like (n = ), and unknown avian (n = ). the sh ⁄ f ⁄ -like lineage (or f ⁄ -like) was previously reported in eastern china and was used previously for vaccine production in an intensive vaccination program. this pb gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b and b , which were also the prevailing reassortants found in other provinces in southern china since . the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h n influenza viruses isolated from live poultry in hong kong markets from a year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt ⁄ , had a g like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h n strains isolated in . g -like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [ ] [ ] [ ] and minor poultry, mainly quail, in southern china. whether the g -like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h n virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt ⁄ strain clustered with other g -like strains isolated previously in minor poultry in southern china, the g -like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b and b persistently occurred in either chickens or other minor poultry species from to . other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ -like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ -like lineages which are becoming dominant in northern and eastern china. , animal experiments have also demonstrated that f ⁄ -like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. since the f ⁄ -like lineage of the pb gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h n progression estimates that ( - ) pigs might be exposed to the virus on the basis of cases among swine owners ( - ). a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april . pig farms were randomly selected from nine communes representative of at risk area of avian h n . in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h , h , and h subtypes in pigs and h and h subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version ae ae (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < ae . herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < ae ) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of ae % at a confidence level of %, in an infinite population within these regions (win-episcope ae ). six hundred-and-nine pig sera were tested in nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h n in the red river delta raised by ae % [ ae ; ae ] in april . but among seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised ae % [ ae ; ae ]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table ) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types and seem to be specialized in fattening, the type produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table . typology of farming system type : large fattening farms largest scale production, with more than pigs per year specialized in fattening, and purchase more than pigs per year type : small fattening farms small scale of production, with less than pigs per year specialized in fattening, and purchase less than pigs per year type : medium breeding-fattening farms medium scale of production, with less than pigs per year breeding and fattening piglets, with rare purchase common variance [$n( ,r herd)]. the univariate analyses were conducted on variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < ae ) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table , the farming system type got a higher seroprevalence of ae % [ ae - ae ] and a higher risk indicator, with or = ae (p-value = ae ) in comparison with type . this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = ae [ ae - ae ]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed %. the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. the pandemic h n virus of (h n pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h n pdm. , the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of and arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. the pandemic was the first to arise for over years and the first to occur after the understanding that pandemics arise from animal influenza viruses. systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h n pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h n pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. while h n pdm has been genetically and antigenically stable in humans, reassortment between h n pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h n and highly pathogenic avian influenza (hpai) h n have also been occasionally identified in pigs in parts of asia. , it has been shown that h n pdm readily reassorts with h n to generate viable progeny in vitro. it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. , virus detection by rt-pcr a subset of recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a -well plate format. total viral nucleic acids were extracted in a -well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, ll of sample was lysed in ll buffer al, supplemented with ae lg carrier rna in a s block (qiagen), which placed the samples into a well plate format. after protease digestion, samples were transferred to silica based membrane in well plate format for binding. following two washing steps, rna was eluted in ll of elution buffer (buffer ave) into a well elution microplate cl (qiagen) . for the synthesis of cdna, ll of purified rna was used in a ll reaction containing ll of · buffer, ae nm of each deoxynucleotide triphosphate (dntp), mm dithiothreitol, lg random primer, u of rnaseout recombinant ribonuclease inhibitor, and u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp thermocycler (applied biosystems) with the following parameters: minutes at °c, minutes at °c, and soak at °c. subsequent to the reactions, ll of cdna was diluted ⁄ by adding ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrÒ green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, ll of ⁄ diluted cdna was amplified in a ll reaction containing ae ll of · power sybr green pcr master mix, nm of forward primer m c ( ¢-ctt cta acc gag gtc gaa acg- ¢) and nm of reverse primer m r ( ¢-agg gca ttt tgg aca aag ⁄ t cgt cta- ¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. real-time pcr was performed in the abi fast system (applied biosystems) with the following cycling conditions: minutes at °c once, seconds at °c, and minutes at °c for cycles, followed by melting curve analysis with seconds at °c, minutes at °c, and seconds at °c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ ⁄ (h n ) were included as standards to perform absolute quantification. a manual baseline was set from cycles - and a manual cycle threshold (ct) was set at ae . samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at bp in length. a total of tracheal and nasal swabs were processed during the years january -april and yielded influenza virus isolates, an overall virus isolation rate of ae %. of these, were subtype h (classical swine, eurasian avian-like swine, and triple-reassortant), were human-like h viruses, and were eurasian avianlike swine h n viruses. culture in mdck cells yielded % of h subtype viruses, % of the human seasonal-like h n viruses, and ae % of the avian-like eurasian swine h n viruses. culture in embryonated eggs yielded ae % of the h subtype viruses, % of the human seasonal-like h n viruses, and ae % of eurasian avian-like swine h n viruses ( figure ). tracheal and nasal swabs each gave comparable overall virus isolation rates ( ae %). however, isolation rates for human-like h n viruses were ae fold higher in nasal swabs ( ae % versus ae % respectively; p = ae ) ( figure ) . a parallel evaluation of rt-pcr and culture was carried out in specimens. rt-pcr detected ⁄ ( %) of the culture positive specimens. rt-pcr was also positive in ⁄ ( ae %) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h n viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h n viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, kong, - introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, while the pandemics of and had acquired the viral haemagglutinin, pb polymerase, and in , the neuraminidase gene segments from the avian gene pool. the major regional outbreaks of highly pathogenic avian influenza (hpai) h n in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out - times weekly from to during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than ducks and grebes congregate every winter. fecal droppings were collected and transported in vials containing ae ml of vtm, which was prepared from m ( ae g ⁄ l), penicillin g ( · u ⁄ l), polymyxin b ( · u ⁄ l), gentamicin ( mg ⁄ l), nystatin ( ae · u ⁄ l), ofloxacin hcl ( mg ⁄ l), and sulfamethoxazole ( g ⁄ l). an aliquot of ll from each swab sample was inoculated into the allantoic cavity of a -to -day-old chicken embryonated egg, and incubated for days at °c. positive ha isolates were subtyped using standard antisera , and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, followed by sequencing on abi prism xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. during the -year surveillance period, a total of influenza viruses were isolated from samples collected, an overall isolation rate of ae %. a total of isolates were obtained from specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was ae % in - . this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h -h and neuramidinase (na) subtypes of n -n , were all from wild waterfowl ( table ) . out of the twelve ha subtypes isolated, h and h were the two subtypes that were isolated frequently every year for h and in six out of seven years for h , respectively. h and h viruses accounted for ae % and ae % of all virus isolated, respectively. on the other hand, h , h , and h were the least prevalence ( ae %) and were only isolated once in years. of the na subtypes, n and n were isolated most often ( ae % and ae % of all isolates, respectively) and n was the least ( ae %). november was the month that had the highest prevalence of influenza virus ( ae % of samples being positive) compared to only ae % in march. the subtype's variation was the most diverse in december during our years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly ( ae %) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h viruses isolated in this study clustered with that of the other lpai h viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h n . satellite tracking of eurasian wigeons and northern pintails in dec and revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h n viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over dead wild birds were tested positive for hpai h n and has been reported elsewhere. our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h n, although dead wild birds have been regularly found to have hpai h n virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h n influenza virus emerged in mexico in april and rapidly spread worldwide, causing the first influenza pandemic of the st century. most confirmed human cases of h n ⁄ influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h n ⁄ viruses tested are resistant to m blockers. therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, but oseltamivir-resistant variants with h y na mutation (n numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. , in view of the high prevalence of oseltamivirresistant seasonal h n influenza viruses in - , the isolation of resistant h n ⁄ viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h n ⁄ clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis ( mg once daily). wild-type influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ ⁄ virus. to assess growth kinetics of viruses, confluent mdck or mdck siat cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately ae pfu ⁄ cell (single-step) or ae pfu ⁄ cell (multi-step). supernatants were collected every h or h p.i. for time points. a modified fluorometric assay using the fluorogenic substrate ¢-( -methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. the drug concentration required to inhibit % of the na enzymatic activity (ic ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every seconds for minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of ae pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism software. young adult ferrets ( - months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h n and h n viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with tcid virus in ae ml sterile pbs . after the donor ferrets were confirmed to shed virus on day p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for days. nasal washes were collected from donors and recipients on day , , , , , , , and p.i. by flushing both nostrils with ae ml pbs, and tcid titers were determined in mdck cells. serum samples were collected weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h n ⁄ viruses differed only at na amino acid position , where the pandemic a ⁄ dm ⁄ ⁄ virus had an h y amino acid mutation caused by a single t-to-c nucleotide substitution at codon . the wild-type a ⁄ dm ⁄ ⁄ was susceptible to oseltamivir carboxylate (mean ic : ae nm), but the a ⁄ dm ⁄ ⁄ carrying the h y na mutation had ic values approximately - times of the wild-type viruses (mean ic : nm). the ic of zanamivir was comparable for both viruses and were uniformly low (mean ic £ ae nm). the h y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = lm) and lower v max (mean = u ⁄ sec) than na of the sensitive virus (k m , mean = lm; vmax, mean = u ⁄ sec). the results suggested that the h y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least - logs in comparison to that of wild-type virus (p < ae ). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ ⁄ or oseltamivir-resistant virus shed virus productively until day or day p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ ⁄ virus (table ). in a ⁄ dm ⁄ ⁄ virus group, two of direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log tcid ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day p.i. duan et al. and of respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ ⁄ group, only of direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table ) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ ⁄ group (table ) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h y mutant of pandemic h n virus, a ⁄ dm ⁄ ⁄ virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h n ⁄ viruses within host, a donor ferret was co-inoculated with a : ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ ⁄ or a ⁄ dm ⁄ ⁄ virus (table ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure ). two of direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ ⁄ virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ ⁄ virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h n ⁄ viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h y mutant of pandemic h n virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h y mutant of h n ⁄ used in this study was the first oseltamivir-resistant h n ⁄ isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. as this study was undertaken, additional h y mutants of h n ⁄ viruses have emerged in the absence of oseltamivir use. , the emergence of these viruses should raise concerns as to whether resistant h n ⁄ viruses will acquire greater fitness and spread worldwide as the naturally resistant h n viruses did during the - season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h n ⁄ clinical isolates in the animal models. , one of the studies, which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. in another study, two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h y mutants of pandemic h n ⁄ virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. , the pandemic h n ⁄ virus (ph n ⁄ ) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [ ] [ ] [ ] a primary question remaining to be answered is whether the ph n ⁄ -like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph n ⁄ first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. , likewise, the trig-like viruses were never reported in europe. in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. , this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h n and h n viruses in pigs. , among them, two representative reassortants showed the most similar genotypic characterization to the ph n ⁄ virus, the sw ⁄ hk ⁄ ⁄ (h n ) and sw ⁄ hk ⁄ ⁄ (h n ), which respectively harbor seven and six gene segments closely related to the pandemic strains. , to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph n ⁄ -like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph n ⁄ virus, a ⁄ california ⁄ ⁄ (ca ), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph n ⁄ -related swine influenza viruses were isolated through our surveillance program in south china as previously described. , the a ⁄ swine ⁄ guangdong ⁄ ⁄ (h n , gd ) virus was a ph n ⁄ -like swine isolate. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), a recent pandemic reassortant progeny, had a ph n ⁄ like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level (bsl- ) containment laboratories. pigs ( - week old, n = - ) and ferrets ( month old, male, n = ) were intranasally infected with pfu of each virus, and mice ( ) ( ) week old, female balb ⁄ c, n = ) with a dose of pfu. naïve uninfected pigs (n = ) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. statistical analysis was performed by mean analysis with pasw statistics (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < ae . the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with pfu of hk experienced the most severe body weight loss ( ae ± ae %) but started to recover after days post-infection (dpi). hk caused similar peak body weight loss ( ae ± ae % on dpi) in mice as did ca ( ae ± ae %, on dpi), but the onset of clinical signs and weight loss (on dpi) was day later than those caused by the other three viruses. the gd -infected group suffered the least body weight loss ( ae ± ae %, dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on dpi (p > ae ), mice inoculated with gd consistently showed the lowest lung index (lung weight ⁄ body weight, %) on , , and dpi (p < ae ), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk group was higher than that from any other groups on and dpi, indicating the marked virulence of hk in mice. notably, virus titer of hk in the nasal turbinate was lower than the other groups both on and dpi (p < ae ), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph n ⁄ or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with pfu of each virus, all groups of ferrets experienced transient body weight loss for - days, except for those infected with gd , which showed no significant weight loss (p > ae ). although ferrets from the ca -infected group reached their peak weight loss ( ae ± ae %, dpi) one day earlier than those from the hk and hk groups, they began to regain body weight quickly thereafter. hk -infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd -infected group at dpi. comparatively, ferrets inoculated with hk had the most retarded body weight recovery, which did not get back to the baseline level until dpi. hk was only detectable in the nasal wash on dpi, whereas the duration of virus shedding for gd , hk , and ca was - days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph n ⁄ virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk ( ae - ae log tcid ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on and dpi revealed that pigs infected with hk had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on dpi also suggested the best replication for hk in the lower respiratory tract. on days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = - ) except for one from the gd contact group. however, on dpc, its hi titer reached , indicating slower seroconversion. this study revealed that both the pandemic h n and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk ), the hk (h n ) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. among the three pandemics that occurred in the last century, we learned that the h n and the h n pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). studies on the emergence of the catastrophic spanish h n virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, , or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. over years since the last pandemic, the first pandemic in the st century arose in and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb , pb , pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. , analysis of the pandemic h n viruses failed to identify known molecular markers predictive of adaptation to humans. the ''triple reassortant'' swine influenza viruses emerged in late s in north america is a reassortant between classical swine (descendent of the virus after adaptation in swine population), avian, and human influenza viruses. the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late s. , while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, , sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h n viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h n , triple reassortant h n , eurasian swine h n , and a range of reassortants between these three virus lineages. , ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [ ] [ ] [ ] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h n influenza virus [a ⁄ wuhan ⁄ ⁄ (wuhan )] and pandemic h n influenza viruses [a ⁄ california ⁄ ⁄ (ca )] were included for the study. swine influenza viruses that are genetically related with the pandemic h n virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk ), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ ⁄ (h n ) (swar ), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk )]. swhk contains seven gene segments (pb ,pb ,pa,ha,np,m,ns) closely related to the pandemic h n viruses. transmissibility was tested in -to -month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £ against human seasonal influenza h n (a ⁄ tennessee ⁄ ⁄ ), h n (a ⁄ brisbane ⁄ ⁄ ), and influenza b (b ⁄ florida ⁄ ⁄ ) prior the experiments. in each virus group, three ferrets were inoculated with tcid of the virus. at day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (> fold rise in the post-sera collected after - days post contact). experiments were performed in the p + laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at tcid inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at dpi. lower peak titers were detected from swhk and swhk inoculated ferrets, however, the differences were not statistically significant (table ) . tissues collected from inoculated ferrets at dpi showed that pandemic h n and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan ) and pandemic h n viruses (ca ) transmitted most efficiently via direct contact route as the virus can be detected on dpi from direct contact ferrets, and the peak titers were detected on dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar and swhk viruses as the virus can be detected from direct contact ferrets at dpi, with peak titers detected at dpi or dpi. classical swine-like swhk showed least efficient contact transmission as virus could be detected from all direct contacts only at dpi, and the peak titer detected on dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan ( ⁄ ), pandemic h n influenza virus ca ( ⁄ ), as well as swine precursor virus swhk ( ⁄ ). transmission of wuhan and ca to aerosol contacts was detected at dpi or dpi, while transmission of swhk was detected later at dpi, suggesting that the swhk virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h n viruses (ca ) or classical swine-like virus (swhk ), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk , which shares a common genetic derivation for seven genes with h n pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avian- like swine viruses also contributes to the transmissibility of h n pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h n subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h or h subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein as well as other virulence factors that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h n that is consistent with systemic infection , and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h n have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h n infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h n infection is lethal in % of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date cases have been reported to the who, although many more people have potentially been exposed to h n through contact with infected bird populations. some studies have suggested that genetic factors may predispose an individual to severe h n disease, but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, swayne et al. observed that exposure to a virulent h n strain a ⁄ vietnam ⁄ ⁄ by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h n following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h n hpai strain a ⁄ vietnam ⁄ ⁄ . as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with egg infectious dose of a ⁄ vietnam ⁄ ⁄ in a volume of ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > % weight loss and ⁄ or evidence of neurological signs, discussed in ; animals that did not reach the humane endpoint were euthanased on day after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ ⁄ virus. the majority ( %) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure ). ferrets infected orally had an improved chance of survival, as only % of animals developed severe disease (figure ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as of surviving ferrets developed h specific antibodies by day post-infection, and they did not have pre-existing antibodies to h n (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > % weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table ) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. viral load in nasal washes and oral swabs taken at days , , and post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table ) , whereas of ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral figure . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ ⁄ by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h n infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. most ferrets infected orally developed an h -specific antibody response which differs from the studies of swayne et al. in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h n into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) ae ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h n influenza viruses in a ferret model < ae b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea < ae ) ) nd ) nd lung ) < ae + + ) + spleen ae ) + + ) + small intestine ) ) ) ) ) + pancreas ) ae + ) + + the pandemic potential of highly pathogenic h n influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h n variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. the most commonly observed mutations are h y and n s in the influenza a n na subtype (n numbering here and throughout the text); e a ⁄ g ⁄ d ⁄ v and r k in the n na subtype; and r k and d n in influenza b viruses. h n influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, although oseltamivir-resistant variants with the h y na mutation have been reported in five patients after , or before drug treatment; and the isolation of two oseltamivir-resistant h n viruses with n s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h n influenza a ⁄ vietnam ⁄ ⁄ (vn-wt), a ⁄ turkey ⁄ ⁄ (tk-wt) viruses, and oseltamivir-resistant viruses with h y na mutation (vn-h y and tk-h y) were generated by using the -plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs; they were inoculated with tk-wt, tk-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days , , and p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h n viruses) was evaluated by the proportion of clones in day- nasal washes that contained the h y na mutation. na mutations were analyzed by sequence analysis of individual clones ($ clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic of the vn-h y and tk-h y viruses was and times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic of the tk-wt virus was $ times that of the vn-wt virus. all four recombinant h n viruses were susceptible to zanamivir. introduction of the h y na mutation reduced $ % and % of the na activity of vn-h y and tk-h y viruses, respectively, as compared to the wild-type virus activity (p < ae ; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day - p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table ) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose ( pfu ⁄ ferret), and the tk-h y virus caused less weight loss and fever than the tk-wt virus (table ) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h y and tk-h y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h y virus and the impaired fitness of tk-h y virus (table ) . although, the trend towards an increase ⁄decrease in the frequency of the h y na mutation relative to the wild-type was statistically significant (p > ae ) for two studied groups only. mutations within the na catalytic (r k) and framework (e a ⁄ k, i l, h l, n s) sites or near the na active enzyme site (v i, i t ⁄ v, q h, k n, a t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i v and e a could exert compensatory effect on the fitness of vn-h y and tk-h y viruses. the lethality and continuing circulation of h n influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h y na mutation affects the fitness of two h n influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ ⁄ -like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ ⁄ -like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day p.i., the fitness of vn-h y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ ⁄ virus died by day - p.i. and were observed once daily for days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e a and n s, which confer cross-resistance to oseltamivir and zanamivir, , can emerge spontaneously in clade . h n influenza virus in ferrets. further, we observed that mutations at na catalytic (r k) and framework (i l and n s) sites and in close proximity to the na enzyme active site (v i, i t ⁄ v, q h, k n, a t) emerged without drug pressure in both pairs of h n viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d v, f s, i v, e a, h l, and f s) that may affect the fitness of viruses with the h y na mutation. we suggest that na mutations at residues i v and e a are of importance. interestingly, we observed differences in predominance of i v and e a na mutations in different genetic backgrounds: i v mutation was identified in a ⁄ vietnam ⁄ ⁄ (h n )-like and e a in a ⁄ turkey ⁄ ⁄ (h n )-like genetic background. moreover, i v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h y viruses, but not in ferrets inoculated with vn-h y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h y na mutation causes a large shift in the position of the side chain of the neighboring e residue, which must form a salt bridge with r to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h n influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r- ) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [ ] [ ] [ ] [ ] [ ] [ ] we discovered a single intranasal administration of laninamivir octanoate (cs- ), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [ ] [ ] [ ] this suggested that cs- works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs- proved noninferiority in adult patients and significantly superior in child patients, compared to an approved dosage regimen of oseltamivir for treatment. cs- has been commercially available as an inhaled drug, inavir Ò , for the treatment of influenza in japan since october . the long-acting characteristics of cs- are explained by several reasons. first, cs- was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. in the following, the tight-binding ability of laninamivir to pandemic (h n ) na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs- in mouse based on a localization of an enzyme that hydrolyzes cs- to laninamivir. the influenza viruses, pandemic(h n ) (inf ), a ⁄ new caledonia ⁄ ⁄ (h n ), a ⁄ panama ⁄ ⁄ (h n ), and b ⁄ mie ⁄ ⁄ were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p- (bio-rad laboratories, hercules, ca, usa). the na substrate, -methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for hours at room temperature by measuring the fluorescence at an excitation wavelength of nm and an emission wavelength of nm. the enzyme which hydrolyzes cs- to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure the enzyme that hydrolyzes cs- to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. the mouse homolog of the rat esterase was carboxylesterase (ces ). the mrna of the mouse ces was shown to be highly expressed in lung and liver by the gene expression profile, and ces was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces gene and the ces gene lacking the signal sequence were exogenously expressed in the cos cells. the cs- -hydrolyzing activity associated with the cos cells expressing ces was recovered from the culture sup of the cos cells expressing ces lacking the retention signal sequence. localization of ces was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs- in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h n ) virus was also observed similar to that of seasonal h n virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs- in mice. we explain the mechanism by clarifying a cs- hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs- may enter inside cells. ces with er ⁄ golgi retention signal hydrolyzes octanoate of cs- figure . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for minutes. the background (only the na substrate [d] ) is also shown. a part of data from. to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs- move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h n ( ) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h n ( ) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in · dmem media (sigma, st. louis, mo, usa) supplemented with % fetal calf serum and antibiotics viz. unit ⁄ ml penicillin and lg ⁄ ml streptomycin at °c ⁄ % co . the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h n ( ) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at ) °c. plaque assay was performed as previously described by hui et al., . briefly, ae · mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for hours at °c ⁄ %co . the monolayer of the cells was inoculated with serially diluted virus samples for minutes at °c ⁄ %co . subsequently, a mixture of agar overlay was added, and the plates were incubated at °c for days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with ae % crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the % cytotoxic concentration (cc ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing % fcs before addition to the preformed monolayer of mdck cells in -well plates. a series of suitable controls for in vitro cc determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide (mtt) at end-point of hours post-incubation. the assay was performed as described by mosman . briefly, mtt stock at a concentration of ae mg ⁄ ml was prepared in · pbs. the media was aspirated from the wells and ll of mtt dye from the stock was added to each well. following incubation at °c ⁄ % co for - hours, the dye was very carefully removed from the wells, and the cells were incubated with ll of stop solution (dmso) per well at °c ⁄ % co for hour. the absorbance of the supernatants from each well was measured at nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a -well tissue culture plate at °c ⁄ % co . the cells were inoculated with various virus dilutions at °c ⁄ % co for minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after - hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid of the virus. the pre-formed monolayer of mdck cells was inoculated with the -fold dilution corresponding to tcid of the virus for hour at °c ⁄ co . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with % fcs) to various concentrations within their respective cc ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and ll media was collected from each experimental well for performing hemagglutination test. after h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at and nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h n ( ) virus. as a control, human glyceraldehyde- -phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. , immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [ ae m nacl, ae m tris cl (ph ae ), ae m edta (ph ae ), m m protease inhibitor cocktail, lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on % polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg ( : dilutions) as secondary antibody. the median cytotoxic concentration for compound meuh came out to be lm, and that for flh was lm. compounds showing potent antiviral effect on the pandemic influenza h n ( ) virus propagation in madindarby canine kidney cells ( figure ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed for the compounds meuh and flh were and lm, respectively. fifty-two percent (meuh) and % (flh) inhibition against the pandemic influenza h n ( ) virus was achieved using ed of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately - %, whereas approximately % inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m ionchannel blockers , (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors , (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs , has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h n ( ) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. and is now licensed in japan, while another, laninamivir, is being developed as an inhaled prodrug. resistance to nais among circulating influenza viruses was previously low (< % worldwide). [ ] [ ] [ ] however, the - influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h n ) viruses with the h y (h y in n numbering) in the na. [ ] [ ] [ ] [ ] [ ] [ ] the prevalence of oseltamivir resistance was even higher in the subsequent - influenza season with many countries reporting up to % oseltamivir resistance, seasonal and pandemic influenza viruses collected globally between october , and september , were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · ( ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic for the inhibition curve. box-and-whisker plot analyses of log-transformed ic s were performed for each virus type ⁄ subtype and nai using sas . software (sas institute, cary, nc, usa) to identify viruses with extreme ic values (outliers). outliers were characterized based on a statistical cutoff of ic greater than three interquartile ranges from the th percentile. outliers were subjected to genetic analysis by pyrosequencing and ⁄ or conventional sequencing to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic data, using sas . software (sas institute) for each nai and virus among seasonal influenza a (h n ) viruses tested for oseltamivir susceptibility (n = ), ( ae %) were outliers for the drug (table ) and harbored the oseltamivir-resistance conferring h y mutation in the na. by contrast, only a small proportion ( ae %) of tested h n pdm viruses (n = ) were resistant to oseltamivir. all influenza a (h n ) viruses (n = ) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, whose ic of ae nm was beyond the statistical cut-value off and > -fold the mean ic for the drug ( ae nm). all influenza b viruses (n = ) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ ⁄ , with d e (d e in n numbering) mutation in the na, whose ic was beyond the cut-off, but only fourfold greater than the mean ic for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table ) , except for some outliers among seasonal influenza a (h n ) and a (h n ) outliers. the seasonal influenza a (h n ) outliers included a ⁄ thailand ⁄ ⁄ (h n ) and a ⁄ hawaii ⁄ ⁄ (h n ), both with combined h y and d d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h and d in seasonal influenza a (h n ) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d are typically cell-derived and not present in clinical specimens. influenza a (h n ) outliers for zanamivir included a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, as well as a ⁄ maryland ⁄ ⁄ and a ⁄ vladivostok ⁄ ⁄ with d g and mixed d d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h n ) viruses with ic beyond the statistical cutoff but < -fold mean ic for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue namely, d d ⁄ g, d d ⁄ n, or d d ⁄ a. mutations at residue d of the na are associated with reduced susceptibility to zanamivir in a (h n ) viruses, but were reported to be cell-culture derived in recent h n viruses. all virus isolates tested for peramivir (n = ) were sensitive to the drug, except for h y variants among seasonal influenza a (h n ) and h n pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h n ) isolate, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, showed reduced susceptibility to peramivir. the ic values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. nevertheless, combining elevated ic values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic values for oseltamivir among seasonal influenza a (h n ) and h n pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h n ) viruses in this study were shown to harbor mutations at d , which were earlier associated with reduced susceptibility to zanamivir, and were cell-culture derived. the effects of d mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic values for oseltamivir and zanamivir than influenza a viruses. viruses exhibiting such mixes are typically excluded from statistical analyses of ic s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. and influenza virus gene segment (m genes) sequencing ( strains-genbank accession numbers: cy , cy , cy , cy , cy , cy , and cy ) and influenza virus gene segment (na gene) sequencing ( strains genbank accession numbers: cy and cy ) by the standard methods with applied biosystems xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by % (ic ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ ⁄ (h n ) with ae nm ic value and a pandemic influenza virus a ⁄ dundgovi ⁄ ⁄ (h n ) with ae nm ic value ( figure ). there was oseltamivir resistance detected in ae % ( ⁄ ) of seasonal a (h n ) and in ae % ( ⁄ ) of a (h n ) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all a(h n ) viruses analyzed for m channel inhibitor resistance by pyrosequencing contained the s n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment sequencing revealed that seasonal a(h n ) viruses possess the common s n mutation. of note, a single strain a ⁄ zavkhan ⁄ ⁄ (h n ) contained an unusual s d change in the m protein. our study shows that the same prevalence [ ae % ( ⁄ )] of seasonal a(h n ) viruses with h y mutation in ⁄ season in mongolia with the published data for ⁄ season from japan. , however the prevalence of oseltamivir resistance in japan has dramatically increased in ⁄ season to % ( ⁄ ). the observed double mutations: h y and d g in a ⁄ ulaanbaatar ⁄ ⁄ (h n ) strain, which have been also found in japan in ⁄ season. the patient from whom the oseltamivir resistant seasonal influenza h n virus has been isolated was a -year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h n )pdm virus was isolated was a year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h n ) viruses has been increasing in mongolia since ⁄ influenza season. all pandemic influenza a(h n ) strains ( ) tested were resistant to m channel inhibitors due to the presence of the s n mutation in the m protein. among seasonal a(h n ) viruses, one contained a s d change whereas the others had s n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s d change in the seasonal a(h n ) viruses. according to the cdc data (unpublished), the s d change conferred the drug resistance in the a(h n ) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s n mutation). of note, the genotype and genotype dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. , the genotype virus was the only dual resistant virus with a nearly complete c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade b na gene. although the detection of dual resistant seasonal influenza a (h n ) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: . % ( of tested in - ), . % ( of in - ) , and % ( of in - ) (v p < . ). while the continued circulation or co-circulation of seasonal a (h n ) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h n ) viruses also confer resistance in the more widely circulating pandemic a (h n ) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h n ) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h y mutation in h n neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star Ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of lm munana, ae mm mes, mm cacl , and ph ae in a ll assay volume, and performing the assay for minutes at °c following a minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of - were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star Ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately ae u ⁄ well and dynamic range of - orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic values similar to nisn-published, munana-based protocols. ic values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure . the large shift in ic values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to hours after stop solution addition when stored at room temperature and for several days when stored at °c (data not shown). ic values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of ae or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. the assay can tolerate up to % dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard minutes ⁄ °c endpoint mode described above or as real-time kinetic assay with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at °c is linear for at least hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically - fold lower than rfus acquired after addition of stop solution at the same time point. ic values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. we have shown that ni ic values for multiple viral strains remain unchanged when the assay is performed in the presence of ae % np- or % triton x- (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to % ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo- to test the prophylactic potency of h -vhhb, mice were treated intranasally with pbs, lg of h -vhhb, or negative control rsv-vhhb at , , or hours before infection with one ld of nibrg- ma virus. body weight loss was monitored daily, and on day mice were sacrificed to determine the viral load in the lungs. all mice that received h -vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h -vhhb at or hours before challenge resulted in undetectable lung virus titers. when animals were treated with h -vhhb hours before challenge, virus titers were fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure ). we next determined if h -vhhb nanobody Ò could be also be used therapeutically. we administered lg of this nanobody Ò intranasally to mice up to hours after chal-lenge with ld of nibrg- ma virus. four days after challenge, animals that received h -vhhb , , or hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h -vhhb nanobody Ò hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure ). to identify the ha amino acid residues that are potentially involved in h -vhh binding, escape viruses were selected by growth and plaque purification of nibrg- ma virus in the presence of h -vhhm or h -vhhb nanobodies Ò . the ha sequences of six independently isolated h -vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position in ha (h numbering). in addition, two h -vhhm escape mutants carried an n d and four carried an n s substitution. the three-dimensional structure of nibrg- ha shows that n d ⁄ s and k e are close to each other as part of the corresponding antigenic site b in h ha. , interestingly, the n d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h n ha as a strategy to mask an antigenic site. escape viruses selected in the presence of h -vhhb carried k n (n = ) or k e (n = ) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h -vhhm ⁄ b nanobodies Ò (figure ). the virus titer was measured in lung homogenates prepared on day after challenge. the x axis refers to the time points in hours relative to the challenge (time = hours) when ha-specific nanobodies (h -vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = - mice per condition: p values < ae (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h n influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h -vhhb has powerful h n -neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg- virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size ( kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k e substitution in ha abolished the neutralizing effect of h -vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h n virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position , which suggests that the conserved positively charged amino acid is important for neutralization by h -vhh nanobodies Ò . interestingly, escape mutants selected with h -vhhm also carried an n d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n in a ⁄ hong kong ⁄ ⁄ ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. , the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h -vhh nanobodies Ò . , we conclude that influenza virus neutralizing nanobodies Ò have considerable potential for the treatment of h n virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m ) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h n ) viruses with the h y substitution in the viral na genes (tables and ). some cases were associated with oseltamivir treatment on the basis of h y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables and ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for % inhibition (ic ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic ae and ae nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y or h (ic ae and ae nmol ⁄ l, respectively). one isolate of pandemic (h n ) virus with an oseltamivir-sensitive genotype (h in its na) was susceptible to oseltamivir (ic ae nmol ⁄ l) and zanamivir (ic ae nmol ⁄ l). patients with oseltamivir-resistant pandemic (h n ) were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found patients of oseltamivir resistance with h y mutation in the na gene of pandemic (h n ) virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph n infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during - : peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph n virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph n shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as pandemic influenza a (h n ), were tested for the h y mutation in the na using pyrosequencing. results were returned to sender within - hours of specimen receipt. from october until july , a total of specimens from patients were submitted for testing. viruses from ( %) of patients had h y mutation in the na in at least one submitted specimen. clinical information was available for patients (table ) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h y mutant; duration of chemoprophylaxis until ph n infection was detected varied ( - days). among the patients with an h y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h y mutation was days (range - days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h y mutants prior to oseltamivir treatment. patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph n virus infections (table ) ; among individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, ( %) had the h y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, ( %) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: ( ae %) of icu patients had oseltamivir resistance detected. during the pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april to july . overall, resistant ph n viruses were identified from april to july in the united states among tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. further studies to understand risk factors for oseltamivir-resistant ph n infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm , -dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star Ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star Ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, - including identification of a ⁄ h n pandemic virus resistant to oseltamivir. , in addition, this assay has been used for identification of new ni compounds, ni characterization, studies of virus transmission, drug delivery, na quantitation of virus-like particles, and cell-based virus quantitation. neuraminidase assays performed with chemiluminescent , -dioxetane substrates, including na-star Ò and na-xtd tm substrates, typically provide -to- -fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over - order of magnitude of neuraminidase concentration compared to - orders of magnitude with the fluorescent assay. the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a , -dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star Ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately hours (not shown), compared to $ minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton Ò x- -containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic values using data collected over hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ ( figure ) . triton x- detergent at % has been shown to inactivate flu virus while increasing neuraminidase activity. the addition of na sample prep buffer (containing % triton x- ) to virus stocks (at ⁄ volume, achieving a final concentration of %) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic values are unaffected by the addition of triton x- to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide -to -fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic determination assays. ic values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents ( ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems Ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star Ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. over hospitalizations and deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. - following the emergence of pandemic h n influenza, severe illnesses have also been observed in children and young healthy adults. the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [ ] [ ] [ ] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. , procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. this -aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. stimuli of proct include tnf-a, endotoxin, and other bacterial products. several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including h n , are associated with only minimal increases. , , of note, proct induction is attenuated by viral-induced interferon-c. a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. , therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡ years of age admitted to rochester general hospital (rgh) from november st to june th for two winter seasons ( - ) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of > neutrophils (pmns) and < epithelial cells per high power field. serum was collected at admission and hospital day for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is ae ng ⁄ ml (normal levels are ae ± ae ng ⁄ ml). mycoplasma and chlamydia pcr real-time pcr targeting the p adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. results fifty-one of ( ae %) illnesses evaluated tested positive for influenza virus. of these, were due to ''seasonal influenza'' ( influenza a ⁄ h n and influenza b), and were identified as ''pandemic influenza'' ( h n ). demographics of both groups were similar: mean ages ± and ± years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group ( % versus %, p = ae ), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in % and % of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in ( %) subjects ( -seasonal and -pandemic), and none were bacteremic. bacterial infections included: -s. pneumoniae, -m. pneumoniae, -s. aureus, and -h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day were: ae ± ae versus ae ± ae and ae ± ae versus ae ± ae , respectively, and were not significantly different (table ) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡ ae ng ⁄ ml than seasonal subjects [ ( %) versus ( %), p = ae ] ( figure a , b). of the four patients with proc-t > ae ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within hours of antibiotics) were collected in only ( %) subjects. of these, proct was ‡ ae ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and < ae ng ⁄ ml in with influenza alone and five associated with bacterial infection. in the with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ ae ng ⁄ ml for bacterial infection was %, specificity %, positive predictive value %, and negative predictive value %. notably, one patient considered to have influenza alone (proct - ae ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < ae ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr ( ae ± ae ng ⁄ ml versus ae ± ae ng ⁄ ml, p = ae ). combining patients with proct values ‡ ae ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were % and %, respectively. notably, antibiotics were administered to % of subjects despite % having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately % of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of - % with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [ ] [ ] [ ] bacterial pneumonia has been suspected or diagnosed in - % of patients in intensive care associated with h n infection and up to % of patients who died. , despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only ( ae %) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of patients with severe h n or bacterial infection necessitating intensive care, a threshold proct level of ae ng ⁄ ml, demonstrated % sensitivity and % specificity for bacterial infection. among patients with h n associated pneumonia, many of whom had respiratory failure, a threshold proct value of ae ng ⁄ ml provided a sensitivity of % and specificity of % for bacterial infection. access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. , elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of < ae ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡ ae ng ⁄ ml and those with a positive bacterial test, approximately % in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january , in a successful attempt to cure a -year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october , ) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. the necessity of an earliest possible diagnosis was confirmed in the pandemic of . large hospitals reported that patients with an h n ⁄ infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date seasonal infections, and in , h n ⁄ ) through clinical diagnosis, and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every - hours for every of our outpatients. among our patients with seasonal influenza, we saw within hours, within hours, and within hours after disease onset. for pandemic influenza, it was patients within hours, within hours, and two within hours. for all patients, we measured crp < ae mg and fibrinogen < mg ⁄ dl ( hours), crp < mg and fibrinogen < mg ⁄ dl ( hours), and crp > mg and fibrinogen < mg ⁄ dl ( hours, only seasonal cases). antibiotics were necessary in cases, heparin and oxygen administration in cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in % of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, ). mrsa and influenza viruses are posing problems worldwide. the case of a -year-old boy with h n ⁄ infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september , when h n infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h n ⁄ virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than ae mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo measurements and checking the humoral inflammation status every hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in outpatients with seasonal influenza and h n ⁄ outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of h n pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed h n pandemic influenza virus infection. there were virologically confirmed cases of pandemic influenza and deaths in the czech republic during pandemic wave. more than influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for h n pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in of fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths ( males, females) were analyzed. the rna of the h n pandemic influenza virus was detected by pcr in cases, while cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of confirmed h n pandemic influenza victims was ae years, age range - years and median ae years. the mean age of influenza negative victims was ae years, age range - years and median ae years. the % ci for the difference in the age between the two groups is ) ae ; ae . the test is statistically significant at the % level. the obtained significance (p = ae ) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the h n pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in , i.e., ae %, of autopsied patients). in the h n pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in , i.e., ae % of autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the h n pandemic influenza victims were younger than the patients who died with suspected but undetected h n pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h n ⁄ were adopted from assays as described previously to discriminate against seasonal human h n and h n viral segments (table ) . the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h n ⁄ . the method we employed in this study was a -step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described , and was converted to cdna in a universal rt-pcr. each ll rt reaction containing ae ll of purified rna, ll of · firststrand buffer (invitrogen), u of superscript ii reverse transcriptase (invitrogen), ae lg of uni ( ¢-ag-caaaagcagg- ¢), ae mm of deoxynucleoside triphosphates and mm of dithiothreitol was incubated at °c for minutes, followed by °c for minutes for heat inactivation. for each segment-specific real-time pcr, the ll reaction contained ll of a -fold diluted cdna samples, ll of fast sybr green master mix (applied biosystems), and ae lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as °c for seconds, followed by cycles of °c for seconds and °c for seconds, and all eight assays were performed simultaneously in a sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay ( - °c; temperature increment: ae °c ⁄ seconds). cdna of a ⁄ california ⁄ ⁄ virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. pcr product for each segment of pandemic h n ⁄ yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure ). reactions with tm value within sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h n ⁄ samples (n = ) were positive in all eight assays, while all seasonal samples (h n = ; h n = ) were negative in all assays, as expected ( figure and data not shown). these results showed that no reassortant of pandemic h n and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among swine viral isolates collected from to september , of them were recognized as pandemic h n ⁄ in all eight segments. they were confirmed to be of pandemic h n ⁄ origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. , the remaining viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h n or h n viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h n ⁄ . their melting curve patterns were very similar to those derived from segments of pandemic h n ⁄ , except for ha of tr lineage. our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses. the ha-specific assay was able to discriminate pandemic h n ⁄ from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h n ⁄ and its sister clade tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h n , h n , and h n ) and classical swine (h n ) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h n ⁄ viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january during the course of this study. this sw ⁄ hk ⁄ ⁄ has a previously unidentified viral gene combination as shown in figure . it was confirmed to be a reassortant between pandemic h n ⁄ and other swine viruses in full genome sequencing characterization. it has a pandemic h n -like n gene, an ea-like h , and the other six internal genes derived from tr swine viruses. , the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h n ⁄ infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h n ⁄ virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific h n influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies ( ll; lg ⁄ ml; abd serotec Ò , uk) in pbs (ph ae ) were dispensed into standard -well plates and incubated for - hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween ae % and pbs. commercial blocking solution ( ll, superblock Ò t ; pierce Ò , usa) was added and incubated for at least hour at room temperature. after successive washing steps with pbs-tween ae %, non-glycosylated histidine-tagged recombinant protein ( ll; lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h n virus. , after hour incubation, wells were washed for at least two alternating minutes cycles with pbs-tween and pbs. a : dilution of the serum or plasma sample to be assayed ( ll) was added to each well and incubated at room temperature for hour. after repeated alternating minutes pbs-tween ae % and pbs washes, anti-human igg antibody solution ( ll ⁄ well; : dilution in pbs-tween ae %) marked with horse radish peroxidase (pierce Ò , usa) was added and incubated for hour at room temperature. after repeated alternate washes with pbs-tween ae % and pbs), substrate solution ( ll; -step ultra tmb-elisa; pierce Ò ) was added to each well. after incubation for minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of m h so ( ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at nm in a biotek Ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction À abs albumin sample : absorbance values were normalized based on the average signal of non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of : in pbs-tween ae %. a recombinant receptor-binding domain of the ha of the influenza a ⁄ h n ⁄ virus, expressed in escherichia coli strains, was used as the elisa antigen. this kda protein, designated here as ha - -rbd, contained amino acids - of the influenza a ⁄ mexico ⁄ indre ⁄ (h n ) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co + or ni + ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h n , h n swine, and h n , was also tested by the elisa method using : dilutions. protein ha - -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h n ⁄ influenza subjects. dubois et al. demonstrated that this protein, produced in e. coli, folds properly into a -d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h n ⁄ virus. ha - -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h n hemagglutinins. the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h n ⁄ . samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h n ⁄ , were analyzed by elisa using the antigen ha - -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of ae units. only ae , ae , and ae % of these samples exhibited normalized absorbance values higher than ae , ae , and ae , respectively. no sample from non-exposed individuals presented an absorbance value higher than ae . variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h n ⁄ patients. exposure to the h n ⁄ influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample À abs albumin sample Þ=ðabs non exposed subjects À abs albumin sample Þ ð Þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure shows the analysis of human serum samples, including samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values ae - ae times higher than negative samples ( figure ). normalized absorbance values above ae suggested exposure to the virus, although, a more conservative threshold value of ae units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of ae , the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than ae , and % of them exhibited normalized absorbance values higher than ae . in general, high hi titers (> ) were correlated with normalized absorbance values higher than ae . figure a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h n ⁄ patients. all samples determined as positive by hi ( samples) were also positive by elisa. while sensitivity of the hi assay was ⁄ = ae %, the elisa method recognized all samples correctly as positive ( % sensitivity) when a threshold of ae or ae was used. figure b shows that sera from ferrets infected with other influenza strains (h n , h n swine, and h n ) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h n ⁄ infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april , a novel h n influenza virus emerged in north america and caused the first influenza pandemic of the st century. [ ] [ ] [ ] [ ] the pandemic h n (pdmh n ) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h n influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h n influenza viruses were first isolated in and continued to circulate in north america with very little antigenic changes (classical swine h n ) until . since , however, the antigenic make up of swine h viruses has shown increased diversity due to multiple reassortment events and the introduction of h n genes from human influenza viruses. currently, four swine h clusters (a, b, c, d) are found endemic in the north american swine population. , these swine h viruses show substantial antigenic drift compared to the classical swine h viruses. cluster d swine h is derived from current human h viruses, and there is a substantial antigenic divergence between classical swine h and human seasonal h viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh n virus. , , phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh n were circulating in the swine population for at least years prior to the emergence of the pdmh n virus in humans, although the pdmh n virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh n infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [ ] [ ] [ ] after the first report of pdmh n infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh n in swine as well. , [ ] [ ] [ ] the ample geographic range of pdmh n outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh n from other h strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh n . in this study, we developed an elisa assay to discriminate pdmh n strains from other swine and human h viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing % fbs. a ⁄ california ⁄ ⁄ ⁄ h n virus (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table . viruses were propagated in mdck cells and stored at ) °c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid ). three monoclonal antibodies ( b , h , and f ) against ha of pandemic h n were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab b , h and f were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl- -(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified h and f were selected as the capture antibody, and biotin-conjugated b was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified h and f ( ae and ae lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph ae ) was coated to -well plates (test well, t) for h at °c. at the same time, a control antibody was coated to -well plates (control well, c). after blocking the plates with % (w ⁄ v) non-fat milk in pbs for hour at °c, the samples were diluted in extract buffer ( %tween- , ae %bsa in pbs) and added to the wells ( ll ⁄ well, each sample was table . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at °c for hour. after four washes, ll biotin-conjugated b ( ae lg ⁄ ml) in dilution buffer ( ae % bsa in pbs) was added to the wells and the mixture was incubated for h at °c. following three washes, ll diluted hrp-conjugated streptavidin ( ae ng ⁄ ml) in dilution buffer was added to the plates. after incubation for h at °c, the plates were washed five times, and the binding developed using the tmb substrate system for minutes. the ratio of the average od value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values > ae were considered positive in the sandwich elisa. we developed three monoclonal antibodies, b , h , and f , against a prototypical pdmh n strain, a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh n strains. purified h and f were used as capture antibodies, whereas the biotin-conjugated b was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh n strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh n from other swine h clusters (a, b, c, d), swine influenza strains spanning these clusters were tested. these viruses were first diluted : in extract buffer, and then added to the coated plates. as shown in table , the t ⁄ c ratios of these viruses were < ae , and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ malaya ⁄ ⁄ (h n ), a ⁄ wsn ⁄ (h n ), and a ⁄ brisbane ⁄ ⁄ (h n ) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h , h , h , h , h , h , h , h , h , h , and h subtypes. more recently, the mutation d g in the ha of some pdmh n strains has been associated with exacerbated disease and altered receptor binding. [ ] [ ] [ ] [ ] [ ] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ ⁄ (h n ) carrying the d g mutation (engineered by reverse genetics). as described in table , our elisa could still capture the d g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh n strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh n viruses to determine the limit of detection (lod). as shown in table , in our elisa the highest positive dilutions of nl ⁄ and ca ⁄ were : and : , respectively. the lod of the sandwich elisa by tcid was ae · and ae · tcid ⁄ ml, for nl ⁄ and ca ⁄ , respectively. it is important to note that the t ⁄ c ratio from nl ⁄ and ca ⁄ viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ (h n ) did not show the same dependence and was always < ae , corroborating the high specificity of the sandwich elisa for pdmh n strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, nasal wash samples ae · ^ )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) -from ferrets, of those previously infected with ca ⁄ and shown positive by virus isolation, were tested. the samples were diluted : in extract buffer and then tested using the sandwich elisa. result showed out of positive samples by virus isolation were positive also by the sandwich elisa (sensitivity ae %). the samples tested that were negative by virus isolation were also negative in the elisa, indicating % specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh n with high sensitivity, [ ] [ ] [ ] [ ] [ ] [ ] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh n strain. the elisa not only shows high specificity for pdmh n strain, but also shows great sensitivity. the elisa could distinguish pdmh n strains from human seasonal h and h viruses and, more importantly, from other swine h viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh n from swine or human h viruses. it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh n is lower than that for human seasonal influenza viruses. , a study by kok et al. showed that sensitivity of the current rapid antigenic tests for pdmh n is only ae %, whereas that for seasonal influenza a is ae %. chen et al. developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between ae · to ae · tcid ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. compared to the ''gold standard''-virus isolation-our sandwich elisa showed ae % sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh n viruses. the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a &mv lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed nasopharyngeal swabs col- the detection of influenza h , h , b, and pandemic influenza (h )pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi fast real time pcr system using primers recommended by cdc, usa, and super-scriptÔ iii one-step rt-pcr and platinum Ò taq dna polymerase kits (invitrogen). the cycling protocol was: minutes at °c, minutes at °c, and cycles of seconds at °c, seconds at °c. rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. we detected ( ae %) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were ae % a(h n ), ae % a(h n ), ae % influenza b, and ae % a(h n )pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in ae % ( ⁄ ) on mdck cell culture and % ( ⁄ ) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table ) . average times needed for the final results for different methods were: hours for rt-rt-pcr, hours for dfa on r-mix and days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the - th weeks of , however the pandemic influenza detection peak was observed in the - th weeks of ( figure ). the outbreaks by seasonal influenza viruses was observed mostly among the children of - years of age, and pandemic influenza virus outbreak was observed mostly in the adults of - years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires times longer, and by mdck cell culturing, times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was % and influenza b viruses was ae %, which is lower than in similar spanish study. however our study illustrates similar results with a canadian study where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists , where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from patients sampled nine times in days (initial design - samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that % of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 's to provide data on virus shedding from the respiratory tract during influenza infection. recently, vk was studied in naturally acquired influenza infection. , these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [ ] [ ] [ ] [ ] however, among the studies used in a meta-analysis about viral shedding kinetics, the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of volunteers from which nasal samples were collected once a day over days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below %). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. we used the fedorov-wynn algorithm implemented in pfim . to maximize this determinant, which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at : am, we chose three possible hours ( : , : , and : ) for each day with respect of the sleep-time. to validate the design, we simulated datasets of volunteers with the optimised design obtained. we then estimated the population parameters using monolix . for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below %. where rse predicted is the highest predicted rse (here rse for w) with participants and n predicted = and rse min is equal to ae . considering that % of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by ae . we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at : , : , and : hours and twice on the third day post-inoculation at : and : ( figure ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that participants shedding virus or participants with ili symptoms are necessary if % of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. ??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted ae days. ??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = %), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure ). this is probably due to the fact that it was measured at day post inoculation, and that the inter-individual variability is much higher than at day . furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at : am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h n ) virus. patients who visited any of the clinics that belong to the influenza study group of the japan physicians association in the - and the - influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of · cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by ( ae %) of consecutive outpatients in the - season, as seen in table the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [ ] [ ] [ ] [ ] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the - influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were ae %, ae %, and ae %, respectively. these results were quite comparable to our results of the - season, in which the overall results of other ic kits were ae %, ae %, and ae %, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were ae % and ae % for influenza a, ae % and ae % for influenza b, % and ae % for pandemic (h n ) . self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h n ) . these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from ae % of the patients. the rate of successful collection was over % in the age groups of - and - years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained from consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h n ) . and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviÔ-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the - and - influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h n ) virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april , a mixed-origin h n influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h n ) virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h n ) virus was the main obstacle to their widespread use in private clinics. , with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h n ) virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h n ) virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ ⁄ ). during december , clinical specimens from patients with ili were collected at sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h n ) . the detection limit of the new kit against ha of a ⁄ korea ⁄ ⁄ virus was confirmed to be pfu ⁄ ml. by contrast, the detection limit against the np protein was pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table . among specimens, were tested positive by rrt-pcr and were tested positive by viral culture. among the rrt-pcr confirmed cases, were positive, and among the viral culture confirmed cases, were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was ae % ( % confidence interval (ci): ae - ae %) and specificity was ae % (ci: ae - ae %). with viral culture as the reference, the rat sensitivity and specificity was ae % (ci: ae - ae %) and ae % (ci: ae - ae %), respectively. when analyzed by the regions tested, the sensitivity ranged between ae % and ae % for rrt-pcr and between ae % and ae % for viral culture as a reference. among patients who had a record of their symptom onset and sample collection date, ( ae %) visited the clinic on the day of symptom onset, and ( ae %) visited day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at days after onset and reasonable on the day of onset or at day after ( table ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h n ) virus. in one recent study, the sensitivity and specificity of the new rat kit was % and %, respectively, and the ha protein for pandemic (h n ) was detected more sensitively than the np protein for influenza a virus. the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h n ) virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate ). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with % fetal bovine serum (invitrogen), ae mg ⁄ l penicillin (invitrogen), and mg ⁄ l streptomycin (invitrogen), with incubation at °c in a % co humidified atmosphere. influenza a ⁄ hong kong ⁄ ⁄ (h n ), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with ae mg ⁄ l penicillin (invitrogen), mg ⁄ l streptomycin (invitrogen), and mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at °c in a % co humidified atmosphere. virus stocks were aliquoted and stored at °c until use, and the % tissue culture infectious dose (tcid ) of the virus stock was determined by titration in mdck cells according to standard procedures, and the tcid of the stock virus was calculated by the method of reed and muench. to perform a microneutralization assay, mdck cells seeded at a density of cells ⁄ well in an e-plate was removed from the xcelligence system after approximately hour; growth medium was then removed, cells washed, and replaced with ll virus-medium. a human serum, which is known to contain high titre antibody against the h n virus was heat inactivated for min at °c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at tcid ⁄ ll. after incubation for h at °c in a % co humidified atmosphere, ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of tcid . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for minutes, the e-plate was then placed back onto the xcelligence system in the incubator and maintain at °c with % co , and the cell index values were measured every minutes for at least a further hour. the same procedures were performed with cells seeded in conventional well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after hour of seeding mdck cells at cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at ae log increments was carried out. wells infected with the undiluted virus ( tcid ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately hour of infection ( figure ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including log ( -folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond log have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h n virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure ). using influenza virus treated with serum dilutions up to and including a dilution of : , the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at hour of infection. at a serum dilution of : , the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around hour post infection, and this was reduced to hour when serum dilution of : was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately hour. for both serum dilutions of : and : , full cytopathic effect was observed microscopically at hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at : as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally tcid ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires - days after infecting the cells, with days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses including the pandemic h n are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from days to two. the saving could be substantial for work involving bio-hazard level ⁄ pathogens such as h n viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h n influenza a virus of swine origin, the pandemic a(h n ) , with transmissibility from human to human in april posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h n ) virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between and belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h n v. the who assay was found to have a slightly lower end-point detection limit ( ) dilution) in comparison to the new protocol ( ) ). specificity of the assays was % as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h n v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april , of a novel h n influenza a virus of swine origin, the pandemic (a(h n ) ), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h n ) . specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h n v; relab, italy) were evaluated by testing selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between and . • six purified a(h n ), a(h n ), and a(h n ) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from > specimens received by the liguria influenza reference laboratory between january st and december st, . since , nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡ aliquots. those not immediately processed were stored frozen at ) °c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between and included specimens positive for: no seasonal a(h n ) have been detected since january st, . furthermore, weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h n ), seasonal influenza a(h n ), new pandemic influenza a(h n ) , and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h n ) positive sample times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex Ò rv ace detection), and all specimens collected between january st and december st, and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h n ). typing and sub typing were performed using the who protocol and ⁄ or sequencing. viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h n v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table ) . amplification conditions were as follows: reverse-transcription °c for minutes, denaturation °c for minutes, then cycles of °c for seconds, °c for seconds. the entire amplification process extended for minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman Ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h , designed to specifically detect the hemagglutinin gene segment from a(h n ) . the seeplex Ò rv ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoÔ (dual priming oligonucleotide) technology and is designed to detect major respiratory viruses, respiratory rna (influenza a and b virus, parainfluenza virus type , and , respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc and e ⁄ nl ) viruses and dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. conventional viral culture was performed inoculating ae ml of each specimen into mdck-siat seeded into -well plates for influenza isolation. virus detection was performed by the hemagglutination test using ae % guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between and belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h n v. among specimens collected between january st and december st, , all fast set infa ⁄ infb and fast set h n v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among low titre a(h n ) positive samples at fast set infa ⁄ infb, ( ae %) were not detected by the who ⁄ cdc assay, but were positive using seeplex Ò rv . the who ⁄ cdc sw h primer-and-probe set works in ae % ( ⁄ ) and ae % ( ⁄ ) of high and low titre a(h n ) positive samples, respectively. all a(h n ) strains collected during and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv . one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: ae , sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: ae , a(h n ) primer-and-probe set, ct values: ae ) was not detected by either seeplex Ò rv or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h n ) showed that the detection limit of fast set infa ⁄ infb, fast set h n v, and seeplex Ò rv was identical ( ) ) and log lower than that using the who ⁄ cdc protocol ( ) ). a similar analysis with respect to a(h n ) and a(h n ) strains indicated that fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv ( ) and ) , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the ) a(h n ) and a(h n ) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than %. since samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h n ) samples collected during did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h n v positivity was always confirmed by seeplex Ò rv , which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex Ò rv is reported by kim who observed a detection limit of copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h n v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h n v primerand-probe set reacted selectively with a(h n ) target. a recent report demonstrated that the sw infa assay is not specific to a(h n ) and is able to detect both human and avian (h n ) influenza a viruses and so there is the potential for misidentification. high titre (ct ae and ae at fast set infa ⁄ infb) a(h n ) viruses did not react with fast set h n v primer-and-probe set (data not shown). available human a(h n ) sequences are similar within the h n v primer-and-probe regions, but having - mismatches in the forward primer and, more notably, two of the mismatches occurred within nucleotides of the end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h n ) . a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (> % of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between and , the publix-usf partnership resulted in administration of over thirty thousand influenza a (h n ) vaccinations, % of which were given to adults over years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h n vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over % of the monetary outlay used for immunization. until , the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over year olds would save an estimated quality-adjusted life years in a cohort of approximately half the world's population. widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. , in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. , recently, in , influenza vaccine administration was reported to reach between % and % of the elderly population, which denotes varying degrees of success within each particular country. , however, there was also a report of a uniform plateau effect at around % of the population, beyond which additional vaccination coverage was difficult to achieve. physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $ for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between and , the publix-usf partnership vaccinated people against influenza a (h n ), of which were florida residents. the age range was - years old with a median age of years old. seventysix percent of the participants were over years old (see figure ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed % of those vaccinated resided in zip code areas where the average household income was <$ per year. of those remaining, % had an average income of $ -$ per year, and % had an income of >$ per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. , conclusion this unique university-corporate partnership successfully delivered h n vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach % of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from scottish health boards, we estimate the joint probability distribution ( figure ) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure ). as shown in the table, for % pandemic case reporting rate and detection specificity of %, our method is % sensitive and has mdt of weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, % and % sensitive with mdt of weeks. at % specificity, our method remains % sensitive with mdt of weeks. although the threshold method maintains its sensitivity of % with mdt of weeks, sensitivity of mov-avg cusum declines to % with increased mdt of weeks. for a two-fold decrease in the case reporting rate ( ae %) and % specificity, the wcr and threshold methods, respectively, have mdt of and weeks with both having sensitivity close to %, while the mov-avg cusum method can only manage sensitivity of % with mdt of weeks. the first cases of the pandemic were reported in scotland in the th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic weeks later in week . the ili threshold method detects it week later in week . both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by week in the retrospective detection of the pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h n ) pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid= &id= &itemid= ) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system , using primers and instructions supplied by cdc, usa. influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. ten representative strains of a(h n )pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula- , and a ⁄ dundgovi ⁄ ⁄ . sequencing of influenza virus gene segments was performed in applied biosystems xl genetic analyzer using primers and instructions supplied by cdc, usa, and bioinformatic analysis was performed with abi ⁄ seqscape v. . and mega programs. the pandemic alert in mongolia was announced by the government on april , , just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h n )pdm virus was detected in mongolia until the beginning of october . around suspected cases, mostly arriving from the a(h n )pdm epidemic countries, tested zero by rt-rt-pcr for a(h n )pdm virus. the first a(h n )pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october , ( st week of ). the reported ili cases escalated rapidly, reached the peak in the - th week of , and gradually decreased thereafter ( figure ). week of . however, the registered ili cases increased again from the th week of , and peaked at the - th weeks of . the viruses isolated during this nd peak were influenza b strains ( figure and table ). for the genetic characterization of the mongolian pandemic isolates, gene segments i (pb ), gene segments ii (pb ), gene segments iii (pa), gene segments iv (ha), gene segments v (np), gene segments vi (na), gene segments vii (m), and gene segments viii (ns) of the representative a(h n )pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy ). all genes of mongolian strains were possessing ae - ae % similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ (h n ). the who declared the pandemic alert phase (phase iv) on april , , and was prompted to announce the pandemic phase (phase v) two days later. after days, the who declared the beginning of the pandemic peak period (phase vi) on june , . however, in mongolia, the pandemic alert period continued for days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the st influenza a(h n )pdm case on october , in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the nd wave of pandemics in many countries of the northern hemisphere (see, picture and table ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per population at the peak period was - times higher above the upper tolerant limit, and - times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over % of the registered ili cases have been in the age group under , it has been observed that over % of the registered cases in this pandemic peak period were in the age group of - . on january , , we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may , we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for months. who announced pandemic vii and viii phases much later. , this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade , according to the classification of a(h n )pdm influenza strains suggested by m. nelson, which has circulated worldwide since july . this is also evidence that the st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in ⁄ infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h n ( ) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. despite all uncertainty around the pandemic flu h n , there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: % of all health facilities are in djibouti city. of the ae % ( ) of the population that seeks medical care on influenza and influenza like illness each year, ae % ( ) and ae % ( ) of them are attended to at the city's public and private clinics, respectively ( figure ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group ( - years). pharmaco-surveillance: % ( ) of total prescriptions were antipyretic and antiflu drugs, ae % ( ) of which were consumed by peripheral regions, the non dji- lab diagnostics: the annual ili lab diagnosis was negligible ae % ( ), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from and . with support of egypt-based naval army medical research unit three (namru ), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least ae % ( ) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least vaccine doses of h n ( ) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers ( ae %). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. , information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h n ) virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡ : ) -has been proposed ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in ⁄ influenza season (medium-scale mixed [a ⁄ h n and b] epidemic in study area, and a ⁄ h n was circulating at the nursing home). the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from november to april in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡ ae °c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡ : (seroprotection level) were ae % ( ae - ae %) for a ⁄ h n , ae % ( ae - ae %) for a ⁄ h n , and ae % ( ae - ae %) for b. during the follow-up period, the a ⁄ h n strain was isolated therein, and subjects experienced sudden-onset fever ( ‡ ae °c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡ : ) had lower incidences of febrile illness (or adj , ae ; % ci, ae - ae ) and rapid kit diagnosed influenza (or adj , ae ; % ci, ae - ae ) than those with a lower titre. thus antibody efficacy ( ) or adj ) against fever related to a ⁄ h n and kit diagnosed influenza were both estimated to be %. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. the emergence of a ⁄ fujian ⁄ ⁄ coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december , to august , showed that the antigenic evolution of the h n fujian strains had periods of rapid antigenic changes, equivalent to amino acid changes per year ( figure ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (> years) before and after the ph n influenza epidemic that occurred during the southern hemisphere winter of , and again prior to onset of the southern hemisphere influenza season. reactivity to the novel h n strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of % to %. a further increase in the seropositive proportion from % to % was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the winter epidemic. together with a final serum collection, planned for late , these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph n . assessment of the extent of disease due to novel influenza a(h n ) virus (ph n ) during the winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, reflecting the considerable geographical distances between state and territory capital cities ( figure ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. rates of reported hospitalisations and deaths appeared fairly similar across states but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph n using residual plasma from healthy blood donors collected before and after the epidemic to estimate ph n exposure. here we report the findings of that first collection, together with new data on seroprevalence of ph n antibody in specimens gathered in march-april . these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph n vaccine program conducted in spring ⁄ summer ⁄ on the proportion of individuals with antibody titres deemed protective. findings informed estimates of population susceptibility to ph n prior to the influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph n in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph n . collection periods were as follows: approximately plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to specimens were identified in each of the following age strata: - , - , - , - , - , and > years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately specimens were collected (i.e., up to specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph n was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). egg-grown a ⁄ california ⁄ ⁄ virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), : (volume ⁄ volume) and tested as previously described. following hour incubation, ll % (volume ⁄ volume) of rbc was added to each well. hi was read after minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h n ), a(h n ), or influenza b viruses and paired human plasma and sera collected from donors before april or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡ ) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season collections. all statistical analyses were conducted in stata . locations of specimen collection are shown in figure , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: at baseline, from the post-pandemic collection, and in early . pared with baseline was % overall, rising from % to % (table ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [ % ( % ci ae , ae )], perth [ % ( ae , ae )], and sydney [ % ( ae , ae )]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or ae ( % ci ae , ae ), p = ae ]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between and years (table ) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of - years [e.g. - years or ae ( % ci ae , ae , p < ae )]. an overall increase in the seropositive proportion from % to % was observed between october and april , distributed throughout all jurisdictions ( ( , ) ]. antibody titres prior to the influenza season rose in all age groups, but remained significantly lower among [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year olds than in the youngest age cohort (table ) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: - years [or ae ( % ci ae , ae )]; - years [or ae ( ae , ae )]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph n during the period between the two plasma collections. this study suggests that, while adult exposure to ph n during the southern hemisphere winter was uncommon at around %, vaccine uptake in the australian population over the period november -may was in the order of %. this latter estimate is in keeping with recently published figures for adult ph n vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over years of age, possibly in a subgroup whose health status may have differed from that of the donor population. no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted season. a final serum collection is planned for the end of the influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' known to be variable from historical reports of past pandemics in disparate populations. in - , the first wave of the swine-origin novel h n flu (h n ) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h n among school children using absentee reports provided by school health teachers in two small administrative districts (population: about in total) in japan. we then examined the epidemiological diversity on the inci-dence of h n within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, and ; elementary schools, and ; junior high schools, and , respectively. the number of school children in each school ranges from to . the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r ) of h n was estimated according to becker. agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version . (xj technologies, st. petersburg, russia). by the end of march , cumulative incidence (ci) of h n among school children in moroyama and sakado reached % and %, respectively. the overall r among school children in this area was ae . vaccination rate of children in this area during the surveillance period was reported to be very low (< %). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march scattered from % to % ( figure ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be : or : . using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of % (the ci as of the end of march ). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from % to %, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h n for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol ; (s ):s . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the a ⁄ h n v pandemic influenza were reported in mexico and the united states in april . given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h n v influenza across different countries by setting up prospective cohorts of households, followed during years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h n v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall . inclusions began on december , and ended on july , . households were sampled using a random telephonic design (mitofsky-waksberg method) in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the a ⁄ h n v virus. the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was ⁄ . geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, , taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: - years (reference group), - years and over years. the definition of ili was that used by the cdc : fever ‡ ae °c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september , (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included subjects belonging to households. results reported hereafter do not account for missing data. participating households were sized - subjects, mean size = ae . in comparison, the mean size of french households is ae according to the latest national census. the median age of subjects at entry was ae years [iqr: ae ; ae ] versus ae [ ae ; ae ] for french population. the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from ae % for subjects over years to ae % for subjects below years (table ) . vaccination with the pandemic strain was the highest in subjects below ( %) whereas vaccination with the seasonal strain was the highest in subjects over ( ae %). detailed data regarding vaccination is given in table . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [ ] [ ] [ ] [ ] [ ] [ ] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the a ⁄ h n v virus. [ ] [ ] [ ] [ ] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h n v infections in seasonal vaccine recipients, as described by several other studies, - the main explanation may be a cross-reaction between pandemic and seasonal strains. , , further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown virulence. infants under years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in . the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under years old was ae ⁄ people ( % ci - ) being higher in infants of years old ( ⁄ people of years ( % ci - ) ( table ) . infants had less risk of getting sick in relation to the rest of the population (rr ae [ % ci ae - ae ]) (p < ae ). the chance of sari in infants was ae ( % ci ae - ae ) compared to the rest of the population. the lethality rate was higher in infants under year old ( ⁄ people [ ⁄ ]). discussion: the evidence suggests that the infants under years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown dissemination and virulence. in june, the world health organization declared the pandemic. , the ili often presents an unspecific clinical picture in infants under years old, from mild symptoms to sari, especially in the newborn babies. infants under years old have higher risks of hospitalization, complications, and rate of death for sari. , on may th, argentina declared the first imported case of ia h n , and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april th to november th, suspected cases of ili in the province of tucumán were reported. the ili rate was ⁄ people, and ia h n comprised ⁄ people. the lethal rate of sari ia h n was ae ⁄ people ( ⁄ ). the objective of this research was to determine the epidemiological characteristics of the pandemic ia h n in infants under years old in the province of tucumán between may and december in . the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of inhabitants of which are infants under years old. the crude birth rate for was ae &. the infant mortality rate was ae &. respiratory pathologies in infants under years old were the third cause of death in the province ( %). the public health system of the province is composed by three sectors: public, private, and welfare. with health facilities as a total, the average of available beds is & per inhabitants and & per neonates. a cross-sectional study was executed from may to december in in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than °c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and % confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, < years old and the rest of the population. it was considered significant a rate of p < ae . the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received ili reports, ae % ( ⁄ ) were infants under years old. twenty seven percent were dismissed ( ⁄ ), and % ( ⁄ ) of suspected cases were confirmed. the first ia h n case was a child of years from the province of buenos aires, in th epidemiological week, and the last suspected case was reported in october , ( figure ). the ili rate in infants under years old was ae ⁄ people ( % ci - ), being higher in infants of years old ( ⁄ people of years, [ %ci - ]). the higher ili rates in confirmed the pandemic of ia h n ( ) was detected for the first time in the province of tucumán. the evidence suggests that infants under years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h n influenza - : behavior of influenza h n in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, were cases of ili in the group aged - years old; % were males. the incidence rate in this group was ae per thousands of inhabitants. the % of laboratory samples were influenza a h n , % were confirmed as unspecific influenza, and % were dismissed. the school aged children group had a high risks of getting sick (r.r. ae [ % c.i. ae - ae ]), especially males. it appeared that school aged children had a protective factor for presenting sari (or ae [ % c.i. ae - ae ], p < ae ). the lethality rate in this group was ae ⁄ thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. it was reported a decrease in the ew coinciding with winter holidays (ew ). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april , different countries reported cases of influenza a h n ; mexico reported a high mortality rate associates with this disease. the world health organization (who) declared the phase influenza pandemic alert on june . several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew and . since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from % in the initiation period to ae % in the acceleration period. in australia, % of h n cases were school aged children ( - years), with a median age of years ( % of cases were aged - years and, and % between - years). in canada, the infection rate was highest in this group. in chile, the incidence rate was ⁄ inhabitants, although in general they had mild desease. school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before % of the population falls ill. school closure can reduce the demand for health care by an estimated - % at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. tucumán is placed in northwest argentina and has a total area of km . the population ( census, projection ) was inhabitants; of wich were - years old. the health system of the province is composed of sectors: public, private, and welfare. it has a total of health facilities with internement available and an average of & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in , with a peak in the ew . the objectives were: general objective to describe the behavior of the influenza a h n epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december . data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children - years old. exclusion criteria: patients treated with antiv- iral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than °c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h n or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi software (epi infoÔ cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from national census projections. an estimation based on the same census was used for the group between and years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications ( ), were cases of ili in the group aged between and years old, % ( ⁄ ) of which were males. the incidence rate was ae , and it differed according to the sexes: ae males and ae females per thousands of inhabitants (p < ae ). of all laboratory samples ( ) % were confirmed as influenza h n , % were confirmed as unspecificied influenza, and % were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr ae [ % ci ae - ae ]), especially males (rr ae ) compared with females (rr ae ). the highest attack rate was observed in the capital of tucumán ( ⁄ inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or ae [ % ci ae - ae ], p < ae ). the lethality rate was ae ⁄ thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or ae [ % ci ae - ae ], p < ae ) and of dying (or ae [ % ci ae - ae ], p < ae ). respiratory comorbidities were the most fre- quent: asthma ae % ( ⁄ ) and % rors ( ⁄ ). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. if we compared the group aged - years with - years old, the epidemic curve of the first group showed a decrease in the ew , coinciding with winter holidays (ew ) (figure ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of ae % in males and ae % in females (p < ae ) at ew . however, after the first week of winter holidays, the curve in males had a significant increased to ae % compared to ew , reaching the highest weekly rate of the epidemic ( ⁄ inhabitants). the reopening of classes coincided with a significant decrease of the rate ( ae %), from to ae ⁄ inhabitants in ew (p < ae ). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of ae % of the rate, from ae to ae in ew ( figure ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh n infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [ ] [ ] [ ] [ ] part of these results are now available in ref. subjects of a community study, - years old between november and october , we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged - were recruited and provided baseline sera in november and december . between september and december a further children aged - were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the sera collected before the first wave and the sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged or younger, and children between the ages of and gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh n from april to november were provided by the hong kong hospital authority (the e-flu database). since may , patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh n virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ ⁄ by viral microneutralization (mn). most individuals infected with influenza develop antibody titers ‡ : by viral microneutralization after recovery. we defined the pdmh n seroprevalence rate as the proportion of individuals who had antibody titers ‡ : . while mn antibody titers of ‡ are not by themselves conclusive evidence for pdmh n infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn infection. the iar was defined as the proportion of individuals infected by pdmh n during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh n infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june , all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with age classes ( - , - , - , - , and ‡ ) to describe the transmission dynamics of pdmh n in hong kong between june and november . we assumed that the mean generation time was ae days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p and p , the reduction in within-age-group transmission for - and - years old during summer vacation (compared to school days during september-december ); (iii) d r , the average time for neutralization antibodies titer to reach ‡ : after recovering from infection; (iv) h a , the age-specific relative susceptibility with - years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [ ] pediatric cohort study ( - april virological surveillance data suggested that the first wave of pdmh n in hong kong occurred from august to october . most of the laboratory-confirmed infections in this first wave occurred in individuals aged below years old accounting for > % of the lab-confirmed cases and hospitalizations, % of icu admissions, and % of deaths. taking into account a delay of - weeks for antibody titers to appear during convalescence, we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the - years old from september to november and a plateau thereafter (data not shown). among individuals aged - years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the - years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table . the severity estimates (ccr, chr, cir, and cfr) are shown in table . in summary, we estimated the iar was ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, and ae % among - years old. overall, we estimated a population-weighted iar of ae % ( - %) among individuals aged - years through the first wave in hong kong. ccr were around ae - ae % among the - years old. chr were around ae - ae % among the - years old. cir increased from ae ( ae - ae ) per infections in - years old to ( ae - ) per infections in - years old. cfr followed a similar trend with ae ( ae - ae ) death per infections in - years old to ae ( ae - ) deaths per infections in - years old. compared to children aged - , adults aged - were ae and times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: . the basic reproductive number was ae ( %ci, ae - ae ). . it took an average of ( - ) days for recovered individuals to develop neutralization antibody titer ‡ : . table . estimated age-specific proportions of individuals with pdmh n infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per infections . compared to - years old, - years old children and - teenagers were ae ( ae - ae ) and ae ( ae - ) times more susceptible to pdmh n infection, respectively. . compared to - years old, - years old older adults and - years old elderly were only ae ( ae - ae ) and ae ( ae - ae ) times as susceptible as the - years old, respectively. . compared to the school period during september-december , summer vacation reduced within-agegroup transmission by % ( - %) among - years old, but only % ( - %) among - years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached % (data not shown). we estimated that during the first wave in hong kong, ae % of school-age children and ae % of individuals aged - were infected by pdmh n . a serologic survey in england found similar iars in london and the west midlands. both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh n surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh n on the iar as the denominator. in most previous studies of pdmh n severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. , our estimated cirs and cfrs are broadly consistent with presanis et al.'s 'approach ' severity estimates, but around - times lower than their 'approach ' estimates. our estimates of chr are - times higher than their approach estimates of symptomatic chr. however, the hospitalization-death ratio was ⁄ = as of november in hong kong, but ⁄ = as of june in new york, suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡ : by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers < : , or if some individuals with baseline titers ‡ : were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡ : . however between august and october , % of influenza a viruses detected in hong kong were pdmh n , and only % of isolated viruses were seasonal h n viruses. third, a minority of severe illnesses associated with pdmh n infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh n . finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around ae % of the population aged - and half of all school-age children in hong kong were infected during the first wave of pandemic h n . compared to school-children aged - , older adults aged - , though less likely to acquire infection, had ae and times higher risk of icu-admission and death if infected. thus, although the iar of pdmh n is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of pandemic influenza (h n ) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh n infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh n virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn c; adb no. n -ai- ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. - to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) ('ultragrivak,' registered ae ae ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) ('influvir,' registered ae ae ) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was generated by : genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ - (h n ) and master donor strain a ⁄ leningrad ⁄ ⁄ ⁄ (h n ). , the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was generated by classical ( : ) reassortment of a ⁄ california ⁄ ⁄ (h n ) with the master donor virus. the vaccine strain contains ha and na genes from a 'wild-type' h n strain and internal genes from the master donor virus. participants were aged to years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was estimated in vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of days at a dosage of ae ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day ); (ii) days after first vaccination (day ); (iii) days after the second vaccination (day ); and (iv) weeks after the second vaccination (day ). serum hai antibodies were measured by standard hai assay using % human red blood cells. test antigens for the assay were a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa using whole purified a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) viruses at hau per ae ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd + cd + ifn-c + and cd + cd + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. pbmcs were prepared with standard histopaque- gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day ), gmts of hai antibodies to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) laivs were ⁄ ae and ⁄ ae , respectively. in addition, gmts of siga against these specific antigens from nasal swabs were ⁄ ae and ⁄ ae , respectively. no hai antibody titers greater than : were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd + ifnc + cells were ae % to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and ae % to a ⁄ ⁄ california ⁄ ⁄ (h n ). for cd + ifnc + cells, initial levels were ae % and ae %, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h n -crossreactive antibodies and t-cells has been observed previously. [ ] [ ] [ ] effect of vaccination antibody immune responses both influenza a (h n ) and influenza a (h n ) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv, % percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, % of volunteers exhibited seroconversion. after the first vaccination, a % conversion rate of siga was observed; after the second vaccination, % showed conversions in levels of siga. the first vaccination with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv showed ae % of hai antibodies seroconversions vaccination, and % seroconversion after second vaccination. for local siga, those results were ae % and ae % following the first and second inoculation, respectively. figure summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h n ) laiv inoculation, significant differences in both cd and cd ifnc-producing t-cells were observed at day after the second vaccination (d ). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h n wild-type viruses were able to respond to the live attenuated h n influenza vaccine. after the first influenza a (h n ) laiv vaccination, reliable increases were observed in cd + cells only. after the second vaccination, increases in both cd + and cd + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd + and cd + cells) were more marked in the a ⁄ ⁄ california ⁄ ⁄ (h n ). considering the long-term circulation of h -subtype viruses among humans in contrast to the novelty of h viruses, such a result would be expected. similar data were also observed following vaccination with the h n laiv. after first vaccination, the percent of people with notable increases in virus-specific cd + and cd + t-cells was % and % to h n and % and % to h n , respectively. after the second vaccination, these results were % and % to h n and % and % to h n , respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡ -fold) in hai antibodies had notable increases in cd + and ⁄ or cd + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was % and % to h n and h n , respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h n laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ ⁄ solomon islands ⁄ ⁄ (h n ) ( table ) . reliable increases to a (h n ) were observed in up to % of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [ ] [ ] [ ] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. , the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged ( months old), adult ( months old), and young ( months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with · pfu of mouse-adapted influenza a ⁄ pr ⁄ ⁄ . clinical disease was measured by body weight changes over a week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with · cfu pseudomonas aeruginosa strain pao . twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of ae lg cdna forward and reverse primers according to optimized conditions and ae ll of · syber green icycler supermix (biorad), in a total vol-ume of ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr f: tgctttcct-gctggagattt- nm, mus_tlr r: tgtaacgcaac agcttcagg- nm, mus_tlr f: atatgcgcttcaa tccgttc- nm, mus_tlr r: caggagcatactggt gctga- nm, mus_tlr f: ggcagcaggtggaattg tat- nm, mus_tlr r: aggccccagagttttgttc t- nm, mus_tlr f: ctggggacccagtatgctaa- nm, mus_tlr r: acagccgaagttccaagaga- nm, mus_tlr f: ggagctctgtccttgagtgg- nm, mus_tlr r: caaggcatgtcctaggtggt- nm, mus_ b-actinf: agccatgtacgtagccatcc- nm, mus_b-actinr: ctctcagctgtggtggtgaa- nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f ⁄ ) , cd + ) or t cells (cd b ) , cd + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. a recent finding by didierlaurent, et al., described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were - weeks of age, where our study included two cohorts of older mice ( months and months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table ) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = ae ) and an increase in ifnc (p = ae ) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table ). we also report a noticeable change in the immune cell populations with respect to b-cells, cd + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr , , , and . only tlr showed differences with respect to age with young mice showing little or no detectable change in tlr mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table ) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table , figure ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h n ) virus (ph n ) emerged in early and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r , is defined as the average number of secondary cases that index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph n and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph n infections ('cases') reported between may and november , to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph n infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. we used a serial interval with mean (standard deviation) of ae ( ae ) days, and in sensitivity analyses, we used serial intervals with mean ae days and ae days. statistical analyses were performed in r version . . (r development core team, vienna, austria). in late april following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph n for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h n in and severe acute respiratory syndrome in . laboratory-confirmed ph n cases were isolated until recovery, and their close contacts were placed under quarantine for days. imported cases were identified sporadically through may and early june . the first case of ph n not traceable to importation (i.e. a local case) was identified on june and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph n , all childcare centres, kindergartens, and primary schools were proactively closed for days (subsequently extended for another - days to summer vacation in early july). any secondary schools in which one or more confirmed ph n case was identified were reactively closed for days. on june the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph n . these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure (a), superimposed on the epidemic curve of laboratory-confirmed ph n cases and ph n -associated hospitalizations. around % of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. figure (b) shows the estimates of r t based on laboratory-confirmed ph n cases. the estimated r t peaked at ae on june , and fell below between june and july (which was within the school closure period). r t fluctuated between ae and ae through the school summer vacations in july and august, it subsequently increased to around ae - ae after schools reopened in september until the epidemic peaked in late september, and then fluctuated below as the epidemic declined. the trends in r t based on h n -associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent days, and closely tracked the final estimates of r t . our results suggest that ph n may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around ae - ae in new zealand and australia. lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. furthermore, in hong kong the influenza virus usually does not circulate after august, and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, and our results show that it was feasible to monitor ph n -associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h n and pandemic h n influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the h n pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in , netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a -year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as %. the challenge in netflix recommendation system is a classic mc problem. [ ] [ ] [ ] [ ] [ ] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, svt, cf, bellkor, pf, and fwls. eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as %. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn ( ) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure (a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be , , , or . low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. in order to overcome the above issues, we incorporated a regularization function into the eqn ( ), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn ( ) . in addition, a temporal mds method is proposed to project the antigens into a or dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h n dataset, low reactors. figure (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported clusters (hk , en , vi , tx , bk , si , be , be , wu , sy , and fu ) were displayed in the core of a spiral s-shape, and bk and be are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk and fu in the projection is ae units, which is close to the distance between hk and bk ( ae units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section . . in comparison, with the temporal model, not only the viruses in clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure b ), the antigenic distance between hk and fu is ae units, where the distance between hk and fu is ae units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure (c) is an antigenic cartography generated using a hi dataset with h n influenza viruses spanning from april of to june of . this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h and h avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [ ] ; (b) the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for a ⁄ h n pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in - are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, , was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd + t-cells potentially facilitates rapid viral clearance, , and lower levels of cd + t-cells perhaps provide partial protection. in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models , incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of or more households are also available. the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum ) who have had been exposed to an influenza virus and the 'naive' population (stratum ) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e i and e i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq i , e i , t i , and t i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r with the force of infection: modulated by a sinusoidal seasonal term with amplitude b with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices , or accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in, = p utq i and s in, = in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q = q and q = . the latent exposed period is fixed to be c = ⁄ ae days, and the rate of recovery is parameterised by m = ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q and move into s at rate u q = ⁄ t wq . recovered hosts (r i ) migrate back to s at a rate u = ⁄ t w . the state p contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p ), ii. waning prior protection (beginning in state q ), iii. permanent acquired protection with probability q (moving into state p ), iv. waning acquired protection with probability ) q, and, v. partial prior protection (beginning in state s ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis assumes that the prior protection is sterilising but temporary, whilst protection hypothesis assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis : sterilising, waning prior protection we fix x e = and fit for q (t = ) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r = ae , a = ae , a = ae , a = ae , t w = ae years, t wq = ae years, we fix q (t = ) ⁄ n = and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure ) . the parameters corresponding to the fit in figure for leicester are r = ae , a = ae , a = ae , a = ae , t w = ae years, p (t = ) ⁄ n = ae , s (t = ) ⁄ n = ae , b = ae , t inf = ae days, q = ae , e a = ae , e i = ae , and e s = ae . our model with protection hypothesis -which, similarly to the model discussed in ref. [ ] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s from an initially large proportion (over %) of hosts with prior protection in q combined with the waning of acquired immunity in around % of cases on a time-scale of months. disease severity as measured by symptomatic proportion increases from % in the first wave to above % for the second and third waves. over a quarter of the population are initially permanently immune, and a large r value of ae drives transmission in the remaining population. protection hypothesis -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis . the parameter estimates suggest that approximately % of the population initially experiences reduced disease severity (e a $ ae ), but similar susceptibility and infectiousness. a larger value for r $ ae is required to drive transmission despite low numbers beginning in p , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in , which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the h n experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h n , have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the h n experience. [ ] [ ] [ ] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. - models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian h n pandemic response. [ ] [ ] [ ] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. in previous work, we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around %. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately % of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure (b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of < % (a substantial reduction from the no intervention case of approximately %) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low ( - %), and consequently it is difficult to universally recommend an antiviral intervention. in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [ ] . we selected a random sample of outbreaks from a total of simulated epidemics ( % of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < %). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [ ] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. matlab a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: . r , the basic reproductive number of the outbreak (assigned values between ae and ae for this example). as the value of r increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. . e t , the relative infectiousness of treated individuals (assigned values between ae and ae ). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. . g, the proportion of infections that are severe (assigned values between ae and ae ), and which in turn determines the presenting proportion (derived values between ae and ae ). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve ( -specificity versus sensitivity, not shown) for the logistic regression model specified in table has an area under the curve of ae , demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of % we still have a specificity of approximately %. evaluation of the pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure and ref. ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table ) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the h n outbreak. , our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in , is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [ ae , ]. that is, nais provided as treatment have a maximum impact of just a % reduction in infectiousness. however, our previous results show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately %) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [ ] [ ] [ ] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model to study the spatio-temporal transmission characteristics of the h n virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r ) in all our simulations is ae , following the suggested range by who of ae - ae . as the latent ⁄ incubation and infectious periods for h n have not yet been reliably ascertained, we adopt the natural history of the and pandemic influenza viruses. , here, the latent period ranged from to days, with a median value of ae days. the infectious periods begin day prior to symptom onset and can continue for - days, with a median value of ae days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. , for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of and an average hours of flight time to taiwan. the average daily passenger number is based on the annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno= ). each simulation lasts days and starts with a baseline simulation of r % ae h n pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april -march , . we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with hours average flight-time, as presented by pitman et al. the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly % of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h n pdm patients. it consists of weekly opd (outpatient department) icd- code (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the taiwan weekly h n opd cases alongside the weekly new clinical cases from our simulation results in figure . our simulations not only capture the epidemic trend, but also pick out the most likely date, may , for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february , as the empirical start date for h n pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as weeks before detection. figure also highlights the importance of the timing for the implementation of mitigation measures; for example, a -day-delay of the identical intervention plan results in nearly an additional % of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to months, and the epidemic peak can be delayed up to days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between % and % suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by day, and reduced car by < ae %. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h n pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an % airport exit screening success rate, % airport entry screening success rate and % symptomatic case tracing success rate, the combined intervention strategy results in: a % reduction in car if the vaccination campaign starts in mid-november; % reduction if the campaign starts in mid-october; % reduction if the campaign starts in mid-september; and % reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h n pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h n pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h n cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h n pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly % of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of - . although the proportion of people reporting symptoms was often greater in the first wave, - a puzzling feature was the much higher mortality in the second wave, in which . % of the population died, compared with . % in the out-of-season first wave and . % in the third wave. an obvious hypothesis to explain the changes in mortality from wave to wave would be that the virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, it has not been possible to recover virus from the first wave in for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave and wave could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [ ] [ ] [ ] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before , , suggests that the - viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves and . the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in , military, school, and urban populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [ ] [ ] [ ] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to , that arguably reduced pandemic mortality for older adults in - . , cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in - for populations in england and wales. we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure ) before the first wave, we assumed that people could be fully susceptible (s ), or partially protected (q ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation to the proportion susceptible (s & q) in each wave, for each population: note: in both figures and , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s = proportion fully susceptible to infection and death before wave ; q = proportion susceptible to immunising infection, but not to death from exposure in wave ; p = proportion temporarily protected against both immunising infection and death from exposure in wave ; n = proportion even more protected against both immunising infection and death from exposure in wave (model b only); r = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the - pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of . as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, , we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. the initial conditions were specified by the parameters: p , q , s and n . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of . accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure ) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the % credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = . - . , s = . - . , q = . - . ; n = . (fixed); p = ) s ) q ) n ; f = . - . ; g = . - . ; d = . - . and r = . - . . this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = . ; d = . ; g = . ) and estimated the % credibility intervals for the others as: h = . - . ; s = . - . , q = . - . ; and as before n = . (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the - influenza pandemic , can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. , the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r estimates of . - . across different populations are consistent with our earlier findings. , if all persons had been susceptible, such r values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s + q , was < % in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r between populations is due to different rates of population mixing. estimates for h indicate that between % and % of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in - was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection or seasonal effects. nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in - , or in , , can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [ , ] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. it has not escaped our notice that although attack-rates in the h n pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. it seems likely that those who were most severely affected in were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before . during the early phases of the influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week ( % ci: , ). results were consistent with the influnet data showing that the peak in italy was reached in week . these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as of april , after the pandemic threat emerged worldwide, it was crucial for policy makers to have early predictions on the possible spread of the pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april , a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. to parameterize the transmission model, we used data derived from the national surveillance system until june and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august . after the first pandemic alert was announced by the who in late april , a national active surveillance system for the pandemic influenza was set up from april to july . however, over the period from april to october , surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july , following who recommendations, the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever > °c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october , following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since , influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of (range - ) general practitioners (including physicians and pediatricians) per year, covering about ae - % of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of ae days), this study is consistent with refs [ , , , ] , but for the proportion of symptomatic individuals, which is assumed to be ae %. the basic reproductive number of the national transmission model was set to ae , according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. , we initialized our simulations through the global homogeneous mixing model in such a way that imported cases were generated until june . this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [ ] . we also considered scenarios accounting for partial immunity in the population. in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for - days, either in hospital or at home) established in the early phase of the pandemic (april-july ), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until july . we also assumed, according to the italian school calendar, that schools were closed from june to september for the summer holidays, and from december to january for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of vaccine doses month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of %. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than years old. the vaccination coverage was assumed %. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect % of symptomatic cases. after july , recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from % to % of the symptomatic cases. consistently with ref. [ ] , both treatment and prophylaxis were assumed to start day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by %, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by %, infectiousness by %, and the occurrence of symptomatic disease by %. as of july , approximately confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from to , we found that the exponential growth rate was ae ⁄ week and thus we estimated the national reproductive number to be r = ae . this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r = ae ). in the absence of intervention measures, the predicted cumulative attack rate was ae % ( % ci: ae , ae ), and the peak was expected on week ( % ci: , ) with a peak day incidence of ae % ( % ci: ae %, ae %). by assuming case isolation, antiviral treatment, and prophylaxis to % of symptomatic cases until july , the peak was expected on week ( % ci: , ). when considering ae % of natural immunity in the population aged more than years, the peak was expected week later than in the previous scenario, i.e., on week ( % ci: , ). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure ). based on model predictions, we estimated the underreporting factor of influnet ranging from ae to ae , considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the % ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table ) . we simulated the planned italian vaccination strategy (begun on october ), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influ- net data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, , we assumed a similar fraction of cases in the different social contexts considered in the model (namely ⁄ in households, ⁄ in schools ⁄ workplaces, and ⁄ in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range ae - ae . if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range - ae %, which is in excellent agreement with the range estimated in ref. [ ] on previous a ⁄ h n influenza seasons, namely ae %- ae %. moreover, based on our results showing that vaccinating % of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age - years) to donate blood if they have experienced influenza-like symptoms more than weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. , the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r between ae and , which is consistent with recent estimates. , [ ] [ ] [ ] logistical model for the passive immunotherapy program we assume that q d (%) of to year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = days after cessation of symptoms. follow-ups of convalescent individuals infected with h n pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around - days after recovery and stays at that level for months after. we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl setting. we assume that five bsl -trained technicians are available to test the blood specimens, each running viral neutralization tests in days. therefore, the capacity and turnaround time of blood screening are u s = and t s = days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of plasma donations per day (assuming -hour daily operation with each donation taking minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = and t p = ⁄ days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h n pdm in hong kong suggested that r t < . we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from ae % to %. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = % and q s = %. in general, the treatment coverage q increases sharply as the basic reproductive number r and the lumped demand parameter r t p h decrease (figure a ). in particular, when r is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r . similarly, when r and r t p h are small, q is very sensitive to both. with a donor percentage of q d = %, the proposed program can supply passive immunotherapy to more than % of severe cases (q > %) if r < ae and r t p h < ae %, but < % if r > ae and r t p h > ae %. in general, the treatment coverage q increases sharply as the donor percentage q d rises from %, but with rapidly decreasing marginal increase ( figure b ). when r < ae and r t p h < ae %, q > % even if q d is as low as %, which is comparable to the current average blood donation rate of ae donations per population in developed countries. when q d is > %, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = % is more than % that for q d = % across all values of r and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond % has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached %, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond - %, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat - % of severe cases in a moderate pandemic (basic reproductive number r < ae , lumped demand parameter r t p h < ae %) when the donor percentage is - %. increasing the donor percentage beyond % has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h n pdm virus infection in hong kong, % of convales- cent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program ( - %) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from % to % of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h n virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h n patients. while early treatment improves survival, approximately % of patients treated within days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h n pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase (cox- ) plays a regulatory role in h n hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox- inhibitor, ns- has a direct antiviral effect against h n infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a ) were pre-treated with ns- or drug-vehicle for hour before h n virus infection. h n viruses at multipicity of infection (moi) of was used to infect the cells. following virus adsorption for mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls for , , , , and hours post-infection. cells were harvested for rna isolation at hours post-infection to study viral matrix (m) gene expression. supernatants were collected for % tissue culture infection dose (tcid ) assay to determine the virus titers at , , , and hours after h n infection. results ns- was found to suppress virus gene transcription and infectious virus yield in h n -infected human cells. conclusion we demonstrate that a selective cox- inhibitor, ns- , shows an inhibitory effect on h n viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox- inhibitors may be a therapeutic target for treating h n disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h n ) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, human cases have been reported with fatalities, an overall case fatality rate of around % (cumulative number of confirmed human cases of avian influenza a ⁄ (h n ) reported to world health organization updated to october ). our previous data demonstrated that cox- was markedly up-regulated in h n -infected primary human macrophages, and that it played a regulatory role in the h n hyperinduced host pro-inflammatory responses. such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h n disease in humans. with the use of selective cox- inhibitors, we found that the h n -hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. selective cox- inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox- , and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox- inhibitor, ns- , against h n infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox- and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h n . such therapy may be combined with conventional antiviral drugs. the h n virus used was a ⁄ vietnam ⁄ ⁄ ( ⁄ ) (h n ), a virus from a patient with h n disease in vietnam during . the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. virus infectivity was expressed as tcid . all experiments were performed in a biosafety level facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi medium supplemented with % heat-inactivated autologous plasma. the cells were allowed to differentiate for days in vitro before use in the infectious experiments. alveolar epithelial cells: a cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with % fetal calf serum, . mg ⁄ l penicillin, and mg ⁄ l streptomycin. differentiated macrophages or a cells were pre-treated with a selective cox- inhibitor, ns- (cayman), at concentrations as indicated or drug-vehicle for hour before infection. cells were infected with h n viruses at moi of . following virus adsorption for min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at hours post-infection to study viral m gene expression. supernatants were collected for tcid assay to determine the virus titers at , , , and hours after h n infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr Ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the real-time pcr system (applied biosystems). the specificity of the sybr Ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached times the standard deviation of the base-line (from cycle to ). the ratio change in target gene relative to the b-actin control gene was determined by the )ddct method as described elsewhere. ns- reduced the viral m gene expression in h n infected human macrophages in a dose-dependent manner ( figure ) . similarly, production of infectious virus yield in h n infected macrophages was found to be suppressed in the presence of ns- at lm compared to vehicletreated cells (figure a) . a comparable effect of ns- was observed in h n -infected human alveolar epithelial cells ( figure b ). we have previously demonstrated that cox- expression was dramatically upregulated following h n infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h n disease. this suggests that cox- may be an important host factor involved in h n pathogenesis and also provide a possible explanation on why h n virus replication is susceptible to a selective cox- inhibitor. cox- was previously reported to play an important role in the pathogenesis of other influenza a viruses. an in vivo study has highlighted the importance of cox- in h n - infected mice. findings showed that infection induced less severe illness and reduced mortality in cox- knock-out mice than in wild-type mice. on the other hand, cox- knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox- knock-out mice. these data suggests that cox- and cox- may lead to opposite totally contrasting effects on influenza h n infected mice. cox- deficiency is detrimental, whereas cox- deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, we have chosen ns- that selectively block cox- but preserve cox- activity and showed that this drug significantly reduced h n virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, we believe that selective cox- inhibitors and cox- signaling pathways deserve investigation as a promising approach for targeting therapy in h n diseases. however, a few reports have suggested the importance of cox- in the late stage of inflammation for the resulution of inflammation, [ ] [ ] [ ] and this raises concern whether inhibition of cox- may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, which is a leading cause of death in h n patients. we previously looked at the autopsy samples of lung tissues from h n patients and found that cox- expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h n infected lung autopsies. taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h n patients, , we may therefore speculate that people who had fatal h n infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (< - days). in conclusion, the roles of cox- in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox- inhibitor therapy in h n infected patients may be extremely critical. therefore a time-dependent study using selective cox- inhibitors on h n -infected animal models will be particularly important in order to address the effectiveness of this drug in treating h n disease. avian antibodies to combat potential h n pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h n with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h n virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h n is mandatory, contain high levels of igy specific for a ⁄ h n virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h n and related h n strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h n could offer an enormous source of valuable biological material that provides protection against a ⁄ h n virus with pandemic potential. the approach could be used to control seasonal influenza. since , hpaiv of the h n subtype has resulted in more than cases of laboratory-confirmed human infection in countries with a death rate of more than % (http://www.who.int/csr/disease/avian_influenza/). h n influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. development of human monoclonal antibodies (mabs) against h n influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h n , phage display, humanized mabs, and human recombinant abs has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. egg igy has been used to prevent bacterial and viral infections (see review ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). the epidemic of hpaiv h n virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h n virus vaccines that controls the h n epidemic to some extent. chickens immunized with recombinant h and ⁄ or inactivated h n reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h n virus challenge. theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h n hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h n vaccination has been implemented, provide prophylaxis and therapy of hpaiv h n infection in mice. six-to -week-old female balb ⁄ canncrl (h- d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. the % egg infectious dose (eid ) was determined by serial titration of virus stock in eggs, and eid ⁄ ml values were calculated according to the method of reed and muench. human virus stocks were grown in mdck cells as described previously , with viral titers determined by standard plaque assay. the % tissue culture infectious dose (tcid ) of virus was determined by titration in mdck cells. the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld ) titers were determined by inoculating groups of eight mice i.n. with serial -fold dilutions of virus as previously described. for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with pfu ( · ld ) of a ⁄ pr ⁄ ⁄ (h n ) virus as previously described, · ld of vn ⁄ (h n ) or · ld a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ) resuspended in ll pbs per animal. ketamine-anesthetized mice were treated intranasally with ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found % of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h -specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h n vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h n has no detectable h -specific hai or vn activity. we first treated naïve mice intranasally with h n -specific igy before infection with hpaiv h n strain, a ⁄ vietnam ⁄ ⁄ , isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure a ). when animals were treated once with h n specific igy after h n inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day after infection; however, % of treated mice succumbed to infection during the second week after inoculation ( figure b) . it is possible that not all the hpaiv a ⁄ h n viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ virus injected intravenously or into the brain can spread to the lungs. to circumvent the virus escape, we administered multiple treatments with h n specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure d) . similarly, the protective efficacy of h n -specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ). this virus shares . % nucleotide sequence homology with ha (h ) but has different na (n ) from the one used for mass immunization in vietnam (reassortant avian h n influenza virus a ⁄ goose ⁄ gd ⁄ -derived, strain re- ). the results indicate that h n -specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h n and the related strain h n . the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h n virus, a ⁄ pr ⁄ ⁄ . we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ igy at - hours before or after infection with lethal dose of pr ⁄ virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr specific igy-treated mice at day after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h n infection offers a practical alternative to immunotherapy using convalescent plasma and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least months storage at °c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. , eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h n vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h n pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi% f . % fjournal.pone. . the polyphenol rich plant extract cystus is highly introduction the ⁄ h n influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h n influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h n and also h n influenza a viruses dramatically increased in the recent past. [ ] [ ] [ ] [ ] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus , which showed antiviral activity against influenza a viruses in cell culture and in mice. , moreover, the antiviral activity of cy-stus against seasonal influenza virus and common colds was also demonstrated in humans. however, the efficiency of cystus against soiv and a ⁄ h n isolates was unknown so far. therefore, we investigated cy-stus effectiveness against the pandemic strain and seven natural influenza a ⁄ h n isolates detected in several avian species during ⁄ avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus treatment was effective in in vitro studies against soiv and a ⁄ h n influenza virus. viruses avian h n isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ ⁄ was obtained from the robert-koch-institut, berlin, germany. all h n viruses were further propagated in embryonated chicken eggs or mdck ii (h n v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, mdck ii cells were infected with different viruses at moi of ae . virus-infected cells were then treated with antiviral compounds cystus from ae to lg ⁄ ml or oseltamivir from ae nm to mm. after incubation for hours at °c and % co , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with % methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells ( · cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba ( ae % ba) mm mgcl , ae mm cacl , penicillin and streptomycin to a multiplicity of infection (moi) of ae for minutes at °c. cystus was added in a concentration of lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. in order to investigate the antiviral potential of cy-stus , ec values based on the inhibition of the cpe on mdck ii cells were determined for cystus and in addition for oseltamivir. the ec values for cystus ranged from ae to ae lg ⁄ ml. cystus demonstrated the highest sensitivity against the soiv, sn and mb isolates with ec values below lg ⁄ ml. compared to these virus strains, cystus showed a slightly increased ec value for gsb ( ae lg ⁄ ml). in contrast the ec values for bb and bb were notably elevated ( ae and ae lg ⁄ ml). thus, the weakest antiviral effect of cystus was observed against these two isolates. the ec values evaluated for oseltamivir ranged from ae to ae lm ( table ), indicating that bb ( ae ) and gsb ( ae lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus to block virus replication as published before. as a control, virus infected cells were treated with oseltamivir as described earlier. in the absence of the drugs all influenza strains showed similar growth properties (figure , black squares) . first progeny viruses were detectable between and hours post infection (figure , black squares) . treatment with cystus resulted in reduction of virus titers of all influenza virus strains (fig. a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h n influenza virus strains (gsb and bb ), supporting the data of ec values ( figure d+h , grey rhombes). we assessed the antiviral activity of cystus against the newly emerged soiv and seven avian h n influenza viruses. cystus showed efficient antiviral activity against the pandemic h n v strain and was effective to a wide range of h n viruses. furthermore, cystus demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h n viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. , , cystus is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. it is well known that polyphenols exhibit protein-binding capacity. however, cystus exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [ ] [ ] [ ] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female ae -week-old weanling balb ⁄ c mice were nasally inoculated with pfu of iav ⁄ pr ⁄ h n at day . immediately after infection, mice were given lg of osv orally or vehicle at -hours intervals for days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. a retrospective clinical study was conducted. for the study, children with acute influenza were recruited and grouped according to the treatment received: days treatment with osv (n = ), cam (n = ), osv + cam (n = ), and untreated (n = ). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. , comparisons were made of the levels of siga against iav ⁄ h n and iav ⁄ h n , total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days and post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table ) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day , the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il- , a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, , we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · . figure shows changes in the anti-ha(h n ) siga ratio (titer) and fold of increase in siga titer in each patient during the -days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were < in % of patients. during the days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; % of patients treated with osv + cam showed > -fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < ae ), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table ) . the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. the membered-and membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, , and to be effective in the treatment of respiratory syncytia and iav infection. , the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. , , , in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in . % of patients in the osv group (those represented by the dotted lines and closed diamonds in figure ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par stimulation alone induced erk activation, par stimulation does not inhibit erk activation in influenza infected cells. thus, par agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. , in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par acts as an antiviral through an interferondependent pathway. , thus, agonists of par are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par induced erk activation, a crucial step for efficient virus replication. , however, par agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par protection is likely to be erk-independent, the use of anti erk molecules in combination with par agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ (h n ) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a cell were obtained from atcc and grown as previously described. for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk ⁄ (t ⁄ y ) and for erk ⁄ antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a cells were infected with iav at an moi of in emem medium, as previously described. , at various time points post infection, cells were collected and proteins were analysed as previously described. , par stimulation was performed at °c in emem medium as previously described. after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at g for min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. , results since activation of the erk pathway is essential for efficient influenza replication, we first investigated the kinetics of erk activation after influenza infection in human a alveolar epithelial cells. for this purpose, a cells were infected with influenza viruses at a moi of at different time point post-infection, and activation of erk ⁄ pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure a ). antibodies against erk ⁄ were used as controls. since erk is activated after influenza infection, we then tested whether activation of par in uninfected cells also leads to activation of this pathway. for this purpose, a cells were stimulated with the selective human (h) or mouse (m) par agonist or a control peptide for the indicated time ( figure b ). when exposed to the par agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par without infection in a cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par stimulation induced erk activation, we then investigated whether par could inhibit erk activation in influenza infected a cells. results in figure showed that in influenza infected cells, par activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par stimulation alone induce erk phosphorylation in a epithelial cells, while erk activation is not inhibited in a infected cells compared to uninfected ones after par stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par , activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, , thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, and it is a well known immunotolerant molecule. indeed, it inhibits the innate immune response as well as the adaptive immune response. , also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. , as shown here, it is unlikely that par protection occurs through an erkdependent pathway. thus strengthening the immune response with par agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the st century have passed. in june , vaccine companies estimated they could produce in months almost . billion doses of pandemic vaccine. instead, they actually produced only million doses, of which % were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., . lg instead of lg ha per dose), an additional billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than % of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on th century public health ''technologies.'' given current understanding of biology in the early st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago and has been reviewed in several publications. [ ] [ ] [ ] [ ] [ ] [ ] the central importance of the host response in the pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the virus that were responsible for its virulence. in doing so, they have overlooked a crucial piece of clinical evidence from the pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. , a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in , could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. , , influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the h n pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h n virus. in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in that statins might be used to treat pandemic influenza. other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). , these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il- was necessary for containing infection, but in another study il- appeared to be harmful. nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr (h n ) h n and pandemic h n viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by - %, often when treatment is started - days following infection. - (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h n -infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice ( - weeks old) to that of ''children'' ( - weeks old). this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. for influenza itself, preliminary evidence presented in october suggests that immunomodulatory treatment of influ- table . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il- , il- ) up regulate anti-inflammatory cytokines (il- , tgfb) up regulate pro-resolution factors (lipoxin a , resolvin e ) up regulate ho- and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl- ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references , , , for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho- , heme oxygenase - ; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by %. in these patients, the cell signaling effects of statin treatment, summarized in the table , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, , similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in ), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $ . (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than % of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $ - million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. , this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc efficiently blocks h n influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h n viruses in humans and the emergence of resistant h n variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. here, we show that the novel nf-kappab inhibitor sc efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h n isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h n and a ⁄ h n viruses, we were able to demonstrate reduced clinical symptoms and survival of sc treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h n -induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h n strains. thus, a sc -based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel Ò was performed in -well plates as described by mastrosovich and colleagues. virus-infected cells were immunostained by incubating for hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and minutes incubation with true blueÔ peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft Ò paint software. the virus titer is given as the logarithm to the basis of the mean value. the detection limit for this test was < ae log pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in ml trizol Ò reagent (invitrogen) at °c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in ll rnase free water and diluted to a working concentration of ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastÔ sybr Ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. ct values were obtained with the smartcycler Ò (cepheid). to answer the question whether the nf-kappab inhibitor sc shows antiviral properties against influenza virus, h n infected mdck cells were treated with different concentrations of the inhibitor (figure ). already treatment with nm of sc led to a reduction of viral cpe of more than %. almost % protection of cells was achieved when cells were treated with lm sc . the results indicated that sc has antiviral properties at concentrations ranging from to nm. we next tested whether sc would also be effective in the mouse model of influenza virus infection. when h n mice were treated i.v. once daily for days with mg ⁄ kg sc , survival rate of the animals increased significantly (p < ae ). the same results were found when h n influenza virus infected mice were treated i.p. with mg ⁄ kg sc (data not shown). moreover, sc treatment was not only effective when the inhibitor was given prior to h n influenza virus infection, but also in a therapeutic setup when sc was applied to the animals days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc or the solvent, and hour later the lungs were prepared to perform qrt-pcr. as shown in figure a the amount of viral mrna was reduced by % in sc treated mice compared to solvent treated controls, indicating that sc leads to a reduced expression of h n specific mrna in the lung of infected mice. since infection of mice with h n leads to hypercytekinemia, we also investigated the expression of cytokines in sc treated mice. as shown in figure b the amount of il- specific mrna was drastically reduced in sc treated mice compared to solvent treated controls. moreover, also the expression of ip- was altered in sc treated h n influenza virus infected mice. here, roughly % reduction of specific mrna was detectable ( figure c ). thus, sc leads to a reduced transcription of il- and ip- in h n infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h n influenza viruses and highly pathogenic avian h n virus strains to oseltamivir and amantadine have been reported. in that respect, the strategy to target cellular factors , might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h n and h n subtypes in the mouse model. next to the reduction of virus sc was also able to reduce h n -induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h n . most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc based drug may serve as a broadly active non-toxic antiinfluenza agent. [ ] [ ] [ ] [ ] [ ] in hong kong, the first confirmed case was a tourist from mexico reported on may , . the local government made its first attempt to contain the spread of h n in the local community by closing the metropark hotel where that tourist was staying, and quarantining guests and staff for days. following identification of the first local case around weeks later on june , , the government closed all kindergartens and primary schools from june until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h n avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [ ] [ ] [ ] [ ] [ ] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph n , and we also examined the factors associated with greater use of preventive measures. a total of surveys were conducted between april and november , covering the entire first wave of the ph n pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over % of hong kong households were used to recruit a total of local adults. one cantonese-speaking adult (age ‡ ) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. statistical analyses were conducted in r version . . (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of telephone calls being made; the response rate among eligible participants was . %. hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi: . /j. - . . .x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june , when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph n cases are shown in figure (a) . the anxiety scores and risk perception of the respondents are shown in figure (b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph n severity (same, more, or much more serious than sars) initially in early may . fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july and a steady decline to . %, while perceived severity of ph n declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph n activity in september (figure c) . as the first wave of ph n progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph n . around - % of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph n throughout the first wave ( figure d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june when local kindergartens and primary schools were closed and the first ph n -associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h n compared to sars (much more or more severe), higher probability to contract h n over the next month (certain, much more, or more likely), higher probability to contract h n over the next month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying possible modes of transmission as the actual modes of transmission of h n . social distancing measures. other significant factors contributing to greater use of preventive measures were worry and knowledge. greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h n could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h n could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. community transmission emerged in hong kong in mid-june , and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph n spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around . on the -point scale, compared to a maximum of . during sars on the same scale. anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; , however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. the differences in findings may be due to the fact that our anxiety measure was not specific to h n , and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. , , , social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, , but the economic impact could be substantial. as community incidence of h n peaked, we did not observe any increase in use of preventive measures (figure ) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph n did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph n transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph n in hong kong. on the other hand, knowledge that ph n could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, , this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover % of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in and avian flu in , our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph n pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph n was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph n suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in will host much of the olympic and paralympic games. along with the rest of the global community, in and early london faced the challenges of responding to the first pandemic of the st century. at the time, nhs in london was composed of organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h n v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april , which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h n v were reported in scotland on april, with the first in london on april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the ⁄ influenza a ⁄ h n v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h n v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h n v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the ⁄ influenza a ⁄ h n v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h n v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h n v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early . it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream : limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream . reducing the risk of emergence of pandemic influenza. • stream . limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream . minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream . optimizing the treatment of patients. • stream . promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream : limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: . transmissibility of influenza across the progression of infection and spectrum of disease . relative contributions of the different modes of transmission for influenza . biological, behavioral, and social host factors that influence the risk of transmission and infection . patterns, drivers, and mechanisms affecting the seasonality of transmission . viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains . strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas . impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions . impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine . role of vaccination in limiting the spread of influenza and strategies for its use . impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream : minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza . identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes . evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects . enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing . development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings . study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings . improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario . research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management . development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression . further clinical evaluation of current antiviral drugs, particularly in populations at risk . integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning . improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care . dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) . increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza . antiviral susceptibility of circulating zoonotic viruses (e.g., h , h , h influenza viruses) . reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: . identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface . develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems . studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives . examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease . quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes . research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk . integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface . identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication . identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts . identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures . understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h n ) in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h n ) to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h n ) was effective from th april- th september : healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers ( - ), school students ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , adults ( - ), and retirees ( ‡ ). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from th july to th september , which covered the period from the start of summer holiday to the end of the th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from st september onwards (st = : t < st september, st = otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version . . a total of ( ae %) pre-schoolers, ( ae %) school students, ( ae %) adults, and ( ae %) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure ) . interrupted time series analysis on the epidemic curve of school students returned an arima( , , ) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: ae (se = ae , p = ae ), ae (se = ae , p = ae ). in short, the model can be interpreted as: the number of infected school students rose by ae per day on average during the entire study period, with a sharp increase by ae coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h n ) and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h n ) . if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november , gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may . one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h n epidemic, and h n pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h n ) pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, , such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h n (reviewed in ). previously, we demonstrated that interferon regulatory factor (irf ) and p map kinase (p ) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h n and seasonal h n infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h n virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda are the key mediators directly induced by the h n virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor (tlr ) plays the predominant role in h n -induced cytokines and chemokines in human macrophages via the regulation of irf and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h n directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h n infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. human h n infection is associated with a mortality rate of more than %. the basis for the unusual severity of h n disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h n (reviewed in ). however, signaling pathways involved in the cytokine induction by h n virus are not fully understood. previously, we demonstrated that irf and p map kinase (p ) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. rig-i and melanoma differentiation-associated gene (mda ) are important cytosolic sensors of nucleic acid of pathogens, while tlr and tlr also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. rig-i was found to be responsible for the recognition of influenza a virus infection, and the transfection of vrnps induces ifn-beta expression. while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h n infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for days in rpmi medium supplemented with % autologous plasma. the cells were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses at a moi of ae . a cells were obtained from atcc and cultured in mem medium supplemented with % fcs and % penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h n and h n viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf and nf-jb was employed to detect the nuclear translocation of these transcription factors after h n infection. rabbit polyclonal antibodies against human irf and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr expression, culture supernatants collected from mock, ⁄ or ⁄ infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with -kda cut-off. virus particles as well as molecules with a molecular weight higher than kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda are the key mediators directly induced by the h n virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. in this study, we demonstrate that knockdown of rig-i or tlr led to the reduction of ifn-beta and tnf-alpha in human macrophages by both ⁄ (h n ) and ⁄ (h n ) infection. as shown in figure a , ⁄ virus induced higher level of ifn-beta mrna expression than ⁄ infection. cells transfected with rig-i or tlr sirna significantly reduced the expression of ifn-beta after ⁄ infection, by % and %, respectively. rig-i silencing also significantly reduced the ifn-beta expression in ⁄ infected cells by %. in contrast, silencing of mda or tlr did not suppress the induction of ifn-beta by either ⁄ or ⁄ infection; in fact, there was a slight ( %) increase of ifn-beta in cells transfected with mda sirna. based on these results we conclude that while both rig-i and tlr contribute to h n -induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf and nf-jb, we next measured the nuclear translocation of irf and nf-jb in cells with rig-i or tlr silencing after h n infection. immunofluorescence staining assay on irf and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure b . we demonstrated that rig-i knockdown led to a significant reduction of irf nuclear translocation after ⁄ infection, whereas the nuclear translocation of nf-jb after ⁄ infection was significantly suppressed by rig-i or tlr silencing. these results suggest that the involvement of rig-i and tlr in the cytokine induction by ⁄ was via the regulation of irf and nf-jb nuclear translocation. since rig-i and tlr are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, ⁄ , or ⁄ infected macrophages. as shown in figure a , ⁄ supernatant differentially induced the mrna expression of rig-i, mda , and tlr compared to ⁄ supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda and tlr . in the presence of lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a cells were also treated with the supernatants collected from macrophages infected with mock, ⁄ , or ⁄ virus. differential induction of rig-i, mda and tlr by ⁄ supernatant compared to ⁄ supernatant treated cells was observed (figure b) . ⁄ supernatant dramatically induced all three innate sensing receptors, while ⁄ supernatant only marginally induced rig-i and mda , but not tlr . as in human macrophages, treatment with lm of jak inhibitor caused a significant suppression of ⁄ supernatantinduced rig-i, mda , and tlr expression in a cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h n directly induced ifn-beta ( figure ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf and nf-kb in human macrophages. these results were consistent with a previous study using beas- b cells showing the essential role of rig-i in ifn-beta reporter activity by h n influenza virus infection. while tlr also played a role in induction of ifn-beta and the activation of irf and nf-kb, it plays a less important role compared to rig-i. the reduction of irf and nf-kb activation was also confirmed with the study by le goffic showing differential regulation of irf and nf-kb by rig-i and nf-kb can also be regulated by tlr . in addition to the direct role of rig-i and tlr in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h n virus, the h n viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda , and tlr was markedly induced by virus free culture supernatants from h n -infected macrophages, while supernatant from ⁄ -infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda , and tlr in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h n infection. a more precise identification of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y (h n ) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy sub (h n ), rgvac sub (h n ), and rgvac ins (h n ), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy sub (h n ) and rgvac ins (h n ) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table ) . chickens intravenously inoculated with rgy sub (h n ) or rgvac ins (h n ) did not show any signs of disease. rgvac sub (h n ) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at day post-infection. the h and h mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in -day-old chicks and -week-old chickens are shown in table . rgy sub (h n ) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy sub-p (h n ) died within days. eventually, over % of the chickens intravenously infected with rgy sub-p (h n ) died by days post inoculation, and its pathogenicity was comparable to that of hpaivs. rgvac sub-p (h n ) was pathogenic to both chicks and -week-old chickens, and mortality increased after one more passage. rgvac ins-p (h n ) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused % mortality among -week-old chickens. the lethal effect of rgvac ins-p (h n ) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac sub (h n ). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three -week-old chickens were challenged intranasally with the viruses at an eid of ae and observed for clinical signs until day post-infection (data not shown). all chickens inoculated with rgy sub-p (h n ) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac sub-p (h n ) and rgvac ins-p (h n ) were pathogenic as in the intravenous experiment, killing two of three chickens by day post-inoculation. one of three chickens were not infected with rgvac sub-p (h n ) or rgvac ins-p (h n ) via intranasal route (data not shown), indicating these p viruses had not been completely adapted to the host. to investigate the possibility of these p viruses to acquire further pathogenicity for chicken, rgvac sub-p (h n ) and rgvac ins-p (h n ) were obtained from the brain homogenates of the chickens that died on days post intranasal inoculation with the p viruses. although mortality rate of chickens inoculated with the p viruses was equal to that with p viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac sub-p (h n ) were infected, and time to death was shortened to - days post inoculation in chickens with rgvac ins-p (h n ) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from -week-old chickens intranasally inoculated with each virus on days post infection ( table ) . rgy (h n ) and rgvac (h n ) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy sub-p (h n ) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac sub-p (h n ) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac ins-p (h n ) showed disease signs, and one died days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p viruses were efficiently replicated in systemic organs of the chickens as compared with p viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy sub-p (h n ) killed % of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table ) . however, chickens intranasally inoculated with rgy sub-p (h n ) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over ae , classified as hpaiv according to the definition by european union. ohuchi et al. reported that the insertion of additional basic amino acids into the h ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h and h has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt cells in the absence of trypsin. , the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h and h has. the intranasal pathogenicity of the h and h mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table ) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac mutants (h n ), and morbidity was closely associated with viral titers in the brain (table ) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy mutants (h n ), explaining why rgy sub-p (h n ) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by hours post allantoic inoculation (data not shown). rgvac sub-p (h n ) and rgvac ins-p (h n ) were more pathogenic to chicken embryos than rgy sub-p (h n ); the allantoic fluid obtained from the embryonated eggs inoculated with the h viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h virus were strictly confined to endotherial cells in chicken embryos or chickens. , therefore, it is suggested that endotheliotropism differed between the h and h viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac sub-p (h n ) and rgvac ins-p (h n ) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy sub-p (h n ) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h and h known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p and k-k ⁄ r-k ⁄ t-r with k at p , and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p . however, replacement of p r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. most of the type ii transmembrane serine protease identified so far recognize a single r at position p , but the newly isolated mspl and its transcript variant tmprss preferentially recognize paired basic residue, particularly r and k at position p , at the cleavage site. [ ] [ ] [ ] thus, mspl and tmprss can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. we used human cell line ecv , which expresses mspl and tmprss at levels below detection, and established the cells stably expressing mspl and tmprss , such as ecv -mspl and ecv -tmprss . to determine the cleavage specificities of mspl ⁄ tmprss and furin, peptides ( lg each) were incubated with ae mu mspl ⁄ tmprss for hour and furin for hours at °c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of -residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ ⁄ (h n ) and a ⁄ fpv ⁄ rostock ⁄ (h n ), and low pathogenic strain a ⁄ aich ⁄ ⁄ (h n ). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h ha peptide with a single r at the cleavage site ( figure a) , both the h ha peptide with the k-k-k-r motif ( figure b ) and the h ha peptide with the r-k-k-r motif ( figure c) were fully processed at the correct positions by mspl within hour. in the case of h ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p , n'-k-k-r-k-k-rfl-g-c' in the presence of mm cacl . these cleavage site specificities of furin were consistent with that reported for the h ha peptide of hpai virus a ⁄ hong kong ⁄ ⁄ (h n ) with r-k-k-r motif. however, the h ha peptide with k at position p ( figure b) was hardly cleaved by furin under the same experimental conditions. tmprss showed similar results (data not shown). these findings suggest that mspl and tmprss cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss transfectant cells. in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss . to test for the generation of infective virus, the conditioned media of -day culture of ecv -wt and ecv -mspl cells infected with wt and mutant hpai h n viruses were inoculated into newly prepared cells and cultured for hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv -wt and ecv -mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv -mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p in a small number of hpai viruses. figure shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ ⁄ (h n ) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ ⁄ ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv -wt cells containing furin ( figure ). these substrate specificities of furin suggest that proteases other than furin and pc ⁄ play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) with k-k-k-r cleavage motif in ecv -mspl cells (figure ) . these results were supported by the data of two cleaved peptides by mspl in figure c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. , in addition, mspl and tmprss are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc ⁄ , but also on the cell surface by mspl and tmprss . the pb -f protein, which is translated from the + reading frame of the pb gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. - a mitochondrial targeting sequence is located in the c-terminal portion of the pb -f open reading frame, and expression of full length pb -f has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. , it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb -f . , a suggested second function of the pb -f protein is that it enhances immunopathology by triggering the inflammatory response. , in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb -f from the pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. , in this report we analyze the pb -f protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb -f proteins from a variety of epidemiologically important iav strains including all pandemic strains from the th century, a highly pathogenic avian influenza virus of the h n subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb -f proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb -f proteins from pr , a ⁄ brevig mission ⁄ ⁄ , ⁄ singapore ⁄ ⁄ , a ⁄ hong kong ⁄ ⁄ , a ⁄ wuhan ⁄ ⁄ , and a ⁄ vietnam ⁄ ⁄ , peptides from the c-terminal end were synthesized as described. an additional n-terminal peptide was synthesized from the pr sequence as a positive control (mgqeqdtpwilstghistqk) as described. a panel of viruses were reverse engineered as described , and included laboratory strain pr , a virus unable to express pb -f (dpb -f ⁄ pr ), or expressing the pb -f of the pandemic strain ( pb -f ⁄ pr ) or the truncated h n strain (beij pb -f ⁄ pr ). , in addition, : reassortants encoding pb gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. highly pathogenic avian influenza of the h n subtype (h n pb ⁄ pr ), or from a human h n strain (h n pb ⁄ pr ) were utilized along with their isogenic deletion mutants for pb -f (h n dpb -f ⁄ pr and h n dpb -f ⁄ pr ). cell lines and cell death assays raw . cells were grown under conditions as described. cells were infected with one multiplicity of infection (moi) of virus for - hours, or exposed to lm (final concentration) of peptides derived from the c-terminal portion of pb -f for hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = - ) in a volume of ll ( ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co inhalation, the trachea was exposed and cannulated with a gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of < ae was considered significant for these comparisons. graphpad prism version . for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb -f to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. , the effect of pb -f expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb -f from pr or expression of the c-terminally truncated beij pb -f had a significantly reduced influx of macrophages ( figure a) . expression of the pb -f caused similar inflammatory effects as the pr virus. disruption of pb -f expression the virus containing the h n pb gene segment in a pr background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb -f ( figure a) . however, no differences were seen that could be attributed to the h n derived pb -f . the lungs of mice infected with the panel of pb -f variant viruses were examined at hours. pathologic changes typical of pr viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure ). in the lungs of mice infected with pr or pb -f ⁄ pr , however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb -f ⁄ pr or beij pb -f ⁄ pr ( figure b, d) . as the function and influence of pb -f protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb -f beij ⁄ pr virus, we sought to elucidate whether the c-terminal domain of pb -f could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ), and (h n ), and the h n virus compared to controls ( figure b) . similar effects were not seen with the peptide derived from a more recent h n strain, a ⁄ wuhan ⁄ ⁄ . when peptide exposed mice were followed for morbidity for days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb -f proteins of viruses containing pb gene segments reassorted directly from the avian reservoir. the pb -f protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb -f proteins from several different iav strains to cause cell death. we therefore infected raw . cells with the panel of recombinant viruses at an moi of for - hours. as has been demonstrated previously, , , pr virus induces significant cell death compared to uninfected controls ( figure c ). when raw . cells were infected with pr virus, necrotic death peaked hours after infection. viruses lacking the c-terminal portion of pb -f , including the dpb -f ⁄ pr and the beij pb -f ⁄ pr were unable to cause cell death ( figure c ). in addition, expression of the pb -f also did not cause significant increases in cell death over controls. expression of pb -f or deletion of pb -f in either an h n or h n pb gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw . to the panel of pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ) and (h n ), and the h n for hours. cell death in raw . cells was caused only by the peptides derived from the laboratory strain pr and the peptide derived from the pandemic strain ( figure d ). viability was not affected by exposure of raw . to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb -f contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb -f protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb -f proteins only from those strains contributing to the formation of all pandemic strains of the th century and from the currently circulating, highly virulent h n strains that constitute an imminent pandemic threat. the iav h n strains circulating in humans since around code for a truncated pb -f . these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h n strains, in contrast to their pandemic forbear from , have lost the capacity to cause pb -f mediated inflammation through mutation of the c-terminus of this protein. in a novel h n iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild in contrast to the three pandemics of the th century. the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three th century pandemics shows that only the hemagglutinin (ha) and pb gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb gene segment in each of the th century pandemics is not yet understood. we show here that the pb -f of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb -f may serve as a marker of the pathogenicity of pandemic strains. since the h n strain codes a truncated pb -f of only predicted amino acids, the lack of pb -f mediated inflammation may account in part for its relatively lower virulence. , of the panel of pb -f proteins studied, only that from the laboratory strain pr was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb -f gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb -f to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il- , and il- b, affect host survival both positively and negatively. the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. in addition, iav infection up-regulates several cellular proteases including ectopic trypsin and mmp- . up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), which is crucial for viral entry and replication and the subsequent tissue damage in various organs. the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged weeks (c bl ⁄ crslc) were infected with iav ⁄ wsn ⁄ ( pfu) with and without treatment of pdtc ( . mg ⁄ kg), nac ( mg ⁄ kg), and ndga ( mg ⁄ kg). these inhibitors were administrated once daily for days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. host cellular responses in the airway after iav infection figure shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp- and matrix degradation. just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il- in the lungs were increased persistently for days after iav wsn infection, and that of il- b peaked at days - post-infection (figure a ). since these cytokine responses are associated with activation of nf-jb and ap- , we treated mice once daily for days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap- activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il- b (p < . ), and nac suppressed tnf-a (p < . ), and il- (p < . ) at day post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp- in mice lung, brain, and heart during infection for days ( figure b ). trypsin and mmp- induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap- binding in the promoter region of the genes. viral rna replication in various organs at day post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day post-infection, the late stage of infection ( figure d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens- (zo- ) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day post-infection, which were partly rescued by pdtc, nac, or ndga (figure e ). no other tight-junction protein, claudin- or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il- b, and il- , when up-regulated by iav infection, induce trypsin and mmp- expression in various organs in mice; (ii) inhibitors of nf-jb and ap- effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp- and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap- , and the janus kinase-signal transducers and activators of transcription signaling pathways. , [ ] [ ] [ ] the in vivo experiments presented here showed that nf-jb and ap- inhibitors markedly suppress the expression of cytokines, trypsin, mmp- , and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, , and increases [ca + ] i atp depletion dissociates zo- from the actin cytoskeleton and thereby increases junctional permeability. endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h n and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor signaling pathway. in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h n and pandemic h n pdm for primary human lung microvascular endothelial cell has not been well defined. in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h n and h n viruses initiated viral gene transcription and viral protein expres- sion, but did not produce infectious progeny, while the highly pathogenic avian influenza h n and the pandemic influenza h n pdm virus could replicate even with the absence of exogenous protease (figure ) . furthermore, when compared to seasonal h n and h n , the h n virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp- , rantes, ip- (figure ) , and il- , in virus infected endothelial cells, whereas h n pdm induced intermediate levels of cytokine and chemokine. avian influenza h n and pandemic h n pdm virus (but not the seasonal h n and h n virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h n and h n pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [ ] [ ] [ ] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h n and h n pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. the aim of the study is to identify cellular interactants of m that play important regulatory roles during influenza infection. to identify cellular partners of m we performed a genome-wide yeast-two-hybrid (y h) screening approach using the cytosolic domain of m as bait and a human placenta random primed cdna library as prey and tested more than million interactions. from the y h screening, an interesting interaction with the human annexin a (anxa ) protein, a member of annexin family proteins that binds to phospholipds in a ca + -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa and viral m proteins coexpressed in hek t cells after transfection and infection confirmed the direct interaction between anxa and m . we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa gene in human lung epithelial a cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax during viral infection (figure ). a novel interaction between m and anxa was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m -pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at °c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen ' · reaction mix': ae mm of each dntp + mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems fast real-time pcr system, 'respiratory multiplex' program. well content ll; thermocycler initial stage ae °c for minutes, then °c for minutes. subsequent cycles of ae °c for seconds followed by °c for seconds for cycles. sequence detection software version . (applied biosystems). of clinical isolates analyzed, all samples produced amplification of pdh material; produced amplification of both swine flu and pdh material. human male dna (lot no. at ng ⁄ l, applied biosystems) at concentration calculated at ae cells ⁄ ll was diluted from ) to ) , yielding mean average ct values of respectively ae , ae , ae , and ae . plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct ae was diluted through ) to ) . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier ( lg ⁄ l, qiagen; cat no. ) in place of rnasefree water. the ) concentration was positive in nine out of assays; this fraction was used in the calculation described by simmonds to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = ⁄ = ae ; )ln ae = ae copies. a control curve was generated with ct values of ae , ae , ae , and ae giving copy values of , ae , ae , and ae , respectively. using excel (microsoft office, ), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was ae , female to male ratio : , and average admission length of days. of the mild group (n = ), nine cases were not admitted to hospital. of the remainder, the mean average admission length was ae days. mean average c value for all samples was ae · , with a standard deviation of ae · ; geometric mean was ae , and median average was ae . log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures a, b respectively) . in each case variation was too great to yield statistical significance. figure shows the range of c values observed in the 'moderate' severity group. > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · in a study by duchamp et al., no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values < · ) to over · . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present ( ae · ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value ( ae versus ae ), it was significantly lower than the median c value when no co-morbidity was documented ( ae ). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of to days, with mean average delay of days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [ ] [ ] [ ] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. , with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h n , with consequently increased shedding. the review by mansfield cites significant findings regarding influenza pathogenesis, including the predilection of h n strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april a novel variant of a(h n ) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d g. resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h n ) virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h n ) cases. from a total of a(h n ) positive cases diagnosed and confirmed, were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h n ) strains were isolated in mdck-siat cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h n ) virus circulating in portugal, of the isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb , pb , pa, ns, and np) were also sequenced for six of this isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, using bigdye terminator v. . technology. nucleotide sequences were determined in a dna automatic sequencer abi prism xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v. . software (dnastar inc, usa) using an average of - overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega . software, against published sequences from the vaccine strain (a ⁄ california ⁄ ⁄ ) and from selected a(h n ) strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h n ) influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all isolates by ic determination through munana fluorescence assays. genotypic evaluation was performed by searching for mutations associated with resistance to nais in all na gene sequences. amantadine susceptibility profile was performed for all isolates by searching on m sequence for the molecular markers associated with resistance to this antiviral drug (l f ⁄ i; v a ⁄ d; a t; s n; g e). genetic characterisation of the ha subunit of ha reveals point mutations in different strains. all analysed strains present p s and i v mutations, which distinguish them from the vaccine strain ( figure a ). thirty-three of the sequenced strains group in the s t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. most of these strains ( ) further subgroup in the d e branch, this mutation being associated with one loop of the receptor-binding site. from the early to the late epidemic period, an increased circulation of virus carrying the mutation s t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed ( of ) presents the mutations n d and v i ( figure b) . as mutation s t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the analysed strains further subgroup in y h branch. additionally, mutation i v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb , pb -f , and ns were detected; (ii) pb -f protein is present in the truncated form of amino acids; (iii) the presence of mutations i v and l q in ns and v i in np; (iv) the described association of mutation i v in ns and v i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ ⁄ and a ⁄ portugal ⁄ ⁄ ) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i r) has been referred to as being associated with a reduction in nais susceptibility. full genome sequence analysis of these strains shows that both strains also present the v i mutation in pb gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all analysed strains present a serine in position , which is a molecular marker of resistance to m inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha subunit have been detected in a(h n ) strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. the occurrence of mutations in the position of the ha subunit of a(h n ) virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h n ) pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. the majority of the a(h n ) strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitor- the efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h -labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h -labs have bsl- facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h n ) pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h n ) specific h and n genes rt-pcrs) were developped and distributed to the lab networks on the th of may . from the early phase, the nics and the h -lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb ) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf sequencing platform of the institut pasteur. the first imported a(h n ) influenza cases were observed from the th of april . a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h n ) virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted weeks between mid-october and the end of december (week to week , figure ). the pandemic wave started week - in the ile-de-france area, and only week - in the rest of france. the peak was recorded week ( figure ). the impact of the pandemic was mainly observed in the - years group of age. overall, severe cases have been admitted to the hospital, and deaths have been recorded by the end of the pandemic wave. the major impact was observed in the - years group of age ( % of deaths recorded). amongst the severe cases and the deceased cases, % and % of cases had no risk factor, respectively. these specimens, were positives for h n , representing ae % of total influenza virus detections. only nine brisbane-like h n , brisbane-like h n , and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from % to %. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d g mutation in the ha. amongst the virus tested for antiviral susceptibility or screened for the h y mutation by or specific rt-pcr, only oseltamivir-resistant viruses related to the na h y mutation have been detected. one of these cases also had an i r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d g mutation in the ha. all these patients had a severe infection; one of these had also a h y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april . although the first cases recorded were as early as the th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. , it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that % of the french population consulted for an ili presentation. the impact was mainly observed in the - years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the - years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded ( % and %, respectively). this data is consistent with the observational data reported by numerous other countries. according to the profile of hospitalized cases, a(h n ) was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited ( cases), the age distribution of the deceased patients was different as compared to seasonal influenza ( % mortality in < years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the h n virus. the virological monitoring of the pandemic was achieved by the community-based and hospital-based sea- sonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the th of november, just when the pandemic started in france. it is estimated that millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april , a novel pandemic influenza virus a ⁄ h n emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h n , a ⁄ h n , and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in , a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. in , a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. both studies were conducted with very similar protocols, involving households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous weeks. follow-up consisted of three home visits that spanned approximately - days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡ ae °c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid ). the details concerning laboratory methods have been described in a previous study. all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡ of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ ae °c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version . . ; r development core team). a total of households and individuals were followed-up in the two studies. of household con-tacts tested by rt-pcr, were found to be influenza positive. among these influenza infections, ( ae %) were asymptomatic (rt-pcr positive plus symptoms recorded), were subclinical (rt-pcr positive plus symptom recorded), and presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table ). there were a lower proportion of males who acquired pandemic a ⁄ h n compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h n and seasonal b, whereas runny nose was most common in seasonal a ⁄ h n and a ⁄ h n cases. cumulatively, fever ( ‡ ae °c) was reported in approximately half ( %) of the secondary cases. patterns of viral shedding were analysed in a subset of influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or day post-ari onset. follow- ing the peak, measured levels of viral shedding declined steadily to undetectable levels over - days. the trend of viral shedding in influenza b infected individuals rose days before ari onset, fluctuated for around days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or day post-ari onset, and gradually declining over a period of - days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h n were similar to trends observed for seasonal a ⁄ h n and seasonal a ⁄ h n infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h n influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h n virus were similar to the seasonal a ⁄ h n and a ⁄ h n viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h n were detected and the secondary attack rate in general was low, though a total of households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h n and h n viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing - g were inoculated intranasally with eid of virus without anaesthesia. transmission studies were then performed hours after inoculation. inoculated animals were housed at % relative humidity and °c in the same cage with noninfected guinea pigs or in cages placed m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at and days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn , indo ⁄ , a ⁄ california ⁄ ⁄ , and nibrg- were isolated in %, % ae %, and % of contact animals, respectively. serological confirmation of virus transmission was higher than virological data ( %, %, %, and %, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ ⁄ were co-caged with animals inoculated with nibrg- , they got infected with both viruses ( table ) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ ⁄ california ⁄ ⁄ laiv candidate was detected in the upper respiratory tract of ae % vaccinated pigs. the mdv was identified in % of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg- reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg- virus (table ) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg- and indo ⁄ viruses were able to spread between cages over the m distance ( % and % naïve animals were successfully infected, respectively). a ⁄ california ⁄ ⁄ influenza and vn viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table ) . pigs with confirmed a ⁄ california ⁄ ⁄ virus replication were also infected with nibrg- virus if h n -and h n -infected animals were separated by a space. thus, influenza virus transmission from h n -to h n -infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg- or a ⁄ california ⁄ ⁄ viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a m distance ( table ) . it was also shown that efficiency of transmission of nibrg- was much higher than of other studied h n viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of - m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ ⁄ virus and co-caged with pigs inoculated with nibrg- . two reassortants possessed different combinations of pr , nibrg- , and a ⁄ california ⁄ ⁄ genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg- , na gene from a ⁄ california ⁄ ⁄ , and other genes from pr became ca and ts. : non-ts reassortant inherited pa gene from pr and seven other genes from a ⁄ california ⁄ ⁄ , gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg- viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb and na genes from mdv, pa gene from pr , and ha gene from nibrg- . the origin of the other gene segments (pb , np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg- and indo ⁄ viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h n virus compared to the nibrg- and indo ⁄ h n strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg- virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h n subtype and a heterologous h n subtype influenza virus between guinea pigs has been shown. interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg- virus led to the generation of triple reassortants inherited pb and na genes of mdv and ha gene of h n virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h n and h n pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic , and the ongoing concerns about human cases of avian influenza h n infections, there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. , previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. , schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [ ] [ ] [ ] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large m diameter parabolic mirror (figure ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages ( - years old). these included males (three of years, one of years of age) and females (one of years, one of - years, and one of - years of age). each volunteer was tested with and without wearing either a standard surgical mask or n mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n mask (supplied by mÔ, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a -year old male, the year-old female and a -year old male, and the - year-old female. this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the m mirror (figure a) . a previous detailed study of one of these images measured a maximum airflow velocity of m ⁄ second for an adult cough. similarly, the effects of wearing surgical and n masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. , discussion from these series of schlieren images presented in this and other related studies, [ ] [ ] [ ] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n -style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, , though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method in a cell culture system employing mdck cells in serum-free ex-cellÔ medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue Ò influenza a + b test. the log inactivation and percent inactivation by bac after a second contact time for the h , h , and h pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ ⁄ h n , ae log ⁄ ae %; a ⁄ swine ⁄ cal ⁄ h n , ae logs ⁄ ae %; a ⁄ j ⁄ ⁄ h n , logs ⁄ ae %; and a ⁄ hong kong ⁄ h n , ae logs ⁄ ae % (table ). comparable results of antiviral efficacy are obtained with the tcid and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ ⁄ virus dilutions into two different vessels with hours adsorption time: -well plate and t- flask, ml inoculum per replicate. virus titers obtained were: ae · pfu ⁄ ml from -well plate and ae · pfu ⁄ ml from t- flask ( table ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t- flask gave higher virus recovery ae versus ae · pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid was · tcid ⁄ ml. rapid diagnostic testing for influenza (quickvue Ò influenza a + b test, quidel) for aj versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated % with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table . comparison of viral titer obtained in different vessels using quantal tcid and plaque assay methods plaque assay tcid assay t- ( cm ) -well plate ( cm ) tcid ⁄ ml tcid ⁄ ml ae · pfu ⁄ ml ae · pfu ⁄ ml · ae · options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h n ) influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples ( weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h n ) influenza and rhinovirus infection. out of residents, three had laboratory confirmed influenza [two with pandemic (h n ) ], and had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡ : ) pandemic (h n ) hai antibody in % ( ⁄ ) subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ) compared with none of the residents aged under years (born after and median age years; gmt ae , p = ae ). the acf was closed to visi-tors for days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h n ) , which may explain the minimal clinical impact of pandemic (h n ) in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. people aged over years are also at higher risk of influenza-related death, and more than % of annual influenza-related mortality is usually confined to this high risk group. in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for persons in . since the world health organization declared an influenza pandemic in june , australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october there were reported deaths due to pandemic influenza in australia, and to date there have been about deaths reported worldwide. although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. early in the pandemic (june ), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h n ) virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june th , the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h n ) outbreak in a local acf. on investigation, it was discovered that days earlier a year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h n ) was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june th, the symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h n ) ] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h n ) influenza in a local army camp just over the border in nearby victoria, where pandemic (h n ) influenza was known to be circulating widely. on june th, the year old lady proved nat positive for pandemic (h n ) , but none of the other samples were pandemic (h n ) nat positive. concern arose that there might be an outbreak of pandemic (h n ) in the facility, and that some of the swabs from other residents might be false negatives. between and june, after consent was obtained, directly or through next of kin in demented residents, all submitted to venipuncture for serology, successfully, and the other as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after weeks on th july from of the residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h n ) ] and b was performed. the acute and convalescent serum samples were tested later (in december ), using haemagglutination inhibition assay (hai) to detect pandemic (h n ) antibody. , interventions the acf was closed to visitors from th until th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was - years with a median of years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table ) testing of acute sera collected from every subject found elevated ( ‡ : ) cross-reacting hai antibody to the pandemic (h n ) in % ( ⁄ ) of subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ). however, the hai titre was consistently < : and significantly lower (gmt ae , p = ae ) in the residents aged under years (range - years, median years) (figure ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from to ). the pandemic (h n ) case that was determined by serology was pandemic (h n ) nat negative. to our surprise, seven of the other asymptomatic residents had rhinovirus detected on extended nat (reported on june th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus ( ae %). the serologically confirmed pandemic (h n ) case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. finding a nat positive case of pandemic (h n ) influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h n ) was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case and perhaps ) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. studies have found that the pandemic (h n ) haemagglutinin (ha) gene is more closely related phylogenetically to the h n virus and classical swine influenza a ⁄ h n viruses than more recent seasonal human influenza a ⁄ h n viruses. it is antigenically similar to the h n pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [ ] [ ] [ ] it is likely that individuals alive during the emergence and initial persistence of the pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h n ) , which would contribute towards better clinical protection. in our investigation, % of the residents born before (aged years or above in ) had pre-existing cross-reacting hai antibody to the pandemic (h n ) . in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused fatalities. another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. a previous influenza outbreak in a nsw aged care facility in caused significant mortality and morbidity. that outbreak resulted in hospital admissions and six deaths. in our investigation we have found that % of the residents had chronic lung disease and % had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡ years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the pandemic (or a close variant occurring before ) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h n ) and also to symptom relief. pandemic (h n ) influenza virus (ah pdm) has spread worldwide since march . in a paper of ah pdm, % of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. , viral load and isolation of ah pdm in cell culture in stool samples has been reported. stool specimens were collected from patients suspected to have pandemic (h n ) infection from november through may . virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from % (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case ) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case by using only one primer at the rt reaction and also assayed neu aca - gal and neu aca - gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu aca - galb - glcnacb-pap or neu aca - galb - glcnacb-pap bond as described. ) nucleotide sequences of the ha gene of ah pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah pdm viral rna was detected in nine ( %) of the subjects from stool samples. among nine subjects, one case (case no. ) was positive for viral isolation. case , a healthy -year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case , influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table ). viral load decreased gradually after symptom onset. however, viral shedding was still present days after symptom onset. positive stranded rna was detected days after symptom onset from the stool specimen ( figure ). above two ah pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu aca - gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene ( bp). ah pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp and n respirators are certified to fil-ter at least % of particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [ ] [ ] [ ] we have developed a proprietary acid-polymer formulation to coat a standard ffp respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h n pandemic strain, seasonal h n , h n , h n , h n , and h n . in each test, suspensions of influenza viruses were prepared to - log tcid ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at °c and % relative humidity for hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp respirator samples were inoculated with ae ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. in one test, after a minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial -fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid or eid . antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after minute of exposure. the viral loads applied to respirators in these experiments ranged from ae to ae log tcid , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range - log tcid ). tables - show that the average viral loads detected on uncoated ffp respirator samples remained high in all conditions tested, ranging from ae to ae log tcid (or ae - ae log eid ). in contrast, the average viral load on coated respirators after minute of exposure ranged from below the limits of detection to £ ae log tcid ( ae log eid ). therefore, the relative antiviral efficacy of the coating ranged from ‡ ae to ae log . table shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of ae log eid (table ) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡ ae log . ‡ ae log viral load with organic contaminants* ae ae ae log viral load after heat, moisture, and simulated secretions** ae ae ae log viral load transferred to glove** ae £ ae ‡ ae log eid *influenza subtype was a ⁄ h n , and strain was vnh n -pr ⁄ cdc-rg. **influenza subtype was a ⁄ h n , and the strain was hong kong ⁄ ⁄ . results are mean log tcid , unless specified otherwise. results are mean log tcid , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. * pandemic strains. **results are mean log eid , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. - ffp respirators are certified in europe to filter at least % of nacl particles ( ae lm in diameter), and ffp and ffp respirators must filter at least % and % of these particles, respectively. influenza a viruses are typically ae lm, and can be carried in aerosolised droplets smaller than lm in diameter, which can disperse widely, remain airborne for hours, and be inhaled deeply into the respiratory tract. we have developed an acid-polymer formulation to coat the outer layer of a standard ffp respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp , ffp , and ffp respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp respirators (glaxosmithkline actiprotect) with three ffp controls (sperian willson easy fit). for each test, suspensions of influenza a (h n ) at ae log tcid ⁄ ml in ae · minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for minutes, at a flow rate of ae l ⁄ minute. constant airflow was maintained for another minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. briefly, serial dilutions of the collection medium (mem with % fbs, % gelatine, and % hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over - days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid . the second assay compared five samples of coated respirators with five ffp controls ( m ) and five ffp controls ( m ). a suspension of influenza a (h n ), at ae tcid ⁄ ml, was nebulized for minute and seconds into the aerosol chamber, at a flow rate of ae l ⁄ minute, followed by constant airflow for minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with % nahco ) was assayed as described above. initial viral loads in the first and second assays were ae and ae log tcid , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range - log t-cid ). table shows that the average viral load that passed through the uncoated ffp respirators in the first assay was ae log tcid . the average viral load that passed through the coated respirators was ae log tcid . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was ae log greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was ae log . in the second study, table shows that the average viral load that passed through the uncoated ffp respirators was ae log tcid . in contrast, ae log tcid passed through the coated ffp respirators. by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log , and the coated ffp by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by ae log more than the ffp respirators. in this second study, the average viral load that passed through the uncoated ffp respirators was also ae log tcid . by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by ae log more than the ffp respirators. table also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by ae log more than the ffp respirators, and ae log more than the ffp respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional ae log infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp respirators were no more effective than ffp respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp and n respirators are certified to filter at least % of nacl particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. , we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co , and saturation with contaminants. eight coated respirators were tested at standard relative humidity ( % rh) for hours, and one at elevated humidity ( % rh) for hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at % rh for hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at °c and ppm co was pumped through the masks by a cyclic breathing machine at l ⁄ minute. a mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at °c for hours. triplicate monolayers of mouse fibroblast cells (l- ) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at °c in % co for hours. after hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to adults. after hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after and hours. the patches were applied three times a week for weeks. to evaluate sensitisation, test patches were applied - days later for hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after and hours. table shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused % cell lysis or toxicity, classified as slight reactivity (grade ), and that uncoated material caused no cell lysis or toxicity (grade ) ( table ) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, , we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in children at six elementary schools over the past two seasons ( - and - seasons) was compared with that of pdmh in children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission , and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, - not only for seasonal influenza, but also for h n pandemic compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and patients were treated with amatadine. no treatment was done for patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was ae % ( of households) for seasonal influenza or ae % ( of households) for pdmh . thus, the household incidence of pdmh was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was ae % ( of individuals) for seasonal influenza or ae % ( of individuals) for pdmh . thus, the household transmission rate was also lower for pdmh than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of - individuals, the incidence of seasonal influenza in order of increasing family size was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, and the incidence of pdmh was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, or ae %, ae %, ae %, ae %, ae %, and ae %, respectively, for pdmh (shown in table ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was the highest in c , followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c to f were ae %, ae %, ae %, ae %, ae %, and ae %, respectively. therefore, as for household incidence, the highest rate ( ae %) was observed in c . the corresponding household transmission rates for pdmh were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, with the highest transmission rates observed for infections from parents (shown in table ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh were ae % ( of individuals) and ae % ( of individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. for pdmh , the corresponding rates in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. these findings indicate that, especially in the case of pdmh , most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was ae % and ae %, respectively, whereas the prevalence determined days after appearance of the first case in school was ae % and ae %, respectively. in the recent season at the same elementary schools, however, the prevalence was a high ae %. since the prevalence at days after the appearance of the first case in school was already ae %, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even days after the school closure action was taken. these findings indicate that pdmh , the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh , the distribution of the non-infectious latent period of pdmh was established as (day , day , day , day ) = ( %, %, %, %) and the distribution of the infectious period as (day , day , day ) = ( %, %, %). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was %. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for days. it was shown that there was an increase in the expected number of cases during the days corresponding to the period of actual school closure and that the expected prevalence increased to %. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure ). if the transmissibility of pdmh virus at home is estimated based on the speed of transmission and the degree to which pdmh is prevalent in schools, it would be expected that the household transmission of pdmh is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. . the number of children aged or more and parents with pdmh influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza ( ae % versus ae %). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh was a low ae % ( of individuals), compared to that for seasonal influenza ( ae % [ of individuals]). in other words, adults are less susceptible to pdmh infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. . the percentage of households with more than one affected individual within the same family was higher for pdmh at ae % ( of households) than for seasonal influenza at ae % ( of households). in the patients secondarily infected with pdmh , ae % of them showed symptoms of infection days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was ae % ( of households). therefore, although the prevalence was higher for pdmh , it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh influenza in its transmissibility at home and in school. in the household transmission of pdmh influenza, both the household incidence and household transmission rate of pdmh were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh , the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when % of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, , and children are at high risk of spreading the disease. with reference to previous evidence, - some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. , during - , a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). the monovalent vaccine against pandemic influenza a (h n ) (ph n ) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph n . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we report findings on the effectiveness of tiv against ph n observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. to be eligible, the household had to include at least one child aged - years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above years and those aged - years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio : ) according a code generated by block randomization with random block sizes of , , and . an independent nurse prepared . one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december ), one month after vaccination (december -january ), and after the winter (april ) and summer influenza seasons (august-october ). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$ for each serum specimen collected, us$ . for each home visit, and us$ for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡ fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡ . °c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ ⁄ (h n ) in another study conducted by our group and was, therefore, used in this study. the sera was initially diluted at ⁄ and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. , fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate % confidence intervals where appropriate. all analyses were carried out in r version . . (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, invitation letters were sent and households were enrolled. personal referrals were made from these parents to enroll additional households. among enrolled households, subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡ against the vaccine strains month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h n % in tiv versus % in placebo group, p < . ; a ⁄ h n % versus %, p < . ; b % versus %, p = . ). none of the study subjects had antibody titer ‡ against ph n following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h n ) ( % versus %, p = . ), a(h n ) ( % versus %, p = . ) and b infection ( % versus %, p = . , although the differences were not statistically significant (table ) . study subjects had higher rate of serologically confirmed ph n infection ( % versus %, p = . ), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, % of subjects in tiv versus % in placebo groups showed ph n infection confirmed by either serology or rt-pcr (p = . ). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili ( % versus %, p = . ) or ari ( % versus %, p = . ). an opposite tendency was seen (ili % versus %, p = . ; ari % versus %, p = . ) during summer when ph n predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph n did not differ statistical significantly between subject who received tiv and placebo ( % versus %, p = . ). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph n infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below years were found to have a higher risk (< years or = . , % ci . , . ; - years or = . , % ci . , . , > or = . ). after adjusting for age, sex, and date of study completion, receipt of tiv for the - influenza season was not found to affect risk of ph n infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had % lower risk of ph n infection (infected with seasonal influenza or = . , % ci . , . ; not infected with seasonal influenza or = . ). as (see table s for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡ . °c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h n ) and a(h n ) infection against ph n . other details of the results from the study were published elsewhere. discussion a non-significantly higher rate of ph n infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph n . seasonal influenza infection within - months observed in our study might have conferred better cross protection than tiv against ph n . this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph n . , , [ ] [ ] [ ] apart from differences in study design and vaccine used, we speculate that a short time interval between ph n and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter - influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the h n pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two h n pandemic influenza vaccines in children in the uk. here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h n influenza vaccines, one as b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules days apart, in a randomised, open label trial as previously reported. the study was age stratified ( months to under years & - years) to ensure adequate data in young children. age appropriate safety data (simplified for under year olds) were collected for days after each vaccine dose and serum was collected at enrolment & days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade ( ‡ mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over years old, ae % versus ae %, p < ae , after dose one; ae % versus ae %, p = ae , after dose two, in children under years old, ae % versus ae %, p = ae , after dose one (non significant, ns); ae % versus ae %, p < ae after dose two. fever ‡ °c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in < year olds ( ae % versus ae %; p < ae ). looking specifically at the adjuvanted vaccine in under year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡ °c (axillary measurement) ( ae % versus ae %, p < ae ), local grade ( ‡ mm) reactions ( ae % versus ae %, p = ae ), pain ( ae % versus ae , p = ae ), use of analgesia or antipyretic medication ( ae % versus ae %, p < ae ), and decreased activity ( ae % versus ae %, p < ae ). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in < year olds, haemagglutination inhibition (hi) seroconversion rates were ae % versus ae %, p < ae . among all general and local reactions measured, only the maximum temperature measured during the days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each °c rise in temperature there was a % increase in titre (p < ae ). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h and h . the attenuated phenotype of h and h laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h n viruses in avian populations and concurrent infections in humans since has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in , the world faced a new h n pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain and ae eid per dose ( ae ml) of a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ , respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of days. one hundred-ninety healthy adults aged - years were randomly divided into groups to receive either pandemic vaccine candidates ( ) or placebo ( ) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £ : to a ⁄ california ⁄ ⁄ (h n ) pandemic virus. in all there were and vaccines and and participants who received placebo, and were further tested for immune responses to h n or h n pandemic vaccine, respectively. another participants vaccinated with h n laiv were children between to years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd + cd + ifnc + and cd + cd + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ ⁄ and a ⁄ duck ⁄ potsdam ⁄ - parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h n pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ ⁄ california ⁄ ⁄ pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, ae % and ae % of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h n and h n vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table ) . post-vaccination geometrical mean titers (gmt) among subjects who received two doses of h n vaccine were significantly higher than pre-vaccination titers. the frequency of ‡ fold antibody rises was significantly higher ( ae %) after revaccination than after one dose ( ae %). the percentage of subjects with post-vaccination serum hai titers to h n ‡ : was ae % and for titers ‡ : , it was ae %. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h n vaccine candidate demonstrated significant increases of ‡ fold rise iga antibodies ( %) compared to one dose. cumulative data of h n vaccination (all applied tests) showed % and % of conversions after the first and the second vaccination, respectively. increasing h n vaccine virus infectivity from ae to ae eid ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h n antigen from ae % to ae % of ‡ fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ ⁄ california ⁄ ⁄ , also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡ : were ae %. after revaccination, this parameter increased to ae %. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was ae % and ae %, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were ae % vs. ae % of iga conversions in nasal swabs, respectively. taking into account cumulative data of h n vaccination (hai and elisa data), the obtained results were here and in the ae % and ae % of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h n laiv, and who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd + and cd + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was ae % (cd + ) and % (cd + ). after the revaccination, the percentage of subjects with significant increases in cd + and in cd + cells was ae %. immunogenicity of h n pandemic vaccine candidate in children hai antibody results among children aged to years proved to be significantly higher when compared to adult subjects: after the first vaccination, ae % of the children seroconverted; after revaccination, seroconversions reached ae % ( table ). the gmt rise to h n vaccine with primary vaccination was : ; after revaccination it increased to : . benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [ ] [ ] [ ] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from to %. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ vaccine candidates increased to ae % and ae %, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h n pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects ( ae % of ab seroconversions after the single vaccination and ae % after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h n pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [ ] [ ] [ ] [ ] [ ] [ ] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf -adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h n ) strains than non-adjuvanted vaccines. [ ] [ ] [ ] the broader immune response showed by mf -adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position (n k), (n k), and (p s) with respect to a ⁄ california ⁄ ⁄ . in particular, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ presents n d amino acid mutation detected in clade a ⁄ wyoming ⁄ ⁄ -like viruses. the ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ fell within the clade represented by the ha of a ⁄ califor-nia ⁄ ⁄ ; among these isolates, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ showed antigenic site sequences very close to that of the ⁄ vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ posses amino changes onto antigenic site a(r k), c(g e) and d(r k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ ⁄ and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ ⁄ , g e and k i, with the exception of a ⁄ genoa ⁄ ⁄ , showing r g and l s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ ⁄ clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. two blood samples were collected from each subject, just before and ± day post-vaccination. all sera were stored at ) °c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [ ] [ ] [ ] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡ iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of : ). post-vaccination gmt was reported as ratio, with the corresponding % confidence interval, of gmts after vaccination with mf -adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate % confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf -adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi > ; seroprotection rate > %, or seroconversion rate > %. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. and were expressed as median titres, with the corresponding °- °i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf -adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all strains (data not shown). post-vaccination gmt ratios between mf -adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding % confidence interval, according to viral strain are shown in figure . both vaccines met chmp requirements for mfi (> ), seroconversion (> %), and seroprotection rate (> %) against a ⁄ wyoming ⁄ ⁄ -like, with the exception of a ⁄ genoa ⁄ ⁄ and a ⁄ california ⁄ ⁄ -like circulating viruses and against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ , and a ⁄ wisconsin ⁄ ⁄ strains; the immune response against a ⁄ genoa ⁄ ⁄ met the requirements for mfi and seroprotection rate only in mf -adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ ⁄ -like virus a ⁄ genoa ⁄ ⁄ and the a ⁄ nepal ⁄ ⁄ -like genoa ⁄ ⁄ viruses and against egg-grown a ⁄ brisbane ⁄ ⁄ strain were reached only in subjects vaccinated with the mf adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf -adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ ⁄ -like, a ⁄ california ⁄ ⁄ -like, a ⁄ nepal ⁄ ⁄ -like and a ⁄ brisbane ⁄ ⁄ like viruses, with the exception of a ⁄ genoa ⁄ ⁄ , and against egg-grown a ⁄ california ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure ). the mf -adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf -adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ ⁄ -like viruses, a ⁄ brisbane ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ , and a ⁄ nepal ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ strain ( figure ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ . among viruses more closely related to a ⁄ california ⁄ ⁄ , subjects immunized with mf -adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ strains compared with the non-adjuvanted vaccine ( figure ) . spearman test showed a clear correlation between the distances and the advantage offered by mf expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [ ] [ ] [ ] in subjects vaccinated with the mf -adjuvanted vaccine containing a ⁄ california ⁄ ⁄ , the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ ⁄ -like and a ⁄ nepal ⁄ ⁄ -like strains, and against egg-grown a ⁄ brisbane ⁄ ⁄ virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf -and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the ⁄ season, that appear more phylogenetically close to ⁄ vaccine strain a ⁄ wyoming ⁄ ⁄ , was higher in subjects vaccinated with mf -adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ ⁄ , against whom the nt post-vaccination gmt is identical in mf and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf vaccine group when evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ . as far as the immune response against a ⁄ california ⁄ ⁄ -like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf vaccine group for all the strains. interestingly, a ⁄ california ⁄ ⁄ -like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf -adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ , which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ ⁄ at molecular and antigenic characterization, respectively. thus, the advantage offered by mf in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , one amino acid was a sufficient change in antigenic sites for -fold decrease of hi titre against homologous vaccine strain to observe -fold higher post-vaccination nt titers (mf ⁄ subunit postvaccination gmt ratio range between ae and ae , figure ) and one-dilution higher corrected post-vaccination hi titers in mf vaccine group ( figure ) . finally, the correlation between the distance and the improvement offered by mf in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h n pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. the natural outbreaks of h n pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistÒ) has been licensed for use in humans since . in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h n pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h n influenza virus, a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ). we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h n pdm virus. safety and efficacy studies of the ty ⁄ att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the h n pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. -t cells and mdck cells were maintained as previously described. a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ) has options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - been previously described and it was kindly provided by yehia saif, ohio state university. a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. the genetic signatures for attenuation were introduced into the pb and pb genes of ty ⁄ . ny : ty ⁄ att is a : reassortant with the surface genes from the a ⁄ new york ⁄ ⁄ (h n ) virus and the ty ⁄ att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = ) and intranasally inoculated with tcid ⁄ animal of either h n : ty ⁄ att or with ny(h n ) : ty ⁄ att vaccines diluted in ml of mem. two other groups were similarly inoculated with h n : ty ⁄ wt and h n : ty ⁄ rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. , efficacy of h n ty ⁄ att vaccine in pigs fourty pigs were divided in four groups (n = )( table ) . group was vaccinated with tcid ⁄ animal of ny(h n ) : ty ⁄ att through intranasal route, whereas group was vaccinated intramuscularly with ml of an adjuvanted uv-inactivated ca ⁄ vaccine (uvadj-ca ⁄ ). group , non-vaccinated and challenged (nv+ca ⁄ ), and group , non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups - were challenged intratracheally with ml of · tcid of ca ⁄ . following challenge, pigs were monitored using methods as previously described. all statistical analyses were performed using graphpad prism software version ae (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < ae . the ty ⁄ att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ viruses developed fever (> °c) that peaked hpi ( figure a) and shed large amounts of in nasal secretions ( figure b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at dpi ranged from to tcid ⁄ ml ( figure c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure d ). in contrast, none of the animals inoculated with h n or h n ty ⁄ att viruses developed clinical signs following vaccination, indicating that the ty ⁄ att viruses were safe for administration to pigs ( figure a) . correspondingly, there was - fold less virus shedding from the nose of pigs vaccinated with ty ⁄ att viruses as compared to unmodified ty ⁄ viruses. in general, ny(h n ) : ty ⁄ att -vaccinated pigs shed less virus than h n : ty ⁄ att inoculated pigs ( figure b ). in addition, viral titers in balf were significantly reduced (p < ae ) in ty ⁄ attvaccinated pigs as compared to ty ⁄ wt-infected pigs ( figure c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung ae ± ae * ± * ± * ± * balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ , uv-inactivated ca ⁄ vaccine; nv+ca ⁄ , non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ control group at p < ae . geometric mean hi titer against ca ⁄ at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log ) measure as tcid per ml. -average viral titer (log ) in balf at dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure e ). vaccination with h n ty ⁄ att-based vaccines provides sterilizing immunity against h n pdm in pigs the clinical performance in pigs of the h n vaccines is summarized in table . nv+ca ⁄ animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h n ) : ty ⁄ att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < ae ) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h n and h n ty ⁄ att vaccines were attenuated in pigs. although the ty ⁄ att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at dpi. ny(h n ) : ty ⁄ att (a virus that carries the surface genes of a ⁄ new york ⁄ ⁄ (h n ) and ty ⁄ att internal genes). all h n viruses have their surface genes derived from ty ⁄ . values are shown as the mean ± sem. * p < ae ; **p < ae ; *** p < ae . options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h n as opposed to an experimental ca ⁄ inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the h n , the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h n vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the h n pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb and pb genes of ty ⁄ retains its safety properties in vivo and can induce excellent protection against aggressive h n challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. , type a influenza causes an acute disease of the upper airways, and affects - million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ lethal for mice (total infection dose corresponded to · ld ) or a⁄ pr ⁄ (total infection dose corresponded to ae - ld ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. protective significance was tested in vivo. mice were preimmunized with ) pbs (controls), ) dbf alone, ) virus alone, and ) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi ⁄ ( years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr ⁄ (h n ) and a ⁄ california ⁄ (h n ) -were used (figures and ) . the mice were challenged with · ld of either b ⁄ lee ⁄ or a ⁄ pr ⁄ as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure ) fell ill (pronounced body mass loss), but almost all survived and recovered. the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). intratracheal dbf ( lg per mouse) given to non-immunized mice hour before influenza infection eliminated the lethal effect in - % of infected animals depending on infection dose ( ae - ld ); in mice infected with lower than lethal doses ( ae ld ), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [ ] [ ] [ ] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of : based on highly pathogenic influenza a ⁄ h n viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ ⁄ (pr ) virus from pr -based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n neuraminidase into the genome of the mdv laiv production were ineffective. : reassortants were not generated. step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv in some cases was the only possibility to generate influenza a ⁄ h n cold-adapted vaccine reassortants. difficulties in generating : reassortants could be explained by a substantial gene constellation in the genome of pr based h n reassortant viruses. strong coupling of pb ⁄ pr and avian n genes in a ⁄ h n -pr -rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. in these cases, like with laiv, vaccine strains are : reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr virus. the pr virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr genes in the genome of candidates for inactivated vaccine subtype h n (a ⁄ h n -pr -rg) with internal genes of the laiv mdv by methods of classical reassortment. len -mdv and a ⁄ h n -pr -rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature ( °c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len -mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for sec while being irradiated with a ge watt germicidal lamp at a distance of cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len -mdv. however, all generated clones contained the na from the mdv. of ten such : reassortants based on vn-pr three reassortants had the pa gene from pr and one had ns gene from pr . : reassortants from the targeted h n composition were not generated. after repeated attempts, : temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n neuraminidase (table ) . in contrast, nibrg- didn't reassort with the mdv at all. twelve unsucsessful attempts to develop : or : reassortants of nibrg- with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at °c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg- , various modifications of the co-infection step were studied, such as: altering the nibrg- to mdv ratio (from : to : tions of anti-mdv serum alone or together with anti-pr serum. it was noted that even if the h n to mdv ratio was : , the clones obtained were presumably parental h n viruses without the transfer of any mdv-genes into genome of nibrg- . in all, clones were isolated, and of them were identical to nibrg- parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg- prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg- with mdv (at ratio : ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg- background (b , c , c , d ) ( table ). some of them (c , c , d ) were chosen for the next round of co-infection. after the second round of co-infection, c , c , and d 'intermediate' reassortants with mdv (at ratio : or : ) : vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h n viruses on the base of rg-reassortants for inactivated vaccine with modified h hemagglutinin and mdv for laiv. replacement of pr based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining : reassortants that contained both the ha and na from the wild type avian h n parental virus. in attempts to reassort the nibrg- with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg- virus. partial uv-inactivation of nibrg- was encouraged in replacement of some pr internal genes with mdv genes in some cases avian-human reassortant viruses with gull h n and human influenza h n genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h and n genes were not isolated despite repeated attempts. the gull pb , np, and ns genes were not present in any of the gull-human h n reassortants generated. it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop : vaccine reassortants may be caused by an observed strong connection of pb and na genes in the genome of a ⁄ h n -pr -rg viruses. in our attempts, each reassortant that possessed avian n neuraminidase inheritied pb gene of pr as well. and vice versa, the 'cold' pb gene always appeared to be coupled with the n neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h n and avian h n viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from to , avian influenza outbreaks among humans have been registered in countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h n among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h n human vaccine. the development of ai a ⁄ h n vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h n vaccine development for public health protection in kazakhstan' was approved and financed from to . in this article we give basic results of the development of a recombinant ai a ⁄ h n inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [ ] [ ] [ ] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ : ⁄ (a ⁄ h n ) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by -fold virus passaging in embryos. , purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and ae % aluminum hydroxide was added in : proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom -well plates produced by 'costar' (usa). vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. , for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. the vaccine toxicity was evaluated in white mice with body weight - gm and in rats with body weight - g both males and females according to glp principles. allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. in the first series of experiments, we conducted work for obtaining influenza a(h n ) recombinant strain. bidirectional expression plasmid phw_b with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h n . sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was cloned without modifications. the other segments pb , pb , pa, np, m and ns were obtained from influenza virus ivr- and synthesized and cloned in two-forked expression plasmid phw_b in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ : ⁄ (h n ) is presented in table . vero cell culture ( passage) (who) was received from european cell culture collection (salisbury, wiltshire sp jg, great britain). the cell culture was grown in dmem ⁄ f medium with the addition of % of fetal bovine serum and mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ ⁄ r- : , vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in -well plates. after hour, dmem ⁄ f medium was changed into ml of opti-pro sfm (gibco) medium adding mm l-glutamine and lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ ⁄ - : was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg ⁄ - : virus stocks was ae log eid ⁄ ml (chicken embryos); ae log tcid ⁄ ml (mdck cells); ha titer : . a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ ⁄ r- : strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ ⁄ - : were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ ⁄ - : ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg ⁄ - : is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg ⁄ - : strain pathogenicity evaluation was conducted in - week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg ⁄ - : (h n ) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to ). h n strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg ⁄ - : strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ ⁄ r- : strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ ⁄ r- : strain belongs to h subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of ae ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ ⁄ r- : strain can be used for influenza a ⁄ h n inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg ⁄ - : in - day chicken embryos. the determined parameters are the following: infection dose, - eid ; cultivation period, hour; incubation temperature, °c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of ae - ae log eid ⁄ cm and : ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of ae %, period of inactivation of days, temperature of inactivation medium of - °c, ph of inactivation medium of - ae . on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg ⁄ - : , and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at circulations per min for minutes, filtration through membrane filters with pore diameter of ae lm, ultrafilatration ⁄ diafiltration, gel filtration in b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of ae lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ : ⁄ (h n ) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h n human vaccine has the trade name kazfluvacÒ. its composition components are presented in table . preclinical testing of the vaccine kazfluvacÒ was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [ ] [ ] [ ] [ ] preclinical tests of kazfluvacÒ vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacÒ vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed % of protective effect of kazfluvacÒ vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h n vaccine kaz-fluvacÒ is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [ ] [ ] [ ] [ ] to date the whole-virion inactivated influenza a ⁄ h n vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacÒ differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. , the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacÒ vaccine in increasing doses. the preparation will be administered to volunteers aged - years for assessment of its safety and immunogenicity in doses of ae and ae lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h n v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h n .'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h n influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg- xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide ( ae %) in the ratio : . the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. the content of the residual formaldehyde, aluminum (al + ) ions, and thiomersal in the vaccine was measured according to the operating instructions. the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in -welled u-bottomed plates (''costar'', new york, usa). , apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. , to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). the toxicity of the vaccine was assayed in white mice weighing - g and in rats weighing - g (male and female) in compliance with the principles of good laboratory practice. allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of -days embryonated chicken eggs at the infectious dose within - eid , incubation temperature ( ± ae )°c, and duration of the incubation period hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within ae - ae log eid ⁄ cm and hemagglutinating activity : and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of ae % final concentration as an inactivating agent; inactivation period of hours at temperature ( ± )°c. these conditions provide the complete inactivation of the virus (nibrg- xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg- xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size ae lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose b, and sterilization of the viral suspension through membrane filters with pore size ae lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h n influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac Ò . the key processing characteristics of the whole-virion inactivated a ⁄ h n influenza vaccine vaccine refluvac Ò are shown in table . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h n vaccine refluvac Ò are shown in table . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over %, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in % polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m ) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces % protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ ⁄ (h n v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h n v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h n vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac Ò based on the recombinant strain nibrg- xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [ ] [ ] [ ] [ ] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h n vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ ⁄ (h n ) nymc x- a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose lg ha at single administration both in adults and elderly persons. the results of the pre-clinical tests allow recommending carrying out phase clinical testing of the refluvac Ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac Ò in doses ae , ae , and ae lg of ha are planned. mid , respectively. the study results confirm that new h n laiv and h n laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h n virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h n , h n , h n , and h n are currently being tested in preclinical and phase i clinical studies. the following paper describes the preclinical study of new h n and h n laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ (h n ) and six other genes from mds, the a ⁄ ⁄ california ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ california ⁄ ⁄ (h n ) and six other genes from a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in days old eggs ( °c, hours). fifty percent egg infectious dose (eid ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. female balb ⁄ c mice, - weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with mid ( % mouse infectious dose) of a ⁄ ⁄ california ⁄ ⁄ (h n ), a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), and a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) mds. viral loads were measured in respiratory and brain tissues collected at and days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with mid or mid of either h n laiv or h n laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at dpi. to assess protection, all animals were infected dpi with either mid of a ⁄ california ⁄ ⁄ (h n ) or mid a ⁄ mallard ⁄ netherlands ⁄ (h n ) virus by the intranasal route. four animals from each group were euthanized at dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days , , , and . body weights were taken daily following challenge through day postchallenge. sera were tested for hi against homologous h n and h n viruses. the h n laiv, h n laiv and h n mds influenza viruses replicate in mice lungs at level ae - ae lgeid ⁄ ml at dpi (figure ). at dpi, replication of the viruses in the lungs decreased to ae - ae lgeid ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ (h n ) demonstrated high level replication in lungs - ae lgeid ⁄ ml. the levels of replication of studied viruses in nasal turbinates were ae - ae lg eid ⁄ ml at dpi (figure ) , and ae - ae lgeid ⁄ ml at dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in terms of replication in the lungs and noses of mice at and dpi. no virus was found in the brain tissue of immunized mice at and dpi (in undiluted samples tested). thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), a ⁄ ⁄ cali-fornia ⁄ ⁄ (h n ) vaccine candidates are identical to a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day to day in all study groups, but the changes did not exceed %. as shown in figure , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals ( ae lgeid in the lung and ae lgeid in the nose), but failed to infect the brain and spleen. on the other hand, in both h n laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either mid or mid h n laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h n virus. both h n and h n laiv candidates were found to be immunogenic. after one dose of mid of h n laiv, gmt of hi antibodies were ae . one dose of mid or mid h n laiv elicited hi antibody level with gmt of ae and ae , respectively. the second dose of h n laiv further stimulated serum hi antibody levels to gmt ae and ae , for mid or mid , respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. a subset of the h viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h n (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. in our current study we demonstrate that a(h n ) and a(h n ) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h n and h n laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h n laiv and h n laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase . therefore, h n inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ ⁄ ). this adjuvant was also tested in ferrets in a h n avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of months received one or two im vaccinations weeks apart of either af -adjuvanted ( ae lg of ha with af ) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of % was recorded in the control group at day of necropsy. body weight loss was reduced to £ % and £ % in animals that had received and doses of either unadjuvanted or af -adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡ ae tcid ⁄ g tissue) in the lungs of all control ferrets days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by and log , respectively. interestingly, ferrets that received either one or two doses of af -adjuvanted h n vaccine, showed significantly greater reduction of lung viral loads (> log ). no virus was detected in the lungs of ⁄ ( %) animals immunized with a single injection of the af -adjuvanted vaccine and in % of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af -adjuvanted a ⁄ h n monovalent vaccine was able to reduce the viral load in the nose and in the throat by ae and ae log , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h n monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h n virus infec- however, a second dose of af -adjuvanted vaccine strongly increased hi and mn titers, which persisted for months (table ). antibody responses cross-reactive to heterologous clade . strain were elicited ferrets vaccinated with the af -adjuvanted clade . vaccine. hi antibody titers ‡ crossreactive to clade . and persistent up to d were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade strain was only detected in a few ferrets that had been vaccinated with the af -adjuvanted clade . . all af -adjuvanted clade . antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day . after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by - °c (depending on the challenge virus strain) h post challenge, with an accompanying mean body weight loss ranging from ae % to ae %. ferrets vaccinated with the af -adjuvanted clade . vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade . or clade viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade . strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals days after homologous challenge with the clade . virus. lung viral loads of all ferrets immunized with the af -adjuvanted clade . vaccine were reduced more than log . vaccination resulted in complete viral clearance from the lungs of % of animals assessed days after challenge. as compared to controls, a reduction of the mean viral load of about log was observed in ferrets vaccinated with the af -adjuvanted clade . vaccine after heterologous challenge with either the clade or clade virus. conversely, vaccination with af -adjuvanted clade . vaccine did not result in reduction of lung viral loads after challenge with the clade . heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade . strain, whereas virus was not detected in any vaccinated animal. similarly, log reduction of viral shedding was seen in vaccinated versus control ferrets following clade heterologous challenge. lower reductions in viral shedding were observed after clade . challenge ( ae log ) and clade challenge ( ae log ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h n virus infection all control animals challenged with the clade . , clade . or clade strains. mild to moderate lung lesions were observed in control animals following challenge with clade virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af -adjuvanted clade . vaccinated animals after challenge with the homologous clade . virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade . and clade virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af , elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af -adjuvanted h n vaccine can provide cross-protection upon challenge with different h n clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af -adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since , outbreaks of h n influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h n isolates revealed that there are three sublineages, consisting of g , g , and korean, among ha genes of the eurasian h n viruses. h n viruses do not cause severe disease in poultry, but co-infection of h n viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. , h n viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in kong in , kong in , and it is, thus, postulated that in the present study, h virus strains were analyzed antigenically and phylogenetically to select a proper h n vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni primer and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h - f and h - r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. h influenza viruses were analyzed by hemagglutinationinhibition (hi) test. chicken hyperimmunized antisera against seven h viruses were prepared according to previous report. virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into -day-old embryonated chicken eggs and incubated for hours at °c. ha titers and % egg infectious dose (eid ) were measured every hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. dk ⁄ hok ⁄ ⁄ was injected into the allantoic cavities of -day-old embryonated chicken eggs and propagated at °c for hours. the virus in the allantoic fluids ( ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. the concentration of protein was measured by od using ultrospec pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with ae % formalin at °c for days. immunization of mice and challenge of immunized mice with hk ⁄ ⁄ four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with , , ae , or ae lg proteins of inactivated dk ⁄ hok ⁄ ⁄ whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. one week after the second vaccination, mice in each group were challenged intranasally with ll of ae eid of hk ⁄ ⁄ under anesthesia. on days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a % (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in -day-old embryonated chicken eggs and expressed as the eid ⁄ gram of tissue. the other five mice in each group were monitored for body weight for days after challenge. the ha genes of h viruses were sequenced and analyzed by the neighbor-joining method. all of the h viruses were classified into the eurasian lineage ( figure ) . eleven, seven, and four strains were classified in the korean, g , and g sublineages, respectively. the h viruses of the korean and g sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h n viruses were analyzed by hi test. the antisera against h viruses belonging to the korean sublineage were broadly cross-reacted to h viruses belonging to the g and g sublineages. h viruses belonging to the korean lineage were reacted to the antisera against h viruses belonging to the g and g sublineage compared with h viruses belonging to the other sublineage (data not shown). thus, it was suggested that h vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h viruses. dk ⁄ hok ⁄ ⁄ replicated efficiently in -day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ ⁄ was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h vaccine strain. to assess the potency of the vaccine against h virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ ⁄ were challenged intra-nasally with hk ⁄ ⁄ . immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected lg protein after the first immunization and detected in the group of mice injected lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were ae - ae eid ⁄ g in the groups of mice injected , and lg protein, and ae - ae eid ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected ae , ae lg protein, and control groups from dpi, and reached to % body weight loss from -to -day post-infection ( figure ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h influenza virus infection in mice. recently, h n viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h n viruses were isolated from pigs and humans in china and korea, suggesting that h n virus would be a potential for a pandemic influenza virus in human population. h n viruses were isolated from pigs in china and korea and were classified into the g and korean sublineage. in human cases, all h n virus isolated from humans in china was classified into the g sublineage. it was suggested that h n viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g , and g sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ ⁄ , which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h virus using mice for mammalian model. the challenge virus, hk ⁄ ⁄ , was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. the present results may facilitate the studies of the vaccine for future pandemic caused by h influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h n pdm grew better in the novartis mdck suspension cell line (mdck pf) than in eggs, isolations from h n pdm positive clinical samples were attempted in both substrates. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h n pdm viruses isolated in mdck pf cells. with the emergence of the swine-origin pandemic h n (h n pdm) influenza in april , the need for efficient production of a suitable vaccine was a high priority. virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs or in certified mammalian cells. , unfortunately wildtype h n pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of conventional and reverse genetics derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x- a and nymc-x- (both based on one of the earliest known viruses a ⁄ california ⁄ ⁄ ) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately % that of seasonal h n reassortants. to determine if more recent wild type h n pdm viruses grew better in the novartis mdck suspension cell line (mdck pf), h n pdm positive clinical samples were cultured in mdck pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck pf isolates, extended passaging of three wild type h n pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h n pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h n pdm positive by real time rt-pcr. these specimens were then cultured in mdck pf cells with serum free medium containing trypzean (optaflu) and also independently inoculated into the allantoic cavity of day-old embryonated hen's eggs. virus cultures in mdck pf cells were sampled at and hour and evaluated by various means including ha titres. at hour, virus cultures were further passaged at varying dilutions ranging from ) to ) up to a total of passages. embryonated hen's eggs were incubated at °c for days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ ⁄ virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. the reverse genetics viruses were rescued in vero cells using the plasmid system. both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c- strain, which was produced by novartis. in this small study with recent h n pdm viruses, the isolation rate was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table ) . no amino acid changes were observed in mdck pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table ) . viruses isolated in mdck pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h n pdm viruses in mdck pf cells, serial dilutions were performed over a number of passages ( figure ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ ⁄ , a ⁄ wellington ⁄ ⁄ , and a ⁄ darwin ⁄ ⁄ , a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ ⁄ (figure ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ ⁄ results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x- a and nymc-x- were also assessed for growth in mdck pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table ) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h n pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h n the ratio of ha to p p p p p p p p p p p p p p p p n and m was ‡ %, for the h n the ratio of ha to n and m was £ %, while for the pandemic a ⁄ h n , the ratio of ha to n and m was much lower at £ % (data not shown). the results of this study has observed the growth of a series of - h n pdm viruses in vaccine suitable mdck pf cells to be generally lower than what has been seen with other seasonal influenza viruses. little improvement in virus yield was seen with extra passaging of h n pdm viruses isolated and passaged in mdck pf cells. passaging up to times in mdck pf cells using dilutions ranging from ) to ) resulted in supernatants with viral ha titres ranging from ha ⁄ ll to ha ⁄ ll. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) compared to allantoically inoculated (and passaged) eggs ( %), a trend also seen in previous work with seasonal influenza viruses. in contrast a study by hussain and colleagues found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ ⁄ gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p , p , p , p , p and when available compared to the original clinical sample ha sequence. mdck pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed - amino acid changes compared to the clinical sample, with an egg adaption change (l i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ ⁄ , was found to have a g e change compared to the a ⁄ california ⁄ ⁄ reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ ⁄ -like viruses, but normal reactivity with ferret antisera to h n pdm a ⁄ bayern ⁄ ⁄ -like viruses. despite this mutation all mdck pf derived viruses appeared to be a ⁄ california ⁄ ⁄ -like by hai. the h n pdm egg-derived reassortants (nymc x- a and nymc x- ) when grown in mdck pf cells were superior to wild type h n pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h n pdm reassortants were still below those seen with sea-sonal h n reassortants as was also seen in eggs. this trend has also been noted in other studies. in summary, attempts to improve growth and yield of the h n pdm wild types for mdck pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h n reassortants have in the past. however, using higher dilutions for the passaging of h n pdm viruses in mdck pf cells did result in higher ha titres (a ⁄ brisbane ⁄ ⁄ ). further work is therefore required to generate pandemic h n seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. ( ) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h n ) reassortant virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of ae , compared to ae in the control group. gmt of ni from human subjects who received two doses of pandemic a(h n ) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h n ) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [ ] [ ] [ ] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n in mice could provide partial protection against a lethal challenge against h n or recombinant pr bearing the avian n . immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. however, the who meeting on the role of na in inducing protective immunity against influenza infection ( ) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. the aim of the current study was to evaluate anti-na antibodies to pandemic a(h n ) influenza virus following laiv immunization. the rn ⁄ -swine a(h n ) reassortant influenza virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the ha of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on - % sucrose step gradient. for the mouse studies, week old cba mice were inoculated intranasally with one dose eid ⁄ ae ml of a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine strain or received ae ml pbs. blood samples were collected on day post inoculation. healthy young adults were immunized twice, or days apart in the fall with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, days after the first vaccination, and days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h n ) were collected in december , to weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ ⁄ (h n ). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h specific antibodies by standard procedures using a ⁄ ⁄ california ⁄ ⁄ (h n ) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. was adjusted to assay ni antibody. briefly, -well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with ll of lg ⁄ ml fetuin. the purified a(h n ) reassortant virus was diluted in pbs with % bsa and mm ca + to give a four times higher optical density at nm (od ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for hour at °c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin ( lg ⁄ ml; sigma, st. louis, mo, usa), incubating for hour at room temperature, washing the plates, and adding ll of peroxidase substrate (tmb). the reaction was stopped after minute by adding ll of n sulfuric acid. od values were measured at nm using the universal microplate reader (el x ; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave % od of positive control (virus, no serum control). in addition we used an igg elisa assay with ae lg ⁄ ml of purified na from a ⁄ california ⁄ ⁄ (h n ) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version ae ) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of < ae was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ ⁄ (h n ) influenza virus vaccine (table ) . geometric mean titers of ni antibody levels from vaccinated mice were ae and were significantly higher compared to those in unvaccinated control animals (p < ae ). elisa igg titers expressed as log were ae compared to ae in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = ae ). in a study during the fall of , % of examined unvaccinated subjects were negative to pandemic a(h n ) (hi titers £ : ). serum hi antibody titers to pandemic a(h n ) ‡ : were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = ae ) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = ae ) serum hi and ni antibodies to a ⁄ california ⁄ ⁄ (h n ) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £ : ( table ) . post-vaccination gmts of a(h n )-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses ( ae %) compared to responses after one dose ( ae %) although the differences were not statistically significant ( table ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < ae compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n -specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡ : to protection of % of the subjects against influenza infection is based on a number of reports published in s. serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h n ) we used the reassortant a(h n ) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h n ), evidently cross-reacted with the n of pandemic influenza virus, perhaps due to the previously reported % of conserved na epitopes in pandemic a(h n ). the peroxidase-linked lectin test using the reassortant a(h n ) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h n virus or with h n laiv. and the cross-antibody response to ph n . for calculation of geometric mean titer (gmt), a titer of < was assigned a value of . statistical significance was determined by paired t-test. cross-reactive antibody response to ph n in vaccinated populations of seasonal influenza virus table shows the antibody response to seasonal influenza viruses and ph n of participants. before vaccination, no or little antibody response to ph n had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over % of subjects, except children aged - years ( %) and subjects aged of - years ( %) vaccinated with - season influenza vaccine and adults aged ‡ years ( %) vaccinated with - season influenza vaccine. seroconversion was detected in over % of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than ae -fold. in contrast, seroresponse to a ⁄ california ⁄ ⁄ after vaccination with - and - seasonal influenza vaccines were detected in only % and % of those aged - years, % of those aged - years, % and % of those aged - years, % and % of those aged ‡ years, respectively. seroconversion in all participants ranged from % to %, and postvaccination to prevaccination gmt ratios were < ae -fold. preexisting antibody response to ph n among subjects born before s in china according to a recent report, people who were born from to had a preexisting immunity to ph n . although only a very low level of cross-reactive antibody response to ph n had been observed among older subjects aged more than years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph n influenza virus. the proportion of seroresponse to ph n with the titer of , , and (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from . similarly, a peak of antibody response and the value of gmt occurred both in subjects born from to and sharply decreased afterward ( figure ). the seroresponse of subjects born in and before is significantly higher than subjects born afterward (p < ae ). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph n had been detected in children and adults. , but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged > years with pre-existing cross-reactive antibodies to ph n , which may possibly ba a result of previous exposure to antigenically related h n influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ may also contribute to the high level of cross-reactive antibody response to ph n among adults older than years in the us. , the peak of the antibody response to ph n in subjects born between and , which is consistent with recent reports, may suggest the previous viral infections of spanish flu or closely related influenza viruses, which is before and little after the year of . recent antigenic report of new ph n viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h n ) viruses. a number of reviews [ ] [ ] [ ] [ ] confirmed that the virus is the likely ancestor of all four of the human and swine h n and h n lineages, as well as the 'extinct' h n lineage. in , a(h n ) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human a(h n ) virus. the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the th century, may have contributed to the ability of these subjects to sustain memory b cells, and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h n ) viruses may not have circulated among populations in china before the outbreak of ph n . our data also suggests the need for vaccination with ph n vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. these patients are also prone to get viral infections, possibly due to defects in toll-like receptors and . because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. , the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h n virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il- , and tnf-a cytokine producing cd + th -cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification : patient # is diagnosed with x-linked agammaglobulinemia, patient # and # are in group ia, patient # is in group ib and patient # is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) to produce a ⁄ california ⁄ ⁄ -like virus (x a). the vaccine was mixed with adjuvant to contain ae lg haemagglutinin (ha) of a ⁄ california ⁄ ⁄ -like virus (h n ), squalene ( ae mg), dl-atocopherol ( ae mg), and polysorbate ( ae mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of ae lg ha, but ae lg ha was inadvertently administered to the patients as the first dose. for patient # this was the second dose as he had received an initial dose of ae lg ha months prior to the study. patient # , # , and # received a second dose of ae lg ha. four healthy controls were immunised with one dose of ae lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with % fbs. the pbmcs were resuspended in lymphocyte medium (rpmi with l-glutamine, ae mm non-essential amino acids, mm hepes ph ae , mm sodium pyruvate, iu ⁄ ml penicillin, lg ⁄ ml streptomycin, ae lg ⁄ ml fungizone and % fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd + t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using ha units and ae % turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres < were assigned a value of for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described with the following modifications. ninety-six well elispot plates were coated with lg ⁄ ml of a ⁄ california ⁄ ⁄ like (x a) h n virus diluted in pbs overnight at °c. after blocking with rpmi ( % fbs), pbmcs were added and incubated ( °c, % co ) for hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanÔ) and immunospot Ò software. the influenza-specific cd + th -cell response was measured by intracellular cytokine production of ifn-c, il- , and tnf-a. peripheral blood mononuclear cells ( per well) were incubated for hour ( °c, % co ) in ll lymphocyte medium containing lg ⁄ ml anti-cd , lg ⁄ ml anti-cd d, ae lg ⁄ ml monensin, lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h n influenza split virus vaccine x a (either ae lg ⁄ ml or lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd , cd , cd , ifn-c, il- , and tnf-a (bd biosciences) as previously described. finally, cells were resuspended in pbs containing % fbs and ae % sodium azide and analysed by bd facscanto flow cytometer ( - cells acquired). flowjo v ae ae (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure a) . the lowest hi titres were obtained in patients # , # , and # , whilst patients # and # and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) seasonal influenza vaccine licensing criteria, by obtaining an hi titre > and a mean geometric increase of ae between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and days post-vaccination with the h x a virus. few or no ascs were detected pre-vaccination (data not shown). at days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < ae ) compared to the healthy controls ( figure b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage ( - ascs). patient # had the highest iga and igg asc numbers, followed by patients # and # , whilst patient # and # had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (# and # ) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients # , # , and # were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th -cell response was evaluated by stimulating pbmcs with the influenza x a virus , , and days post-vaccination. stimulation of healthy control pbmcs with x a days after vaccination, induced ifn-c, il- , and tnf-a production by an average of ae %, ae %, and ae % cd + t-cells, respectively. patient # and # had higher responses than the healthy controls and stimulation with x a induced ae %, ae %, and ae % of t-cells from patient # to produce ifn-c, il- , and tnf-a, respectively (figure a) . the response of patient # was further boosted by a second vaccine dose, which resulted in ae %, ae %, and ae % cd t-cells producing ifn-c, il- , and tnf-a, respectively at day ( figure b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd + th cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th -cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient # and # ) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient # and # . these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [ ] [ ] [ ] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd + th -cells to be in the same range (for patient # , # , and # ) or higher (for patient # and # ) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of ae lg ha, whilst the controls received ae lg ha. in addition, the patients received a second booster dose, which influences the day and months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day (a), (b), and day (c) and stimulated for hour with x a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd + cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il- producing cd + t-cells in the patients compared to the healthy controls (students t-test p < ae ). titres are presented as the geometric mean titre ± % confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < ae ). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. , because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. , previous studies have investigated the potential of the m e. , m e has remained highly conserved since it was first isolated in . several studies have examined the use of m e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. , [ ] [ ] [ ] [ ] [ ] [ ] although m e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. some articles found that vaccination with m e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. we aimed to address the poor immunogenicity of m e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [ ] [ ] [ ] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. tim family members share a common motif, including an igv domain. they are differentially expressed on th cells and th cells with the ability to regulate the immune system. , the igv domain of human b - is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. soo hoo et al. vaccinated with tim- antibody and inactivated influenza and found enhanced vaccine-specific immune response. we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet a (novagen, madison, germany). the resultant construct pet a-igv has a histidine (his) tag ( his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with ug m e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein , , ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with % ethanol and different groups of mice were injected bilaterally with , , ug igv plus ug m e-hbc in ul phosphate buffer saline per mouse using a ml syringe with attached ⁄ ¢¢ g needle. the immunization was given at weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at ) °c prior to use for assays. mouse-adapted a ⁄ pr ⁄ ⁄ (h n ), a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ xinjiang ⁄ ⁄ (h n ), and a ⁄ guangzhou ⁄ ⁄ (h n ) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at °c for - days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at °c overnight. to identify igg, igg , igg a against m e, elisa assays were used. in brief, -well (nunc, brunei, denmark) were coated with ul ⁄ well of m e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph ae ) overnight at °c. immediately before use, the coated plates were incubated with blocking solution ( % bsa in pbs) for h at °c and washed four times with pbs containing ae % tween (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg , or igg a ( figure ) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h n , h n , h n , and h n antigens. m e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h n viruses and swine-origin h n have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m e makes it a promising immunogen. a vaccine based on coupling of the m e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m e forms a virus like particle (vlp). vaccination with m e coupled to hbc induces protective antibody, whereas the contribution of t cell protection was negligible. protection induced by vaccination with m coupled to hbc was weak overall. in order to improve the vaccination effect of m e-hbc, new adjuvant igv was evaluated in combination with the m e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m e-hbc vlp. previous papers reported that vlp immunized mice can induce the th and th immune response. different adjuvant combined vlp can produce biased immune response th ⁄ th mixed immune response, or th -preferred th ⁄ th profile. thus, the response following the use of igv as a new adjuvant combined with m e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure . t cell proliferation assay. mice were immunized twice with , , , ug ⁄ ml igv plus m e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h n , (b) h n , (c) h n , (d) h n were added and cocultured with different group splenocytes for h. quick cell proliferation assay kit was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m e groups was determined using the student's t-test. all significance level is p < ae . response. we also evaluated the cross-protection produced by igv combined m e-hbc. we challenge with mouseadapted strain pr and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h , h , h , and h , and t cell proliferation was measured. results indicated that after immunization with igv plus m e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m e-hbc vlp immune response to a th profile. igv induced the m e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the - season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. , collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. , during the circulation of pandemic influenza viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to primary care practitioners of the grog network ( general practitioners and pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of < days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group - years (gps and pediatricians); - years (gps and pediatricians); - years (gps); years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult Ò , viralpack Ò , utm copan Ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un ). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h , seasonal and pandemic h ) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = )or (odds ratio). an exact % confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at % level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls ( ). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in - , gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october , , and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was ae % in patients swabbed in the systematic group ( ae % on imputed data) and ae % [ ae - ae ] in the ad hoc group ( ae % on imputed data). at the national level, vaccine coverage is estimated at ae %. due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < ae ): the rate of - years old is lower in the systematic sampling group ( ae %) than in the ad hoc sampling group ( ae %). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the - pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the - influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the - pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march , and it was declared to be pandemic by mid-june. as of august jul , the pandemic virus had caused more than deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/ _ _ /en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h n pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h n pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb , pb , pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ ⁄ ). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. , the ns protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. , mutations and ⁄ or deletions in ns typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus - and can enhance immune recognition. [ ] [ ] [ ] [ ] in this study, we created a panel of experimental h n pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h n pdm influenza a virus a ⁄ new york ⁄ ⁄ (ny ) was created by reverse-genetics directly from a human swab specimen collected in new york state in april . deletions were introduced into the ny ns plasmid to create three mutant ns segments: ns - , ns - , and nsd . nucleotides - (cdna of ns segment) and - were replaced by stop codons to generate ns - and ns - ; nucleotides - were deleted to generate nsd , whose open reading frames for ns and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into t ⁄ mdck cocultured monolayer adapted from hoffmann et al. , mouse studies experiments were performed in a biosafety level laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of -week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with tcid of each recombinant virus in ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for days. nine mice in each group were euthanized on , , and days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid assay in mdck cells. at dpi, mice per group were challenged intranasally with · tcid ( ld in -week-old mice) of a mouse-adapted variant of ny (a ⁄ ny ⁄ ⁄ -ma ) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for days, and four mice from each virus group were euthanized at and days post challenge. lungs were removed and homogenized for virus titration by tcid assay. the mice that became moribund or lost > % of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), - months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of or ferrets were inoculated intranasally with ae tcid of one of the viruses: ny wt (n = ), ns - (n = ), ns - (n = ), or nsd (n = ). ferrets were monitored for clinical signs through dpi as previously described. nasal washes were collected on , , , and dpi and were titrated in mdck cells by tcid assay. serum was isolated from blood collected ae weeks after immunization and used for neutralization assays. the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ ae weeks postimmunization and monitored for clinical signs of disease through dpi. nasal washes were collected on , , , and dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at dpi, and the mice did not recover until dpi (figure a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mock- inoculated mice ( figure a ). viral titers in the lungs of ns - , and ns - infected mice were $ -fold lower than titers from wt virus-infected mice at all the time points analyzed ( , , and dpi) ( figure b) . notably, the nsd laiv was cleared from the mouse lungs very rapidly, and the mean titers were $ -fold and -fold lower than the titers of the wt virus at and dpi, respectively ( figure b) . the vaccinated mice were challenged with a mouseadapted variant of ny (accepted, journal of virology) on dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by dpc (figure c ). high titers of virus ($ tcid ⁄ ml) were present in the mock-immunized mice at dpc and at dpc ( figure d ). in contrast, virus was not detected in the lungs of immunized mice ( figure d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd , induced sterilizing immunity that protected mice from a lethal ny h n pdm variant. groups of ferrets were intranasally immunized with ae tcid of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from ae to ae tcid ⁄ ml through day in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure a) . the viral titer of all of the ns-laivs is at least -fold lower than that of wt in the nasal wash collected at dpi. the ns - laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns - laiv showed -fold reduction in titer, and the nsd laiv was below the limit of detection (at least fold reduction) at dpi. sera from blood collected ae weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses ( - ) that were similar to the titer elicited by wt virus infection ( figure b ). the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ (h n pdm) ae weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ ⁄ in their nasal washes at dpc ( figure c ). the ferrets immunized with the ny ns-laivs had $ -to -fold lower viral titers than did the naïve animals ( figure d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h n pdm virus under stringent challenge conditions ( pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h n pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns - was the least attenuated and does not represent a good vaccine candidate; whereas, nsd and ns - were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd has a subtle in-frame deletion ( nt) that affects both the ns (residues - ) and nep (residues - ), and is analogous to a naturally attenuated variant of a normally highly pathogenic h n virus (a ⁄ sw ⁄ fj ⁄ ). the analogous ns deletion in a ⁄ sw ⁄ fj ⁄ (residues - ) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns protein stability, and enhance the type i ifn response of this h n virus. our study indicates that deletion of these nt in the ns vrna of the h n pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k , ip , and mxa (data not shown). the role of the deletion of residues - from nep has not been elucidated, but the induction of ifn and isgs by nsd was similar to, or slightly lower than, their induction by ns- , suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny , and ferrets from challenge with a ⁄ mexico ⁄ ⁄ (h n pdm). currently, a total of approximately million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally - internal genes from the a ⁄ pr ⁄ ⁄ (pr ) strain, an highly egg adapted high growth donor strain. influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. , since in this plasmid-based technology, a : reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the : configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ was important in producing high yield b reassortants. however, b ⁄ lee ⁄ is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ ⁄ for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ ⁄ was used, which is one of the recommended influenza b virus vaccine strains for the ⁄ and ⁄ seasons. we found that b ⁄ panama ⁄ ⁄ is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. b reassortant viruses were prepared by the classical reassortment method described by kilbourne. the antiserum to b ⁄ panama ⁄ ⁄ hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ ⁄ ; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ ⁄ as a donor for transferring high yield phenotype. b ⁄ panama ⁄ is a yamagata lineage strain with high growth phenotype (ha titer: - ). b ⁄ panama ⁄ ⁄ itself was a recommended b virus vaccine strain for ⁄ - ⁄ seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ ⁄ , was used, which is a recommended b virus vaccine strain for use in the ⁄ and ⁄ seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ , progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx- , bx- b, bx- d, and r- a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table , all of these reassortants contained the pb of b ⁄ panama ⁄ ⁄ . other genes of b ⁄ panama ⁄ ⁄ (np of bx- , m of bx- b, and pb of bx- d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb of b ⁄ panama ⁄ ⁄ is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ np and b ⁄ panama pb could work together to produce even higher yields. to test this possibility, bx- b ( : reassortant: pb and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ . despite some difficulty in removing the na gene of b ⁄ lee ⁄ (r- c, b, b in table ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx- , which contains the np gene from b ⁄ lee ⁄ and pb and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ ⁄ (table ). in comparison with bx- b, no significant growth enhancement (nor reduction) in eggs was found for bx- over that seen for bx- b. nevertheless, bx- stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the - season by one or more vaccine manufacturers. there are contradictory reports - about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor, and have been able to generate higher virus yield than wt strains. in this study, we found that b ⁄ panama ⁄ ⁄ serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ ⁄ (a recommended vaccine virus of victoria lineage for ⁄ - ⁄ seasons), and that the pb of b ⁄ panama ⁄ ⁄ is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ . as shown in table , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ ⁄ , not from the donor, b ⁄ panama ⁄ ⁄ . this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as : and even : reassortants that we obtained in this study. if this is the case, reverse genetics based on : configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ ⁄ reassort together with the ha and na genes of the same parent (table ). in our recent study on a reassortment between b ⁄ lee ⁄ and b ⁄ panama ⁄ ⁄ , it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr , it is generally believed that reassortant with : or : configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h n v has spread rapidly in all parts of the world in as a true pandemic. epidemic events in russia occurred during the last week of september starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december , clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h seasonal, and h sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h -swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism -avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti . (invitrogen) and mega . (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, ). statistical reliability of tree branches was evaluated by bootstrap test ( replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h n v rna was detected in patients with severe form of influenza-like illness and fatal cases. out of pcr-confirmed flu recovered cases % were patients under years of age, % were aged - years, and % were older than years. mean age of recovered patients was ae years (from month to years). viral rna in postmortem materials was detected mostly in lung tissue ( % of specimens) and trachea fragments ( %), and less commonly in spleen ( %). mean age of the deceased with confirmed flu (h n v) infection was ae years with age ranging from months to years. in % of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was days. according to our data, % of patients died had diabetes, ae % were obese, and % were pregnant women in the nd or rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h n v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of strains of pandemic virus, strains obtained from clinical specimens, and from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s n substitution in m protein, which indicates resistance to the adamantane antivirals, and have no h y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ ⁄ and a ⁄ california ⁄ ⁄ (ha similarity ae %). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ ⁄ , and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s t, na n d, v i, and ns i v (figure ). bootstrap support was . the isolates with ha s t substitution can be classified in one of the five minor genome variants of a ⁄ h n v viruses found in the united states and mexico in . several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position of ha was found in eight of eleven isolates ( %) from postmortem lung and trachea samples and two of forty isolates ( %) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d e and d g ⁄ e) could be associated with influenza virus passaging on eggs. five strains that contained g at position of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h n )v, strains isolated on the territory of russian federation during the pandemic, full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after sept ). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s p, s n, d g, v i, v i) reside in ha, two in na (i v, n k), two in pb (k n, t i), and one in pa (f l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = ae ). if v > ae , than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, ) fully described here (http://www.jstatsoft.org/v /i /paper). we have selected positions with statistically significant amino acid changes in late strains (p-value ae ). according to full genome analysis of influenza virus a ⁄ h n v strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. (figure ). besides the strain a ⁄ perth ⁄ ⁄ also contains substitution s f in the same ha antigenic site. according to data obtained, the epidemic in russia was caused only by influenza virus a ⁄ h n v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under years and among elderly people aged over years, the first wave of pandemic due to influenza virus a (h n )v resulted in increased level of mortality mainly among the people aged - years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h n v strains circulating in the southern hemisphere in the new epidemic season revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb , pb , np, ns ) and sa antigenic site of ha (n d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in ml mem and frozen at ) °c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., > ae °c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of novel h n outbreaks as a global pandemic on june , . viral isolates were obtained from inoculating cultures of mdck cells with ae - ae ml viral suspensions collected in mem originated from patients after - days incubation. [ ] [ ] [ ] [ ] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, - times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., ll starting viral supernatant used to yield final ll rna in molecular grade water (invotrogen inc.) and stored at ) °c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of novel h n viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting different rt-pcr primer pairs covering all rna segments to ensure % amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. , submitted for publication). extracted viral rna ( ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (> infectious units per ml) was added to primer-free rt-pcr total master mixture ( ae ll) for each virus followed by adding primer pair ( ll, pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps ( °c, minutes and °c, minutes); cycling amplifications ( °c for seconds, °c for seconds, °c for ae minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled cdna products ( - lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a · mm picotiterplate. for each individual viral genome containing multiple assemblies ( rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, ae - ae % of - million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche gsmapper (v. . and . ) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest h n genomes of california origin, a ⁄ california ⁄ ⁄ (h n ), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro . . software (http://www.geneious.com). trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro . . . phylogenetic trees of the h n genomes were constructed by importing fasta files containing specific concatenated target sequences of pb , pb , pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled novel h n cdnas by roche flx system up to viral cdnas could be routinely sequenced to completion for different full viral genomes from a single roche flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled h n viral genomes. for each pyrosequencing experiment, approximately - passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only - passed key reads had an average sequencing read length of > bps, defined as 'long reads' ( bps · reads = total of million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, - million bases of raw sequencing data from each single roche flx experiment would provide sufficient sequencing bases to cover full genome sequences with approximate - · of sequencing depth coverage of influenza a with average genome size of bps for the total of eight segmented rnas. so far, more than full h n genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of novel h n outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of pandemic infections, early-mid summer and fall of . phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb , pb , pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of h n are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [ ] [ ] [ ] [ ] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of pandemic, as shown in fig-ure . it is noteworthy that all viruses of mexico and california origins (clade shown at the top of figure ) were isolated at the beginning of pandemic prior to the isolation of all other viruses belonging to the second genetic clade . , discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the pandemics, wrair accomplished large number of representative h n full genomes of worldwide origins via roche flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of h n that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [ ] [ ] [ ] [ ] following the mexican and us reported cases, confirmed outbreaks of swine h n rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. , it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [ ] [ ] [ ] [ ] here we demonstrate through the phylogenetic relationship of sequenced h n full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade genetic group contains only viruses isolated at the beginning (march ⁄ april , mexico and california) of pandemics and the rest of other viruses all belong to the nd genetic group, clade . thus, it's likely that the currently circulating h n of clade causing worldwide infections is genetically different from the initial h n isolates that caused the early infections in mexico and california. , introduction a pandemic influenza virus ( h n ) was recently introduced into the human population. the hemagglutinin (ha) gene of h n is derived from 'classical swine h n ' virus, which likely shares a common progenitor strain with the human h n virus that caused the pandemic in . since antigenic changes of influenza virus ha occur more slowly in swine than in humans, we hypothesized that h n might still retain an antigenic structure similar to that of h n or the early isolates of its descendants. in this study, we compared ha antigenic structures of h n and human h n viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of h n and the h n virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the h n ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of h n . sequence data of ha genes modeller v was used for homology modeling of ha structures. after models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio . software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to ae kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify- d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand ) in ha sequences. it is known that the h ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. , as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h n viruses that have been subjected to antibody-mediated immune pressure since its emergence in , although it was absent in humans from to . to investigate the structures of these antigenic sites of h n , d structures of the ha molecules of sc , the recent seasonal human h n virus (br ), and h n (ca ) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc ha (data not shown). we found that most of these antigenic sites of br ha predominantly contained altered amino acid residues if compared with sc . by contrast, amino acid residues at these positions were relatively conserved in ca ha when compared with sc ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table ), suggesting that antibodies raised by natural infection with sc or its antigenically related descendant viruses play a role in specific immunity against ca . these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that h n would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h n viruses during its epidemic period (i.e. those that have been substituted since ) (figure ) . we then predicted possible amino acid substitutions of h n from the sequence similarity of the antigenic sites. for example, both sc and ca had an asn residue at position in the sa site. for sc , the residue at this position has altered from asn to lys since . combining these two facts, it seems reasonable to hypothesize that ca will also undergo an amino acid substitution from asn to lys at position in the future. interestingly, we found that some of the recent variants of the h n virus have indeed undergone substitutions identical to those predicted in figure . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. therefore, we investigated the number and location of potential n-glycosylation sites and cand sites in h n ha. we found that ca also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h n virus during the early epidemic of this virus. we also found that ca ha possessed three cand sites that were present at the same position in sc ha (positions of the first asn residue, , , and ). of these, the cand sites with positions at and had actually become potential n-glycosylation sites in human h n viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of h n ha. notably, some of the recent h n variants (as of march , ) have an additional n-glycosylation sequon at position , where the h n virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of h n ha is similar, at least in part, to that of the h n ha. the and h n has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of h n , and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of h n in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na -na ) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, a. means na subtype, lineage a, and sub-lineage . the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. in all cases, we employed the gtr + u nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first % trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure a ). within influenza a, two subgroups were found, one consisting of na , na , na , and na and the other consisting of the remaining five subtypes. subtype na was found to be a sister subtype of na , na being a sister subtype of na , and na a sister subtype of na . finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around years ago ( figure b ). however, it had large % hpd values which ranged from years to years ago. the na subtypes of influenza a diverged from more than to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years to (figure b ). in addition, the tmrca for influenza b viral na was dated back to . a total of lineages were identified in influenza a (table ) . three lineages, a, b, and c, were identified for na based upon the tree topology. linage a originated from avian viruses and was further divided into sub-lineages: a. , a. , a. , a. , and a. . linage b consists of north american swine influenza viruses whereas c is a human lineage. two large lineages, a and b, were identified in na . lineage a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage b is an avian-specific lineage, and consists of sub-lineages, b. , b. , and b. . three lineages were found in na . lineage a was found in north american avian, b in eurasian ⁄ oceanian avian, and c also in avian, but it does not show any geographical pattern. for na , na , and na , each was classified into lineages, one found in north american avian ( a, a, a) and the other in eurasian ⁄ oceanian avian ( b, b, b). three lineages identified respectively in na and na are north american avian ( a, a), equine ( b, b), and eurasian avian ( c, c). na was also found to have lineages: north american avian ( a), eurasian ⁄ oceanian avian i ( b), and eurassian ⁄ oceanian avian ii ( c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. within influenza a, two sub-groups were found, one consisting of na , , , and and the other consisting of the rest of five subtypes (na , , , , ) . this observation does not agree with the result described by liu et al., where na subtypes , , , , and formed one group and the remaining four subtypes (na , , , and ) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h n in , h n in , the pandemic of human h n virus in , the crisis of h n hpai in hong kong in , and swine-origin h n influenza in , all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h n ) isolated during the - pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the ⁄ season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. the all ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november , were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. , however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of hemagglutinin (ha) sequences (approximately nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, . sequences were aligned with muscle and mafft. the genetic distance matrix of all pairwise sequences was computed using the k p model under mega . . we then used the distance matrix as input to the cmdscale module in r . . for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r . . (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version . . ) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage occurs earlier than lineage ); the digit is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, a. ae means ha subtype, lineage a, and sub-lineage , and sub-sub-lineage . the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h equals , regardless of which mode we choose ( figure a ). therefore, based on bic, the optimal number of clusters for the h subtype is . as a result, a total of clusters based upon the vvv model were identified ( figure b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure b,c) . lineages a and b were identified for h , which correspond to north american avian and eurasian avian, respectively. lineage a was further divided into sub-lineages, a. , a. , where a. is the ancestral sub-lineage in a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of distinct lineages were identified among subtypes, averaging out to be ae lineages per subtype ( table ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, a, b, and c, were identified for ha . lineage a was further divided into two sub-lineages, a. and a. . the a. is swine-specific, whereas a. is a human pandemic h n sub-line- how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of distinct lineages and sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. we are conducting further analyses of other influenza a segments and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, muscle, and t-coffee. second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), likelihood, or bayesian inference. bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than taxa, as is the common case for influenza studies. alternatively, blast is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. , we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. the application of this approach in the analyses of pb genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, distance matrices were pre-computed by ccv, and they include for ha (h to ), for na (n to n ), and one for each of the internal gene segments (pb , pb , pa, np, ns, and mp). these pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select viruses or % of the cluster size if this cluster has more than viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes < min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluÔ database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluÔ database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante Ò system by a systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september , . among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluÔ database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october , , the rapidly growing gisaid dataset comprises nucleotide sequences (from isolates) with (from isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in , germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - the pandemic h n virus emerged in and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h n , and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in that were positive for pandemic h n and samples collected in that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version . (qiagen) pathogen screen. the resplex ii assay detects common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv - ), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl , hku , e, oc ), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at ) °c until rna was extracted. rna was extracted from ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of ll and stored at ) °c until used. a ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of or below was considered positive. resplex ii panel ver . detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used ( ) to obtain maximum sensitivity. from the clinical specimens that were positive for influenza from by real time pcr, there were ( %) a(h n ) seasonal influenza viruses, ( ae %) a(h n ) viruses, ( %) b viruses, and ( ae %) viruses which were influenza a positive, but could not be typed. clinical samples from selected to study were all influenza a(h n ) pandemic positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of units) for pandemic influenza a virus ( ⁄ ; sensitivity ae %) and to a lesser extent for seasonal influenza a ( ⁄ ; sensitivity of ae %) and b viruses ( ⁄ ; sensitivity of ae %) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h infections in ( ae %) or seasonal influenza infections in ( ae %) ( table ). the most common dual infection seen with pandemic h n viruses and seasonal b viruses was with cvev ( ⁄ ; and ⁄ ; , respectively) while for a(h ) viruses there were no dominant co-infecting viruses ( table ). in one case was detected with three respira- tory pathogens in the same sample, a year old female who had pandemic h n , cvev, and rhv, and in a seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv and influenza b). the median age of subjects with co-infections was younger for both pandemic h n with a median age of years (range: months to years), compared to the full sample set which had a median age of years (range: months to years), while for the patients from with seasonal influenza viruses with co-infections they had a median age of ae years (range: months to years) compared to all samples which had a median age of years (range: months to years). there was good concordance in detecting influenza a and b in respiratory samples collected in between real time rt-pcr and the resplex ii system ( % versus > ae % for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. who found that resplex ii had ae % sensitivity and % specificity for seasonal influenza a viruses and ae % sensitivity and % specificity for influenza b viruses. in contrast, the present study found only ae % sensitivity for the resplex ii detection of influenza a with the samples that were positive for pandemic h n by real time rt-pcr. a recent study by rebbapragada et al. also showed lower sensitivity for pandemic h n viruses in nasopharyngeal samples with the resplex ii system ( % sensitivity and % specificity) compared to other commercial platforms seeplex rvp ( % sensitivity and % specificity) and luminex rvp ( % sensitivity and % specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h n virus (mexico ). in terms of detection of other respiratory viruses such as piv- , piv- , rsv and hmpv, high sensitivities ( ae %, ae %, ae %, and %, respectively) and specificities ( ae - %) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. in both the seasonal influenza positive and the pandemic h n positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only of the samples had another virus detectable and one had two other viruses, while in out had another virus and one had two other viruses detected from a total of influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in with dual infections having a median age of only years compared to the study groups years; and similarly for , the median age for subjects with dual infections was only ae years compared to the study groups' median age of years. a study by chong et al. on nasopharyngeal swabs collected during - using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in ⁄ ( ae %) of samples and only ( ae %) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected ( ae % of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h n with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. , low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck -cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza ) should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at ) °c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level -enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing individually punchable storage tubes. each tube ( ae ml) is individually identifiable with a d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at ) °c. each module has a capacity of remp plates or $ samples. the automated freezer system can assemble requested samples as -well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets ( , , ) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of and were believed to arise from avian influenza viruses. the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h n disease, influenza a virus were found to infect alveolar epithelial cells and macrophages. in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h n and h n ), low pathogenic avian influenza viruses (h n , h n ) with that of the highly pathogenic h n viruses isolated from human h n disease. a range of human seasonal influenza a viruses of subtypes h n and h n viruses were included in this study from to . two isolates of low pathogenic avian influenza a (lpai) (h n ) viruses from different virus lineages isolated from poultry in hong kong in , a low pathogenic influenza a (h n ) virus isolate from wild ducks in hong kong in , and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h n were included. fragments of human bronchi and lung were cut into multiple - mm fragments within hours of collection and infected in parallel with influenza a viruses at a titer of tcid ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for hours and washed twice with pbs and incubated for , , and h at °c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h n virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h n did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h n ( figure a ) and hpai h n ( figure f ) viruses. lpai, such as h n ( figure c -d) and h n ( figure e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h n viruses ( figure b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h n virus can infect and productively replicate in the lower lung, which may account for the severity of human h n disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h n viruses being able to replicate efficiently while h n viruses do not. this may be related to the more strict siaa - gal binding preference of h n viruses. on the other hand, the efficient replication of influenza h n viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h n and avian h n virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns or c ⁄ ns enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , whereas it was inhibited by that of a ⁄ ns . these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. , the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns protein is associated with spliceosomes and inhibits pre-mrna splicing. , cis-acting sequences in the ns transcript also negatively regulate splicing. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. the yamagata ⁄ ⁄ strain of influenza c virus was grown in the amniotic cavity of -day-old embryonated hen's eggs. cos- and t cells were cultured in dulbecco's modified eagle's medium containing % fetal calf serum. subconfluent monolayers of cos- cells were transfected with pme s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at °c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ ⁄ virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). briefly, a [ p]-labeled influenza c virus rna -specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at °c overnight. hybrids were digested with rnase a ( ae u) and rnase t ( u) at °c for minutes and then analyzed on a % polyacrylamide gel containing m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ ⁄ and cos- cells transfected with pme s expressing influenza c virus ns were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m encoded by a spliced m mrna to cm encoded by an unspliced m mrna in influenza c virusinfected cells was about times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns or c ⁄ ns enhances viral mrna splicing. immunofluorescent staining showed that ns localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , though it was inhibited by that of a ⁄ ns ( figure b ). these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only - % of that of unspliced mrnas. , the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns protein associates with spliceosomes and inhibits pre-mrna splicing. , two cis-acting sequences in the ns transcript (positions - in the intron and positions - in the ¢ exon region) inhibit splicing. by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns (c ⁄ ns ) and ns (c ⁄ ns ) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns , whereas it was reduced by co-expression with influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns , though it was inhibited by co-expression with a ⁄ ns ( figure b ). these results suggest that influenza c virus ns can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns on pre-mrna splicing. , the mechanism for splicing enhancement by c ⁄ ns also remains to be determined. we speculate that c ⁄ ns may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m protein, which plays an important role in virus formation and determines virion morphology. , therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm ion channel, which is permeable to chloride ions, and also has ph-modulating activity. although the role of the influenza c virus cm ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. if this is the case, efficient splicing of m mrna may control the amount of cm synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m expression and the reduced expression of cm , thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns facilitates the splicing of m mrna. furthermore, c ⁄ ns may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns and c ⁄ ns proteins in infected cells. c ⁄ ns plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, suggesting that c ⁄ ns is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns is provided during infection to accomplish these functions. in conclusion, c ⁄ ns , which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m and cm proteins, and that of ns gene-derived ns and ns proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed ). brookes et al. recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ ⁄ (h n pdm) and its two egg-adaptation mutants with single amino acid substitutions d g and q r ( and in h numbering system). only the original virus and the variant with g were detected in the directly inoculated animals, indicating that the variant with r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d g mutant failed to transmit. in contrast, there was an apparent selection of the d g mutant in the lower respiratory tract samples from directly inoculated pigs. the d g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in - - and . here we compared phenotypic properties of the original clinical isolate of h n pdm virus a ⁄ hamburg ⁄ ⁄ and its d g and d r mutants to explain observed effects of these mutations on virus replication in swine and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ ⁄ (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in -day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d g and q r were aliquoted and designated ham-e and ham-e , respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a - -linked sialic acid ( - -fet) or a - -linked sialic acid ( - -fet). in brief, viruses adsorbed in the wells of -well eia micro plates were incubated with serial dilutions of - -fet or - -fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a ⁄ c versus a ), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). , to determine cell tropism, cultures were infected at a moi , fixed hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope ( · objective with oil immersion) in the epithelial segment that included - consecutive microscopic fields containing between % and % ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with plaque-forming units of the viruses followed by incubation at °c under airliquid interface conditions. at , , and hours postinfection, we added dmem to the apical compartments of the cultures and incubated for minutes at °c. the apical washes were harvested, stored at ) °c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. the non-egg-adapted h n pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ ⁄ (h n ), bound to - -fet ( figure a ) and did not show any significant binding to - -fet. this result contrasted with the binding of h n pdm viruses to several - -specific probes in carbohydrate microarray analysis. reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d g mutant ham-e differed from the parent virus by its ability to bind to -fet and by its reduced binding to -fet. the q r mutant only bound to - -fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ ⁄ (h n ). the viral cell tropism in htbe cultures ( figure b ) correlated with receptor specificity. ham and mem ⁄ showed a typical human-virus-like tropism , with preferential infection of non-ciliated cells (< % of infected cells were ciliated). the mutant with r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ . observed alteration of receptor specificity and cell tropism ( figure ) suggested that egg-derived mutations can affect replication of the h n pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e infected at least times less cells (data not shown). this result indicated that the mutation q r markedly impaired the ability of ham-e to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers hours post infection than did the d g mutant ( figure ). the d g mutation in h n pdm virus facilitates virus binding to - -linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d g mutant in the lower respiratory tract in humans - and pigs and correlation of this mutation with severe pulmonary disease. [ ] [ ] [ ] [ ] [ ] the d g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with g in humans and pigs. egg-derived mutation q r abolishes virus binding to - -linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q r mutant in pigs and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m ) protein, cm , and the non-structural proteins ns and ns . , cm is the second membrane protein of the virus and is encoded by rna segment (m gene). [ ] [ ] [ ] [ ] [ ] [ ] it is composed of three distinct domains: a -residue n-terminal extracellular domain, a -residue transmembrane domain, and a -residue cytoplasmic domain. , , it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. , cm forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [ ] [ ] [ ] analyses of a number of cm mutants revealed the positions of the amino acids involved in the posttranslational modifications. , evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm to the cell surface. in the present study, in order to investigate the effect of cm palmitoylation on influenza c virus replication, we generated a cm palmitoylation-deficient influenza c virus, in which a cysteine at residue of cm was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. t and hmv-ii cells were maintained as described previously. , llc-mk cells were maintained at °c in minimal essential medium with % foetal bovine serum and % calf serum. monoclonal antibodies (mabs) against the hef, np, and m proteins of c ⁄ ann arbor ⁄ ⁄ (aa ⁄ ), and antisera against the aa ⁄ virion and the cm protein were prepared as described previously. , [ ] [ ] [ ] the seven pol i plasmids for the expression of viral rnas of aa ⁄ , and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. , plasmid dna, ppoli ⁄ cm -acy(-), in which -tgt- of the m gene was replaced with -gct- , was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned plasmids were transfected into t cells as described previously. to rescue a mutant virus, rcm -c a, a recombinant influenza c virus lacking a cm palmitoylation site, the plasmid ppoli ⁄ cm -acy(-), instead of ppoli ⁄ m, was transfected together with the other plas-mids. at hours posttransfection (p.t.), the respective culture medium of the transfected- t cells was inoculated into the amniotic cavity of -day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ s]methionine or [ h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on ae % gels containing m urea, and processed for fluorography. flotation analysis was performed according to the procedure described previously. to examine whether the cm protein without palmitoylation is synthesized in rcm -c a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm serum and analysed by sds-page. as shown in figure , the cm protein was synthesized both in the rwt-and rcm -c a-infected cells, but no incorporation of [ h]palmitic acid into the cm proteins synthesized in the rcm -c ainfected cells was observed, indicating that cm in the rcm -c a-infected cells was not palmitoylated. the rwt or rcm -c a viruses were infected to hmv-ii cells at an m.o.i. of and incubated at °c for up to hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately -to -fold higher than those of rcm -c a at - hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm -c a. thus palmitoylation of cm appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m , and cm proteins between the rwt-and rcm -c a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm serum in the presence of mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm a-monomer, -dimer, and -tetramer, as well as cm b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm and authentic cm in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m , and cm proteins were observed between rwt-and rcm -c ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm -c a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions and was % of the total np in the rwt-infected cells, which was higher than that ( %) in the rcm -c a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm -c ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm is structurally similar to m , an influenza a virus membrane protein known to be involved in infectious virus production, [ ] [ ] [ ] [ ] [ ] it is possible that the cytoplasmic tail of cm participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm -c a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m protein is involved in the interaction between the cm cytoplasmic tail and np remains to be clarified. we showed that cm synthesized in rcm -c ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm to the cell surface in cm -expressing cos- cells. however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with % paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa - gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa - gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss lsm. the optical cross-section pictures were reconstructed by zeiss lsm meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h n virus that has a strict siaa - gal tropism suggests that there are siaa - gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h n virus is associated with fulminant viral pneumonia and mortality rates in excess of %. cytokine dysregulation is thought to contribute to its pathogenesis. , we previously found delayed onset of apoptosis in h n infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h n disease in humans. as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h n and seasonal influenza h n infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h n virus (a ⁄ hk ⁄ ⁄ ), a low pathogenic avian influenza h n lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g ⁄ ), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ ⁄ and a ⁄ vn ⁄ ⁄ ) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of and the cell monolayer was collected at , , and hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple - mm fragments within hours of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. these tissues fragments were infected for hours and incubated for hours at °c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h n virus induced a high percentage of apoptotic cells by tunel assay at , , and hours post infection with a peak of (figure ) . a similar observation of delayed onset of apoptosis was found in influenza h n and h n infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h n and h n virus induced trail expression compared with mock-infected tissue (approximately folds) at hours post infection, but influenza h n virus infected lung induced significantly more trail ( folds compared to mock infected cells), albeit with a limited viral replication ( figure ). influenza h n virus infected lung also elicited more tnf-alpha and fasr transcription than either h n or h n . these observations can account for the greater apoptotic response in influenza h n virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc by influenza h n virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h n and low pathogenic avian influenza h n virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay % crbc suspension was treated by mu a , -specific sialidase at °c for minutes. complete elimination of a , -receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with % crbc or % sialidase-treated crbc were performed in bsl- facility. synthetic ¢sln-paa-biotin(pa ), ¢sln-paa-biotin(pa ), ¢sln-ln-paa-biotin(pa ) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, generally, serial dilutions of sialyglycopolymers were coated in -well-flat-bottom polystryrene plates, and hau live virus ⁄ well were added. alternatively, the plates were precoated with lg ⁄ ml sialyglycopolymers, and then , , , , hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ ⁄ diluted in pbs containing % bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at nm. a total of h n virus strains were obtained from to . the name and passage history of influenza viruses used in the study are listed in table . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ ⁄ and a ⁄ js ⁄ ⁄ , only a ⁄ js ⁄ ⁄ was tested here. three amantadine-resistant variants with m mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table . complete ha with sialidasetreated crbcs, which were only with a , -receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ ⁄ , h n ) and two human h n virus strains, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . high binding of a , oligosaccharides to h n viruses was detected ( figure a -c). and enhanced a , -binding preference was also detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . the a , -binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ ⁄ of both short-and long-a , recognition, a ⁄ gx ⁄ ⁄ prefers to bind to long-a , six oligosaccharides at low viral titer ( figure b,c) . however, both of them showed strong affinity to short-and long-a , oligosaccharides at high viral loads ( figure d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. binding of influenza virus to the a , -or a , -linked sialylated glycans on cell surface is important for host range restriction, and the preference to a , of h n virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ , which are of clade ae ae . although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade ae ae into the adjacent countries of china, the finding of h n virus with - binding in human should be of concern. though h n virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m mutations of a s, a t, and s n cloned from one isolate a ⁄ hb ⁄ ⁄ suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a - -binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m . with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. , we demonstrated differential a , -binding property of two human h n viruses, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . though minor effect of short-a , -binding was detected in viruses a ⁄ gx ⁄ ⁄ at low virus titer, both were of high affinity to long-a , glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of in china (table ) . however, higher affinity to the long-a , glycans was observed in bj ⁄ ⁄ , gz ⁄ ⁄ , and xj ⁄ ⁄ (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a - with long branches on crbc, as demonstrated in a recent study. therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h n viruses is required. influenza a viruses of migrating wild aquatic birds in north america towards improved influenza a virus surveillance in migrating birds european union council directive ⁄ ⁄ eec the neighbor-joining method: a new method for reconstructing phylogenetic trees confidence limits on phylogenies: an approach using the bootstrap prospects for inferring very large phylogenies by using the neighbor-joining method mega : molecular evolutionary genetics analysis (mega) software version . the influenza virus resource at the national center for biotechnology information characterization of low-pathogenic h subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h n viruses h n virus outbreak in migratory waterfowl a ⁄ h and a ⁄ h influenza viruses: different lines of one precursor evolution and ecology of influenza a viruses evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis antigenic and genetic conservation of h influenza virus in wild ducks biologic characterization of chicken-derived h n low pathogenic avian influenza viruses in chickens and ducks genetic and pathogenic characterization of h n avian influenza viruses isolated in taiwan between and experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity h n avian influenza virus field isolate evolution and ecology of influenza a viruses is china an influenza epicenter genesis of 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antigenic and genetic characterization of h n swine influenza in china cocirculation of avian h n and contemporary ''human'' h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? h n influenza a viruses from poultry in asia have human virus-like receptor specificity characterization of h subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? bioedit: a user-friendly biological sequence alignment editor and analysis program for window ⁄ ⁄ nt genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion phylogenetic analysis using parsimony (and other methods) . beta a novel genotype h n influenza virus possessing human h n internal genomes has been circulating in poultry in eastern china since characterization of h n influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern china during a year period characterization of avian h n influenza viruses from united arab emirates phylogenetic analysis of influenza a viruses of h haemagglutinin subtype h n subtype influenza a viruses in poultry in pakistan are closely related to the h n viruses responsible for human infection in hong kong diversified reassortants h n avian influenza viruses in chicken flocks in northern and eastern china genotypic evolution and antigenic drift of h n influenza viruses in china from the nucleoprotein as a possible major factor in determining host specificity of influenza h n viruses pigs as the ''mixing vessel'' for the creation of new pandemic influenza a viruses origins and evolutionary genomics of the swine-origin h n influenza a epidemic pandemic (h n ) outbreak on pig farm reassortment of pandemic h n ⁄ influenza a virus in swine from where did the 'swine-origin' influenza a virus (h n ) emerge? substitution of lysine at position in pb protein does not change virulence of the 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circulating worldwide from oseltamivir-resistant influenza viruses a (h n ), norway, - influenza activity -united states and worldwide, - season emergence of resistance to oseltamivir among influenza a(h n ) viruses in europe oseltamivir-resistant influenza virus a (h n ), europe, - season widespread oseltamivir resistance in influenza a viruses (h n ), south africa and composition of the - influenza vaccine emergence of h y oseltamivir-resistant a(h n ) influenza viruses in japan during the - season pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza a viruses neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in a(h n ) viruses neuraminidase receptor binding variants of human influenza a(h n ) viruses due to substitution of aspartic acid in the catalytic site -role in virus attachment? neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective update: drug susceptibility of swine-origin influenza a (h n ) viruses comprehensive assessment of pandemic influenza a (h n ) virus drug susceptibility in vitro detection of molecular markers of drug resistance in pandemic influenza a (h n ) viruses by pyrosequencing pandemic (h n ) and oseltamivir resistance in hematology/oncology patients fluview: a weekly influenza surveillance report prepared by the influenza division development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network surveillance for neuraminidase inhibitor resistance among human influenza 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for complex multipathogen interactions in acute respiratory infections performance comparison of res-plex ii and xtag rvp fast for detecting respiratory viruses clinical virology symposium communityacquired respiratory co-infection (carc) in critically ill patients infected with pandemic influenza a (h n ) virus infection bacterial co-infections in lung tissue specimens from fatal cases of pandemic influenza a (h n ) -united states a quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine john's hopkins bloomberg school of public health design of an automated laboratory for high-throughput influenza surveillance human influenza surveillance: the demand to expand influenza: an emerging disease avian-to-human transmission of the pb gene of influenza a viruses in the and pandemics proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? influenza h n and h n virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation mapping of the two overlapping genes for polypepetides ns and ns on rna segment of influenza virus genome sequences of mrnas derived from genome rna segment of influenza virus: collinear and interrupted mrnas code for overlapping proteins influenza virus ns protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the influenza virus ns protein: a novel inhibitor of pre-mrna splicing identification of cis-acting intron and exon regions in influenza virus ns mrna that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes identification of a second protein encoded by influenza c virus rna segment influenza c virus ns protein upregulates the splicing of viral mrnas identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein detection of ion channel activity in xenopus laevis oocytes expressing influenza c virus cm protein evidence that the cm protein of influenza c virus can modify the ph of the exocytic pathway of transfected cells the ion channel activity of the influenza virus m protein affects transport through the golgi apparatus intracellular localization of influenza c virus ns protein (nep) in infected cells and its incorporation into virions receptor specificity, host range and pathogenicity of influenza viruses replication, pathogenesis and transmission of pandemic (h n ) virus in non-immune pigs world health organization. preliminary review of d g amino acid substitution in the haemagglutinin of pandemic influenza a (h n ) viruses 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specificity of pandemic influenza a (h n ) virus determined by carbohydrate microarray fields virology. philadelphia, pa: lippincott williams & wilkins the molecular virology and reverse genetics of influenza c virus identification of a second protein encoded by influenza c virus rna segment identification of a amino acid protein encoded by rna segment of influenza c virus influenza c virus cm protein is produced from a amino acid protein (p ) by signal peptidase cleavage a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein influenza c virus cm integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence evidence that the matrix protein of influenza c virus is coded for by a spliced mrna functional properties of the virus ion channels the cm protein of influenza c virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza a virus m and influenza b virus nb proteins characterization of a second protein (cm ) encoded by rna segment of influenza c virus phosphorylation of influenza c virus cm protein the sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza c virus cm protein identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a human melanoma cell line highly susceptible to influenza c virus antigenic characterization of the nucleoprotein and matrix protein of influenza c virus with monoclonal antibodies construction of an antigenic map of the haemagglutinin-esterase protein of influenza c virus the synthesis of polypeptides in influenza c virus-infected cells new low-viscosity overlay medium for viral plaque assays the influenza virus m protein cytoplasmic tail interacts with the m protein and influences virus assembly at the site of virus budding the cytoplasmic tail of the influenza a virus m protein plays a role in viral assembly the influenza a virus m cytoplasmic tail is required for infectious virus production and efficient genome packaging distinct domains of the influenza a virus m protein cytoplasmic tail mediate binding to the m protein and facilitate infectious virus production influenza virus m ion channel protein is necessary for filamentous virion formation influenza h n virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells das inhibits h n influenza virus infection of human lung tissues receptor binding specificity of recent human h n influenza viruses differential onset of apoptosis in avian influenza h n and seasonal h n virus infected human bronchial and alveolar epithelial cells: an in vitro and ex vivo study human influenza virus a ⁄ hongkong ⁄ ⁄ (h n ) infection induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h n -and h n -infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h n influenza a viruses to humantype receptors an avian influenza h n virus that binds to a human-type receptor evolution of highly pathogenic h n avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h n influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h n ) virus recent avian h n viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h n virus hemagglutinin h n chicken influenza viruses display a high binding affinity for neu acalpha - galbeta - ( -hso )glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h n virus the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab# ) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ), hong kong sar; and by niaid contract (sjceirs, hhsn c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ ⁄ virus survived the infection and were observed once daily for days. below lower limit of detection (< ae log eid ⁄ ml).statistical cutoff of ic values for nai susceptibility, determined by x ae + iqr. outliers with ic above this cutoff and > times the mean ic for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h y were classified as resistant and analyzed separately. h wildtype, oseltamivir-susceptible isolates. h y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant ( u ci ), niaid-nih grant, (r ai ), csrees-usda grant ( - ), and niaid-nih contract (hhsn c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp project flu-modcont (no. ). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid rc ai . cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid ( ) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan ( ) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai- (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud- authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ ⁄ and for providing antisera against h n pdm. this study was supported by the european union fp global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h n ) influenza virus circulating in portugal. genetic profile analysis was performed in isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb , pb , pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h n compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after - days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h n infections were broadly similar to seasonal influenza a ⁄ h n and a ⁄ h n . only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h n . the dynamics of pandemic influenza a ⁄ h n were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h n and a ⁄ h n . the coated respirators inactivated a broad range of influenza strains within minute, including the pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). , matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. sequence analysis was performed using dnastar and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h n in children and adults before and after vaccination with [ ] [ ] [ ] [ ] influenza season vaccines as part of the rapid public health response to the emergence of ph n and to provide evidence for ph n vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph n had been detected among participants of all age groups before and after they had been vaccinated with - , - influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph n in all age groups, compared with - % of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph n was observed in % and % of participants born from to before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph n is required in all age groups and that older populations born before may be associated with a lower infection rate of ph n . on april and april , , cases of ph n were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph n after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph n , but some degree of preexisting immunity to the virus existed, especially among adults aged ‡ years. in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph n in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi- serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph n exempt from human-subjects review. a total of serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects ( - , - , - , ‡ years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [ ] [ ] [ ] as with h n components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ ⁄ and a ⁄ brisbane ⁄ ⁄ . the ph n influenza virus used in this study was a ⁄ california ⁄ ⁄ provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by & paraformaldehyde. the criteria recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepÔ, shen zhen, china). the cells were washed and resuspended in complete rpmi- containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, um b-me. splenocytes were cultured in vitro in the presence of inactivated h n , h n , h n , and h n influenza virus antigen for h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of < ae . to evaluate the adjuvant effect of recombinant igv, the anti m e antibody subclasses was measured. igg and igg a were detected after the first and second immunization ( table ). the ratio of igg a ⁄ igg was calculated. immunization with only m e-hbc showed a lower igg a ⁄ igg ratio < ae . igv combined with m e-hbc led to a high igg a ⁄ igg ratio of up to - after first and second immunization. these igg subclass distributions indicated that igv can induce a th immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m e-hbc antigen immunization, h n , h n , h n , h n inactivated antigen was used table . the serum igg, igg , igg a, and igg a ⁄ igg ratio were measured by elisa after first and second immunization. m e were coated on the wells plate overnight, and serial dilution sera of day , , after first and second immunization were added , ae , , , , ug ⁄ ml of igg, igg , igg a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg , or igg a was then added, washed, and the optical density was read at nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns - , ns - , nsd , were generated by rationally engineering mutations directly into the genome of a pandemic-h n virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd -laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around years ago, and subsequently the na subtypes of influenza a emerged around years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of lineages identified. in addition, five and three sub-lineages were further identified in lineage a of na and lineage b of na , respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified distinct lineages and sub-lineages in all ha subtypes through large-scale analyses of influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately ⁄ of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in -day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july , up to april , , we have obtained swabs from clinics and out-patients in st. petersburg and swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h n v. thus, we received from bvls postmortem materials from deceased patients which manifested pcr+ influenza h n v-specific rna. all materials were tested for a possibility of isolation of influenza virus h n v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised ae % of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season - except the pandemic flu. from the swabs purchased from bvls, strains were isolated, which compose ae % of the pcr+ samples, and strains from the post-mortem materials ( ae % of the pcr+ samples).altogether in the season - , we isolated, retrieved, and analyzed in hai influenza strains. ae % of them were pandemic strains a(h n )v, and only ae % influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season - in russia for pandemic influenza a(h n )v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h n v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at + °c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h n v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ (h n )v and the antisera to the strains a ⁄ new jersey ⁄ ⁄ -the virus isolated during the epidemic in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ ⁄ and a ⁄ iowa ⁄ ⁄ . the hai results of representative strains are given in figure . table shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h n )v and also with the antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ up to - ⁄ homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ ⁄ because they reacted with the appropriate antiserum up to ⁄ homologous titer; these strains were a ⁄ pskov ⁄ ⁄ and a ⁄ belgorod ⁄ ⁄ . it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ ⁄ and a ⁄ sw ⁄ ⁄ to ⁄ - ⁄ , and some particular strains even to ⁄ homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in up to ⁄ - ⁄ homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey years had gone (and for the strain iowa this period is nearly years) the ha of these viruses and of the pandemic influenza share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to and to °c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure ). russian isolates of divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than were disclosed in antigenic sites, so the degree of similarity to this strain is %. a new site of glycosylation was also discovered in the position of ha. essential distinctions of the aminoacid sequence of ha and antigenic properties of the h n v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a( fi ) and a( fi ) of the sialic acid of cellular receptors. both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. according to the data of the european center of disease control and prevention (ecdc), the varieties g of the h n v virus were isolated in countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s n in the m protein that determine the resistance to adamantanes. there was no substitution in the position of neuraminidase (na), which determines the resistance to oseltamivir (h y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. thus all studied russian h n v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in and that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h n respectively were assessed for other respiratory viruses using the resplex ii panel ver . system distributed by qiagen. results showed that co-infections with another respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm -c a, a recombinant influenza c virus lacking cm palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm -c a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm -c a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb , pb , p , haemaggluti- both the a , linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h n ha to the a , -linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h n viruses from a , to a , -glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of human h n virus strains isolated from to was demonstrated. dual binding preference to a , and a , -glycans were found in a ⁄ guangdong ⁄ ⁄ and a ⁄ guangxi ⁄ ⁄ . furthermore, both of them showed a high affinity to the long-branched a , -glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h n virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h n viruses have low affinity to neu aca , gal (human-type) receptor, recent findings suggested that the adaptation of h n virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [ ] [ ] [ ] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ ⁄ , diverse genotypes were presented in currently circulating h n virus, accelerating evolution and widespread occurrence. , to date, distinct phylogenetic clades ( - ) were identified based on h n ha, and the confirmed human infections were caused by clade , , ae , ae , ae , and . in china, human h n disease was mostly caused by clade ae ae , which was identified in isolates from confirmed patients from provinces since . clade and clade ae are responsible for the case in and , respectively. two current cases of and were due to clade ae ae . now information on receptor property has been documented in some h n viruses of clade , ae , and ae . [ ] [ ] [ ] little is known about h n virus of clade ae ae , particularly from human.recently, a , -specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h n virus. , the a , or a , -binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h n viruses, , the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h n viruses. furthermore, the long-branched a , sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, ¢sln-ln is the key determinant for the human-adaptation of influenza a virus. here, we analyzed the receptor-binding specificity of human h n viruses isolated in china from to . since , a total of h n infection cases were confirmed in china from provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within days after disease onset, maintained in viral-transport medium, and tested within hours.options for the control of influenza vii