key: cord- -y ws u x authors: delisle, sylvain; south, brett; anthony, jill a.; kalp, ericka; gundlapallli, adi; curriero, frank c.; glass, greg e.; samore, matthew; perl, trish m. title: combining free text and structured electronic medical record entries to detect acute respiratory infections date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: y ws u x background: the electronic medical record (emr) contains a rich source of information that could be harnessed for epidemic surveillance. we asked if structured emr data could be coupled with computerized processing of free-text clinical entries to enhance detection of acute respiratory infections (ari). methodology: a manual review of emr records related to , outpatient visits uncovered reference cases of ari. we used logistic regression with backward elimination to determine which among candidate structured emr parameters (diagnostic codes, vital signs and orders for tests, imaging and medications) contributed to the detection of those reference cases. we also developed a computerized free-text search to identify clinical notes documenting at least two non-negated ari symptoms. we then used heuristics to build case-detection algorithms that best combined the retained structured emr parameters with the results of the text analysis. principal findings: an adjusted grouping of diagnostic codes identified reference ari patients with a sensitivity of %, a specificity of % and a positive predictive value (ppv) of %. of the additional structured clinical parameters considered, two contributed significantly to ari detection: new prescriptions for cough remedies and elevations in body temperature to at least °c. together with the diagnostic codes, these parameters increased detection sensitivity to %, but specificity and ppv declined to % and %, respectively. adding text analysis increased sensitivity to %, but ppv dropped further to %. algorithms that required satisfying both a query of structured emr parameters as well as text analysis disclosed ppvs of – % and retained sensitivities of – %. conclusion: structured emr parameters and free-text analyses can be combined into algorithms that can detect ari cases with new levels of sensitivity or precision. these results highlight potential paths by which repurposed emr information could facilitate the discovery of epidemics before they cause mass casualties. as the outbreak of severe acute respiratory syndrome (sars) [ ] and the continued threat of novel influenza strains that could produce pandemics illustrate [ , ] , human populations remain vulnerable to epidemics of acute respiratory infections (ari). these outbreaks must be discovered as soon as possible if control measures are to be implemented in time to curb morbidity, mortality and socioeconomic disruptions [ ] . the increasing availability of electronic health data offers the opportunity to enhance public health surveillance for infectious diseases, a process that has historically relied on slow and incomplete paper-based reporting [ ] . early electronic surveillance efforts have collected codified diagnoses, chief complaints, school absenteeism, over-the-counter medication sales and other independent data streams to detect the early stages of infectious illnesses, when mild signs and symptoms may not alarm clinicians [ , , , , , ] . while the utility of these pre-diagnostic, so-called ''syndromic'' surveillance systems (sss) has thus far been less than persuasive, there is now increasing evidence that the electronic transfer of diagnostic test results can lead to more complete and timely reporting of notifiable diseases to the public health authorities [ , , ] . these results encourage the continued search for new approaches aimed at improving the timeliness and usefulness of surveillance alerts. by assembling a rich array of clinical data around individual patients, a surveillance system rooted in the electronic medical record (emr) could provide early and nuanced illness detection compared to current-generation sss. notwithstanding the challenges of protecting patient privacy, an emr-based surveillance system could also support the efficient flow of information necessary to promptly investigate alerts and manage evolving epidemics [ , , ] . to gain insight into the potential of the emr as a pre-diagnostic surveillance tool, we asked how information automatically retrieved from a comprehensive emr could be arranged to detect patients with symptoms suggestive of ari. more specifically, we hypothesized that structured and freetext clinical entries into the veterans administration's (va) comprehensive emr could be automatically extracted, transformed and combined to improve ari detection compared to the sole use of icd- diagnostic codes, a common basic avenue to electronic disease detection. the maryland (n = , ) and the utah (n = , ) study samples were representative of their respective outpatient populations and included mostly male veterans. they differed in racial composition: the utah population was more than % caucasian, whereas almost half of the maryland veterans were african american (table ) . our review of the , sampled records identified a total of ari cases. ari incidence during the -month study period was lower at the ut site ( . %) than at the md site ( . %). on average, ari cases were younger than the study population at both sites, with patients over the age of representing % of the study sample but only % of ari cases ( table ). the distribution of ari cases across outpatient or telephone care areas is shown in table . two-thirds of ari cases were seen in the emergency room or in the same-day/walk-in primary care areas, where they made up , % of the visits. comparatively, ari cases amounted to less than % of visits to routine care areas. cough was the most commonly documented symptom of ari ( % of cases), followed by fever/chills/night sweats ( %) and sore throat ( %). eight ( ) percent of ari patients were febrile at the time of the encounter. table displays the frequency with which these and other less common symptoms and signs occurred, either alone or in selected combinations. note that gastrointestinal symptoms were common complaints, even though they were not part of the ari case definition. six of the emr parameters considered were either not found or present in only one ari patient, and did not contribute to ari detection at either site. these included orders of diagnostic tests for influenza or for other respiratory tract pathogens, prescriptions for antivirals, antidiarrheals or antiemetics, and orders for sinus xrays. the overall frequency at which other emr parameters occurred at both sites is shown in table . providers wrote prescriptions aimed at relieving cough or cold symptoms in % of ari patients, and blood tests or imaging in %. rates at which cbc, chest imaging and blood cultures were obtained were low (table ) but increased with the number of abnormal vital signs ( fig. ) . cdas that did not include icd- codes achieved a peak sensitivity of % and specificity of %, and were not retained (data not shown). table reports the composition and performance of illustrative cdas that used icd- codes either alone (cdas - ) or a combination with other structured emr parameters (cda - ). icd- -only cdas used by the biosense (cda ) or the essence (cda ) sss during our study period could be improved by adding three ari-compatible icd- codes (acute maxillary sinusitis ( . ), acute sinusitis nos ( . ), and bronchitis nos ( )) to the essence code set. the resulting icd- code set, labeled ''va'' (cda ), was used as a starting the text-only cda was as sensitive as a cda that best combined icd- codes with other structured clinical parameters ( table , compare cda to cda ). the ''va icd- codes'', ''cough remedies'' and ''text'' cda components could be coupled through an or operand to bring sensitivities up from % (cda ) to - % (cdas and ). while this maximized the estimated area under the roc curve, gains in sensitivity were accompanied by losses in specificity (from to %, cda to cda ). because of the low incidence of ari in this broad outpatient population (an average of . % during our -month study period), these losses in specificity halved the positive predictive value (ppv) that had originally been obtained with cda that used icd- codes alone ( table , compare cda to cda ). we could design cdas with high ppv ( - %) using only structured emr parameters, for example by requiring that both the ''icd- codes'' and the ''temperature $ uc'' parameters be satisfied (cda ). however, the resulting cdas exhibited low sensitivities (e.g. %, cda , and %, cda ) . cdas that had to satisfy both a query of structured emr parameters as well as the text analysis exhibited similarly high ppvs ( - %), but retained much higher sensitivities ( %, cda , and %, cda ). the centers of disease control and prevention (cdc) defines an influenza-like illness (ili) as an acute febrile illness (temperature of $ . uc) accompanied by a cough and/or a sore throat. thus defined, patients with ili represent a subset of our broad ari population. a text-only cda achieved a higher sensitivity for ili than its icd- -only counterpart ( table , %, cda vs. %, cda ) . despite specificities of - %, both detection approaches yielded ppvs in the - % range. as could be expected, restricting cases retrieved through icd- codes or text analysis to those patients who also have a temperature of at least . uc increased the ppv more than -fold (cdas and ) without decreasing sensitivity. selecting febrile patients who also satisfied the [icd- -or-text] clause (cda ) identified all ili cases and yet maintained a ppv of %. alternatively, requiring patients to simultaneously satisfy the icd- codes, text and temperature parameters maximized the ppv ( %) while keeping sensitivity above % (cda ). in this work, we combined selected structured emr entries with a computerized analysis of free-text documentation to detect reference patients with acute respiratory infections with sensitivities up to %, or ppv up to %. the results support our working hypothesis that judicious use of information contained in the emr can improve early disease detection compared to the sole use of diagnostic codes, and raise the possibility that harnessing the emr as a data source may improve the overall performance of automated outbreak detection systems. case definitions used for influenza surveillance vary widely [ ] , reflecting both the variability of influenza's clinical presentation and differing views on what distinguishes an influenza-like illness from other ari [ , ] . absent a standard, we crafted a case definition that would identify a broad population of patients with ari. most ( %) of our ari patients had normal vital signs and thus probably suffered from mild, uncomplicated ari [ ] . nevertheless, twothird of them felt ill enough to visit urgent or same-day care areas. thus, the majority of patients who satisfied our case definition were seeking health services despite a mostly mild illness. these patients lie at the crosshair of syndromic surveillance. we used a manual abstraction of respiratory symptoms from the emr as a reference standard to uncover cases of ari. this casefinding method may have underestimated the absolute incidence of ari encounters, as busy providers may not document symptoms that are impertinent to a focused outpatient visit. nevertheless, the abstraction, which was done on a large random sample of records, relied on information committed to a real-world emr and thus represents a realistic benchmark against which to compare the performance of ari detection algorithms relative to each other. the reference review allowed an evaluation of a stalwart component of sss, icd- diagnostic codes. our data support the suggestion that an early step toward system enhancement is to scrutinize the icd- codes used to categorize cases [ ] : additions of only a few codes improved both detection sensitivity and specificity. while our observed level of performance may not be duplicated in health systems where diagnostic codes are assigned by non-providers and/or have reimbursement repercussions, our findings reinforce results obtained in adult and pediatric emergency room populations [ , ] . note that the icd- code parameter was retained statistically in all of the most successful ari and ili detectors, suggesting that diagnostic codes are not made redundant with the availability of a broad array of structured emr parameters. our results therefore suggest that icd- codes represent a valuable, perhaps indispensible component of automated ari detection algorithms, and bolster their widespread use. some of the non-icd- parameters considered in this study had previously emerged as potentially useful for ari surveillance. in particular, sales of selected over-the-counter or prescription medications [ , , , , , , , ] and requests for diagnostic tests for influenza or for other respiratory pathogens [ , ] have been shown to correlate with the seasonality of influenza and respiratory syncytial virus infections. our study revisits similar parameters, adds new ones not yet formally investigated, but evaluates them in a different way i.e. for the accuracy with which they can detect reference ari cases not already found by alternative parameters. this evaluation was made possible because emr-derived parameters were not utilized as independent data streams. rather, emr parameters were related to each other at the single-patient level. amongst a broad field of candidate structured emr entries, we found two that could complement icd- codes to detect patients with ari: new prescriptions directed at the symptom of cough and an elevation in measured body temperature recorded in an emr field dedicated to vital signs. with % of ari patients experiencing a new onset of cough, it is not surprising that prescriptions for cough remedies uncovered new ari cases. while these results lend further support to the use of medications-related data elements for biosurveillance [ , , ] , the practical difficulty is to devise database queries that, over time, will continue to reflect the concept of ''cough remedies''. cough suppressants belong to a variety of drug classes (opioids, non-opioid antitussives, decongestants, antihistamines) that are often variably combined with each other and with other agents to relieve flu-like symptoms. efforts to standardize competing drug terminologies and to develop methods to automatically update medication groupings [ , ] must be encouraged if symptom-relief medications are to be routinely incorporated into surveillance systems. though significant statistically, the added contribution of the non-icd- structured emr parameters to ari detection was admittedly modest. we attribute this low marginal utility at least in part to the mildness of the disease in the majority of our ari patients. to wit, providers did not order any blood tests or chest imaging in % of these encounters. our efforts at devising cdas that target the subset of ari patients with ili hint at how fruitful incorporating structured non-icd- emr parameters may become as disease severity increases. for example, a parsimonious ili cda (va icd- codes and temperature . . uc, cda ) increased ppv by an order of magnitude with little losses in sensitivity compared to its icd- codes-only counterpart. while we acknowledge that an icd- code set optimized for ili could narrow this performance gap, our observations that more diagnostic tests are ordered with increasing vital signs abnormalities ( fig. ) raise the prospect that as disease severity increases, so will the value of the structured emr data. in contrast to the potential utility of the ''cough remedies'' and the ''temperature'' parameters, specific tests and therapies targeting viral ari were not utilized, and thus did not contribute detection value. perhaps the simplest explanation for why providers didn't order tests for viral respiratory pathogens or anti-influenza drugs is that neither were felt to offer a meaningful therapeutic impact in non-hospitalized patients during our study period. because these practice patterns could change quickly with the emergence or fear of a more severe illness, and for the important situational and logistical insight they may provide during an epidemic, we would certainly retain disease-specific diagnostic and therapeutic emr entries in ari cdas meant for an operational surveillance system. for surveillance purposes, text analyses have been applied to limited data sources within the emr, including ''chief complaint'' fields [ , , , , ] , laboratory or imaging reports [ , , , , ] or dictated hospital discharge summaries [ , ] . we are aware of only one published study where ari surveillance was performed by applying text analysis to the full outpatient notes typed in by the providers themselves [ ] . this study illustrated the feasibility of using the full narrative text for surveillance in the ambulatory setting, and showed a temporal correlation between total daily ili counts obtained using text analysis, queries of structured emr data, categories of chief complaints or influenza isolates. our work extends this knowledge in two significant ways. first, our reference review allowed us to provide insight into the performance of free-text mining per se. despite ongoing challenges with non-standard grammar and abbreviations, spelling mistakes, lack of punctuation, copy-and-pasting, personal templates and checklists, our simple approach to free-text analysis discovered ari cases with a sensitivity ( %) and a specificity ( %, cda ) not dissimilar to what could be reached with structured parameters (e.g. cda ) . second, the relational nature of emr data allowed us to observe the extent to which free-text and structured data sources could complement each other for case detection. on the one hand, we found that combining the results of the two mining approaches through an ''or'' logical operand yielded near-perfect sensitivity, albeit at the cost of a much reduced ppv. these results support the common-sense belief that, at least with a disease characterized by relatively mild symptoms, significant information resides exclusively in the provider's note. on the other hand, we found that combining the two approaches through an ''and'' logical operand could increase ppvs well above % while retaining sensitivities in the % range (cdas , , ) . these results suggest that cross-validating information obtained from structured data with that obtained from clinical narrative can reduce false-positive findings from either data sources and improve the noise environment through which a surveillance system must operate to uncover disease clusters. whether or not the achievable gains in signal-to-noise ratio will outweigh the losses in detection sensitivity to improve the overall ability of the surveillance system to detect disease outbreaks will have to be determined empirically. several factors, some of which have already been mentioned, may limit the generalizability of our results: ) factors related to the performance of our study at the va health care system: a) the veterans study population is mostly male and excludes the pediatric population, a key target for ari surveillance [ ] ; b) veterans health care utilization may differ from that observed in uninsured or privately insured individuals; c) clinical practices, documentation and coding habits by va practitioners may differ from those observed in solo or group practices or in health systems subject to different financial or quality-control incentives; ) factors related to our study period: optimal cdas could differ outside the respiratory infection season, or during periods of heightened apprehension for an influenza epidemic; ) factors related to our iterative cda development process, which may have over adapted cdas to va's particular emr implementation and to our sample dataset in particular, this despite our efforts to maintain a separation between development and validation data subsets; ) factors related to our text mining approach: a) we did not employ a spell checker prior to applying the negex algorithm. instead, we added common misspellings of negation and search terms (e.g. ''deneis'' instead of ''denies'', ''cocuphing'' instead of ''coughing'') identified during the chart review process. thus, unencountered misspellings are left out of our final umls concept table and negation list; b) our adaptation of the negex algorithm is specific to the types of emr note from only two va medical centers. further adaptations could therefore be needed for text documents extracted from the vista systems at other va facilities. our results confirms that it is feasible to harness the emr for biosurveillance purposes and that judicious parameter selection coupled with free-text analyses can yield case-detection tools with performance characteristics not previously available through diagnostic codes only. much more work remains to be done both to replicate our results under different health delivery environments and to determine which case-detection strategy shortens outbreak detection delay and/or minimizes the burden that false alarms impose on public health professionals. this study was approved by the institutional review boards (irbs) of the participating va health systems and those of the university of maryland, the university of utah and the johns hopkins university. the risks were limited to information confidentiality involving data generated during the course of routine patient care. the study did not interfere with such care and thus did not adversely affects the rights and welfare of the participants. the irbs granted a waiver of consent on that basis and because, given the large number of participants, the work would not have been practicable otherwise. the data collected are specified in the ''description of procedures'' section below. we randomly sampled outpatient clinical encounters from october , through march , at va maryland (vamhcs) and at va salt lake city (vaslchcs) health care systems. while we aimed at sampling as broad an outpatient visit population as possible, it was nevertheless not useful to include specialized areas of care that neither diagnose nor treat ari, such as psychiatry or ophthalmology. we therefore limited our sampling to outpatient care areas where at least two encounters were assigned icd- codes related to ari (see below) during the study period. individual patients were sampled only once. reference record review. a manual review of emr entries constituted the reference standard for ari case detection. the unit of analysis was the calendar day of an index outpatient encounter. a trained abstractor reviewed all emr entries during the calendar day of each index encounter for documentation indicative of ari. predefined ari symptoms and signs were recorded individually on an abstraction instrument (ms access, microsoft corp., redmond wa). ari was defined as follows: [ ) positive influenza culture/antigen; or ) any two of the following, of no more than days duration: a) cough; b) fever or chills or night sweats; c) pleuritic chest pain; d) myalgia; e) sore throat; f) headache] and [ ) illness not attributable to a noninfectious etiology]. all uncovered ari cases and a % random sub-sample of negative records were re-reviewed by a physician, who validated the ari-defining elements, and who could also access any part of the emr for documentation that would indicate that a non-infectious disease could best explain the current illness. a panel of specialists in pulmonary and infectious diseases arbitrated cases where there was disagreement. sample size was adjusted so that we could reach , reference ari cases at each study site, a goal estimated by the need to have sufficient power to perform a regression analysis that included our planned candidate explanatory variables (epiinfo, version . , centers for disease control and prevention). development of ari case-detection algorithms (cda) that use structured emr parameters. practicing clinicians from the research team systematically reviewed and adjucated structured or semi-structured emr parameters that could potentially identify patients with ari and that would be available within hours of an index encounter. the parameters chosen were: a) icd- diagnostic code(s) included in the existing ''respiratory'' groupings from sss of national scope (either the centers for disease control and prevention (cdc) biosense [ ] or the department of defense's essence [ ] systems) or the icd- code for fever ( . ). note that icd- codes are assigned by va health care providers when they complete their clinical note for an outpatient visit in emr, and are thus rapidly available for surveillance; b) vital sign abnormalities (temperature $ uc, respiratory rate $ breath per minute, heart rate $ beats per minute); c) orders for tests (complete blood count (cbc) with or without differential cell count, influenza antigen or culture, diagnostic tests for respiratory organisms other than influenza (respiratory syncytial virus, adenovirus, legionella), streptococcal throat screen, sputum culture or gram stain, gram stain for other respiratory specimens, blood cultures); d) requests for diagnostic imaging (chest x-ray, chest computerized tomography, any respiratory sinus imaging); and e) new prescriptions (none similar in the last days), selected from the va national formulary and grouped into the following parameters by expert consensus: cough remedies (from va national formulary (vanf) drug classes re- , - , - , - , - , - , - , - , - , or codeine), ''other cold remedies'' (from vanf cn- , ms- , nt- , - , - , - , re- , - ); antiemetics (from vanf ga- ), antidiarrheals (from vanf ga- ), influenza-targeting antivirals (neuraminidase inhibitors or adamantanes), and alternative groupings of antibacterials (i: antibiotics that could be prescribed for ari (from vanf am- , - , - , - , - , - , - , - , - , - and - ); ii: antibiotics commonly prescribed for ari (clarithromycin, erythromycin, azithromycin, clindamycin, amoxicillin/clavulanate, amoxicillin, penicillin v, gatifloxacin, levofloxacin, cefaclor, tetracycline and doxycycline); iii: the three antibiotics most commonly associated with an encounter ascribed an icd- code from the essence ''respiratory'' grouping at both sites (amoxicillin/clavulanate, azithromycin and clarithromycin). all of the data elements required to construct the above emr parameters in the study population were transferred from the veterans integrated service technology architecture (vista) hierarchical database to a structured query language (sql) relational database using the mumps data extractor software (strategic reporting systems inc., peabody, ma). data elements were included if they were entered in the emr within the calendar day of the index encounters. subsequent data transformations and database queries were implemented using sql server (microsoft corp., redmond, wa). ari cdas were developed independently for each study site. for a given site, the clinician-selected emr parameters were reduced to include only those that contributed significantly to detection of reference ari cases using backward elimination logistic regression with % confidence intervals [ ] . supplemental statistics in those analyses included akaike's information criterion, wald's chi square test, likelihood ratio test statistic, and drop-in-deviance. explanatory parameters were explored for multicollinearity with variance inflation factors and spearman correlations. the performance of retained cdas was summarized with standard statistical descriptors (sensitivity, specificity, positive and negative predictive value, and an estimate of the area under the receiver-operating characteristic (roc) curve [ ] ). bootstrap analysis with % confidence intervals was conducted to test the reliability of the most successful ari cdas [ ] . for a given cda, the dataset was divided by placing a random sample of % of the data in a first subset, and the remaining % in a second subset. the previous analyses for the selected case detectors were performed on the % subset. the statistical descriptions for the case detectors in each dataset were compared and the results were found to be similar to those for the entire dataset. the case detectors were then used to predict the ari cases in the % subset. data for each case detector was resampled times to ensure consistent results. all statistical analyses and roc estimates were performed using splus (version . , insightful corp., seattle, wa). development of ari cdas that use unstructured clinical text. of the many free-text data sources within the emr, we focused on the notes typed in by providers to document outpatient visits. our goal was first to apply simple methods using character string matching coupled with negation detection to extract information documenting ari symptoms. if a clinical note related to an index visit contained non-negated strings related to two or more symptoms from our ari case definition, then the index visit was labeled as ''positive'' for the presence of ari. to develop a list of search strings, we began by mapping ari symptoms from our case definition to the unified medical language system (umls) using the national library of medicine umls metathesaurush search tool [ ] . the umls incorporates many common source vocabularies (including icd- , mesh, and snomed) and maps concepts to a standard vocabulary represented by concept unique identifier (cui) codes [ , , , , , ] . we examined all of the umls-supplied lexical variants and semantic types related to ari [ ] to build the final list of strings. this list included synonyms, term variants, and common misspellings (table s ) . we searched for the text strings identified above in the full text of all emr clinical notes completed on the calendar day and related to each index encounter ( , notes related to , encounters). to determine if concepts identified by the text strings were affirmed or negated, for example whether a patient was ''coughing'' or denied ''coughing'', we used the publicly available negex version algorithm [ ] . we sought to improve the performance of the native algorithm by iteratively reviewing - % of notes associated with false negative and false positive cases, then correcting problems with negation detection. reusable text templates and checklists, commonly imported into va note documents, drove most of the modifications to the negex algorithm, which included: ) adding pre-processing steps to remove extraneous white spaces, carriage returns and line feeds; ) identifying special characters commonly associated with imported templates (e.g. series of ----------------, ********** to indicate section breaks) or indicating embedded data-mining software objects used to automatically retrieve vital signs or medication lists; ) identifying headings of templates that often contained a list of non-negated ari-related strings (e.g. ''cough assessment'', ''clinician instructions'', ''problems to report to your doctor'', ''needed for'', ''observe for''); ) modifying the term list used by negex for possible negation status (e.g. adding the term ''absent''); ) using regular expressions to identify non-textual patterns where the concept identified was either affirmed or negated by special characters or abbreviations (e.g. neg, pos, a novel coronavirus and sars update on avian influenza a (h n ) virus infection in humans emergence of a novel swine-origin influenza a (h n ) virus in humans strategies for containing an emerging influenza pandemic in southeast asia outbreak investigations-a perspective what is syndromic surveillance? essence ii and 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bootstrap methods and their application effective mapping of biomedical text to the umls metathesaurus: the metamap program the unified medical language system umls concept indexing for production databases: a feasibility study indexfinder: a method of extracting key concepts from clinical texts for indexing evaluating the umls as a source of lexical knowledge for medical language processing a simple algorithm for identifying negated findings and diseases in discharge summaries the authors would like to thank shawn loftus and robert sawyer, md for technical assistance with the institutional databases, shobha phansalkar for help with automated text analyses, guillermo giangreco, md for help with the validation of the record review, and kathleen speck for coordinating our scientific and administrative activities. key: cord- -php aj r authors: koller, d.; almenara, s.; mejia, g.; saiz-rodriguez, m.; zubiaur, p.; roman, m.; ochoa, d.; wojnicz, a.; martin, s.; romero-palacian, d.; navares-gomez, m.; abad-santos, f. title: safety and cardiovascular effects of multiple-dose administration of aripiprazole and olanzapine in a randomised clinical trial date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: php aj r objective: to assess adverse events and safety of aripiprazole and olanzapine treatment. methods: twenty-four healthy volunteers receiving daily oral doses of mg aripiprazole and mg olanzapine in a crossover clinical trial were genotyped for polymorphisms in genes by qpcr. drug plasma concentrations were measured by hplc-ms/ms. blood pressure and -lead ecg were measured in supine position. adverse events were also recorded. results: aripiprazole decreased diastolic blood pressure on the first day and decreased qtc on the third and fifth day. olanzapine had a systolic and diastolic blood pressure, heart rate and qtc lowering effect on the first day. polymorphisms in adra a, comt, drd and htr a genes were significantly associated to these changes. the most frequent adverse drug reactions to aripiprazole were somnolence, headache, insomnia, dizziness, restlessness, palpitations, akathisia and nausea while were somnolence, dizziness, asthenia, constipation, dry mouth, headache and nausea to olanzapine. additionally, htr a, htr c, drd , drd , oprm , ugt a and cyp a polymorphisms had a role in the development of adverse drug reactions. conclusions: olanzapine induced more cardiovascular changes; however, more adverse drug reactions were registered to aripiprazole. in addition, some polymorphisms may explain the difference in the incidence of these effects among subjects. aripiprazole (ari) and olanzapine (ola) are atypical antipsychotics commonly prescribed for patients with schizophrenia or schizoaffective disorders (robert r. conley and kelly, ) . ola has a higher serotonin ( -ht) a/dopamine d receptor binding ratio compared to typical antipsychotics. additionally, it has antagonistic activity at dopamine d and d , -ht and -ht , histamine h , α -adrenergic and muscarinic m - receptors (bhana et al., ; bymaster et al., ) . on the contrary, ari is a partial agonist at dopamine d , d , d and serotonin -ht a, -ht c and α -adrenergic receptors and it is a -ht a and -ht antagonist (shapiro et al., ) . ari is predominantly eliminated through hepatic metabolism by cytochrome p (cyp) isozymes a and d . its main active metabolite, dehydro-aripiprazole (dari) has similar affinity for the dopamine d receptor and represents approximately % of ari exposure in plasma (kim et al., ) . ola is mainly metabolized by direct glucuronidation via the udpglucuronosyltransferase (ugt) enzyme family, by oxidation via cyp a and secondarily by cyp d and cyp a (callaghan et al., ) . several typical and atypical antipsychotics increase the risk of heart rate-corrected qt (qtc) prolongation and, as a consequence, torsades de pointes and sudden cardiac death (ray et al., ) . in previous studies with schizophrenic patients, mean qtc interval was decreased with ari and qtc prolongation risk was lower with ari and ola compared to other antipsychotics (czekalla et al., ; polcwiartek et al., ; ray et al., ; vieweg, ) . however, although ari usually does no produce qtc prolongation, some studies reported the contrary in patients and healthy volunteers (belmonte et al., ; hategan and bourgeois, ; suzuki et al., ) . additionally, patients with impaired cyp d enzyme activity (i.e. poor metabolizers, pms) may be at a greater risk of qtc prolongation (dorado et al., ) . neuroleptic malignant syndrome, although it is rare, may occur with the administration of all antipsychotics. one of its symptoms is fluctuation in blood pressure (bp) (sarkar and gupta, both ari and ola can cause an increase in heart rate (hr) (belmonte et al., ; tajiri et al., ) . however, when changing the therapy from ola to ari, a significant decrease was detected in hr (tajiri et al., ) . therefore, ola had a stronger hr increasing effect compared to ari and it was dose-dependent (tajiri et al., ) . the most common adverse drug reactions (adrs) to ari in schizophrenic patients are akathisia, extrapyramidal symptoms, somnolence and tremor (ribeiro et al., ) . on the contrary, the most common adrs to ola in schizophrenic patients are constipation, weight gain, dizziness, personality disorder, akathisia, postural hypotension, sedation, headache, increased appetite, fatigue, dry mouth and abdominal pain (r. r. conley and meltzer, ) . the aim of the current study was to evaluate the safety of days treatment with ari and ola and their effects on electrocardiogram (ecg) and bp. moreover, we aimed to correlate these factors with sex, plasma drug concentrations and genetic polymorphisms. twenty-four healthy volunteers ( males and females) were enrolled in a multiple oral dose, open-label, randomized, crossover, two-periods, two-sequences, single-centre, comparative, phase i clinical trial performed between june -april . five doses of mg/day ari tablets or mg/day film-coated ola tablets were administered to each volunteer during consecutive days. block randomization was used to assign a treatment to each volunteer on the first day. the drug was administered at : h each day under fasting conditions. after a days washout period, each volunteer received the opposite drug. the random allocation sequence, the recruitment of participants and their assignment to interventions were performed by investigators of the clinical trials unit. the clinical trial was performed at the clinical trials unit of our hospital. the protocol was approved by its research ethics committee authorized by the spanish drugs agency and under the guidelines of good clinical practice and the declaration of helsinki (eudra-ct: - - ). all subjects were adequately informed about the study and, if agreeing to all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted . . https://doi.org/ . participate, signed an informed consent form before inclusion. the inclusion and exclusion criteria were reported in our previous publication (koller, saiz-rodríguez, et al., ) . twenty-two blood samples were collected in edta k tubes for pharmacokinetic assessments during each treatment period of the study. samples were centrifuged at rpm ( g) for minutes and then plasma was collected and stored at − °c until determination of plasma concentrations by the analytical laboratory. ari, dari and ola plasma concentrations were quantified by a high-performance liquid chromatography tandem mass spectrometry (hplc-ms/ms) method developed and validated in our laboratory . the calculation of pharmacokinetic parameters was explained in our previous study (koller, saiz-rodríguez, et al., ) . bp was measured in supine position with an automatic monitor at screening, predose, times during treatment and times after the last drug administration. likewise, the -lead ecg was obtained at the same time points. qtc and hr were automatically calculated by the ecg device. bazett correction formula was used to correct the qt interval (bazett, ) . according to the international council for harmonisation e clinical guidance (international council on harmonisation, ), a qtc interval greater than milliseconds or a change from baseline greater than milliseconds were considered as qtc interval prolongation. safety and tolerability of ari was assessed by clinical evaluation of adverse events (aes) and other parameters including vital signs, physical examinations, and -lead ecgs. during the development of the study, volunteers were asked if they had experienced any ae. moreover, the ramsay sedation scale (ramsay et al., ) was evaluated times/day and on each safety for statistical analysis. intensity (mild, moderate, and severe), time sequence and outcome of aes were also registered. adrs were classified using system organ class allocation as general (asthenia, fatigue, tiredness and gait alterations), cardiovascular (palpitations), gastrointestinal (constipation, nausea, vomiting, hyposalivation, hypersalivation, dry mouth and diarrhea), nervous system (akathisia, headache, difficulties with concentration, dizziness, paraesthesia, presyncope, syncope, tremor, somnolence and restless legs), psychiatric (restlessness, insomnia, anxiety, abnormal orgasm and nightmares), respiratory (epistaxis, hiccups, cough and sore throat), endocrine (galactorrhea), metabolic (lack of appetite, increased appetite and hyporexia), reproductive (dysmenorrhea, mastalgia and menstrual irregularity), skin (hair loss, pruritus, rash and sweating), musculoskeletal (shoulder pain, knee pain, neck pain, upper limb weakness, lumbalgia, cramps, back pain and leg pain), infections (cold), eye (photophobia) and investigations (increased liver enzymes) (brown et al., ) . dna was extracted from ml of peripheral blood samples using a magna pure lc dna isolation kit in an automatic dna extractor (magna pure® system, roche applied science, indianapolis, indiana). thenceforth, it was quantified spectrophotometrically in a nanodrop® nd- spectrophotometer (nanodrop technologies, wilmington, delaware, usa) and the purity of the samples was measured by the a / absorbance ratio. samples were genotyped with taqman assays using the openarray platform on a quantstudio k flex instrument (thermo fisher scientific, waltham, massachusetts, usa). the genotyping array included snps, whereof the following were analysed in genes based on their importance in the metabolism and mechanism of action of ari and ola: cyp a * c (rs ), * f (rs ), * b t>c (rs ), cyp d * (rs ), * (rs ), * (rs ), * (rs ), * (rs ), * (rs ), * (rs ), * (rs ), * (rs ), * (rs ), cyp a * (rs ), rs , rs , cyp a * (rs ), * (rs ), abcb c t (rs ), g t/a (rs ), c t (rs ), rs , - g>t (rs ), + c>t (rs ), - c>t (rs ), + t>c (rs ), - t>g (rs ), - g>a (rs ), - a>g (rs ), - a>g (rs ), adra a rs , bdnf all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint val met (rs ), comt rs , rs , drd taqia (rs ), c>t (rs ), - ins/del (rs ), drd ser gly (rs ), htr a t c (rs ), c t (rs ), rs , htr c - c/t (rs ), - g/c (rs ), rs , oprm rs and ugt a rs . genotyping results were analysed within both the quantstudio™ k flex and thermo fisher cloud softwares (thermo fisher scientific, waltham, massachusetts, usa). finally, a matrix of genotypic calls was exported for each polymorphism. statistical analyses were performed with spss . software (spss inc., chicago, illinois, united states). p values lower than . were considered statistically significant. the hardy-weinberg equilibrium was estimated for all genetic variants. deviations from the equilibrium were detected by comparing observed and expected frequencies using a fisher exact test based on the de finetti program (available at http://ihg.gsf.de/cgi-bin/hw/hwa .pl). changes in bp, hr and qtc were analysed by repeated measures anova. pharmacokinetic parameters and polymorphisms were analysed as covariates. estimate of effect size (partial eta squared, ηp ), i.e. the proportion of the total variance that is attributed to an effect, is reported for each anova test to avoid type ii errors. chi-square test was used to compare incidence of adverse effects between different genotypes. bonferroni post-hoc test for multiple comparisons was applied for each analysis. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint cyp d * , * , * , * , * , * , * , * , * , * , * and * were classified in phenotypes based on the functionality of the alleles (caudle et al., ; gaedigk et al., ) . cyp a * , * , * , and cyp a * and * genotypes were merged into a cyp a phenotype (sanchez spitman et al., ) . cyp a * c, * f and * b variants were also merged into a phenotype . abcb variants were merged into haplotypes: - mutant alleles were assigned to group , - mutant alleles were assigned to group and - mutant alleles were assigned to group . another abcb haplotype was assembled by only considering c t, g t/a and c t polymorphisms due to greater impact on the transporter's activity or expression levels (vivona et al., ) . zero or one mutant allele carriers were assigned to group , carriers of or mutant alleles were assigned to group and bearing , or mutant alleles were assigned to group . comt rs and rs polymorphisms were merged into a haplotype: carrying no mutant allele was assigned as wild-type, carrying one mutant allele was considered as heterozygous while carrying more than one mutant allele was considered as mutant. demographic data are shown in table . ten subjects were caucasian and were latin. age was similar between males and females. males had greater weight and height than females (p < . ), however, bmi values did not differ significantly (p = . ). genotype frequencies of the analysed genes are shown in table s . mean and standard deviation (sd) of ari, dari and ola pharmacokinetic parameters are shown in table . females had lower dari/ari ratio than males (p = . ). the remaining pharmacokinetic parameters were not statistically different between sexes. aripiprazole ari had a dbp lowering effect on the first day of treatment ( mmhg, p = . , ηp = . ) and a qtc lowering effect on days and ( ms, p < . , ηp = . ; ms, p = . , ηp = . , respectively) ( table s ) . none of the volunteers had a qtc value higher than ms or showed more than a ms change. hr incremented from predose to h after drug administration on the th day in males, while it did not change in females (p = . , ηp = all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint . ). dbp on the first day decreased more in htr a rs c allele carriers and in adra a rs c/c subjects compared to t/t and c/g subjects ( vs + mmhg, p = . , ηp = . ; vs mmhg, p = . , ηp = . , respectively). ola had a sbp, dbp, hr and qtc lowering effect on the first day h after drug administration ( mmhg, p < . , ηp = . ; mmhg, p < . , ηp = . ; bpm, p < . , ηp = . ; ms, p = . , ηp = . , respectively) ( table s ) . none of the volunteers had a qtc value higher than ms or showed more than a ms change. hr increased from predose to h after drug administration on the th day in males, while it did not change in females (p = . , ηp = . ). on the th day of drug administration, qtc values increased in males and the opposite effect was detected in females (p = . , ηp = . ). sbp on the first day decreased more in drd rs ser/ser and ser/gly and in adra a rs c/c subjects compared to volunteers with gly/gly and c/g genotypes ( and vs mmhg, p = . , ηp = . ; vs mmhg, p = . , ηp = . , respectively). moreover, dbp on the first day diminished more in comt wild-type subjects compared to those with heterozygous and mutant phenotype ( vs and mmhg, p = . , ηp = . ). additionally, hr on the first day decreased more in drd rs ser/ser and ser/gly subjects compared to those with gly/gly genotype ( and vs mmhg, respectively, p = . , ηp = . ). the effects of ari and ola on bp, hr and qtc are compared in figure . changes in sbp, dbp and qtc were not statistically significant between ari and ola (p = . , p = . and p = . , respectively). however, although it did not reach the statistically significant level, ola lowered both sbp and dbp to a greater extent compared to ari. after the th day, tolerance was developed for the hypotensive effect of ola. additionally, hr was significantly lower during ola treatment compared to ari (p < . , ηp = . ). during the study, no serious or life-threatening aes were documented. all volunteers experienced at least one adr. the most frequent adrs were somnolence ( %), headache ( %), insomnia ( %), dizziness ( %), restlessness ( %), palpitations ( %), akathisia ( %) and nausea ( %) ( table ) . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint the number of adrs was similar between males and females ( . ± . and . ± . adr/ subject, respectively). palpitations were only registered in females ( volunteers, p = . compared to males). the incidence of akathisia was higher in drd rs ser/ser homozygotes compared to gly allele carriers ( . % vs %, respectively, p = . ). moreover, only drd rs g/-subjects experienced asthenia compared to g/g homozygotes ( . % vs %, respectively, p = . ). the incidence of headache was significantly higher in htr c rs t carriers than in c/c homozygotes ( . % vs . %, respectively, p = . ). additionally, an association was found between cyp a nm/rm phenotype and the incidence of insomnia ( . % vs % in um, p = . ). finally, somnolence was detected more frequently in htr a rs c/c and oprm rs a/a subjects compared to c/t subjects and g carriers, respectively ( . % vs %, p = . and . % vs %, p = . , respectively). the most frequent adrs were somnolence ( %), dizziness ( %), asthenia ( %), constipation ( %), dry mouth ( %), headache ( %) and nausea ( %) ( table ). the number of adrs was similar between males and females ( . ± . and . ± . adr/ subject, respectively). constipation was detected more frequently in htr a rs t allele carriers, htr a rs a allele carriers and ugt a rs t/t subjects compared to c/c homozygotes, g/g homozygotes and c allele carriers ( . % vs . %, p < . ; . % vs . %, p = . and . % vs %, p = . , respectively). moreover, only drd rs ser/ser subjects experienced dry mouth compared to gly allele carriers ( . % vs %, respectively, p = . ). the incidence of insomnia was higher in htr c rs g/g homozygotes compared to c allele carriers ( . % vs . %, respectively, p = . ). in addition, nausea was only detected in htr c rs c/t heterozygotes and not in c/c and t/t homozygotes ( . % vs %, respectively, p = . ). finally, palpitations were only reported in cyp a um and htr a rs a allele carriers and not in nm/rms and g/g homozygotes ( . % vs %, p = . and . % vs %, p = . , respectively). adrs to ari and ola classified by system organ class allocation are shown in figure . the number of registered adrs was significantly higher after ari administration ( vs , p < . ). likewise, more psychiatric and cardiac adrs were detected during ari treatment compared to ola ( vs and vs , respectively, p < . ). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint ari caused both hyper-and hypotension in previous studies. hypertension was reported in case reports. the elevated blood pressure dropped immediately after ari withdrawal (yasui-furukori and akira fujii, ). in contrast, in other studies, dose-related hypotension was also reported: when ari was reduced to mg/day (wang, ) or discontinued (torgovnick et al., ) , the blood pressure returned to normal range. on the contrary, ola has little effects on the cardiovascular system, if any (khasawneh and shankar, ). nevertheless, it can cause hypotension (lee et al., ) . bradycardia was also reported previously during ola treatment (lee et al., ; markowitz et al., ) . in our study, ari decreased sbp (without reaching significance) and dbp on the first day of treatment, but this effect was not repeated on the rest of the days. the bp lowering mechanism may be due to blocking the α -adrenergic receptors. moreover, its -ht a antagonism could induce vasodilation and its -ht a agonism could produce hypotension and bradycardia (lin et al., ) . regarding ola, sbp, dbp and hr significantly decreased after the first dose. these changes could be explained by its α -adrenergic antagonism (beasley et al., ) . these effects were only significant on the first day of treatment and progressively diminished on the following days ( figure ) as tolerance was developed. it was reported previously that only the first dose of ola caused hypotension and bradycardia (bever and perry, ) . our study is the first to report this association with ari. sbp and dbp decreased more in adra a rs c/c subjects compared to c/g subjects during ola and ari treatment, respectively. α a-adrenergic receptors also have important roles in sympathetic cardiovascular regulation. mice that do not express adra a had increased bp and hr (kurnik et al., ) . consequently, rs mutant allele carriers should have increased bp compared to wild-type homozygotes. dbp decreased more in htr a rs c allele carriers compared to t/t subjects during ari treatment. carriers of the wild-type allele of htr a rs could have induced vasodilatation, and therefore a decrease in blood pressure (lin et al., ) . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint during ola treatment, sbp and hr decreased more in drd rs ser/ser and ser/gly subjects compared to those with gly/gly genotype. dopamine causes cardiac stimulation and therefore vasoconstriction and increase in bp. ola blocks dopamine receptors, therefore less dopamine binds to them what can result in decreased bp and hr (bangash et al., ) . d dopamine receptor blockage depends on the genotype, what explains that carriers of the mutant genotype may have a less efficient interaction between the drug and the receptor, causing smaller variations in bp and hr. in previous studies, comt rs a (val, wild-type) allele carriers had lower sbp and dbp throughout the study (ge et al., ; htun et al., ) . our study confirmed these findings: subjects with the comt wild-type phenotype (including comt rs ) had significantly higher dbp decrease after ola administration. however, another study found the opposite: the wild-type allele was associated with sbp elevation (hagen et al., ) . thus, there is no clear consensus about the role of comt polymorphisms in its bp-lowering capacity. nevertheless, this association could be due to its role in modulating dopamine function (tunbridge, ) . habitually, females show higher hr and qtc than males (li et al., ; lutfi and sukkar, ) . in our previous study both were higher in females after a single dose of ari (belmonte et al., ) . in our current study we found the contrary with ari and ola, however, as this difference could only be seen on the th day of drug administration, it may be considered an artefact. the mean qtc interval normally decreases with ari and the qtc prolongation risk is lower compared to other atypical antipsychotics (polcwiartek et al., ) . the qtc interval was overall decreased in case reports and clinical trials including schizophrenic patients and healthy volunteers, however, qtc prolongation events were also discovered (polcwiartek et al., ) . our study confirms that ari induces qtc decrease what started on the second day of drug administration and was maintained forth until the last day. therefore, based on our results, ari does not seem to cause qtc prolongation and consequently torsades de pointes and sudden cardiac death. ola does not induce qtc prolongation (czekalla et al., ) . based on the authors knowledge, the current study is the first to report qtc decrease. however, it was produced only on the first day of drug administration alike bp and hr. to date, the clinical all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint significance of its qtc-shortening is uncertain. nevertheless, it may induce proarrhythmia (shah, ) . according to the drug label, the most common adrs to ari in schizophrenic patients are akathisia, extrapyramidal symptoms, somnolence and tremor (ribeiro et al., ) . somnolence and akathisia were among the most frequent adrs that we observed. the most common adrs in clinical trials in healthy volunteers and psychotic patients were nausea, vomiting, constipation, headache, dizziness, akathisia, anxiety, insomnia, and restlessness (ribeiro et al., ) . all these adrs were detected in our clinical trial in , , , , , , , and % of the volunteers, respectively. there is no evidence on sex differences in the prevalence of adrs (montero et al., ) . our study confirms this hypothesis. in general, the most common adrs to ari were nervous system and psychiatric conditions. according to the drug label, the most common adrs to ola in schizophrenic patients are constipation, weight gain, dizziness, personality disorder, akathisia, postural hypotension, sedation, headache, increased appetite, fatigue, dry mouth and abdominal pain (r. r. conley and meltzer, ) . constipation, dizziness, headache and dry mouth were among the most frequent adrs that we observed in , , and % of the volunteers, respectively. adrs that we could not detect in our clinical trial were personality disorder, fatigue, sedation and abdominal pain. the absence of these findings could be due to the short treatment; the majority of these adrs usually appear after at least weeks of treatment (r. r. conley and meltzer, ) . a mild weight gain was observed during our study which was reported in our previous article (koller, almenara, et al., ) . there are possible sex differences in the prevalence of adrs to ola (seeman, ). however, we did not see a difference between the two sexes in our study. the lack of association could be due to the short-time treatment and the low sample size. the most common adrs to ola were metabolic disorders. palpitations are considered infrequent adrs to ari (ribeiro et al., ) . however, female volunteers and no males experienced it in our study. palpitations may also occur during ola treatment, however, they are not considered as frequent adrs (chaves et al., ) . our study confirms this finding; only volunteers were registered with palpitations. interestingly, both all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint were females, similar to ari. we are the first to report these findings. palpitations can be associated with arrhythmias. the proarrhythmic effect, drug-induced torsade de pointes occurs more frequently in females than in males (wolbrette et al., ) and was observed with ari (polcwiartek et al., ) , but not with ola (glassman and bigger, ) . therefore, as in the current study, palpitations, a proarrhythmic sign, may occur more frequently in females. in addition, during ola treatment palpitations were only reported in cyp a ums and htr a rs a allele carriers. ola is metabolized by cyp a (callaghan et al., ) . ums may reach high concentrations of ola metabolites rapidly, which could be related to the development of palpitations. additionally, palpitations were only reported in htr a rs mutant (a) allele carriers. serotonin can induce the development of palpitations (trindade et al., ) . therefore, a allele carriers may have reduced -htr a blockage and consequently higher serotonin levels. akathisia is commonly associated with first generation antipsychotics. it would be expected that ari had a low incidence of akathisia being an antagonist at -ht a receptors (thomas et al., ) . however, ari seems to increase the risk of akathisia. therefore, its pathophysiology seems complex, involving several neurotransmitters including dopamine, acetylcholine, gaminobutyric acid, norepinephrine, serotonin and neuropeptides (iqbal et al., ) . consequently, drd mutant (gly) allele carriers could be more protected from developing akathisia. the fact that drd rs g/-subjects experienced asthenia but not g/g homozygotes strengthens the dopamine theory: polymorphisms in its receptors seem to have a role in developing nervous system adrs. on the contrary, ola does not cause akathisia (thomas et al., ) what was confirmed in our study. it appears that its sedating properties could be responsible for attenuating the effects of akathisia (thomas et al., ) . -ht receptors are related to the development of somnolence and headache during antipsychotic treatment, but the molecular background is unknown to date (fang et al., ; laporta, ) . ari relates to a lower risk of somnolence and headache compared to other atypical antipsychotics (fang et al., ) , however, they are still among the most common adrs (ribeiro et al., ) . somnolence was detected more frequently in htr a rs wildtype subjects while headache was observed more in mutant allele carriers of htr c all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted . . https://doi.org/ . rs . our study is the first to report these findings. it strengthens the hypothesis that -ht receptor variability can lead to the development of these adrs. in our previous study, oprm rs mutant (g) allele carriers were associated with the increased likelihood of somnolence to fentanyl in healthy volunteers (saiz-rodríguez, ochoa, herrador, et al., ) . in the current study we observed the contrary: wild-type (a/a) homozygous subjects developed somnolence more frequently during ari treatment. the g variant is reported as a protective allele against adrs (matic et al., ) . however, its role in developing adrs to ari is currently unknown. opioid receptor activation inhibits gabaergic interneurons in order to increase dopamine release (hirasawa-fujita et al., ) . therefore, higher dopamine concentration in oprm rs wild-type homozygotes may increase the risk of somnolence. in addition, cyp a nm/rm subjects showed a higher prevalence of insomnia during ari treatment than um subjects. based on current knowledge, ari is not metabolized by cyp a . however, in our population, cyp a ums showed lower ari and dari disposition compared to the other phenotypes (koller, saiz-rodríguez, et al., ) . therefore, nm/rm subjects were under prolonged ari exposure what could cause the development of insomnia. regarding ola, insomnia is not among the most common adrs to ola (zyprexa (olanzapine), fda., ) . however, volunteers experienced it during our study, both carrying htr c rs g/g genotype. the lack of serotonin can cause insomnia (murray et al., ) . htr c rs mutant (g/g) homozygotes may have less -htr c receptor blocking effect and consequently higher risk to experience insomnia. serotonin and acetylcholine activate the colonic smooth muscles inducing their contraction. ola, being a -ht antagonist, inhibits their contraction and consequently causes constipation (zhang et al., ) . carriers of htr a rs and rs mutant alleles (t and a, respectively) displayed a higher incidence of constipation, therefore, in these subjects, ola possibly has higher binding affinity to -ht a receptors. in our previous study, the prevalence of fatigue in ugt a rs t/t subjects was significantly higher compared to wild-type (c) allele carriers (cabaleiro et al., ) . in the current study, we found the same association, but with constipation. this enzyme may be responsible for the phase ii metabolism of ola (koller, saiz-rodríguez, et al., ) . these associations were not found with ari what we expected as it is not metabolized by the ugt enzyme family. however, similar to ola, it is a all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint -ht a antagonist. nevertheless, this mechanism should be more complex; dopamine or other neurotransmitters could have a role in the development of constipation. normally, a balance is maintained between acetylcholine and dopamine. when this balance is disturbed, acetylcholine levels increase, while dopamine levels decrease (aosaki et al., ) . dry mouth is an anticholinergic side effect of ola, but not to ari given its special mechanism of action (bhana et al., ) . drd rs wild-type (ser/ser) subjects may have higher dopamine level due to the low amount of acetylcholine, therefore experiencing dry mouth after ola. nausea during ola treatment was only detected in htr c rs c/t heterozygotes and not in c/c and t/t homozygotes what can be due to the low sample size. this association was not found with ari. the main limitation of our study is the low sample size. therefore, it is of importance to interpret these results with caution. our results should be confirmed in further studies with healthy volunteers to increase the sample size as well as in schizophrenic patients to demonstrate their clinical utility. notwithstanding, our conditions were perfectly controlled and we administered both drugs to each volunteer, what can reduce the influence of other factors, such as comorbility, smoking and nutrition. additionally, the duration of this study was short and several adrs to ari and ola could appear later than days. however, the ethics committee would rarely authorize more than days treatment with antipsychotics in healthy volunteers. ola had more cardiovascular effects than ari. however, bp, hr and qtc decreased significantly only on the first day of drug administration. therefore, it seems that a rapid tolerance is developed to the drug. we suggest that htr a, adra a, drd and comt polymorphisms establish the interindividual variability of the cardiovascular effects of ari and ola. contrastively, more adrs were registered to ari than to ola, especially psychiatric and nervous system disorders. additionally, we propose that htr a, htr c, drd , drd , oprm , ugt a and cyp a polymorphisms have a role in the development all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint of adrs to ari and ola. consequently, some polymorphisms may explain the difference in the incidence of adrs among subjects. figure . effects of aripiprazole and olanzapine on blood pressure, heart rate and corrected qt interval. the shaded section shows the values after the end of treatment. the results are shown in mean ± sd. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint values are shown as mean ± sd unless otherwise indicated. *p < . versus males. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint table . pharmacokinetic parameters of aripiprazole, dehydro-aripiprazole and olanzapine after days administration of aripiprazole mg/day or olanzapine mg/day. hiccups ( ) - hyporexia ( ) -increased appetite - ( ) increased liver enzymes ( ) ( ) palpitations ( ) ( ) upper limb weakness ( ) -left shoulder pain - ( ) photophobia - ( ) all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint high frequency and founder effect of the cyp a * loss-of-function allele in the spanish population classifies cyp a as a polymorphic enzyme use of inotropes and vasopressor agents in critically ill patients: inotropes and vasopressors in the critically ill an analysis of the time-relations of electrocardiograms olanzapine versus placebo and haloperidol olanzapine: a serotonin-dopamine-receptor antagonist for antipsychotic therapy olanzapine: an updated review of its use in the management of schizophrenia the medical dictionary for regulatory activities (meddra) radioreceptor binding profile of the atypical antipsychotic olanzapine polymorphisms influencing olanzapine metabolism and adverse effects in healthy subjects olanzapine. pharmacokinetic and pharmacodynamic profile comt val met polymorphism is associated with blood pressure and lipid levels in general families of bama longevous area in china antipsychotic drugs: prolonged qtc interval, torsade de pointes, and sudden death high systolic blood pressure is associated with val/val genotype in the catechol-omethyltransferase gene. the nord-trøndelag health study (hunt) aripiprazole-associated qtc prolongation in a geriatric patient genetic variation of the mu opioid receptor (oprm ) and dopamine d receptor (drd ) is related to smoking differences in patients with schizophrenia but not bipolar disorder the clinical evaluation of qt/qtc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs akathisia: problem of history or concern of today olanzapine-induced orthostatic hypotension light reflex and its relationship with pharmacogenetics in a randomised multiple-dose trial simultaneous determination of six antipsychotics, two of their metabolites and caffeine in human plasma by lc-ms/ms using a phospholipid-removal microelutionsolid phase extraction method for sample preparation genetic variations in the α( a)-adrenoreceptor are associated with blood pressure response to the agonist dexmedetomidine relief from migraine headache with aripiprazole treatment severe cardiovascular side effects of olanzapine in an elderly patient: case report drug-induced long qt syndrome in women severe orthostatic hypotension after adding low-dose aripiprazole to clozapine. archives of clinical psychiatry the uku side effect rating scale: a new comprehensive rating scale for psychotropic drugs and a cross-sectional study of side effects in neuroleptic-treated patients the effect of gender on heart rate variability in asthmatic and normal healthy adults hypotension and bradycardia in a healthy volunteer following a single mg dose of olanzapine analgesia and opioids: a pharmacogenetics shortlist for implementation in clinical practice clinical trials with a new atypical antipsychotic (aripiprazole): gender specific information analysis insomnia caused by serotonin depletion is due to hypothermia the cardiac safety of aripiprazole treatment in patients at high risk for torsade controlled sedation with alphaxalone-alphadolone atypical antipsychotic drugs and the risk of sudden cardiac death efficacy and safety of aripiprazole for the treatment of schizophrenia: an overview of systematic reviews polymorphisms in cyp a , cyp c and abcb affect agomelatine pharmacokinetics polymorphisms associated with fentanyl pharmacokinetics, pharmacodynamics and adverse effects effect of cyp a * , cyp a * , and cyp a combined genotypes on tamoxifen metabolism all rights reserved. no reuse allowed without permission. perpetuity preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version drug information update. atypical antipsychotics and neuroleptic malignant syndrome: nuances and pragmatics of the association secondary effects of antipsychotics: women at greater risk than men drug-induced qt interval shortening: potential harbinger of proarrhythmia and regulatory perspectives: drugs, qt shortening and regulatory perspectives aripiprazole, a novel atypical antipsychotic drug with a unique and robust pharmacology dose-dependent increase in the qtc interval in aripiprazole treatment after risperidone effects of olanzapine on resting heart rate in japanese patients with schizophrenia the incidence of akathisia in the treatment of schizophrenia with aripiprazole, asenapine and lurasidone: a meta-analysis all rights reserved. no reuse allowed without permission. perpetuity preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version aripiprazole-induced orthostatic hypotension and cardiac arrhythmia adverse effects associated with selective serotonin reuptake inhibitors and tricyclic antidepressants: a metaanalysis the catechol-o-methyltransferase gene: its regulation and polymorphisms new generation antipsychotic drugs and qtc interval prolongation. primary care companion to the abcb haplotypes are associated with p-gp activity and affect a major molecular response in chronic myeloid leukemia patients treated with a standard dose of imatinib dose-related reversible hypotension during aripiprazole treatment gender differences in arrhythmias all rights reserved. no reuse allowed without permission. perpetuity preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version worsened hypertension control induced by aripiprazole. neuropsychiatric disease and treatment olanzapine may inhibit colonic motility associated with the -ht receptor and myosin light chain kinase zyprexa (olanzapine). highlights of prescribing information. u s food and drug administration the authors are grateful to the volunteers as well as the effort of the staff of the clinical trials unit of our hospital, especially to alejandro de miguel-cáceres for sample processing and key: cord- -jm nxc authors: delisle, sylvain; kim, bernard; deepak, janaki; siddiqui, tariq; gundlapalli, adi; samore, matthew; d'avolio, leonard title: using the electronic medical record to identify community-acquired pneumonia: toward a replicable automated strategy date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jm nxc background: timely information about disease severity can be central to the detection and management of outbreaks of acute respiratory infections (ari), including influenza. we asked if two resources: ) free text, and ) structured data from an electronic medical record (emr) could complement each other to identify patients with pneumonia, an ari severity landmark. methods: a manual emr review of outpatient ari visits with associated chest imaging identified x-ray reports that could support the diagnosis of pneumonia (kappa score = . ( % ci . ∶ . )), along with attendant cases with possible pneumonia (adds either cough, sputum, fever/chills/night sweats, dyspnea or pleuritic chest pain) or with pneumonia-in-plan (adds pneumonia stated as a likely diagnosis by the provider). the x-ray reports served as a reference to develop a text classifier using machine-learning software that did not require custom coding. to identify pneumonia cases, the classifier was combined with emr-based structured data and with text analyses aimed at ari symptoms in clinical notes. results: reference cases with possible pneumonia and with pneumonia-in-plan were identified. the x-ray report text classifier increased the positive predictive value of otherwise identical emr-based case-detection algorithms by – %, while retaining sensitivities of – %. these performance gains were independent of the case definitions and of whether patients were admitted to the hospital or sent home. text analyses seeking ari symptoms in clinical notes did not add further value. conclusion: specialized software development is not required for automated text analyses to help identify pneumonia patients. these results begin to map an efficient, replicable strategy through which emr data can be used to stratify ari severity. effective responses to epidemics of infectious diseases hinge not only on early outbreak detection, but also on an ongoing assessment of disease severity. indeed, the proportion of infected patients who develop severe illness often governs public perception and is a key factor in deciding whether or not to trigger interventions that can cause harm and exact significant social and financial costs. for surveillance systems aimed at epidemics of acute respiratory infections (ari), the rationale for incorporating information about disease severity is particularly compelling: ) doing so could help discover outbreaks that involve only a small number of very sick patients, such as what initially occurred with sars [ ] or what could be anticipated shortly after a criminal release of plague [ ] or tularemia [ ] ; ) such systems could help adjust ongoing responses to seasonal or pandemic influenza, where severity can vary by orders of magnitude between epidemics [ ] or even between waves of the same epidemic [ , ] . to be useful, information about ari severity needs to be both timely and specific [ , ] . current methods of monitoring influenza-related hospitalizations or deaths fall short of meeting these requirements [ ] . electronic medical records (emr) are fast becoming commonplace, and form a rich source of information that could be secondarily used for surveillance purposes. in the past, we initiated a project to unravel how emr data could be combined to identify outpatients with ari [ ] . in this work, we sought to develop casedetection algorithms (cda) aimed at pneumonia, a key landmark in the severity spectrum of ari. in particular, we asked how information retrieved from the free-text of chest imaging reports and clinical notes could complement structured data to uncover pneumonia cases. this study was approved by the institutional review boards at the university of maryland and the va maryland health care system. research-related risks were limited to maintaining the confidentiality of data generated during routine patient care. a waiver of consent was granted because the research-related risks were minimal and did not adversely affect the rights and welfare of the participants, and because the work would not have otherwise been feasible, given the large number of participants. we applied a previously validated ari case-detection algorithm (cda) [ ] to emr-derived information related to outpatient visits at the veterans administration maryland health care system, from january , through december , . this ari cda was chosen as a screening tool because it identifies % of outpatients that satisfied a broad definition of ari: positive respiratory virus culture/antigen or any two of the following symptoms, of no more than days duration: a) cough; b) fever or chills or night sweats; c) pleuritic chest pain; d) myalgia; e) sore throat; f) headache and illness not attributable to a non-infectious etiology [ ] . the ari cda flagged an outpatient visit if the provider assigned it an ari-related international disease classification, th revision, clinical modification (icd- ) diagnostic code or issued a prescription for a cough remedy or documented at least two symptoms from the above ari case definition in his/her clinical note, as retrieved by computerized text analysis [ ] . visits flagged by the ari cda were included if chest imaging was obtained within hours of clinic registration time. participants were sampled only once, at their first eligible visit during the study period. the methods to validate the performance of selected pneumonia cda on a separate population are described in the next section. reference chest imaging report review. a pulmonary disease physician read all eligible chest imaging reports (n = , in unique patients). reports were labeled ''negative'' if they did not support the diagnosis of pneumonia. this category included all images within normal limits or showing no evidence of active pulmonary disease. reports with comments on shrapnel or bullet fragments, pleural plaques or other abnormalities outside the lung parenchyma, calcified granulomas, old nodules, scars or chronic emphysematous changes were put in this category. reports were labeled ''non-negative'' if they could possibly support the diagnosis of pneumonia. these reports described a wide range of abnormalities, from ill-defined densities where the diagnosis of pneumonia could not be excluded, to frank infiltrates characteristic of pneumonia. all ''non-negative'' reports and a % sample of the ''negative'' reports were blindly reviewed by a second pulmonary physician (n = ). kappa score between the two independent reviewers was . ( % ci . : . ). ''nonnegative'' reports containing wording typically used to describe abnormalities indicative of pneumonia were also flagged and used as an alternative training set in the development of the automated imaging report classifier (see below). reference clinical record review. reference cases with pneumonia were identified by manually reviewing all emr entries made during the calendar day of index visits that corresponded to the reference, manually reviewed, ''non-negative'' chest imaging reports outlined above. symptoms and diagnostic impressions were abstracted by a pulmonary physician, entered into a data collection instrument (ms access, microsoft corp., redmond wa) and recombined into two case definitions: ) ''possible pneumonia'': non-negative chest imaging report and at least one of the following symptoms, new or changed within the last days: a) cough; b) sputum; c) fever or chills or night sweats; d) dyspnea; e) pleuritic chest pain and illness not clearly attributable to a noninfectious etiology; ) ''pneumonia-in-plan'': a non-negative chest imaging report and pneumonia listed as one of the top two diagnostic possibilities in a physician's or nurse practitioner's note. cases with possible pneumonia or pneumonia-in-plan were labeled ''admitted'' if they gained admission to the hospital within hours of index visit registration. otherwise, they were labeled ''outpatient''. development of chest imaging report classifier. we used open-source automated software that couples a clinical nlp pipeline (clinical text analysis and knowledge extraction system (ctakes) [ ] ) with an implementation of a conditional random fields probabilistic classifier [ ] to develop the text analyses that could separate non-negative from negative chest imaging reports (automated retrieval console (arc) software, v. . [ , ] ). in a preliminary effort to improve the performance of the classifier, the reference imaging reports were presented for machine-learning as four alternative training sets where: a) the text of the reports was fed either whole or scrubbed from the characters preceding the string ''impression'' when the latter was found; b) targeted reports were either all of the non-negative reports (n = ) or only those that described abnormalities typical for a pneumonia (n = ). text classification models with the highest f-measure were retained for each training set. the four retained models were then separately combined with other emr-derived data and performance of the resulting cdas at identifying patients that fitted our case definition compared (see next paragraph). the text classification models trained with reports that contained typical pneumonia descriptions and whose text was restricted to the ''impression'' field led to the best performing pneumonia cdas, and were those used for this report. candidate components for cdas included those previously found useful to identify patients with ari: ari-related icd- codes (labeled as ''ari icd- codes''), cough remedies [ ] , and clinical notes identified as positive for ari symptoms by text analysis [ ] (''text of clinical notes''). we also considered the following cda components, when related to the index outpatient visit: ) a subset of the ari icd- codes whose narrative included the string ''pneumonia'' (''pneumonia icd- codes'': - , - ); ) a new prescription for antibiotics of a class of commonly used to treat pneumonia (cephalosporins, fluoroquinolones, macrolides, penicillins); ) admission to the hospital, for any reason, within hours of the index outpatient visit (''(not) admitted to hospital''); ) chest imaging performed (''imaging obtained''); ) whether at least one chest imaging report related to the index visit was labeled ''non-negative'' by the automated text classifier described above (''text of imaging reports''). performance measures. the performance of the pneumonia cdas was summarized with standard test descriptors (sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and f-measure ( * ppv * sensitivity/(ppv + sensitivity)). denominators used to calculate these tests were either the whole study population (n = ), those patients who were hospitalized for any reason following their index visit (n = ) or those who were not (n = ). validation of selected cdas. the ari cda and imaging report classifier were applied to emr-derived databases for a year period anterior to the original study period i.e. / / - / / . a random, % sample of the visits flagged by the [ari cda and text of imaging reports] query were manually reviewed. cases identified served as the reference to validate the ppv of selected pneumonia cdas. the ari cda flagged , first visits from unique patients during the algorithm development phase of the study period. of these, , were associated with at least one report for chest imaging performed within hours of check-in time. the study population was % male, older ( years old, mean standard deviation) and . % african american (table ) . a manual review of emr entries on the day of the , index visits identified cases that satisfied at least one pneumonia case definition, with possible pneumonia and with pneumoniain-plan. most patients with a pneumonia-in-plan also had possible pneumonia ( / ), including nearly all ( / ) patients admitted to the hospital. patients who satisfied either case definitions were therefore merged into a common target group for the development of the ''admitted pneumonia'' cdas. ninety percent of all index visits occurred in urgent/same day care settings. patients with possible pneumonia and pneumonia-in-plan had similar demographics (table ) and symptoms and signs (table ) , with the possible exception that the latter population had more febrile symptoms. compared with their outpatient counterparts, admitted pneumonia patients were overrepresented in the older age groups ( - years old, table ) and appeared to have more dyspnea, fever-related symptoms, and clinical signs of lung consolidation ( table ) . the composition and performance of illustrative cdas for cases with possible pneumonia or pneumonia-in-plan are shown for all locations of care in table , and for those cases that remained outpatients or were admitted (tables and , respectively). structured emr information ipso facto included as components of the relevant cdas included: ) that chest imaging was obtained (''imaging obtained'', tables - ); ) whether or not a case was admitted to the hospital (''(not) admitted'', tables - ). an icd- code set for pneumonia diagnoses (''pneumonia icd- codes'', tables - ) helped identify pneumonia with ppvs of . - . % but had limited sensitivity ( . - %, cdas , , , , and , tables - ), even when providers had indicated that pneumonia was a likely diagnosis in their clinical notes i.e. in pneumonia-in-plan or admitted pneumonia cases (cdas , and , tables - ). a broadly inclusive ari icd- code set (''ari icd- codes'', tables - ) increased detection sensitivity to - %, but degraded ppv ( . - . %) and overall performance, as reflected by lower f-measures (compare cda to , to in table , to , to in table , and to in table ). cdas that did not include icd- diagnostic codes were not among the most successful (data not shown). prescriptions for medications aimed at ari symptoms and various groupings of antibiotics that could be used to treat bacterial pneumonias did not add value (data not shown). we retrieved information from free-text emr entries according to two different strategies. in the first strategy, text analysis routines were used to search for ari symptoms in the providers' clinical notes (''text of clinical notes'', . coupling positive results of text of clinical notes analyses to ari icd- codes using an or logical operand increased detection sensitivity over otherwise comparable cdas. however, specificity and ppv decreased and overall performance either did not improve or worsened (compare cda to and to , table ; cda to and to , table ; cda to and to , table ). coupling the text of clinical notes analysis to ari icd- codes using an and logical operand further increased ppv, but severely reduced sensitivities and overall performance (cda , , , and , tables - ). in the second strategy, text analysis was used to flag chest imaging reports that could support the diagnosis of pneumonia (''and text of imaging reports'' component, . adding this component increased the ppv of otherwise identical cdas by - absolute percentage points (compare cda to , to , to and so on, tables - ). despite attendant losses in sensitivity, results from the ''text of imaging reports'' classifier increased the f-measure of all cdas that included the broad ari icd- code set. with the possible exception is cda , whose fmeasure was the highest achieved in this study, the or text of clinical notes component did not add further value to cdas that already included analyses of the chest imaging reports (compare cda to and to , table ; cda to and to , table ; cda to , table ). table ) and was in large part due to flagging of follow-up rather than initial pneumonia visits (data not shown). ppvs actually increased for patients admitted to the hospital (cda , , table ). discussion automated text analyses of chest imaging reports improved the performance of emr-based cdas that included structured data elements and free-text search for ari symptoms. this contribution persisted across pneumonia case definitions, applied to outpatients and hospitalized patients alike, and helped cdas reach precisions of - % while maintaining sensitivities of - %. these data support our working hypothesis that selected free text analyses can supplement structured emr data to assess the severity of ari outbreaks. this work benefits from prior efforts to combine emr data to identify patients with ari. the ari cda used as an initial screen for the current study had been developed and validated against a population-based sample of over , emr records, where it recognized % of cases that satisfied a broad definition of ari [ ] . this screening algorithm forms a practical starting point for an emr data flow intent on monitoring the incidence and severity of aris, and is likely to have flagged most symptomatic pneumonia patients. pneumonia is seldom a definitive diagnosis, even when histological information is available [ ] . absent a standard, we sought clinically acceptable case definitions that could be reliably abstracted from clinical records. as is both customary and recommended by treatment guidelines [ ] [ ] [ ] [ ] , our case definitions required supportive chest imaging. to this common imaging requirement, the possible-pneumonia definition added clinical symptoms whereas pneumonia-in-plan relied solely on the provider's final diagnostic assessment. despite these differences, more than % of patients with pneumonia-in-plan also satisfied the more permissive possible pneumonia definition in both our development and validation reference populations, indicating that the two definitions addressed related clinical conditions. given that independent emr abstractors could identify respiratory symptoms [ ] , pneumonia diagnostic impressions and supportive chest imaging with a high degree of agreement, our data suggest that the possible pneumonia and the pneumonia-in-plan case definitions can serve as useful tools to reproducibly retrieve pneumonia-related information from an emr. prior attempts to automatically identify pneumonia patients through medical records have concentrated on diagnostic codes assigned after hospital discharge. discharge codes have been found to be good markers for hospitalized pneumonia patients, whether benchmarked against retrospective record reviews [ ] [ ] [ ] or prospective data acquired for clinical trials [ ] [ ] [ ] [ ] . discharge codes, however, are of limited value in epidemic surveillance because they are untimely and do not distinguish between community-and hospital-acquired pneumonia [ ] . in this study, we evaluated diagnostic codes assigned by providers at the conclusion of outpatient visits, as is practiced at the veterans administration health system. we found these codes to represent a key component of pneumonia detection, even if they proved less accurate at finding pneumonia patients who were sent home rather than hospitalized [ ] . while the utility of diagnostic codes vary when they are assigned by third parties or have reimbursement repercussions, our results nevertheless provide an impetus for diagnostic codes to be made available as soon as possible following outpatient services, so that they can be used for surveillance, decision support and quality control. the chest imaging report has long been recognized as a fruitful context in which to mine for evidence of pneumonia. over the last years, various combinations of approaches, including natural language processing [ ] [ ] [ ] [ ] , expert rules [ , ] , bayesian [ , ] or neural networks [ ] and machine-learning [ ] , have held their own compared to physicians for their ability to find pneumonia-related concepts in report narratives. imaging report analyses have been compared to discharge diagnostic codes [ , ] , but have seldom been evaluated for their added value against a broader reference standard for clinical pneumonia [ ] [ ] [ ] . to our knowledge, only one previous publication used imaging report analyses to detect outpatients with communityacquired pneumonia [ ] . besides bolstering the evidence for the utility of these text analyses, our data illustrate the importance of targeting them properly: in the course of this study, classifying , imaging reports did more to improve detection performance than extracting ari symptoms from almost million clinical notes. although an assessment of the significance of the performance gained through imaging report text analysis must await purpose-specific evaluations, our data nevertheless support the notion that a generalized machine learning approach can perform well across information retrieval tasks [ , ] . also significant, in our view, is the ease with which we could develop the classifier. clinical users focused on the document-level classification needed to create the reference training set. once the latter was fed to the arc software, model development required little further user interaction, and there was no need for custom programming. such an efficient workflow makes it possible to quickly rebuild the classifier elsewhere, should it proves less robust than our validation exercise suggests. our study is subject to limitations beyond those already mentioned. first, we did not evaluate cda components that have been associated with pneumonia in the past such as abnormalities in vital signs [ ] , white blood cell count [ ] or oxygenation [ ] , and microbiological results. while these data elements could be missing in some patients [ ] , they could provide an opportunity to further improve detection performance. second, our work was performed in a health system whose population and health care practices may not be generalizable. even if diffusion of our approaches was initially restricted to va institutions, at least some automated pneumonia surveillance could nevertheless be deployed across all states. third, sampling was not random but instead based on a screening algorithm. while this algorithm has been validated using a random, population-based sample, our study sample remains subject to verification bias [ ] such as the systematic exclusion of pneumonia patients for whom chest imaging was not obtained [ ] . fourth, the retrospective nature of the record review coupled with shortcomings of clinical acumen and chest imaging [ ] imply that we may have missed pneumonia patients whose symptoms, signs or imaging abnormalities were absent [ , ] , missed, atypical, inadequately documented or miscoded [ ] . despite these potential failings, our results do reflect information committed to a real-world emr, and thus represent a realistic environment in which to compare the relative performance of alternative detection approaches. in summary, our results indicate that an emr-based approach that couples queries of structured data with text analysis of imaging reports can be used to assess disease severity in outpatients with ari. by identifying high-performing yet parsimonious cdas that could be replicated without creating customized software, our results begin to map an efficient strategy by which pneumonia surveillance could be more widely implemented. severe acute respiratory syndrome plague as a biological weapon: medical and public health management. working group on civilian biodefense tularemia as a biological weapon: medical and public health management 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subject to selection bias testing strategies in the initial management of patients with community-acquired pneumonia high-resolution computed tomography for the diagnosis of community-acquired pneumonia influence of age on symptoms at presentation in patients with community-acquired pneumonia the authors would like to thank robert sawyer md for technical help with the institutional databases. conceived and designed the experiments: sd ag ms ld. performed the experiments: bk jd ts. analyzed the data: sd ts. contributed reagents/materials/analysis tools: sd ld. wrote the paper: sd ag ms ld. 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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grimwood, k.; sloots, t.p.; whiley, d.m.; acworth, j.p.; phillips, n.; goyal, v.; chang, a.b. title: prevalence, codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom date: - - journal: clin microbiol infect doi: . /j.cmi. . . sha: doc_id: cord_uid: aii tj x most studies exploring the role of upper airway viruses and bacteria in paediatric acute respiratory infections (ari) focus on specific clinical diagnoses and/or do not account for virus–bacteria interactions. we aimed to describe the frequency and predictors of virus and bacteria codetection in children with ari and cough, irrespective of clinical diagnosis. bilateral nasal swabs, demographic, clinical and risk factor data were collected at enrollment in children aged < years presenting to an emergency department with an ari and where cough was a symptom. swabs were tested by polymerase chain reaction for respiratory viruses and seven respiratory bacteria. logistic regression was used to investigate associations between child characteristics and codetection of the organisms of interest. between december and august , swabs were collected from ( . %) of enrolled children, median age . months (interquartile range . – . months). overall, ( . %) of specimens were positive for any organism. both viruses and bacteria were detected in specimens ( . %). factors associated with codetection were age (adjusted odds ratio (aor) for age < months = . , % confidence interval (ci) . , . ; age to < months = . , % ci . , . ; age to < months = . , % ci . , . ), male gender (aor . ; % ci . , . ), child care attendance (aor . ; % ci . , . ) and winter enrollment (aor . ; % ci . , . ). haemophilus influenzae dominated the virus–bacteria pairs. virus–h. influenzae interactions in ari should be investigated further, especially as the contribution of nontypeable h. influenzae to acute and chronic respiratory diseases is being increasingly recognized. the importance of virus-bacteria interactions in childhood acute respiratory infections (ari) remains uncertain [ ] . molecular methods enabling simultaneous detection of bacteria and viruses from a single specimen [ ] allow these relationships to be investigated. until recently, however, studies have focused mainly on either viruses [ ] or bacteria alone [ ] . studies that have examined both viruses and bacteria during an ari have typically been limited in their scope, including short duration (e.g. year during the influenza a/h n pandemic [ ] ), linkage to specific diagnostic criteria [ ] and small sample sizes [ ] . there is a substantial body of work in the literature that has examined aetiologic associations between respiratory microbes and ari that has used upper airway specimens, particularly lower ari [ ] . while upper airway specimens (nasopharyngeal swabs) are controversial because they cannot reliably distinguish between carriage and disease [ ] , they continue to be widely used in observational and experimental studies of ari in children, including those attempting to identify associations between organisms and clinical symptoms and/or severity. notably, the association between viral ari and the later development of asthma has been based on upper airway specimens [ ] . while a causal association between human rhinovirus (hrv) and respiratory syncytial virus (rsv) infections in early life with future asthma has been proposed by cohort studies [ ] , these studies did not report on upper airway bacteria that are likely to be important in early immune development and viral infections [ , ] . indeed recent larger studies which examined for both bacteria and viruses found that after adjustment for potential confounding factors it was the number of respiratory episodes rather than hrv or rsv infections in early life that were associated with future asthma [ ] . in the context of the above limitations, we focused on relating the virus and bacteria detections with epidemiologic data instead of attempting to assign causal associations between upper airway microbes and clinical disease. we describe the upper airway bacteria and viruses in children presenting to a tertiary paediatric emergency department (ed) with an ari that included cough as a symptom. we sought to describe the frequency and child-specific predictors of virus and bacteria codetection in this population and to describe the seasonal distribution of each organism and its codetections. the royal children's hospital (rch), brisbane, australia (now the lady cilento children's hospital), is the largest tertiary pediatric hospital in the state. its ed annually services over children. brisbane has a subtropical climate with maximum temperatures averaging °c in summer and °c in winter. the average monthly rainfall is almost mm, with summer the wettest season. we conducted a prospective study of children aged < years presenting to the rch ed with an ari including cough as a symptom between december and august . the primary objective of the overall cohort study was to determine the prevalence and predictors of chronic cough after ari in children; its full study protocol has been published previously [ ] . here we focus on the microbiologic aspects of that primary study. the children's health queensland (hrec/ /qrch/ ) and queensland university of technology research ethics committee ( ) approved the study. children were excluded if they had known chronic medical conditions (excluding asthma); were immunocompromised or receiving immunomodulating drugs (other than short-course (< weeks) oral or inhaled steroids) in the preceding days, or had insufficient english. written informed consent was obtained from parents/guardians of the child and from adolescents (aged > years). bilateral anterior nasal swabs were obtained using the virocult specimen collection system (medical wire and equipment, wiltshire, england, uk). protocol-specific criteria with respect to the adequacy of collection technique helped assess sampling quality [ ] . swabs were stored at − °c within hours of collection and were transferred to the research laboratory for virus and bacteria identification by validated pcr assays, as described previously [ , ] . viruses of interest included hrv, rsv a and b, influenza a and b, parainfluenza - , adenovirus, human metapneumovirus, human coronaviruses (oc , e, nl + hku ), human bocavirus, enterovirus and human polyomaviruses ki and wu. respiratory bacterial pathogens of interest included streptococcus pneumoniae, nontypeable haemophilus influenzae (nthi), moraxella catarrhalis, staphylococcus aureus, bordetella pertussis, mycoplasma pneumoniae and chlamydia pneumoniae. descriptive analyses were performed with data expressed as proportions and/or means of the selected characteristics with the corresponding % confidence intervals (cis). where continuous data were not normally distributed, medians with accompanying interquartile ranges are presented. logistic regression was used to assess the relationship between codetection of virus and bacteria and specimen quality, age, gender, length of illness (days), antibiotics in the past days, oral steroids in the previous days, household tobacco smoke exposure, child care attendance, siblings, household pets, breastfeeding history and season of enrollment. validated vaccination histories were not available and hence are not included in the analysis, although parent-reported influenza vaccination in the preceding months was included. factors in univariable analyses with p < . were entered into a backwards selection regression model to identify characteristics independently associated with virus and bacteria codetection; adjusted odds ratios (aor) and their corresponding % cis were calculated; p < . was considered statistically significant. model goodness of fit was assessed by the pearson chi-square likelihood ratio test. all analyses were performed in stata v se (statacorp, college station, tx, usa). of the children screened for participation, ( . %) were enrolled. reasons for nonenrollment included ineligibility ( . %), refusal ( . %) and other reasons ( . %) (e.g. ed staff workload, discharged before consent obtained, critically ill children). nasal swabs were collected from ( . %) enrolled ( . % male) children; median age was . months (interquartile range . - . months). parent-reported receipt of an influenza vaccine in the preceding months occurred in % of children in the study. ten swabs were not tested because of poor sample quality; hence, children were included in this analysis. overall, ( . %) of specimens were positive for any organism. four hundred ninety-seven ( . %) of the swabs had at least one virus; ( . %) of had only one virus and ( . %) of had two or more viruses detected. the most commonly detected viruses were hrv ( . %) and rsv ( . %) (supplementary table ). six hundred sixteen ( . %) of the swabs had at least one bacterial pathogen identified; ( . %) of tested positive for only one, while ( . %) of had two or more bacteria detected. the most commonly detected bacteria were m. catarrhalis ( . %), s. pneumoniae ( . %) and nthi ( . %) (supplementary table ). in contrast, only ten specimens were positive for m. pneumoniae and two for c. pneumoniae; these were not considered further in the analysis. both viruses and bacteria were codetected in ( . %) of specimens, and of these, swabs ( . %) tested positive for two or more bacteria and two viruses together. univariate analyses of associations between child characteristics and virus-bacteria codetections identified age, gender, child care attendance, siblings and enrolling season for inclusion in regression models ( table ). factors that remained significantly associated with codetection in the final model (likelihood ratio χ = . , p . ) were age (aor for age < months = . , % ci . , . ; age to < months = . , % ci . , . ; age to < months = . , % ci . , . ), male gender (aor . , % ci . , . ), child care attendance (aor . , % ci . , . ) and winter enrollment (aor . , % ci . , . ). table presents the unadjusted and adjusted (for age, season and antibiotics in the past days) associations between individual viruses and codetection with respiratory bacterial pathogens. of note was that rsv was significantly associated with nthi, s. pneumoniae, m. catarrhalis and s. aureus (table ) . given the associations between rsv and each bacterium, we constructed a model to identify predictors of rsv that included all four bacteria of interest, age, season and antibiotics in the past days. in the final model, nthi (aor . ( % ci . , . ), age (< months: aor . , % ci . , . ; to < months = . , % ci . , . ; to < months = . , % ci . , . ) and autumn enrollment (aor . , % ci . , . ) remained significantly associated with rsv detection. associations with season are presented in fig. we investigated the child characteristics associated with nasal codetection of viruses and bacteria in children with ari with cough using molecular methods. in children presenting to a tertiary paediatric ed with an ari and cough, at least one virus or bacterium was detected in nasal swab specimens from . % of cases, while viruses and bacteria were codetected in . %. identification of organisms in isolation was uncommon. factors significantly associated with virus-bacteria codetections were young age, male gender, child care attendance and winter season. differences emerged between clinically important viruses [ ] . for example, rsv was associated with age, the autumn months and nthi, while influenza was associated with winter and s. pneumoniae. other than for s. pneumoniae with influenza or rsv, little published data exist describing the frequency of virus-bacteria coinfections in nasal specimens during an ari [ ] . h. influenzae dominated the virus-bacteria pairs in our study despite not being the most common bacteria detected. indeed, the role of nthi in the pathogenesis of ari may be underestimated [ ] . there is a growing body of evidence suggesting both beneficial and detrimental synergies [ ] that may play important roles in regulating host responses, clinical severity [ ] and treatment responses [ ] . further, the availability of vaccines, now and in the future, that may affect h. influenzae necessitates the need for it to be a focus of ari research. risk factors associated with virus-bacteria codetections are similar to those observed for ari elsewhere [ ] . a study of hospitalized children with ari in china found those aged to years and boys were more likely to have virus-bacteria codetection [ ] . however, the prevalence of any bacteria identified in that study was only %, and codetection was just %. it also did not include b. pertussis, s. aureus or m. catarrhalis and relied solely on culture for identifying bacteria. while our overall detection rate of at least one organism in > % of subjects was substantially higher than some studies reporting on upper airways organisms in association with disease, our high detection rate is similar to other studies. a community-acquired pneumonia study [ ] and an american indian child cohort of ari [ ] described detection rates of % in the former [ ] and % in the latter [ ] . the seasonal distribution of most organisms correlated with the patterns of childhood ari in subtropical climates, particularly over autumn and winter months, although all organisms were identified year round. of note in our study is the relatively low frequency of rsv ( . %) and influenza ( . %) detected despite significant rsv and influenza seasons reported by state surveillance systems over the study period (https://www.health.qld.gov.au/ph/cdb/ sru_data.asp). similarly, only seven children ( . %) were found to be positive for b. pertussis despite the presence of a waning pertussis epidemic in the first year of the study [ ] . the low prevalence of rsv may partly be related to the relatively low number of enrolled children aged < months, in whom rsv is a dominant ari pathogen [ ] , and those who were critically ill. the prevalence of influenza virus in our study is consistent with other australian paediatric studies that included both low and high influenza activity seasons [ , ] . an australian observational study of influenza vaccine effectiveness in children aged months to < years in (a low influenza activity season) found ( . %) of influenza-like illnesses with specimens were influenza virus positive [ ] . similarly, in an australian cohort study of aris in children aged < years followed for months that coincided with increased influenza activity, influenza virus was detected in % of aris where parent-collected nasal specimens were obtained [ ] . however, a study from the united states of paediatric ed visits over a -year period found the proportion of ari/fever visits involving confirmed influenza infections ranged from to % (median %) [ ] , while a french study of a rapid influenza diagnostic tests in a paediatric ed setting reported % of children presenting with fever without source were positive for influenza virus during an epidemic period [ ] . the difference between these and our study are likely to reflect differences in case definitions and that the study was not conducted during an influenza pandemic, during which parents may be more likely to present to an ed if their child is ill. with respect to b. pertussis, the ratios of notifications of pertussis cases against the -year means in queensland for , and were . , . and . respectively (https://www.health.qld.gov.au/ph/cdb/sru_data.asp), and the declines were evident in all age groups (lisa mchugh, personal communication, ); hence, it is likely that the study period incorporated an interepidemic period, consistent with pertussis trends over time. a strength of our study is the detail of potential factors associated with detection rates. we accounted for prior antibiotic and oral steroid use and illness duration at presentation and did not limit recruitment to children with specific clinical entities, such as pneumonia or wheezing illnesses. the lack of an effect of prior antibiotic exposure likely reflects our reliance on pcr assays rather than culture for bacteria identification. the major limitation of the study is the proportion of children with cough who were screened but not enrolled, particularly those in the younger age groups and those with mild or severe disease, and our results may thus not reflect the population of children with ari attending an ed. further limitations include the inability to investigate causality, given the absence of controls and the cross-sectional design, meaning that temporal relationships between codetected organisms could not be examined. however, that was not the intent of our study. the use of anterior nasal rather than nasopharyngeal swabs may have led to an underestimation of bacteria species. however, anterior nasal swabs are less traumatic in young children and facilitate bilateral sampling; a loss in sensitivity for bacteria was considered acceptable for the purposes of the overall study [ ] for which the samples were collected. the infrequency of detecting just a single bacterium with a virus precluded investigating the interactions between organisms in greater detail. our study in children with cough highlights several things. firstly, relating the upper airway microbial epidemiology in relation to ari in children is complex and suggests that reports focusing solely on either bacteria or virus should be interpreted cautiously. secondly, studies that relate upper airway pathogens to clinical data should take into account factors that influence bacteria and virus detection, such as age, gender, season, child care attendance, duration of illness, specimen quality and prior antibiotic and steroid use. thirdly, our finding of the significant association of nthi with rsv should be further investigated in the context of the increasing appreciation that the contribution of nthi to acute and chronic respiratory diseases is receiving [ ] . we are now in an era of recognizing these complexities and the interactions of individual constituents and how this might influence host-specific factors, including immune responses and clinical severity [ ] . making substantial inroads into reducing the ari burden in children therefore requires high-quality studies in several different population settings. viral bacterial co-infection of the respiratory tract during early childhood cough formation in viral infections in children is virus coinfection a predictor of severity 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and bacteria in sputum samples of children with community-acquired pneumonia acute respiratory infection in children from developing nations: a multi-level study. paediatr int child health detection of viral and bacterial pathogens in hospitalized children with acute respiratory illnesses australian vaccine preventable disease epidemiological review series: pertussis lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics epidemiology of respiratory viral infections in children enrolled in a study of influenza vaccine effectiveness community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens influenza-related hospitalization and ed visits in children less than years impact of rapid influenza diagnostic test on physician estimation of viral infection probability in paediatric emergency department during epidemic period we thank the following for their support with study implementation and recruitment: m. lang, p. key: cord- - iqpl p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: - - journal: viral infections of humans doi: . / - - - - _ sha: doc_id: cord_uid: iqpl p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons [ , ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ ] and with signifi cant direct and indirect healthcare expenditure [ , ] . ari incidence is highest in the fi rst years of life, with up to episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ , - ] . in preschool-aged children, nearly % of general practitioner visits are for ari [ ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ , ] , and smooth muscle cells [ ] but not in monocytes [ ] or dendritic cells (dcs) [ ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. . ). furthermore, many modern studies ( ) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and ( ) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ - ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ , ] . in the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ , ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ ] . the ccu functioned for years ( - ) , and it was here in that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. . ) [ , ] . propagation failed once this tissue was expended [ , ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ - ] . in renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in [ - ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in , few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ ] . later, price ( ; the jh strain) and then pelon and co-workers ( ; , strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ , ] . the early hrvs were initially classifi ed as echoviruses (echo ; later hrv- ) [ ] . at the same time, propagation of the hgp (hrv- ) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ , ] . despite the challenges [ ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from to tcid /ml [ - ] and growth differences among the different virus types. additionally, cell age after plating (< h), inoculum volume (relevant to the culture vessel), medium ph ( . - . ), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ - ] . the hrvs can grow at temperatures above °c (some prefer that under certain conditions) [ ] , but rolling at °c, preceded by a - -h stationary incubation period [ ] , has historically provided the highest yield and fastest in vitro hrv growth [ , , , ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ , ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in [ ] . the fi rst classical strains were officially named in [ ] , the last in [ ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ , - ] . in hrv-c and w (another hrv-c) were shown to grow using organ culture [ ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc ) [ ] . isolation identifi ed hrvs in ~ % of adults with aris, associated with . illnesses per year [ ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ - ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ - ] . with reliable detection levels that extend from as few as tcid /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ , ] . the common understanding of the systemic [ - ] or symptomatic [ , ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ ] . the ′ untranslated region (utr; figs. . and . ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in [ ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ , , , , - , , - ] . the ′utr is comprised of a number of conserved sequence "islands" (fig. . ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ , ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ ] . the pcr primers of broadly reactive conventional rt-pcr [ , ] and rt-rtpcr [ , ] assays are shown these primers or changing the method of employing them [ - ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ ] , and by comparison of multiple primer sets using the same specimens [ ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying -fold fewer tcid /ml or fold fewer genome copies than agarose gel detection of amplicon [ , , ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ , , - ] , and some have gone on to be approved for clinical laboratory use [ ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd - per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ , ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp , ′utr+vp +vp (called hereafter vp /vp ), or ′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ ] . the vp /vp region (fig. . ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~ hrvs from a challenging biological substrate, clinical specimens [ ] . screening of airway specimens for hrvs is not routine [ ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. . ) [ ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells h after infection [ , ] . single-stranded infectious rna forms after ris start to accumulate [ ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ ] . virions are synthesized from to h after infection and reach maximum release levels at - h [ ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease a ( a pro ) of hrv-b may directly cleave eukaryotic initiation factor g (eif g) when bound to eif e [ , ] . the eifs have key roles in initiation and rate control of host cell translation [ ] . host cellular protein production is virtually replaced by hrv-b proteins after only h of infection [ ] . hrv-b -infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf- , stat , nf-κb) and shutting down host cell protein synthesis. protease c ( c pro ) from hrv-a targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ , ] . recombinant a pro protein from hrv-a ,hrv-a , hrv-b , hrv-b , hrv-c , and hrv-c exhibited differing specifi cities and kinetics against eif g as well as npc components demonstrating functional diversity between hrv types [ ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately kb positive sense rna genome (fig. . ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by a pro and c pro into the proteins ( p -p ) and structural peptides ( vp -vp ; vp and vp derive from the vp precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. . ). there are complete hrv polyproteins on the genbank database ( fig. . ). the fi rst complete hrv genome sequence (hrv-b ) was described in [ ] followed by hrv-a in [ ] and hrv-a b in [ ] (fig. [ ] . sequencing of the vp /vp region was completed for all classical strains in [ ] , and the complete set of d regions were available in [ ] . currently there are at least named hrv-c vp regions the current spectrum of complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v . [ ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version (poisson model, bootstraps with consensus support shown at the nodes where space permitted [ ] ) (reprinted with permission from miller and mackay [ ]) available and complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many ′utr and vp /vp sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of ′utr, vp , and vp /vp subgenomic regions for useful representation of hrv species and types [ , ] . recombination, the process of genetic exchange which results in a chimeric genome [ ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ ] have been made based on examination of the available set of hrv coding and noncoding regions [ ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ , , ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ , , , ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the ′utr or at the ′utr/vp junction [ , , ] but rarely in coding sequence ( a [ ] or c [ ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the - nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a a, hrv-a , hrv-b , hrv-b , and hrv-a ), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b , was described in [ ] followed by hrv-a a in [ ] , hrv-a in [ ] , hrv-a in [ ] , and hrv-a in [ ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. . ) [ ] . the hrv capsid shell is composed of protomers, each comprising one copy of the viral proteins vp , vp , vp , and vp . vp , vp , and vp (each ~ kda) are to some extent exposed on the capsid surface, whereas vp (~ kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp , vp , and vp are each formed by a convoluted set of protein sheets and loops [ ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b and hrv-a : nim- a (located in vp ), nim- b (vp ), nim-ii (vp and vp ), and nim-iii (vp and vp ) [ ] . antigenic sites identifi ed on hrv-a are called a, b, and c [ ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. . ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of > viral types (table . ). this accounts for the combination of hrv-a a and -a b, exclusion of hrv- , which is actually ev-d despite confusion over acid liability [ - ] and combination of hrv-hanks which is actually hrv-a [ ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ ] . species within the genus share > % amino acid (aa) identity in the polyprotein and in c+ cd and > % aa identity in p ( fig. . ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than . %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ ] . a variant of the same hrv type shares - % aa identity or more in vp [ ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least % different in its amino acid identity from any other genus. in a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in , and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c , formerly nat ; hrv-c , f. nat ; hrv-c , f. qpm; hrv-c , f. c , etc.; table . ) [ ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp sequence or provisionally ("c_pat," table . ) based on vp /vp [ ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the ′utr can be and still is used [ , ] for hrv genotyping, but it is a more problematic region than vp or vp /vp because of the recombination activity that affects this region, especially among the hrv-cs [ ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥ % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ ] . there are currently types within hrv-c (which includes the types once grouped together under hrv-"a ," hrv-x, and hrv-ny clades), hrv-a types, and hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c versus hrv-a simplot data projected onto the hrv-c pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a , gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ ] . attachment of the vldl-r involves adjacent vp molecules. magnifi ed vp area represents one half of a vldl-r footprint [ ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature ( - °c versus °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ , - ] . these two groups correlate with receptor usage [ ] (table . ) and possibly with the titer of the inoculum employed [ ] . in it was proposed to abandon this terminology in favor of a sequential numbering system [ ] . picornaviruses recognize a variety of cellular receptors [ , , ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ , ] . the capsid of the majority of classical hrvs ( n = ) [ ] interacts with the amino-terminal domain of the kda intercellular adhesion molecule (icam- ; cd ) [ - ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ , , ] . icam- interacts with its receptor, leukocyte function antigen- (lfa- ), and plays a role in recruitment and migration of immune effector cells [ ] . the minor group [ ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ , , ] . in andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ , ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ ] . when sequences from other subgenomic regions, including p , c, and cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table . ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ , ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ , ] . hrv-a and hrv-a are also likely the same serotype [ ] . a full list of genetically close serotype pairings was presented by ledford et al. [ ] hrv-c nomenclature was defi ned in and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp /vp data can be confi rmed with vp sequence and the provisional number removed (e.g., c_pat to c_pat have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ , , , - ] . for the hrv-cs, fi rst described as the "hrv-a " clade (not to be confused with the single virus, hrv-a , this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in [ ] , sequencing of ′utr and vp /vp has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. . ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ ] ; hence, prior to , detection of the full spectrum of ≥ hrvs did not occur. after , the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over months examined hrv frequency and diversity in specimens from healthy children ( - years of age) [ ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between and distinct hrvs at a single location [ , , ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ , , ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ ] . in populations of mixed age, the majority of hrv detections occur in children [ ] . among specimens from healthy children, over a third ( %) were hrv positive. children less than years of age ( % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than years old ( % hrv positive) [ ] . healthy adults in the military [ , ] , at university [ ] , at home [ - ] , and in the workplace [ ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ , ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ ] including those with febrile illnesses [ ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as - years of age) with % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. . ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. . ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ , , ] (and, peculiarly, on a monday [ ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ ] and hrsv [ ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" - days after return to school from holidays, in a range of climates [ - ] . this was particularly obvious among asthma hospitalizations of children ( - years of age) in ontario, canada, which peaked at weeks - across a decade [ ] . upon investigation, hrvs were the most prevalent of the viruses found in a -year analysis of emergency room presentations in ontario [ ] . hrvs also predominate during "hay fever season" [ ] . although a defi ned seasonality is not always found in the tropics [ ] , this may sometimes be due to testing that does not include hrvs [ , ] or only some hrvs [ ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during - months, or more types can co-circulate [ , ] [ ], dropping [ , ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for . % of any virus detected in a birth cohort followed for months [ ] and, in another cohort, occurred twice in two children, within a -month period [ ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ , , , - ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ , - ] or have no impact on the clinical outcome in their host [ - ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet % or more of hrsv, hmpv, ev, and ifv detections and % of hcov-nl detections can be found in the company of another virus [ ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ , ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ , ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ , ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for days postvaccination [ ] . this study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by days or more, during times of a prolonged epidemic [ ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by % overall [ ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ ] . the mechanism underneath these opposing outcomes is unclear. during the heyday ( s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv- [ , ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to respiratory viruses (fig. . ) [ , ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ . ). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ ] . during an -week period that spanned peak h n pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as h n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ ] . reports of subjects with continuous and extended (greater than - weeks) periods of hrv positivity [ , ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ ] . hrv rna has been detected days prior to symptoms commencing and for as long as or more weeks after they cease [ , - ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ ] will not detect overlapping serial infections (fig. . ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ ] . hrv shedding normally ceases within - days, after signs and symptoms have stopped [ , , , , , ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ , , , ] . few studies [ ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ , ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of versus days [ ] . multiple detection of the same hrv type ( % identical hrv- a sequence in each patient over time) extended to months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ , , ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ , ] . some studies employ sensitive symptom scoring systems [ ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ ] . infants frequently exposed to other children have more asymptomatic viral infections [ ] . among infected adult family members, asymptomatic infections are more likely [ ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ ] . it is clear that a proportion, around fi ve to % of study-defi ned asymptomatic control populations [ , , ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ , ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ , , ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ , , ] . a birth cohort of children enrolled and sampled when ill and every months for months identifi ed hrvs - % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ ] . the childhood origins of asthma (coast) birth cohort followed infants at high risk for allergies and asthma for months and identifi ed hrv infections as preceding (mean age of fi rst detection, months) those of hrsv (mean age at least months), and hrvs were found in % of asymptomatic versus % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ ] . in a study of children with asthma sampled weekly for weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ ] . a convenience population of healthy children ( - years old) without asthma were followed during at least three seasons, and picornaviruses were detected in % of specimens ( % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = , for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( - ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although of the infections came from households with an infected sibling [ ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ , ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ , ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ ] . an hrv was once isolated from feces [ ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ ] , with gastroenteritis [ ] , and in a child with pericarditis [ ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ ] , regardless of inoculation route [ ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ , ] . the incubation period between infection and onset of virus shedding into nasal secretions is - days with shed viral titers peaking in adults between days and [ , ] . the time until successful hrv transmission among adults in a childless family setting is usually - days and requires the donor to be shedding at least tcid at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ ] . the lungs have been shown to host replicating hrv [ ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of . ml to ml of suspension [ - , , - ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ , ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, % (donor to objects to recipient) and % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ ] . even under observation, eye rubbing ( . h − - . h − ) and nosepicking ( . h − - . h − ) occur frequently [ , ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ , ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ ] despite viral loads in nasal washings peaking at . × tcid /ml [ ] . experimental transmission was also reportedly ineffi cient [ ] . in contrast, "naturally" close-quartered military populations, interacting over - weeks, experienced rapid spread of hrvs to > % of the group [ ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches - % [ , ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva ( % compared to % of hand washes and % of nasal swabs) [ , , ] and from only . % of participants exposed to large-particle aerosols [ ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ , ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ , ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ ] . when exposed to liters of a small-particle aerosol, tcid of hrv- was associated with fever and prominent tracheobronchitis in antibody-free (< : ) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ , ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from % of ~ -hourold fi lters placed to sample air in offi ce buildings [ ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than days after they were removed for servicing [ ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ , , ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. . ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ ] and do so despite not hosting hrv replication directly [ ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ ] . the type iii ifns (ifn-λ or il- , ifn-λ or il- a, and ifn-λ or il- b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ , ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), ′ ′oas/rnasel, and the mx proteins [ ] . ifn-α upregulates expression of mxa, ′ ′-oas, and pkr [ ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, ′ ′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene (mda , specifi c for picornaviruses and others) [ , ] . these rna helicases recognize either rna with a ′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ , ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a [ ] , ifn regulatory factor (irf)- homodimerization is interfered with hrv-b which limits ifn-β induction [ , ] , and mda is degraded by hrv-a a but not hrv-a [ ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr , tlr , tlr , and tlr recognize nucleic acids and are also involved in innate antiviral responses. tlr and tlr identify g/u-rich ssrna from endocytosed viruses, while tlr recognizes unmethylated cpg dna present in dna viruses [ , ] . tlr and tlr are found on the cell surface and recognize hrv or hrsv proteins, respectively [ , ] , and tlr recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ ] . tlr activation can reduce ′ ′oas and mxa mrna expression and ip protein in adolescents with asthma compared to healthy controls [ ] . tlr activation did not result in a similar disparity [ ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table . ) [ , , ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h - -like and t h- -like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h - cellular response is important in managing cellular immunity and producing interleukin (il)- and ifn-γ. the t h - cellular response manages humoral immunity and stimulates b cells via il (initiating production of ige), il (infl uencing eosinophils), and il (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ ] . t h- lymphocytes, opposing t h- lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ ] . the t h- response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h- state in mice and probably in humans [ ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h - response is comparatively defi cient in people with asthma [ , ] . this is problematic as an increased t h - -like cytokine response, deduced from higher sputum mrna ifn-γ/il values, speeds clearance of hrv and symptom amelioration [ ] . one possible cause of the t h - defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ ] . the "hygiene hypothesis" [ , ] posits a pathway for an asthma etiology described [ ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h- -like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a a infection revealed some signifi cant differences that were found between cell types and response to infection [ ] . these included immune response genes (il b, il , il , il f , il ) and airway remodeling genes (loxl , mmp , fn ) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ , - ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b was found to promote release of il from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il (drives t h - development) [ ] . however, neither il nor il was signifi cantly induced in asthmatic adult volunteers in response to hrv-a compared to healthy subjects [ ] . while ifn-α was detected after transfection of dcs with hrv-b ssrna, low tnf-α and il levels were also noted [ ] . it was posited that the reduced il could indicate negatively affected local immunity possibly predisposing to secondary infections [ ] . infection of stromal lung cells by hrv-b triggered exaggerated levels of the pleiotropic il (an il type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a . g-csf and il (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ , , ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam- than those of nonatopic adults [ ] as do normal subjects infected by the major group hrv-b [ ] . by contrast, ifn-γ and il , which appear later postinfection, downregulate icam- expression in infected cells [ ] and encourage infi ltration of neutrophils [ ] , respectively. changes in icam- levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il- β proinfl ammatory properties; enhances adhesion molecule expression including icam- ; induces il- receptor gm-csf a granulocyte and monocyte growth factor il- stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il- t h differentiation, promotes ige synthesis il- activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il- /cxcl- neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il- anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il- , il- , and tnf-α irf a master hub, regulating antiviral immunity ip /cxcl chemoattractant for activated t h and nk cells tnf-α proinfl ammatory activity similar to il- β; activates neutrophils; induces vascular permeability mpc- a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp an il- -like cytokine that activates myeloid dcs to induce naive t cells into t h cells producing il- , il- , and tnf-α; induced by hrv in the presence of il- bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ , ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least days to weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least weeks [ , - ] , falling faster than serum levels [ , ] . there is also some evidence for a degree of nasal immune memory [ ] . volunteers with pre-study serum antibody could still be infected in some studies [ , , ] , but not in others [ ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ , ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ , ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥ : existed; as levels grew from , so did levels of resistance to infection [ , ] . adults were protected by serum titers of : - : [ , ] . the trend was interrupted by adults in the - year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ , ] . however, if it were possible to identify "master" strains [ ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ , ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ , , , ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ , ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a and hrv-a [ ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a sera had a higher titer for hrv-a than anti-hrv-a sera did for hrv-a [ ] . over strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a and hrv-a are linked via hrv-a , hrv-a , and hrv-a (anti-a serum neutralizes hrv-a , anti-a neutralizes hrv-a , and anti-a neutralizes both hrv-a and hrva-a [ , ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with to > % of colds positive for an hrv [ , , ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the h n pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ , , ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ , ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ , ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day and was more persistent up to days later [ , ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ , ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ , ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ , ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ , ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h type response; and (iv) hrvs may simply exacerbate that which already exists [ ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ , , ] , or a mix of both. in children with asthma, viruses have been detected in at least % of exacerbations ( % picornaviruses, probably hrvs [ ] ) and in % of adults [ ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ , - ] . bacteria are not major factors in wheezing exacerbations [ ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ , , ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ , ] . hrvs even predominated in some culture-based studies of wheeze [ , ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ , , ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th -type t cells (th cells) [ ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il is signifi cantly higher in serum and nasal aspirates than for hrsv [ ] . ip was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ , , , ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ , , , - ] although, in some instances, an exacerbation is the only sign of infection [ ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ ] . bacteria were often present with ifv, but not with hrvs [ ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ ] although hospitalizations may be fewer for hrv-cs than the other species [ ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ , ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ - ] . since a longitudinal day-care study in , the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ , ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction ( %) due to the target bacteria [ ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ , , ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ , ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ , ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of - % of children with aom [ , ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ ] . picornaviruses have been detected in % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from % of children with recurrent aom and/or adenoid hyperplasia [ , ] . in a cohort of children followed from to months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ ] . viruses, most often hrvs ( . % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes ( % of aom without ari) [ ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between and months of age ]. hrvs accounted for % of viruses detected and % of specimens with a single virus detected. this dominance was maintained even through the h n infl uenza pandemic [ ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ ] . the presence of hrv-b was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ ] . hrv infections result in more copd exacerbations (~ % of cases [ ] ) than any other virus identifi ed to date [ , ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ ] . adding the measurement of an infl ammatory marker in the serum, like il- , further improves the speed of predicting an infectious etiology for exacerbations of copd [ ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ , ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of years [ ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ , ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ , ] . infections causing pneumonia vary with age and vaccination status [ ] . viruses can be detected in up to % of infants ( - months of age) with pneumonia, and these cases follow a seasonal pattern [ , ] . bacteria can also be detected in over % of infants and older children, the elderly, and those with severe cap [ , ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising - % of childhood pneumonia cases [ , ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across or more years, which presumably would account for seasonal variation in virus prevalence [ , , , ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ , , , ] . there are laboratory data which support this [ ] as well as observational data showing a high proportion of hrvbacterial co-detections [ , ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ , , , ] . viral-bacterial coinfections can comprise % of patients, while viral-viral ( - %) and bacterial-bacterial ( - %) are much less common [ , , , , ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ , , ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ ] . acute bronchitis (less than -week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ , ] . despite testing, a third of cases remain without a viral etiology [ ] . tracheobronchitis resulted from some hrv-a infection of volunteers [ ] . chest pain and cough have been reported in half or more of adults with hrv infection [ ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ , ] . bronchiolitis occurs seasonally, especially in winter, in infants ( - months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ , ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a -year prospective cohort of very low birth weight infants in buenos aires, argentina [ ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ , ] . the precise role for viruses and bacteria in sinusitis is still unclear [ ] . sinusitis is a common comorbidity in those with asthma [ ] . the in situ presence of hrv-b rna in maxillary sinus epithelium was reported in seven of adults with acute sinusitis [ ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ , ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a [ ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ , - ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ , ] , albeit in very low numbers by nonmolecular studies [ ] and without a significantly different clinical outcome from non-hrv aris in these patients [ ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ , , ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ , ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ ] despite good results in experimental tests [ ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ - ] . however, continual hand washing with extra ingredients resulted in skin irritation [ ] . the experimental testing [ , ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ ] . anticholinergic agents have proven useful to reduce rhinorrhea [ ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ ] . when initiated early in the illness, a combination of antiviral (ifn-α b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ ] . antiviral agents (table . ) require early application to effectively precede the pathogenic immune response to hrv infection [ ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. . ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented in hrv detections. we do not yet know their niche or clinical impact. it may be possible that hrv-bs are the most well adapted of the hrvs, causing little to no detectable clinical impact, or they may create a different impact than that which we expect, or they may be a species in decline. the jury remains out on whether hrvs cause or are involved in the development of asthma or merely trigger exacerbations once asthma is established. with a very high healthcare impact from asthma around the world and atopic conditions that may be exacerbated by hrvs on the rise, this is an important area for 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azithromycin induces antiviral responses in bronchial epithelial cells suggested reading newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord- - sb moe authors: lagare, adamou; ousmane, sani; dano, ibrahim dan; issaka, bassira; issa, idi; mainassara, halima boubacar; testa, jean; tempia, stefano; mamadou, saidou title: molecular detection of respiratory pathogens among children aged younger than years hospitalized with febrile acute respiratory infections: a prospective hospital‐based observational study in niamey, niger date: - - journal: health sci rep doi: . /hsr . sha: doc_id: cord_uid: sb moe background and aims: in niger, acute respiratory infections (aris) are the second most common cause of death in children aged younger than years. however, the etiology of ari is poorly understood in the country. this study aims to describe viral and bacterial infections among children aged younger than years hospitalized with febrile ari at two hospitals in niamey, niger's capital city, and the reported clinical procedures. methods: we conducted a prospective study among children aged younger than years hospitalized with febrile ari at two national hospitals in niamey between january and december . clinical presentation and procedures during admission were documented using a standardized case investigation form. nasopharyngeal specimens collected from each patient were tested for a panel of respiratory viruses and bacteria using the fast track diagnostic plus kit. results: we enrolled and tested children aged younger than years, of whom ( . %) were aged younger than year, and ( . %) died during the study period. overall, / ( . %) specimens tested positive for at least one respiratory virus or bacterium; of these, ( . %) tested positive for respiratory viruses, ( . %) tested positive for respiratory bacteria, and ( . %) tested positive for both respiratory viruses and bacteria. the predominant viruses detected were respiratory syncytial virus (rsv) ( / ; . %), human parainfluenza virus (hpiv) types to ( / ; . %), human rhinovirus (hrv) ( / ; . %), human adenovirus (hav) ( / ; . %), and influenza virus (inf) ( / ; . %). streptococcus pneumoniae ( / ; . %) was the most frequently detected bacterium, followed by staphylococcus aureus ( / ; . %) and haemophilus influenzae type b ( / ; . %). chest x‐rays were performed at the discretion of the attending physician on ( . %) case patients. of these patients, ( . %) had abnormal radiological findings. a total of / ( . %) and / ( . %) children received antibiotic treatment prior to admission and during admission, respectively. conclusion: a high proportion of respiratory viruses was detected among children aged younger than years with febrile ari, raising concerns about excessive use of antibiotics in niger. acute respiratory infections (aris) are responsible for elevated childhood morbidity and mortality globally, , accounting for approximately four million deaths among children aged younger than years in and resulting in substantial burden of healthcare systems. , in developing countries, aris account for % of all deaths among children aged younger than years and . % of all disabilities and premature deaths. , therefore, data on the epidemiology and seasonality of ari are important to develop control and prevention strategies. in niger, the estimated mortality rate among children aged younger than years was per in (unicef child mortality estimate), and more than % of these deaths were associated with malaria, respiratory infections, and diarrhea. according to the niger health statistics yearbook, the estimated case fatality proportion associated with respiratory infections was . %. in the absence of laboratory diagnosis, it is difficult to clinically differentiate between ari-related causative pathogens, due to similarities of symptoms. , the main causative agents of ari are viruses and bacteria. [ ] [ ] [ ] in niger, there is scarcity of data on aris, although a routine influenza surveillance system exists. inf has been detected in % of samples from children aged younger than years hospitalized with severe acute respiratory illness (sari). this study aims to describe the viral and bacterial infections among children aged younger than years hospitalized with febrile ari at two national hospitals of niamey, the capital city of niger, and the reported clinical procedures. we conducted a prospective study among children aged younger than years hospitalized with febrile ari between january and december . niger has four distinct seasons as categorized by the national directorate of meteorology: the cold season (mid-december to mid-february), the dry season (mid-february to may), the rainy season (june to september), and the hot season (october to mid-december). this study was part of the "total niger infection respiratoire aiguë" (tonira) project, which was funded by the total corporate foundation in order to strengthen medical care of febrile ari among children aged younger than years. the study was conducted at the pediatric wards of two national tertiary hospitals situated in niamey, namely, the hôpital national de niamey (hnn) and the hôpital national lamordé (hnl). these two hospitals provide general health care to the population of niamey estimated at . million in , as well as to patients referred from across the country. laboratory detection of respiratory viruses and bacteria were conducted at the centre de recherche médicale et sanitaire (cermes), niamey, niger (the national reference laboratory for influenza). demographic and clinical data, as well as the use of antibiotics before and during admission, were collected using standard data collection forms. malaria testing was implemented on site using thick blood film microscopy. chest x-rays were performed at the discretion of the attending physician, and typical abnormal radiological findings assessed included pulmonary opacities, acute bronchiolitis, and enlargement of intercostal spaces. malnutrition was determined using the ratio of weight for height, and its level was classified as moderate or severe by the attending physician using national standards based on the technical guidelines for integrated disease surveillance and response in the african region (retrieved from https://www. afro.who.int/sites/default/files/ - /idsr-technical-guidelines_ final_ _ .pdf). febrile ari in conjunction with malaria, malnutrition, and diarrhea were assessed during admission using a standardized case investigation form. in-hospital outcome (ie., discharge, referral, or death) was recorded for all enrolled patients. a febrile ari case was defined as a hospitalized child aged younger than years with onset of fever c or higher and cough within days prior to admission and at least one of the following signs: inability to drink or breastfeed, lethargy, vomiting, convulsions, nasal flaring, chest indrawing, stridor in a calm child, or tachypnea. nasopharyngeal swabs were collected from all enrolled patients, placed in universal transport medium, stored at to c, and trans- amplification. the fluorescence was assessed at the amplification step. the positive and negative virus plasmid controls provided in the kit were included in all runs to monitor assay performance. viral and/or bacterial detection was reported as percentage positive, overall and within selected categories (eg, age groups and seasons). children aged younger than years were stratified into two age groups: infants (aged younger than year) and young children (aged - years), for comparability with other studies from africa. [ ] [ ] [ ] [ ] in addition, the majority of enrolled children were aged younger than year hindering our ability to categorize age in smaller age groups. stata version . (statacorp, college station, texas, usa) was used for the analysis. the protocol was approved by the national ethical consultative com- overall, / ( . %) specimens tested positive for at least one respiratory virus or bacterium (table ) overall, respiratory viruses were detected in / ( . %) speci- figure a ). overall, respiratory bacteria were detected in / ( . %) specimens. s pneumoniae ( / ; . %) was the most frequently detected bacterium, followed by s aureus ( / ; . %) and hib ( / ; . %) ( table ) . m pneumoniae and c pneumoniae were detected individually in less than % of specimens. of the specimens that tested positive for at least one respiratory bacteria, two or more respiratory bacteria were detected in / s pneumoniae and s aureus were mostly detected during the rainy season (june to september) ( table and figure b ). we report the detection of respiratory viruses and bacteria among children aged younger than years hospitalized with febrile ari in t a b l e (continued) , and asia (range %- %). [ ] [ ] [ ] the hpiv were also the most predominant viruses detected in children aged younger than years in a previous study conducted in niger. rsv is considered to be a major cause of ari in children aged younger than years, , [ ] [ ] [ ] and it was the most commonly detected virus also in this study ( / ; . %). among the cases in which hpiv and hcov were detected, hpiv type and hcov type oc were the predominant types, and this is consistent with previous studies. , in this study, infs were detected in . % of the cases, which is in agreement with findings from the national influenza surveillance our study presents some limitations. first, the study spanned over a period of only months, limiting our ability to fully assess the temporal circulation pattern of the investigated agents. due to the small sample size, we may also have been underpowered to detect small variations in the temporal distribution of the investigated agents. second, attribution of causality remains challenging due to the lack of controls in our study. the association of pathogen detection with illness could not be well assessed, although most of the viral and bacterial pathogens identified in this study have been described in previous case control studies as causative agents of ari. , in addition, we did not collect blood samples to assess whether the detection of common bacterial colonizers of the nasopharynx, such as s pneumoniae, were associated with invasive disease. third, we did not record the number of patients with febrile ari that refused enrollment, hindering our ability to estimate underenrollment. last, given that only patients with febrile ari were enrolled, this could have resulted in an underestimation of the rsv burden since a large proportion of rsv-related illness is not associated with fever. in this -year prospective study, both viral and bacterial pathogens were detected in high proportion among hospitalized children aged younger than years with febrile ari in niamey, niger. however, further investigations should be carried out to assess risk factors and association of pathogens with illness. although national guidelines for ari treatment recommend systematic empiric antibiotic therapy, our results suggest that antibiotic use might be unnecessary in most cases, given the predominance of viral infections as potential cause of febrile ari. furthermore, other predisposing diseases such as malaria, malnutrition, and diarrhea may be contributory factors to febrile ari exacerbation among hospitalized children. 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, usa. the authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. acute respiratory infection case definitions for young children: a systematic review of community-based epidemiologic studies in south asia 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sensitivity profile of streptococcus pneumoniae carried in healthy toddlers before pcv introduction in niamey response to conjugate haemophilus influenzae b vaccine among infants in niamey, niger etiology and factors associated with pneumonia in children under years of age in mali: a prospective case-control study performance of surveillance case definitions in detecting respiratory syncytial virus infection among young children hospitalized with severe respiratory illness-south africa molecular detection of respiratory pathogens among children aged younger than years hospitalized with febrile acute respiratory infections: a prospective hospital-based observational study in niamey we are thankful to the moh, cermes, and clinicians and nurses at the hnn and hnl for their contributions. this work was funded by the total corporate foundation through a partnership with pasteur institute of paris and cermes. the funder was not involved in the study design, collection, analysis, and interpretation of data, writing of the report, and decision to submit the report for publication. adamou lagare had full access to the data and takes responsibility for the integrity of the data and the accuracy of the data analysis. the lead author/manuscript guarantor (adamou lagare) affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. all authors declare that they have no commercial or other associations that may pose a conflict of interest. https://orcid.org/ - - - stefano tempia https://orcid.org/ - - - x key: cord- -y g yak authors: choi, eunjin; ha, kee-soo; song, dae jin; lee, jung hwa; lee, kwang chul title: clinical and laboratory profiles of hospitalized children with acute respiratory virus infection date: - - journal: korean j pediatr doi: . /kjp. . . . sha: doc_id: cord_uid: y g yak purpose: despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (aris) has proven difficult as the same viruses are often detected in asymptomatic children. methods: multiplex reverse transcription polymerase chain reaction assays were performed to detect common respiratory viruses in children under years of age who were hospitalized with ari between january and december . viral epidemiology and clinical profiles of single virus infections were evaluated. results: of , patients, viruses were identified in , ( . %), with the assay revealing a single virus in , cases ( . %). while major pathogens in single virus-positive cases differed according to age, human rhinovirus (hrv) was common in patients of all ages. respiratory syncytial virus (rsv), influenza virus (if), and human metapneumovirus (hmpv) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hrv and adenovirus (adv) were detected in every season. patients with aris caused by rsv and hrv were frequently afebrile and more commonly had wheezing compared with patients with other viral aris. neutrophil-dominant inflammation was observed in aris caused by if, adv, and hrv, whereas lymphocyte-dominant inflammation was observed with rsv a, parainfluenza virus, and hmpv. monocytosis was common with rsv and adv, whereas eosinophilia was observed with hrv. conclusion: in combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral aris and help us to establish more efficient therapeutic and preventive strategies. acute respiratory infections (aris) are a main cause of morbidity and mortality in children, with viruses being responsible for more than % of aris worldwide. , ) clinical manifestations of ari can hardly differentiate bacterial from viral etiologies, which can lead to the unnecessary use of antibiotics. recent development of the multiplex reverse transcription polymerase chain reaction (rt-pcr) assay makes noninvasive identification of respiratory pathogens possible. however, a more accurate diagnosis of causative ari pathogens does not decrease hospital admissions or antibiotic use in children with ari. ) since viruses are often detected in asymptomatic children, ) identification of a virus by rt-pcr does not always imply that it is the culprit for a current ari. because these viruses are sometimes asymptomatic carriers and sometimes pathogens, a diagnosis should not be based solely on viral identification in respiratory samples. moreover, new respiratory viruses have been increasingly recognized, but their clinical significance remains unclear. , ) thus, in addition to multiplex rt-pcr assays, korean j pediatr ; ( ) : - a better understanding of these viruses is required to improve clinical management. in this study, we assessed the viral epidemiology of ari in hospitalized children under age of years, and we characterized the virusspecific clinical and laboratory profiles as well as clinical outcomes. together with rt-pcr results, this data will help us understand the clinical course of viral ari and to establish more effective preventive and therapeutic strategies. this retrospective cohort study was approved by the institutional review board of korea university guro hospital (kugh). hospitalized patients under years of age with a discharge diagnosis of ari from january to december were enrolled. ari was diagnosed based on one of symptoms or signs; fever of more than . °c, cough, rhinorrhea, sore throat, tonsillar injection, wheezing, crackle, chest wall retraction. nasopharyngeal aspirates from all patients were obtained within hours of admission for multiplex rt-pcr assay to detect the following common respiratory viruses: influenza virus a and b (ifa, ifb), respiratory syncytial virus a and b (rsv a, rsv b), parainfluenza virus - (piv , piv , piv , piv ), human coronavirus e and oc (hcv- e, hcv-oc ), human rhinovirus (hrv), human enterovirus (hev), adenovirus (adv), human bocavirus (hbv), and human metapneumovirus (hmpv). only a single sample was taken from the patients during the admission. rt-pcr results were used to evaluate the incidence of respiratory viruses. laboratory parameters on the first day of admission, as well as overall clinical profiles including baseline characteristics, presenting symptoms and signs, treatments and clinical outcomes, were analyzed in patients with single-virus infections. any possible bacterial coinfections were excluded by sputum and blood culture and clinical course. mycoplasma coinfections were excluded by sputum pcr or by a serial increase of antibody titer or a high initial igm titer without history of recent respiratory disease. respiratory samples from children were collected by nasopharyngeal aspiration. the presence of each virus was determined using the seeplex rv rt-pcr assay (seegene, inc., seoul, korea). two sets of primers were designed based on conserved regions of genetic sequences for the respiratory viruses mentioned above. amplified pcr products were analyzed by electrophoresis on % agarose gel and were compared with the reference band size provided by the manufacturer. ) statistical analysis was performed using ibm spss statistics ver. . (ibm co., armonk, ny, usa). data were expressed as mean± standard deviation or as percentages, as appropriate. a multipleway analysis of variance was run to test differences between viral groups. then, each viral group was compared with the other viral groups using chi-square tests for categorical data and unpaired student t tests for continuous data. a p-value < . was considered statistically significant. from respiratory samples of a total of , patients, viruses were detected in , patients ( . %). after excluding patients with bacterial coinfections (mycoplasma in cases, other bacteria in cases, and mycoplasma with other bacteria in cases), , patients ( . %) were found to have viral aris. in those cases, the most common were hrv ( of , , . %), adv ( of , , . %), and rsv a ( of , , . %) (fig. viruses were detected more frequently in younger cases (≤ year old vs. > years old was . % vs. . %, p= . ). cases with multiple viruses tended to be male-dominant when compared to single-virus cases ( . % vs. . %, p= . ) ( table ) . a total of , single-virus cases were analyzed for their distribution at different ages (fig. ) . seventy-five percent of single-virus cases were identified in patients younger than years old. while rsv a, rsv b, hbv, and piv were major pathogens in patients younger than one year of age, ifa, ifb, hev, and adv were common in patients older than three years of age. hrv was a predominant single virus that was seen across all ages. although not as common as hrv, hmpv was also prevalent across all ages. during the observation period, many viruses showed seasonal patterns. ifa and ifb were most common from winter to spring, while hmpv was predominant in the spring, and piv and hev common from spring to summer. rsv a and b peaked during fall and winter biennially. although not common, hbv was prevalent during the spring and hcv was seen during fall and winter. hrv and adv did not show a discrete seasonal pattern (fig. ). although each virus prevailed throughout preferred seasons during the observation period, there was annual deviations. there were no differences in comorbidities such as premature birth, congenital heart disease and chronic lung disease among viral ari cases. rsv a, rsv b, and hmpv were more associated with lower ari compared with other viruses. croup was most commonly associated with piv and hcv ( . %, . %), while pneumonia was associated with rsv a, rsv b, and hmpv ( . %, . %, and . %). additionally, bronchiolitis was associated with rsv a and b ( . % and . %), while asthma exacerbations were associated with hrv ( . %, p< . ) only. most cases of ari presented with cough and rhinorrhea (data not shown). while fever was associated with many viruses, rsv a, rsv b, and hrv were not significantly associated with fever ( . %, . %, and . %, p< . ). adv and piv were associated with fever lasting more than days ( . %, headache was significantly associated with hev and adv ( . % and . %, p< . ). antibiotics were used more frequently in cases with adv and hmpv ( . % and . %, p< . ), and steroids were used more frequently in cases with hrv ( . %, p< . ). rsv was associated with greater nebulizer use, o supplementation and intensive care unit care, and it was also associated with a longer hospital stay ( table ). cases of adv, hev, and hrv had a greater tendency to show leukocytosis over , /l when compared to other viruses ( . %, . %, and . %, p< . ). although leukopenia less than , /l was observed more with ifa, ifb ( . %, . %, p< . ), an absolute neutrophil count less than , /ml was more associated with piv ( . %, p< . ). while neutrophil-dominant inflammation, represented by a neutrophil-to-lymphocyte ratio ≥ , was more associated with ifa, ifb, adv, hrv, and hev, lymphocyte-dominant inflammation with a neutrophil-to-lymphocyte ratio ≤ was more associated with rsv a, rsv b, piv, and hmpv. monocytosis greater than , /l was observed more with rsv a, rsv b, and adv ( . %, . %, and . %), while eosinophilia greater than /l was observed more with hrv than with other viruses ( . %, p< . ). although not common, thrombocytopenia less than , /l was observed more with hcv-oc ( . %, p= . ). ele vated levels of aspartate aminotransferase and alanine aminotransferase were associated more with piv and rsv a. c-reactive protein levels less than mg/l were associated with ifb, rsv a, rsv b, and piv ( . %, . %, . %, and . %, p< . ), and an erythrocyte sedimentation rate less than mm/hr were associated with ifa, rsv a, and piv ( . %, . %, and . %, p< . ). both c-reactive protein levels greater than mg/l and erythrocyte sedimentation rates greater than mm/hr were associated with adv (p< . ) ( table ). in this study, viruses were detected in . % of all ari cases, but when cases of bacterial coinfection were excluded, viruses were detected in . % of cases. bacterial infection may be erroneously estimated because we counted all possible cases, even if not confirmed. additionally, we did not routinely test for bacterial infection at presentation. nevertheless, viral detection rates in this study were comparable to those of other studies. , ) although hrv, adv, and rsv are the most prevalent viruses, rsv was more present as a single pathogen, whereas hrv and adv were more seen with multiple pathogens. rsv, hrv, and piv were the most prevalent single pathogens in patients younger than years old. after the age of three, the prevalence of rsv decreased while the prevalence of if and adv increased, making hrv, if, and adv the most prevalent after three years of age. the younger age group shows higher virus positivity compared with the older age group, which may reflect prolonged shedding of respiratory viruses in young children. ) cases with multiple detected viruses are more likely to be males, while the sex ratio in single-virus cases is concordant with that of cases examined. the incidence of infectious disease is also reported higher among males. , ) it is possible that after viral infection, viral clearance may be delayed in males, resulting in a greater chance for viruses to accumulate. the significance of multiple viral coinfections has been controversial. while it is unlikely to be associated with more severe illness, , , ) specific pathogen pairs such as rsv with influenza virus may be associated with increased severity. ) studies report strong causal attribution of rsv, if, and hmpv, less strong evidence for hrv and no attribution of adv, hbv and hcv, [ ] [ ] [ ] which is in agreement with the ratio of single to multiple detections of each virus in our study. quantitative viral analysis may help to distinguish active infection from viral shedding in cases with multiple viral coinfection. ) because the clinical significance of multiple viral infections remains controversial and hard to interpret, we compared single-virus cases to understand characteristics of each virus. seasonality of some respiratory viruses is an important clue for early recognition. in addition to the strong seasonal patterns observed with if, rsv, and hmpv, it is interesting that rsv a and b have an apparent biennial seasonality. while hrv and adv are prevalent throughout the year, seasonal patterns of viruses are discrete. each respiratory viruses differ in seasonal onset as well as activity, but this can slightly vary from year to year. knowing the seasonality of each virus will help to plan effective control strategies and to make epidemiological diagnosis. analysis of virus-specific clinical profiles showed that cases of rsv a, rsv b, hbv, piv, and hcv affect patients who are signifi cantly younger than those with ifa, ifb, adv, and hev aris. premature birth history, and presence of congenital heart disease and chronic lung disease are not associated with any specific virus. while fever is a common symptom of ari, cases of rsv and hrv are significantly afebrile. infants younger than months with rsv infection tend to be afebrile, which may be due to a lack of a pyrogenic cytokine response. , ) while patients with afebrile rsv cases are significantly younger than febrile rsv cases ( . months vs. . months, p= . for rsv a, . months vs. . months, p= . for rsv b), there is no age difference between afebrile and febrile hrv cases ( . months vs. . months, p= . ) in this study, suggesting that pyrogenic immune responses might be different depending on the virus. some viral respiratory infections are associated with asthma and asthma exacerbation. ) cases of rsv and hrv ari present more commonly with wheezing, but eosi nophilia and a diagnosis of asthma exacerbation are associated only with hrv infection in this study. a study performed in children with acute wheezing illness reports that rsv is the predominant virus in patients with no previous wheezing, whereas hrv is predominant in patients with a history of wheezing. ) whether rsv infection causes asthma is still debatable, but it is possible that lower ari with pathogens such as hrv and rsv could precipitate the development of asthma, especially in children with atopic features. ) the total leukocyte, neutrophil, lymphocyte and monocyte counts, and specific associations between blood cells, especially the neutrophil-to-lymphocyte ratio have been used as markers of inflammation and infection. , ) viral aris show leukocytosis rather than leukopenia and furthermore, this study shows that if, hrv, hev, and adv are associated with neutrophil-dominant inflammation whereas rsv, piv, and hmpv are associated with lymphocyte-dominant inflammation. of note, monocytosis is observed with rsv a, rsv b, and adv aris. other inflammatory markers such as erythrocyte sedimentation rate and c-reactive protein levels are not significantly increased in most viral aris except for adv infection. thus, adv ari present with neutrophildominant leukocytosis, a high erythrocyte sedimentation rate and a high c-reactive protein level, which mimics the response to bacterial infections. the decision to use antibiotics is determined by clinical and laboratory evaluation. in this study, antibiotics are used more often in cases of hmpv and adv, and they are also used more when infection in young infants who are potentially immune-incompetent, manifests as a lower ari. concerns of bacterial superinfection often cause us to continue the use of antibiotics, even after identification of a viral pathogen. application of a clinical rule without consideration of virus-specific clinical and laboratory characteristics may lead us to make a wrong decision. ) this study has some limitations. not all patients were examined for a bacterial etiology at presentation, and the use of antibiotics might affect bacterial detection. thus, it is possible that this study overestimates the number of viral aris. also, the rt-pcr assay used in this study did not quantify the viral load. it is still possible that even the single virus detected in a patient with a symptomatic ari might not be the causative pathogen. in conclusion, the epidemiology of predominant viral pathogens associated with aris among children differs seasonally as well as regionally. using routinely available hospital laboratory data will be a great addition to current virus surveillance and will help to identify the viruses circulating in the community and to predict their timing, trends and impact. ) in addition to viral identification, recognition of the virus-specific clinical and laboratory patterns presented in this study will expand our understanding of the epidemiology of viral ari and will help us to improve management and prevention of viral infections. respiratory virus infections aetiology of acute respiratory tract infections in hospitalised children in cyprus clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial respiratory pathogens in children with and without respiratory symptoms new respiratory viral infections the role of infections and coinfections with newly 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primary care with acute respiratory tract infection and cough: a prognostic cohort study a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from no potential conflict of interest relevant to this article was re ported. key: cord- -hs wfffs authors: lambert, stephen b; allen, kelly m; carter, robert c; nolan, terence m title: the cost of community-managed viral respiratory illnesses in a cohort of healthy preschool-aged children date: - - journal: respir res doi: . / - - - sha: doc_id: cord_uid: hs wfffs background: acute respiratory illnesses (aris) during childhood are often caused by respiratory viruses, result in significant morbidity, and have associated costs for families and society. despite their ubiquity, there is a lack of interdisciplinary epidemiologic and economic research that has collected primary impact data, particularly associated with indirect costs, from families during aris in children. methods: we conducted a -month cohort study in preschool children with impact diary recording and pcr testing of nose-throat swabs for viruses during an ari. we used applied values to estimate a virus-specific mean cost of aris. results: impact diaries were available for % ( / ) of community-managed illnesses between january and january . the mean cost of aris was au$ ( % confidence interval $ to $ ). influenza illnesses had a mean cost of $ , compared with rsv, $ , the next most expensive single-virus illness, although confidence intervals overlapped. mean carer time away from usual activity per day was two hours for influenza aris and between and minutes for all other ari categories. conclusion: from a societal perspective, community-managed aris are a significant cost burden on families and society. the point estimate of the mean cost of community-managed influenza illnesses in healthy preschool aged children is three times greater than those illnesses caused by rsv and other respiratory viruses. indirect costs, particularly carer time away from usual activity, are the key cost drivers for aris in children. the use of parent-collected specimens may enhance ari surveillance and reduce any potential hawthorne effect caused by compliance with study procedures. these findings reinforce the need for further integrated epidemiologic and economic research of aris in children to allow for comprehensive cost-effectiveness assessments of preventive and therapeutic options. respiratory virus infections are a major cause of morbidity and healthcare usage in children, resulting in substantial costs for families and society [ ] [ ] [ ] [ ] [ ] . given their ubiquity, there has been surprisingly little research examining the costs associated with childhood respiratory infections that has involved collecting primary data from families. even for influenza, the most studied of all respiratory viruses, cost-of-illness and vaccine cost-effectiveness evaluations in children have tended to rely on assumptions or use retrospectively collected estimates, often from surveys, for resource utilisation, such as carer time away from work in seeking healthcare or caring for an ill child [ ] [ ] [ ] [ ] . there are three pieces of evidence required by those developing health policy in assessing whether to recommend or implement a publicly-funded vaccination program, or any intervention, against respiratory viruses: epidemiology of the targeted illness, the efficacy of the intervention, and the cost-effectiveness of the intervention [ ] . all interventions to prevent or treat infections will be associated with a cost of implementation, but cost-effectiveness is determined not only by the cost of the intervention, but also by costs arising from the illness. getting these data for respiratory viruses, particularly information on indirect costs incurred by families, requires a conjunction of epidemiologic and economic research [ ] . the prospect of new and improved influenza vaccines [ ] , the hope of new vaccines against other respiratory viruses [ ] , development of novel therapeutic possibilities [ ] , and the possible use of nonpharmaceutical interventions to contain virus transmission [ ] [ ] [ ] [ ] all underline the need to more critically weigh the costs and benefits of prevention and treatment for common respiratory tract viruses. we present here findings from a community-based cohort study using parent-collected specimens for etiologic assignment and diary recording of impact data. these data have been used to calculate virus-specific costs of illness from a societal perspective, including often neglected indirect costs. the study cohort and acute respiratory illness surveillance details of recruitment, composition, and maintenance of the dynamic study cohort have been published elsewhere [ ] . ethics approval for the study was given by the royal children's hospital ethics in human research committee, melbourne, and written informed consent was obtained from parents before participation. this dynamic cohort consisted of one healthy child less than five years of age at time of recruitment from each study family. children involved in this study were recruited from a number of sources. in victoria, australia, maternal and child health nurses (mchns) provide support to families during the early childhood years, particularly on issues to do with general health and vaccination. based on a model used by our group for community vaccine studies [ ] , mchns from local councils assisted with recruitment by providing study information to parents of eligible children. advertising material for the study was placed in child care and playgroup centers and, because of proximity, we also used bulletin boards and staff e-mail lists at the royal children's and the royal women's hospitals in melbourne. details about the study child and household demographics were collected at an enrolment home visit, including annual gross household income collected in / australian dollar values (aud$). income was separated into four bands, roughly dividing the study households into quartiles: band , less than $ , ( % of study households); band , $ , to $ , ( %); band , $ , to $ , ( %); and band , $ , or greater ( %). the approximate proportions for australian households during the same period were: band , %, band , %, band , %, and band , % [ ] . parents undertook daily respiratory symptom surveillance of the study child using a diary card and collected a combined nose-throat swab (nts) and completed a summary impact diary when the child had an acute respiratory illness (ari). for this study we used a sensitive ari definition that had previously been used in an influenza vaccine efficacy study [ ] and our pilot study [ , ] . symptoms were classified as category a (fever, wheezing, shortness of breath, pulmonary congestion or moist cough, pneumonia, or ear infection) and category b (runny nose or nasal congestion, sore throat, cough, muscle aches, chills, headache, irritability, decreased activity or lethargy or weakness, or vomiting). an ari of interest required one category a or two category b symptoms on a single day [ ] . other than pneumonia, which we asked parents to record only if supported by a health care professional's diagnosis, no illness or symptom details, including a report of otitis media, were validated by study staff or health care professionals. a new ari could not commence unless there were three symptom free days since the end of the previous ari. this meant an ari could contain no more than two consecutive symptom-free days. study families were asked to continue normal healthcare seeking behaviour and treatments, and were not alerted about the start of the influenza season or asked to alter surveillance during the winter season. pre-stamped envelopes were provided and families were asked to return all completed study documents (daily symptom diary, impact diaries) at the end of each month. ari duration was calculated using symptom diary data and aris were classified by study staff as being simple (no fever or otitis media recorded), or occurring with fever, otitis media, or with both fever and otitis media [ ] [ ] [ ] . the nts was couriered to the victorian infectious diseases reference laboratory (vidrl) where it was tested for a number of common respiratory viruses using a polymerase chain reaction (pcr) method for adenoviruses and reverse transcription (rt) pcr for rna viruses: influenza a virus, influenza b virus, respiratory syncytial virus (rsv), parainfluenza viruses i, ii, and iii (pivs), and picornaviruses [ ] . a letter outlining these test results was sent to parents when these details became available. at completion of the study all specimens were transported to the queensland paediatric infectious diseases (qpid) laboratory where they were tested for human metapneumovirus (hmpv) and human coronavirus nl (hcov-nl ) using rt-pcr [ ] . a summary impact diary was used to collect details of resources used during the study child's ari and was based on an impact diary used in a pilot study [ , ] , with some simplification. the units of resource use requested were: ▪ health care visits: number and timing of primary care (general practice) visits, hospital presentations and admissions, and visits to other providers (such as naturopaths, homeopaths); ▪ use of prescribed antibiotics; ▪ laboratory tests performed to investigate the illness; ▪ carer time consumed during the illness seeking health care; and ▪ excess carer time during the illness spent caring for the ill child. we did not collect information about some items that were shown not to be major cost drivers in the pilot study: non-antibiotic prescription medication, over-the-counter and other medication, paid childcare for other children whilst normal carers were spending time caring for the ill study subject, and travel costs seeking health care. the average total cost for these items in the pilot study [ ] was aud$ per ari. time values were captured in hours and minutes. parents were not given instructions about when or how frequently they should capture time data during an ari. for both carer time spent seeking healthcare and excess time spent caring for an ill child, time was recorded as a total value for the ari in two categories: time away from work and time away from usual, non-work activities. all costs were incurred over a day period between january and january . costs are reported in this manuscript using australian dollar values, with taken as the reference year for reporting unit prices. the mean exchange rates for major currencies during the study were: united kingdom (uk) pound £ = aud$ . , euro € = aud$ . , and united states (us) $ = aud$ . [ ] . discounting costs for time preference is not routinely considered for periods of time less than months, and as this study period barely exceeds this time frame, no costs have been discounted. details of the source and value for all costs are provided ( table ) . applied costs were retrieved, where possible, from published sources, and where no standard published cost was available we used costs derived from the pilot study. resource costs were allocated as being borne by either the 'patient and family' sector, the 'healthcare' sector, or the 'employer' of absent staff. the proportions of time away from work seeking healthcare or time away from work caring for an ill child that were incurred by either the patient and family sector or met by an employer were not collected, and these values have been derived from the same proportions in the pilot study, based on illnesses (table ) [ ] . we applied a sex-weighted hourly wage rate derived from the australian bureau of statistics average weekly full-time adult total earnings for all reported times [ ] . we calculated mean costs (total and by categories) with % confidence intervals ( % ci) and median costs with interquartile ranges for aris in study children. data were analysed using stata . for windows (statacorp, texas, usa). there were children, one from each study family, progressively enrolled in the study and we identified aris in , child-days of follow-up [ ] . of these, aris ( %) had at least one specimen and an impact diary available, ( %) had an impact diary returned but no specimen, ( %) had at least one specimen returned but no impact diary available, and ( %) had neither a specimen nor impact diary returned. children aged between one and two-years of age contributed the most person-time to the study ( % of all child-days) and had the highest acute respiratory illness (ari) incidence rate ( . aris per child-month). contribution by males and females was equivalent, and children who attended some form of out-of-home care were responsible for % of all person-time [ ] . the illnesses with a diary returned that did not involve a hospital admission had a total cost of $ , (table ) , and mean cost of $ ( % ci $ to $ ). as our particular interest is in the cost of communitymanaged aris, that is, those illnesses that do not require the mean and median costs by virus identification, including co-identification and specimen availability, are provided ( table ). the differences between the mean values and the median values demonstrate the right-skewed nature of these data, similar to other health-related costs [ ] . whilst confidence intervals overlap, the point estimate of the mean cost of an influenza a ari, $ , is three times higher than the next most expensive single virus ari: rsv $ . of the aris where more than one virus was identified, influenza a virus was present in four: two illnesses with co-identification with a picornavirus alone, one illness with hcov-nl alone, and one illness with a picornavirus and piv. these four illnesses had a mean cost of $ . there were no illnesses where influenza b virus was identified. three children had received influenza vaccine in the year prior to the study and none had an influenza-positive ari. as the difference in mean cost between the most expensive (rsv: $ ) and least expensive (hmpv: $ ) noninfluenza single virus ari falls within a comparatively narrow band ($ ) we collapsed these data into a single category for further comparisons ( there was little difference in the mean duration of influenza a illnesses and other single virus illnesses, but coidentifications were . and . days longer than each of these respectively ( table ). the mean delay between illness onset and a result letter being sent was shortest in influenza illnesses at . days (table ). in this study we present the costs associated with community-managed respiratory viral infections in healthy preschool aged children. these costs are based on the direct recording of impact information captured by parents when the study child was unwell. the study has a unique combination of features including a sensitive definition for ari, parent-collected specimens, laboratory testing for respiratory viruses using sensitive molecular methods, and, based on findings from our pilot study, comprehen- sive collection of costs, including the previously neglected indirect cost, time away from a usual, non-work activity. we found, from a societal perspective, the point estimate for the mean cost of all aris ($ ; % ci $ to $ ) was not dissimilar to the mean value we calculated from the pilot study ($ ; % ci $ to $ ) [ ] using the same ari definition and a slightly modified impact diary. the use of pcr testing for diagnosis on collected specimens allowed us to assign impact and costs to specific viral agents. for all but influenza a illnesses, the cost of community-managed aris in healthy preschool-aged children fell within a relatively narrow $ range. despite overlapping confidence intervals, the finding of most note in this study was the dramatically higher point estimate of the mean cost of influenza a aris, being three times higher than illnesses caused by rsv and the other common respiratory viral infections of childhood. the presence of fever and/or otitis media generally increased the mean cost of illness; but despite having a high prevalence of fever, a longer mean duration, and higher primary care usage [ ] , adenoviral infections, for example, did not have the cost burden of influenza infections. this highlights the pivotal contribution of excess carer time away from usual non-work activity to total costs, making it the key cost driver for all aris in children and differentially amplifying the total costs of influenza illnesses. whilst the confidence intervals for mean cost of influenza a aris and other single virus aris overlap, due to the relatively small number of influenza illnesses available for costing, we believe it is unlikely that chance could account for such an extreme difference. the availability of preventive vaccines and specific therapeutic options makes influenza the most studied of respiratory viruses in all age groups; no other virus is more predictably disruptive year-on-year than annual interpandemic influenza [ ] [ ] [ ] [ ] . studies conducted in the second half of last century [ ] [ ] [ ] [ ] and recent observation [ , ] and intervention [ ] [ ] [ ] ideally further studies in other countries should be conducted to allow for an examination of how impact and cost data vary with the nature of the healthcare system, local virus epidemiology, and other societal factors, including household structure. despite lower mean costs than influenza illnesses and the lack of population-based prevention options, the importance of working towards the prevention of other respira-tory viral infections is obvious. picornavirus aris, though typically milder and more difficult to be certain of a causal association with illness [ , ] , were associated with the highest overall costs of any viral group totalling over $ , or one-third of all costs, for the -month study period. in the absence of specific vaccines and therapies for other viruses, the application of nonpharmaceutical interventions at a population level, such as improved hand and respiratory hygiene, may have an important place in reducing illness due to respiratory viruses [ ] . our findings reinforce the importance of virus testing in such studies to accurately estimate epidemiology and costs [ ] . these data add to accumulating evidence that laboratory confirmation of influenza, in particular, is required, rather than less specific influenza-like illness (ili) definitions or hospital coding. other recent studies have found laboratory-confirmed influenza hospitalizations were two to four times more costly [ ] [ ] [ ] than shown in previous studies using coding-based estimates [ , [ ] [ ] [ ] . when ili definitions or coding are used, rather than laboratory confirmation, a lack of specificity results in influenza illnesses being mixed with other agents, thereby considerably diluting cost differences [ , ] . a direct comparison of parent-collected nts specimens with collection of a more invasive specimen, such as a nasopharyngeal aspirate, by a healthcare worker at the time of an ari was beyond the scope of this study. any reduction in sensitivity caused by the type of specimen used is likely to minor: our finding that % of all specimens collected from children in this study were positive for at least one virus is within the range of values from recent home visit studies which also used pcr for diagnosis and nasopharyngeal aspirates ( %) [ ] or nasal lavages ( %) [ ] . there are clearly some issues about the cost of illnesses caused by respiratory viruses in children unresolved by our study, and some issues that need to be considered before interpretation. despite being a relatively large cohort the number of illnesses on which to make costing estimates for some virus types is quite small. further community-based estimates are required to not only confirm our findings but to improve precision around point estimates. compared with the australian population, households with lower incomes were under-represented in our study sample, and, despite overlapping confidence intervals around income band point estimates of mean costs, this may have lead to an overestimation of total costs. however, this may be balanced somewhat by the over-representation of households from the top income band which had a relatively lower mean ari cost ($ ). this pattern of household income distribution was similar to that found in the pilot study [ ] . for this study we sought to make our study sample more representative of the general community by focusing our recruitment efforts in local council areas with a higher proportion of lower income households. we have no empiric data available that would allow us to quantify the effect of any potential bias resulting from this skewed sample. other recent burden studies do not report similar household level income data to allow for comparison [ ] [ ] [ ] . it may be the case that lower income households are under-represented as they do not have the spare capacity required, in time or other resources, to allow for study involvement. we received impact diaries for just over % of all aris identified by daily symptom surveillance. aris without a diary were more likely to be shorter and without fever or otitis media; any information bias resulting from this would likely be in the direction of inflating mean illness costs. our study only captured information from a single season with higher than normal influenza activity with h n influenza a (drifted strain subtype a/fujian/ / -like) being the predominant circulating type [ ] . variations in incidence and severity year-by-year for all respiratory viruses make it difficult to directly translate our findings to other years. we believe documenting all time spent on caring for an ill child is important, even when taken away from a usual activity. we appreciate that applying standard wage rates to leisure time is not a straightforward issue in economics. this approach values carer leisure time and non-paid working time in a similar way to a worker's time consistent with neoclassical theories of labour economics [ ] . in attaching value to leisure time and using sex-weighted wage rates, we have made our assumptions explicit, and provided sufficient detail (table ) so that others can adjust unit prices using different approaches. previous burden data [ ] have been used to assess the cost-effectiveness (c/e) of using influenza vaccine in children [ ] . if our cost values, incorporating these indirect costs, were used in the numerator of c/e calculations, there is a distinct possibility of double counting [ ] . double counting is likely where the denominator is a utility measure that incorporates a quality assessment (such as the quality adjusted life year or qaly), and most economists would see leisure time as a logical component of the qaly. there is also debate [ ] about the inclusion, measurement, and valuation of lost working time in economic evaluations, with the debate centring on whether in practice qaly instruments capture income effects related to absenteeism. for all illnesses where a specimen was tested, parents received a result letter by mail. the delay between illness onset and posting the letter was shortest for influenza illnesses, but for most illnesses parents would have been aware of the result before illness end. pandemic influenza was not being widely discussed in australia during , but interpandemic influenza does receive media coverage annually encouraging vaccine uptake, and this may have caused parents to overestimate key parameters associated with their child's influenza-positive illness. however, if such a bias was in operation it might also be expected that time values for illnesses where no virus was identified may be relatively understated when compared to aris with one or more viruses present. we did not find such a phenomenon; aris with no virus identified had a higher mean cost than those with a single virus present, and for the key cost driver of excess carer time away from a usual activity, no cause illnesses had higher values than both single and multiple virus aris. despite the impact of respiratory viral infections in children there are relatively few burden comparisons available that collect primary data from ill children. an italian study examining the impact of hmpv, rsv, and influenza in children less than -years of age presenting to an emergency department found hmpv illnesses to be significantly more burdensome than rsv, having a similar impact to influenza [ ] . in our study hmpv was the least expensive single-virus illness. this finding may be due to the different nature of illnesses that result in hospital presentation or hospital admission, compared with community managed illness. of the aris in this study only . % (n = ) prompted hospital presentation, with less than % (n = ) requiring admission. an excellent community-based finnish study describing the burden of influenza in children -years of age or younger over two seasons, with child-seasons of data, also contrasts this imbalance between community-managed and hospitalized cases of influenza, with only three emergency department referrals and one hospital admission in children less than three years of age with influenza [ ] . this study differed from ours in that whilst it used laboratory confirmation, it did not employ more sensitive molecular diagnostics [ ] , families were required to visit the study clinic when the study child had fever or signs of respiratory infection, indirect costs did not include nonwork time away from a usual activity, and the study did not provide a comparison with other viral acute respiratory illnesses [ ] . the findings from the finnish study reinforce the need to follow children for aris over more than one season, with different rates of influenza infection from year-to-year in each age group. these differences extended to changes in likelihood of infection between age groups: for example, the rate of laboratory-confirmed influenza increased by one-third from season one ( ) ( ) to season two ( ) ( ) for children less than three years of age, but the rates for three to six year olds and seven to year olds fell % and %, respectively. a german study, recruiting children less than three years of age with lower respiratory tract infection (lrti) through office and hospital-based paediatricians, collected cost of illness from a societal perspective, including loss of work days by caregivers [ ] . this study showed that non-hospitalized cases of influenza lrti had twice the cost of piv lrti and were one-third more costly than rsv lrti, with this difference made up entirely by indirect costs [ ] . whilst methods vary, previous cost effectiveness studies of influenza vaccine in children are characterised by two findings: first, that cost-effectiveness is unsurprisingly enhanced by taking a societal perspective through the inclusion of indirect costs [ , , , , , ] . our findings reinforce the importance of indirect costs [ ] , and highlight a previously inadequately measured layer of burden -carer time away from a usual, non-work activity. second, the potential cost-effectiveness of implementing a vaccination program is improved by flexible or non-individual based delivery programs [ , ] . vaccine delivered through pharmacies for a small service fee -improving access and negating the time and costs associated with a primary care visit -or large school-based programs, are likely to be acceptable to parents and providers. it is likely that the cost benefits of preventing influenza in children would extend beyond the targeted age-group [ ] , similar to the indirect effects in older age groups seen following the introduction of childhood conjugate pneumococcal vaccination in the us [ ] . our study reinforces the costly impact of all respiratory viruses, but particularly interpandemic influenza, on children, their families, and society. efforts to further explore the costs associated with community-managed illness over a number of seasons for all respiratory infections are needed. similar to recent hospital-based findings, using laboratory-confirmation to specifically identify influenza appears to increase the cost of illness many fold; a finding that may make population-based vaccination programs a more cost-effective proposition. we believe the use of parent-collected specimens may have important effects in reducing bias in both the epidemiologic and impact data collected. not requiring parents to either present with their ill child to a health clinic or host a home visit by study staff may result in enhanced ari surveillance, but more importantly, allows for the reporting of impact data uncontaminated by compliance with study procedures, thereby reducing any impact a hawthorne effect may have. further studies that collect primary, integrated epidemiologic and economic data, particularly indirect costs, directly from families about community-managed aris in children, are required. such data would allow for a more informed exploration of the cost-effectiveness of vaccine programs and other interventions designed to reduce the morbidity associated with aris in children. the economic burden of non-influenza-related viral respiratory tract infection in the united states socioeconomic impact of influenza on healthy children and their families burden of influenza in healthy children and their households clinical and socio-economic impact of influenza and respiratory syncytial virus infection on healthy children and their households clinical and economic impact of influenza vaccination on healthy children aged - years 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schoolchildren infection with influenza a/victoria virus in houston families influenzavirus infections in seattle families, - . i. study design, methods and the occurrence of infections by time and age influenza and the rates of hospitalization for respiratory disease among infants and young children the effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children effectiveness of influenza vaccination of day care children in reducing influenza-related morbidity among household contacts effectiveness of influenza vaccination of children with recurrent respiratory tract infections in reducing respiratory-related morbidity within the households the effect of mass influenza immunization in children on the morbidity of the unvaccinated elderly human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms predominance of rhinovirus in the nose of symptomatic and asymptomatic infants cost of influenza hospitalization at a tertiary care children's hospital and its impact on the cost-benefit analysis of the recommendation for universal influenza immunization in children age to months epidemiology, complications, and cost of hospitalization in children with laboratory-confirmed influenza infection direct medical cost of influenza-related hospitalizations in children the economic impact of pandemic influenza in the united states: priorities for intervention cost-effectiveness analysis of an intranasal influenza vaccine for the prevention of influenza in healthy children an economic analysis of annual influenza vaccination of children influence of clinical case definitions with differing levels of sensitivity and specificity on estimates of the relative and absolute health benefits of influenza vaccination among healthy working adults and implications for economic analyses icd- codes for identifying influenza hospitalizations in children role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study frequency of detection of picornaviruses and seven other respiratory pathogens in infants burden of influenza in children in the community impact of human metapneumovirus in childhood: comparison with respiratory syncytial virus and influenza viruses economic impact of community-acquired and nosocomial lower respiratory tract infections in young children in germany higher than normal seasonal influenza activity in victoria the role and estimation of productivity costs in economic evaluation costeffectiveness of influenza vaccination of healthy children avoiding double-counting in pharmacoeconomic studies diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza the efficacy, effectiveness and cost-effectiveness of inactivated influenza virus vaccines herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (caiv-t) in children control and prevention: direct and indirect effects of routine vaccination of children with -valent pneumococcal conjugate vaccine on incidence of invasive pneumococcal disease -united states medicare statistics: average patient contribution per service medicare benefits schedule book operating from november medicare benefits schedule book operating from november content/health-pbsgeneral-pubs-manual-appendix .htm] . national hospital cost data collection national hospital cost data collection the work was supported by project grants from the victorian department of human services, the murdoch children's research institute, and the university of melbourne. stephen lambert was the recipient of a national health and medical research council public health postgraduate scholarship. we thank all children and families who volunteered to participate in the study. this study would not have been possible without the generous terence nolan and stephen lambert have, in the past five years, received research grants for epidemiological and vaccine related research from csl limited, medimmune, gsk biologicals, wyeth, and merck. kelly allen and robert carter have no competing interests to declare. all authors were involved in the study design and approach and sbl and tmn developed the original protocol. kma and sbl were responsible for the day-to-day conduct of the study. sbl performed the analysis and drafted the article. all authors contributed to and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge http://respiratory-research.com/content/ / / key: cord- -q kw n o authors: jean, sim xiang ying; conceicao, edwin philip; wee, liang en; aung, may kyawt; wei, sylvia seow yi; yang, raymond teo chee; qing, goh jia; ting, dennis yeo wu; jyhhan, kuo benjamin; lim, john wah; gan, wee hoe; ling, moi lin; venkatachalam, indumathi title: utilizing the electronic health records to create a syndromic staff surveillance system during the covid- outbreak date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: q kw n o objectives since december , covid- has caused a worldwide pandemic and singapore has seen escalating cases with community spread. aggressive contact tracing and identification of suspects has helped to identify local community clusters, surveillance being the key to early intervention. healthcare workers have contracted covid- infection both at the workplace and community. we aimed to create a prototype staff surveillance system for the detection of acute respiratory infection (ari) clusters amongst our healthcare workers (hcws) and describe its effectiveness. methods/results a prototypical surveillance system was built on existing electronic health record infrastructure. over a -week period, we investigated ari clusters amongst departments. one of the ari clusters was later determined to be related to covid- infection. we demonstrate the feasibility of syndromic surveillance to detect ari clusters during the covid- outbreak. conclusion the use of syndromic surveillance to detect ari clusters amongst hcws in the covid- pandemic may enable early case detection and prevent onward transmission. it could be an important tool in infection prevention within healthcare institutions. late in december , reports of an unknown respiratory virus arose from a seafood market in wuhan, china. since then, sars-cov- , the causative agent of covid- has sparked a global pandemic. ( ) healthcare workers (hcws) in singapore have not been exempt from infection, with exposure arising from the community and the work place. ( , ) there have been various methods described for the surveillance of our hcw groups including regular intermittent pcr testing versus active symptom monitoring. ( ) within local institutions, use of real time location tracking devices and institutional temperature recording systems have been utilized as a method of identifying at risk individuals. ( , ) surveillance is one of the key pillars of infection prevention, enabling early detection and institution of downstream practices that allow reduction in pathogen spread and containment of infection. syndromic surveillance may allow for an even earlier mobilization of response before the causative pathogen is identified and enhance disease containment. increasingly so, institutions are moving away from traditional laborious methods of healthcare surveillance. utilizing the electronic health records, we have created a prototypic surveillance system in the detection of acute respiratory infection (ari) clusters amongst staff and aim to describe its effectiveness in this study. our institution is a tertiary hospital in singapore with approximately patient beds and employs around , staff. we implemented the staff surveillance system prototype on rd february and analyzed data for the first -week period till nd may . the aim of this descriptive analytic study is to describe the effectiveness of a prototypic staff syndromic surveillance system in identifying acute respiratory infection (ari) clusters amongst the staff population in the hospital. the main outlets for staff to report sick at our institution are the department of emergency medicine (dem) and the staff clinic (sc). laboratory tests were ordered by physicians in dem or sc at their discretion. we combined data from the electronic health records (ehr) and human resource (hr) data sources to build the syndromic staff surveillance system. using the ehr we identified staff at risk of an ari using the snomed diagnosis codes present on clinical documents. the snomed codes included were, 'upper respiratory tract infection', 'lower respiratory tract infection', 'bronchitis', 'pneumonia', 'tonsillitis' and 'pharyngitis'. given the concerns for an atypical presentation of covid- infection ( ) at a time when disease had not been sufficiently characterized, 'undifferentiated fever' and 'gastroenteritis' was also included in the analysis. hr databases were utilized to obtain the department of at-risk staff mapped by their associated cost centers. the laboratory database contains respiratory virus multiplex panel that tests for rhinovirus, adenovirus, parainfluenza sub-types , , and , influenza subtypes a and b, human coronavirus subtypes e, nl and oc , metapneumovirus, respiratory syncytial virus subtypes a and b, pcr swabs and covid- pcr swabs. the system is be able to differentiate inpatient from outpatient orders based on accounting codes. the institution uses a nation-wide temperature surveillance for outbreak monitoring called s . this requires all employed staff to input their temperatures within the system twice daily. the s system is accessible via the hospital's intranet and to enable easy access, an internet-based form via the formsg platform was created ( ) . staff with temperatures of more than . degrees celsius were deemed as at-risk individuals for purposes of this surveillance system. as staff interaction is not limited to persons within their own department, in order to group them according to their risk association, these at-risk staff presenting to dem or sc were prompted to submit a self-administered questionnaire via formsg separate from the s fever declaration system (available at: https://form.gov.sg/#!/ e b b f e ). to improve uptake of this questionnaire, it was made accessible through the above link as well as a qr code that was displayed prominently in sc and dem. the fields collected included date of symptom onset, type of symptoms, whether their work involved direct patient contact or exposure to a clinical area in the weeks prior to symptom onset, travel history weeks prior to symptom onset, close contact with other healthcare workers within the institutional campus who had been unwell and close contact with any other persons outside the institution who had been unwell. this provided further granularity to their work locations prior to onset of symptoms as well as work and social contact with persons known to be ill. a network analysis is then applied to information from the formsg to obtain the general distribution of staff with specific symptoms, and their social circles. this network analysis might be able to identify super-spreaders, in particular allied and ancillary health workers and physicians who may not provide location-centric care. for the duration of the study, all at-risk staff, were mapped in time and location by their various departments and locations. the data was aggregated and presented in a heat map for further visualization. baseline data was obtained for the first weeks, following which data was reviewed on a daily basis (monday to friday) and a twice weekly aggregated output was used to set alert thresholds for the emergence of any clusters. a cluster was defined as a signal of > % the previous baseline or an upward trend for weeks of aggregated results of > standard deviation. the workflow is summarized in figure . the longest lag time between detection of a threshold of a defined cluster till study team detection was three days. upon identification of a cluster, the departmental heads of those clusters were contacted for further information such as rostered location of work, communal eating areas and other symptomatic staff. staff who were symptomatic at the time of review and who had not been assessed at the sc or dem were advised to go to staff clinic where they were seen and swabbed for sars-cov- . the results of these swabs were tracked by the epidemiology team. during this -week period, a total of unique at-risk staff records were identified, ( . %) staff with at-risk symptoms who presented to dem or sc and staff with fever as self-recorded in the s system. of those with at-risk symptoms, ( . %) presented to sc and (table ) . for the network analysis, no outliers were reported because of the restrictions placed to minimize cross-covering of duties. a total of ari clusters in departments were identified amongst staff and later investigated. these involved the following departments in the hospital: radiology, ambulatory endoscopy centre, outpatient pharmacy, medical social services, central operating theatre, emergency department and physiotherapy. amongst these identified departments, ( . %) staff attended sc or dem, were swabbed for sars-cov- , of whom were positive and ( . %) were negative. one staff from the ambulatory endoscopy centre was identified with rhinovirus infection on respiratory virus pcr testing (figure a and figure b ). of the staff with covid- , two were within the medical social worker cluster. the first patient presented on th of march , and the large contact tracing and case finding exercise that followed, lead to a surge in staff from this department presenting to sc. this was picked up by the newly initiated surveillance system as a potential cluster. further investigation of the cluster, also revealed a workplace transmission of covid- to a colleague within the same department. ( ) the remaining covid- positive staff were attributed to community acquisition. four of the cases were identified through mass screening of asymptomatic staff as part an essential worker screening exercise. they were not identified by the staff surveillance system as they did not present to dem or sc but were identified after laboratory confirmation of covid- . the concept of innovative electronic surveillance systems is not novel, clinicians and administrators have long wished to improve upon more traditional methods of gathering and analyzing data. the push in our institution to a fully electronic system has allowed an ehr driven ari cluster surveillance system to develop. these have been utilized within emergency departments and hospitals to act as early warning systems ahead of traditional laboratory surveillance to help reduce the time lag between detection and diagnosis. ( ) others have evaluated the feasibility of syndromic surveillance in detecting lower respiratory tract infections and were able to demonstrate detection of legionnaires' disease clusters in a timely fashion. ( ) given the time pressure to create a staff surveillance system during the covid- outbreak, our system draws from the existing infrastructure of the ehr and building upon various existing data sources. as this staff surveillance system was created early in the covid- pandemic when we did not have baseline data to set thresholds, we set arbitrary thresholds based on the prior week results. whilst we managed to demonstrate feasibility, the thresholds will be revised based on cumulative data in the next phase of this study. this initial feasibility study shows that the use of a syndromic surveillance system has the ability to identify ari clusters amongst staff populations that would initiate downstream investigation and active screening. the ari cluster surveillance system managed to identify an ari cluster in the medical social worker group. active contact tracing efforts within the department after the detection of a positive case resulted in heightened alertness of staff towards mild symptoms that may have otherwise gone unnoticed and resulted in increased testing and an increase in attendances to sc and dem by the msw cohort. this was subsequently picked up on our staff surveillance system. it is important to emphasize that having a robust staff surveillance system, does not eliminate the need for good infection prevention practices, active/passive screening and a thriving staff culture aimed at safety and quality. the staff surveillance system requires the presentation of staff to our institutional health services such as sc and dem, those who report sick outside of institution were not captured by this surveillance system. during the covid- pandemic, our institution mandated that all staff presented to the institution sc or dem to report sick, only ancillary staff who are not under direct employ were exempt from this rule. as they were a specialized group of staff, specialized measures were undertaken including mass screening. this mandate coupled with strong action taken against presenteeism and data access provided by management aided in the robustness of the system resulting in a near complete data capture. this situation however is unique to the covid- pandemic and new thresholds will need to be established as the institutional policies change according to the state of the ongoing pandemic. asymptomatic infections will also not be picked up by this staff syndromic surveillance system. although a large proportion of staff were found to work in inpatient locations based on the formsg survey, no ari clusters were found in staff in inpatient locations. this may be in part attributed to infection prevention measures that were instituted including universal masking for staff as well as use of appropriate ppe and timely patient isolation in inpatient locations. whilst not performed for staff, we also managed to demonstrate a reduction in health-care associated respiratory viral infections during a similar period in the covid- pandemic. ( ) this may have further reduced the risk of transmission of ari illnesses from patients to staff and indirectly result in a reduction in ari clusters amongst staff in inpatient locations. in one of the investigated clusters, a staff was found to be positive for rhinovirus suggesting a possible etiology for the ari cluster other than covid- . however, cost concerns resulted in lack of widespread testing for other respiratory pathogens, thus limiting the discovery of other respiratory viral clusters other than covid- . furthermore, respiratory virus testing resulting in identification of another pathogen may in part help to relieve the on-ground anxiety of staff. due to initial studies suggesting that a gastroenteritis illness may be an atypical presentation of covid- , to increase data capture this was included as an at-risk presentation. based on our institutional data, and available current literature, such symptoms are low in covid- and this will be revised moving forth. future directions to create a more robust surveillance system within the institution include creating a patient syndromic surveillance system, meshing output from both systems and incorporating geospatial mapping to allow for better visualization of ari clusters. further review of thresholds with the current collected data is also planned for the next phases of the staff surveillance system. our study demonstrates the feasibility in utilizing the ehr in the detection of ari clusters amongst hospital staff. this will aid in the early detection of aris including covid- . hence, the staff syndromic surveillance system is an important component of the hospital's infection prevention efforts to prevent healthcare associated covid- . world health organisation coronavirus disease (covid- ) pandemic containment of covid- cases among healthcare workers: the role of surveillance, early detection, and outbreak management healthcare workers in singapore infected with covid- monitoring approaches for health-care workers during the covid- pandemic use of a real-time locating system for contact tracing of health care workers during the covid- pandemic at an infectious disease center in singapore: validation study responding to the covid- outbreak in singapore: staff protection and staff temperature and sickness surveillance systems overview of sentinel systems for hospitalized severe acute respiratory infections (sari) represented in the weekly euroflu surveillance bulletin covid- and the gastrointestinal tract: more than meets the eye emergency department syndromic surveillance providing early warning of seasonal respiratory activity in england syndromic surveillance for local outbreaks of lower-respiratory infections: would it work reduction in healthcare-associated respiratory viral infections during a covid- outbreak our team would like to acknowledge all staff within the institution who have contributed in covid- pandemic efforts. this work was not grant funded. the authors have no conflict of interest to report. waiver of consent was obtained from institutional irb ( key: cord- - vxqr mc authors: shi, ting; arnott, andrew; semogas, indre; falsey, ann r; openshaw, peter; wedzicha, jadwiga a; campbell, harry; nair, harish title: the etiological role of common respiratory viruses in acute respiratory infections in older adults: a systematic review and meta-analysis date: - - journal: j infect dis doi: . /infdis/jiy sha: doc_id: cord_uid: vxqr mc acute respiratory tract infections (ari) constitute a substantial disease burden in adults and elderly individuals. we aimed to identify all case-control studies investigating the potential role of respiratory viruses in the etiology of ari in older adults aged ≥ years. we conducted a systematic literature review (across databases) of case-control studies published from to that investigated the viral profile of older adults with and those without ari. we then computed a pooled odds ratio (or) with a % confidence interval and virus-specific attributable fraction among the exposed (afe) for common viruses: respiratory syncytial virus (rsv), influenza virus (flu), parainfluenza virus (piv), human metapneumovirus (hmpv), adenovirus (adv), rhinovirus (rv), bocavirus (bov), and coronavirus (cov). from the studies included, there was strong evidence of possible causal attribution for rsv (or, . [ % ci, . – . ]; afe, %), flu (or, . [ % ci, . – . ]; afe, %), piv (or, not available; afe, approximately %), hmpv (or, . [ % ci, . – . ]; afe, %), adv (or, not available; afe, approximately %), rv (or, . [ % ci, . – . ]; afe, %) and cov (or, . [ % ci, . – . ]; afe, %) in older adults presenting with ari, compared with those without respiratory symptoms (ie, asymptomatic individuals) or healthy older adults. however, there was no significant difference in the detection of bov in cases and controls. this review supports rsv, flu, piv, hmpv, adv, rv, and cov as important causes of ari in older adults and provides quantitative estimates of the absolute proportion of virus-associated ari cases to which a viral cause can be attributed. disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report. acute respiratory tract infections (ari), including pneumonia, constitute a substantial disease burden in adults and elderly individuals. respiratory viruses are detected more frequently than bacteria in adults with pneumonia [ ] . the substantial contribution of viruses to ari hospitalizations among adults is being increasingly recognized [ , ] . although influenza virus (flu) is the most widely recognized viral infection associated with respiratory illness, > viruses have been linked to pneumonia, causing a substantial disease burden in adults and elderly individuals. these include common pathogens such as rhinovirus (rv), respiratory syncytial virus (rsv), flu, human metapneumovirus (hmpv), parainfluenza viruses (piv), and human coronaviruses (covs) [ ] . rsv is associated with a substantial disease burden in adults, especially among older adults (aged ≥ years) [ ] . moreover, adults hospitalized with rsv disease can develop severe respiratory complications [ ] . rv has been associated with severe respiratory disease outbreaks in adults in long-term care facilities in several settings [ ] . despite advances in diagnostic technology, defining the specific causes of viral pneumonia is challenging, particularly among older adults who may have lower viral loads and for whom viral diagnosis is frequently not considered and/ or testing is not performed [ ] . therefore, it is necessary to measure concurrently the background prevalence of nasopharyngeal viral infection in a control (asymptomatic) group, to investigate the etiological role of viruses in older adults with ari to help inform decisions on prevention and management strategies. previously, we have conducted a systematic review to understand the etiological role of common respiratory viruses, focusing on children aged < years [ ] . to the best of our knowledge, similar estimates for adults are lacking. therefore, we aimed to conduct a similar systematic review to identify all case-control studies since investigating the potential role of respiratory viruses in the etiology of aris in older adults aged ≥ years. we conducted a systematic review across databases (including chinese databases) following the approach detailed in the prisma (preferred reporting items for systematic reviews and meta-analyses) guidelines [ ] . tailored search strategies were developed and used to search the medline, embase, global health, lilacs, china national knowledge infrastructure (cnki), wanfang data, and chongqing vip databases (appendix). we further searched the reference lists of relevant articles for eligible articles. all searches were limited to between january and august . no publication status criteria or language restrictions were used. we included studies that fulfilled the following selection criteria (supplementary figure ) . three investigators (t. s., a. a., and i. s.) conducted independent searches of the english-language literature and extracted data by using standardized data extraction templates. one investigator (t. s.), whose first language is chinese, searched and extracted data from chinese-language databases (ie, cnki, wanfang, and cqvip). the protocol of this review was published in the prospero database (no. crd ). the case group was defined as older adults with ari or pneumonia aged ≥ years, adapted from world health organization integrated management of adolescent and adult illness [ ] . the details of the definitions are displayed in supplementary table . the control group was defined as older adults aged ≥ years who were either healthy or did not have any respiratory symptoms. we categorized countries as either industrialized or developing, on the basis of criteria from the united nations children's fund [ ] . we calculated odds ratios (ors) as the ratio of the odds of detecting each virus in older adults with ari or pneumonia to the odds of detecting each virus in healthy or asymptomatic controls, with accompanying % confidence intervals (cis). we used a continuity correction of . if a virus was detected in one group but not the other [ ] . this allowed calculation of an or for these instances and enabled inclusion in subsequent meta-analyses. using stata (version . ), we performed a meta-analysis of virus-specific ors and reported pooled meta-estimates with corresponding % cis, using the random effects model (ie, the dersimonian-laird method) because the included studies are heterogeneous in various aspects and are thus assumed to have different effect sizes [ ] . the virus-specific attributable fraction among the exposed (afe) was used to quantify the etiological role of each virus in patients with ari. this is an estimate of the percentage of aris that can be attributed to each virus, in absolute terms [ ] , and was calculated as * [or − ]/or, with a % ci (from the corresponding % ci of the or). moreover, for a specific virus, if all included studies did not report any virus detection in one group consistently (usually the control group), we assumed that a strong association indicating a possible causal role for this virus in ari could be concluded. in these circumstances, we considered that there was no need to run a meta-analysis that would only result in an extremely high or point estimate and an afe approaching %. we identified ( from chinese databases) records through the literature search and records from the reference lists of relevant articles. among them, only studies (including from chinese databases) fulfilled our inclusion and exclusion criteria ( figure ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . forty-three studies were excluded for a variety of reasons: no data specific to older adults ≥ years old were available (n = ), the case or control definitions were not fulfilled (n = ), no applicable data for cases and controls were reported (n = ), or serum was used as the clinical specimen (n = ). seven studies were conducted within developing countries, while were from developed countries (supplementary table ). although the search was performed for articles published since , all included studies were published since . all included studies were case-control studies with adults who had ari or pneumonia in the case group and asymptomatic or healthy adults in the control group. methods varied among studies. among the case definitions used, studies used ari or acute lower respiratory tract infection, while the others used (severe) pneumonia (n = ). all studies investigated a control group, which had no respiratory symptoms, and in studies healthy older adults (without acute illness) served as controls. of the case ascertainment methods used, articles recruited the cases from inpatients; , from outpatients; , from general practices; , from the community; and , from mixed settings (outpatient settings and the emergency department, and both outpatient and inpatient settings). in studies, controls were ascertained in hospital-based outpatient or clinic sites, whereas in studies, controls were identified from the community. all studies collected a mixture of nasopharyngeal swab specimens, nasopharyngeal aspirates, nasopharyngeal washes, oropharyngeal swab specimens, and nasal/throat swab specimens as the clinical specimen. all studies used polymerase chain reaction analysis (pcr; in some, pcr was combined with serologic analysis or culture) as the diagnostic test. meta-analyses of virus-specific ors are reported as well as the corresponding attributable fractions among the exposed (supplementary table ). rsv, flu (including flu a), hmpv, rv, and cov (also cov oc and e) were significantly more common in older adults with a diagnosis of ari or pneumonia than in asymptomatic or healthy controls (ors, these viruses had statistically significant positive afes, which showed clear associations between these viruses and ari or pneumonia in older adults. moreover, piv (including piv and piv ; data for piv and piv were not available), flu b, and adv were only identified in cases consistently across all included studies ( , , and studies, respectively) and absent in control groups. thus, these viruses were all assumed to have strong associations with ari. only studies had data available for bov, and although both reported virus detection in a greater proportion of cases than controls [ , ] , the association remains a question for further research. a subgroup analysis was performed to explore the roles of viruses in ari with respect to region: developing countries and industrialized countries. the meta-estimate or was higher in industrialized countries as compared to developing countries in the case of flu (with overlapping % cis), while it was similar for piv, adv, and rv. there were insufficient studies to conduct a similar subgroup analysis for other viruses. a sensitivity analysis was performed to investigate the roles of these common viruses in older adults admitted to hospitals with ari or pneumonia [ , , , , , , , ] . eight studies were included, and results are presented in supplementary table . the meta-estimate or did not differ significantly from the previous estimate, in which cases from other settings (ie, outpatient and general practice settings) were also included. and cov in ari and pneumonia in older adults, thereby indicating a potential for substantive reductions in the number of ari cases if older adults were vaccinated against these viruses or treated with antivirals. for the other respiratory viruses studied, the role of bov in ari and pneumonia was uncertain because of the limited evidence available from the published literature, requiring more research to clarify its role in older adults with ari. a sensitivity analysis focusing only on older adults who were admitted to hospitals with ari or pneumonia did not differ significantly from our estimate, in which patients from all settings were considered. this might result from the limited number of studies available to provide a more robust sensitivity analysis. no studies calculated adjusted ors to account for confounding effects from age or season, which might compromise the actual association and should be considered in the study design in future research. these findings should inform the results of studies that seek to estimate the global, regional, and national burden of disease due to these viruses in older adults [ ] . they show that rsv, flu, piv, hmpv, adv, rv, and cov are important causes of ari in older adults, and disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report, rather than the afe estimates in children aged < years. there is considerable international attention on rsv-associated ari in older adults at this time, during which novel vaccine and antiviral strategies are being evaluated and prioritized [ , ] , and more-accurate disease burden estimates (using these results) would help to inform future policies and interventions. the prevalence of virus detection from etiologic studies of pneumonia in adults is substantially lower than the detection rate in studies of children. the epic (etiology of pneumonia in the community) study team showed that the viruses were detected in % of adults who had been hospitalized with community-acquired pneumonia, compared with % of children who were admitted to the hospital [ , ] . there are several reasons for such low levels of detection, such as the inability to obtain lower respiratory tract specimens, the use of diagnostic tests with insufficient sensitivity, the absence of appropriate diagnostic testing methods, the undetectability of the virus at the time of the study, and the presence of unknown pathogens that were not identified. the low rate of virus detection among adults who were hospitalized for pneumonia highlights the need for more-sensitive diagnostic approaches, innovative discovery of pathogens, and assessing viruses in the past history (weeks before the presence of disease) [ ] . moreover, chronic obstructive pulmonary disease (copd) exacerbation is a very important cause of aris and hospital admissions [ ] . only of studies included copd in the etiologic data, and this information was unclear in the remaining studies, which might have underestimated the role of viral infection in these patients. a previous etiological review focusing on young children aged < years [ ] showed that rsv, flu (including flu a), piv, hmpv, and rv were significantly more common in children hospitalized with acute lower respiratory tract infection than asymptomatic controls. the associations of these viruses (except rsv) with ari and pneumonia were stronger among adults. this is in part because, in comparison to young children, the detection of viruses in the control group (ie, among individuals without respiratory symptoms or healthy controls) was less common in older adults, with the exception of rsv. several methodological issues could affect our results: sample size, age group, case ascertainment, clinical specimen, and diagnostic testing. although a thorough search has been performed across databases, including chinese-language databases, only studies from the published literature were identified, which met our selection criteria. not every virus of interest was tested in each study. the number of studies available was even smaller when subgroup analyses and sensitivity analyses were performed. moreover, the sample size varied from to adults in the case group and from to adults in the control group. the small sample size undoubtedly contributed to the imprecise % cis around the ors. thus, we may have failed to detect clinically significant ari-virus associations, owing to small sample sizes. we aimed to stratify the association between common respiratory viruses in adults with ari or pneumonia by age. however, most articles did not stratify and report data by age group. instead, they summarized the result for the entire age group, usually in adults aged > years. therefore, some of our meta-estimate ors may not be representative of older adults who are aged ≥ years. since age might be a risk factor for ari in adults (the rate of severe ari increases as age advances), this could potentially affect the viral profile detected, introducing further heterogeneity [ ] . fifteen of studies used passive clinic or hospital based case ascertainment. among them, cases were recruited from inpatients, outpatients, emergency departments, or general practices, which might reflect different healthcare behavior and disease severity. also, since the episodes of ari and pneumonia were only diagnosed through routine care, this introduced bias, considering that testing was only done when the clinicians deemed it necessary to test. similarly, studies used community-based controls, while another studies recruited older adult controls from hospitals or general practices. hospital or clinical ascertained controls may not reflect the general population and may have other health conditions potentially affecting their viral carriage. moreover, recruiting controls who were selected as healthy or without respiratory symptoms could favor those who were not exposed to the respiratory virus (yielding a falsely high or). therefore, we consider that the ideal control group for these studies would be a random sample of an age-and sexmatched population of older adults who are from the same area of residence and studied at the same time as cases. all included studies obtained upper respiratory tract specimens (described as nasopharyngeal secretions). assays might have specimen-specific sensitivities and specificities for detecting viruses, which could lead to heterogeneity in the estimation of virus-specific rates. the sensitivity of using nasopharyngeal washes for detecting any virus in adults was found to be higher than that for using nasopharyngeal swab specimens, which in turn was higher than that for using oropharyngeal swab specimens ( . %, . %, and . %, respectively) [ ] . the limited use (due to ethical concerns and feasibility) of invasive procedures to obtain lower respiratory tract specimens directly from the lung also influenced the diagnosis of viral infection in adults with ari [ ] . pcr and serology-based diagnostic testing are more sensitive for detecting respiratory viruses than other methods, such as antigen detection and culture. high sensitivity is important for accurate assessment of etiological contribution, particularly in older adults who may have a lower nasopharyngeal viral load and an atypical clinical presentation [ ] . moreover, despite being uncommon, detection of viral coinfection (range across studies, %- %) may tend to overstate the contribution of individual respiratory viruses (although dual or multiple infections, in which both or several viruses have etiological importance, are possible). bacterial coinfections (range across studies, %- %) were also reported. with improving diagnostic methods, multiple etiological agents are increasingly identified simultaneously in older adults with ari, making the individual contribution of each agent difficult to define. viruses can be detected in individuals with no respiratory symptoms. this is often seen in volunteer challenge studies and in some community surveys [ , ] . the detection of viruses in control groups without respiratory symptoms might be due to a nascent infection or a persisting colonization from a previous infection [ ] . these factors will tend to result in true associations being attenuated. the fact that molecular detection of viruses in older adults with ari is higher than the detection rate in controls without respiratory symptoms may not necessarily indicate causation. alternative explanations should be considered first before causality can be concluded. these include the respiratory viral infection acting as a so-called innocent bystander, without a causal role, and serving only as a predisposing risk factor for ari. similarly, the absence of a positive association (and afe) does not mean that a virus is not a cause of ari. moreover, without establishing the temporal sequence of exposure and outcome, determinations of causality are less secure. therefore, the association between viruses and ari and pneumonia should be interpreted carefully. in conclusion, this review provides clear evidence that is suggestive of the potentially causal role of rsv, flu, piv, hmpv, adv, rv, and cov in older adults with ari and presents the first estimate of the proportion of ari cases that can be attributed to virus exposure. etiological studies, which simply report rates of viral identification as causal, should make attempts to interpret findings in terms of the proportion of ari cases among older adults in whom a respiratory virus is identified that can be attributed to this viral exposure. the resceu investigators are as follows national institute for public health and the environment) penta foundation) jeroen aerssens, veronique wyffels, and matthias cleenewerck (janssen) cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. adults viral pneumonia respiratory syncytial virus infection in elderly and high-risk adults modelling estimates of the burden of respiratory syncytial virus infection in adults and the elderly in the united kingdom 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novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay viral respiratory tract infections in adult patients attending outpatient and emergency departments respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care respiratory virus is a real pathogen in immunocompetent community-acquired pneumonia: comparing to influenza like illness and volunteer controls incidence and characteristics of viral community-acquired pneumonia in adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection viral etiology of acute lower respiratory infection in adult inpatients detection on non-bacterium pathogen in cases of acute respiratory infection human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in countries: a systematic analysis for the global burden of disease study accessed drug candidates and model systems in respiratory syncytial virus antiviral drug discovery cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. children better tests, better care: improved diagnostics for infectious diseases respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia time lines of infection and disease in human influenza: a review of volunteer challenge studies flu watch group. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections we thank joris menten from janssen for reviewing the manuscript.financial support. this work was supported by the innovative medicines initiative joint undertaking (grant ), which in turn receives support from the european union's horizon research and innovation programme and efpia. is the elected president of the british society for immunology, which is an unpaid appointment but provides support for travel and accommodation at some meetings. all other authors report no potential conflicts. 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. key: cord- -hdjbxah authors: mcerlean, peter; greiman, alyssa; favoreto, silvio; avila, pedro c. title: viral diversity in asthma: immunology and allergy clinics of north america: asthma and infectious disease date: - - journal: immunology and allergy clinics of north america doi: . /j.iac. . . sha: doc_id: cord_uid: hdjbxah asthma exacerbations are precipitated primarily by respiratory virus infection and frequently require immediate medical intervention. studies of childhood and adult asthma have implicated a wide variety of respiratory viruses in exacerbations. by focusing on both rna and dna respiratory viruses and some newly identified viruses, this review illustrates the diversity and highlights some of the uncertainties that exist in our understanding of virus-related asthma exacerbations. peter mcerlean, phd a, *, alyssa greiman, bsc a , silvio favoreto jr, phd, dds b , pedro c. avila, md c asthma is a heterogeneous inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperresponsiveness, inflammatory cell infiltration, thickening of the lamina reticularis, and the accompanying symptoms of chest tightness, wheezing, coughing, and shortness of breath. , asthma now affects an estimated . million adults and . million children ( . % and . % of the population, respectively) within the united states , and is additionally indicated as a "contributing factor" in nearly deaths each year. whereas chronic asthma results from the daily inhalation and response to allergen (eg, dust, pollen, animal dander) and is by and large successfully managed by the individual, acute asthma attacks or exacerbations are precipitated primarily by respiratory viral infections and frequently require immediate medical intervention. in the united states alone, severe asthma exacerbations lead to over , hospitalizations each year, at a cost of one-third of the total $ . billion in annual asthma-related health care expenditures. although the exact mechanism(s) by which respiratory viral infection causes asthma exacerbation remains to be determined, the respiratory viruses implicated in exacerbations have themselves been largely identified and well characterized ( table ) . traditionally associated with acute respiratory illness (ari) or symptoms of the "common cold," the respiratory viruses implicated in asthma exacerbations predominantly possess rna genomes with a distinct genome organization (positive [ ] or negative [À] sense), virus particle (virion) morphology (enveloped or nonenveloped), host cell receptor interaction, and well-defined annual or seasonal prevalence. the full extent of respiratory virus involvement in exacerbation has recently been revealed in asthma studies with the implementation of molecular methods of viral detection, specifically the reverse transcriptase polymerase chain reaction (rt-pcr). , molecular methods of viral detection have superior sensitivity and specificity compared with cell culture-based methods and additionally allow for the improved identification of multiple viruses (and other pathogens), revealing the role dual or multiple infections play in asthma exacerbations. indeed, molecular methods have also been invaluable in identifying new respiratory viruses, the majority of which were described after the emergence of the severe acute respiratory syndrome-associated coronavirus (sars-cov) that prompted research into the etiology of ari. these "newly identified viruses" (nivs) including human metapneumovirus (hmpv; described pre-sars), the human rhinovirus (hrv) species c (hrv-cs), human coronaviruses (hcovs)-nl and -hku , human bocavirus (hbov), and the ki and wu polyomaviruses (kipyv and wupyv) are now the focus of intense research, and their involvement in asthma exacerbations is slowly beginning to be determined. because those respiratory viruses most associated with exacerbations-the hrvs, respiratory syncytial virus (rsv), and hmpv-are reviewed elsewhere in this issue by miller and colleagues, this review discusses some of the other respiratory viruses implicated in childhood and adult asthma exacerbations, including additional rna viruses and those with dna genomes. by also encompassing some of the recently described nivs, this article illustrates the diversity that exists in virus-related asthma exacerbations. influenza viruses (ifvs) are probably the best known of all the respiratory viruses, due to their ability to cause annual epidemics and potential pandemics of serious respiratory disease. ifvs pose the greatest risk of morbidity and mortality to young children and the elderly, and as such have been the focus of repeated public health and vaccination campaigns. however, despite their potential to cause serious respiratory illness in otherwise healthy individuals, most asthma studies describe relatively low levels of ifvs in exacerbations, accounting for approximately % to % of all virus-related asthma exacerbations (see table ). , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ifvs constitute the family orthomyxoviridae and are segmented Àsingle stranded (ss) rna viruses. the ifv virion has a pleomorphic envelope derived from host cell membranes and incorporates viral encoded surface proteins: hemagglutinin (ha), neuraminidase (na), and matrix protein (m ). under the envelope and encased within the matrix protein (m ), the core of the ifv virion contains the ribonucleoprotein complex, consisting of the viral rna segments, polymerase proteins (pb , pb , and pa), and the nucleoprotein (np). there are types of ifvs, -a (ifav), -b (ifbv), and -c (ifcv), which are divided further in subtypes and strains based on the combination of ha and na proteins (eg, h n , h n ). ifav and ifcv have subtypes known to infect both animals (eg, birds, pigs) and humans, whereas ifbv is predominately a human virus. although all types of ifvs can cause ari in humans, ifav and ifbv subtypes and strains are of the most prominent in the annual epidemics or "flu season" that occurs during the winter months. ifvs initiate infection via the ha protein attaching to sialic acid (sa) linked to galactose (gal) sugars on the terminal ends of host cell surface glycans. it has been determined that the ha interaction is dependent on the sialic acid-galactose linkages, with ha from subtypes that infect humans recognizing a , linkages (saa , gal), whereas ha from subtypes infecting other animals mostly recognize a , linkages (saa , gal). , although ifvs are implicated in . % to . % of wheezing illnesses in children , , , and have been detected in . % of asthmatic adults during emergency department (ed) visits, uncertainty remains as to whether ifvs are specifically responsible for the production of asthma exacerbations. , the main contention arises from the role that vaccination has played in preventing exacerbations. because, unlike other respiratory viruses, vaccines against ifv subtypes and strains are available and asthmatics frequently have increased vaccination rates due to their at-risk status during flu season, ifv-related exacerbations should be reduced or indeed eliminated among asthmatic children and adults. , however, some studies have shown that despite their three-to fourfold greater odds of having an influenza vaccination (as reported by parents), asthmatic children have higher rates of ifv-related hospital visits and that vaccination does not affect the number, duration, or severity of ifvrelated asthma exacerbations compared with placebo. other similar studies have shown that influenza vaccination also fails to reduce severe and fatal complications in adults with asthma and chronic obstructive pulmonary disease (copd). the association of ifvs with exacerbations in vaccinated asthmatics questions the efficacy of seasonal influenza vaccines in this group and also suggests a role for other respiratory viruses in ifv-related exacerbations. if influenza vaccinations are effective, then it is likely that ifv-related exacerbations are caused by infection with another respiratory virus, such as those that peak in prevalence during the same time as ifvs (eg, rsv and hmpv). however, if influenza vaccination of asthmatics fails to reduce ifv-proven exacerbations (ie, by rt-pcr during symptomatic illness) then alternative vaccination strategies may be required to increase efficacy. ensuring that comprehensive respiratory virus testing is employed during both influenza vaccination and asthma studies and that vaccination rates among asthmatics remain high, the contention over vaccination and ifv-related exacerbations will be better clarified. some recent studies focusing on ari suggest that infection with other respiratory viruses, particularly hrvs, may actually provide "protection" against ifv infection. , although such claims remain to be fully substantiated, viral competition may be a contributing factor in the relatively low rates of ifv-related asthma exacerbations being reported. it remains to be determined whether the asthmatic phenotype preferentially facilitates infection with respiratory viruses other than ifvs or whether, owing to vaccination in the wider community, the overall prevalence of ifvs is reduced making infection with another "uncontrolled" respiratory virus more likely. the association of ifvs with asthma exacerbation is complicated by the emergence of more virulent pandemic strains, such as the novel swine-origin ifav h n (s-oiv), which may pose a greater risk to asthmatics than seasonal strains. although the full impact of s-oiv remains to be determined, initial studies have indicated that among both children and adults hospitalized with rt-pcr-identified s-oiv, asthma accounted for the largest percentage ( %) of all underlying medical conditions. however, it is unclear whether s-oiv evokes a specific immune response among asthmatics or whether, owing to increased awareness of this particular strain, asthmatics were more likely to seek medical care when respiratory symptoms initially began. hcovs were initially described in the s in studies aiming to determine the etiologic agent responsible for ari, but gained notoriety after the emergence of sars-cov in . since that time, additional hcovs have been described in ari studies, viral diversity in asthma indicating that there are several hcovs potentially associated with respiratory illness in humans. because of the severity of associated illness, this review excludes sars-cov and focuses solely on the hcovs associated with ari and currently implicated in % to % of virus-related asthma exacerbations: hcov-oc , hcov- e, and the nivs, hcov-nl and hcov-hku (see table ). , , , hcovs are classified within the family coronaviridae, subfamily coronavirinae, with the genus alphacoronavirus containing hcov- e and hcov-nl and the genus betacoronavirus containing hcov-oc and hcov-hku . hcovs possess a ssrna genome, which is associated with nucleocapsid (n) phosphoprotein within the core of a host cell-derived enveloped virion. the hcov virion also comprises viral encoded proteins (s, e, and m), and characterization has indicated that binding to host cell receptors is mediated predominately via the spike (s) glycoprotein, with each hcov employing a specific host cell receptor during infection (see table ). the virion of betacoronavirus also contains a hemagglutinin (he) protein, which is thought to be involved in either host-receptor interactions or release of virus from infected cells. [ ] [ ] [ ] the molecular and receptor interaction differences existing between hcovs are reflected in their seasonal prevalences. studies of ari have indicated that hcov-nl can be detected in the spring, summer, or winter whereas hcovs -hku , -oc , and - e infections mainly occur during the autumn and winter months. [ ] [ ] [ ] [ ] a recent study of hcovs -nl , -oc , and - e also found fluctuations occurring between their yearly prevalence. the clinical impact of each hcov also appears to vary, but all present the greatest disease burden within the childhood population. in a retrospective study of clinical samples taken over a -year period from young children (median age . months), the percentage of lower respiratory tract illness (lrti; including asthma exacerbations and bronchiolitis) associated with any hcov, hcov-nl , or hcov-oc was estimated to be . %, . %, and . %, respectively. although this study failed to include hcov-hku in the testing panels, other studies have associated hcov-hku with wheezing illness in children. , among asthmatic adults, hcovs have been detected during both ed visits and welldefined episodes of exacerbation prompted by ari. atmar and colleagues reported that hcovs were detected in of ed visits during a -year study of asthmatic adults, and kistler and colleagues reported detections of hcovs, oc , hku , and nl in asthmatic adults with ari, of which hcov-nl occurred most in episodes of exacerbations. despite being associated with ari, hcov- e appears to be the hcov least associated with asthma exacerbations. recent studies using rt-pcr have failed to find hcov- e in either adult or childhood episodes of asthma exacerbation. , , although some previous studies detected hcov- e in asthmatic children, detection occurred with hcov-oc and individual hcov detection rates were not reported, despite the investigators using hcov-oc -and hcov- e-specific primers and antibodies. another early study of asthmatic adults detected hcov- e through analysis of paired sera in instances where exacerbation could be objectively measured. however, hcov- e detections could not be associated with a peak expiratory flow rate (pefr) mean decrease of greater than l/min. the reason for the lack of hcov- e detections in asthma exacerbations remains unclear. parainfluenza viruses (pivs) are primarily associated with bronchiolitis and laryngotracheobronchitis or croup, in children younger than years of age. , pivs also pose a serious risk to immunocompromised individuals, and outbreaks of viral pneumonia among transplant recipients and patients undergoing chemotherapy have occurred. , the pivs consist of serotypes ( - ) and subtypes ( a and b), and as a group are responsible for % to % of virus-related asthma exacerbations (see table ). , , , , , like rsv and hmpv, pivs are nonsegmented Àssrna viruses belonging to the family paramyxoviridae. pivs to , a, and b are classified within of the genera of the subfamily paramyxovirinae, genus respirovirus (piv- and piv- ) and genus rubulavirus (piv and piv- a and - b). in contrast to the g glycoprotein encoded by rsv and hmpv, pivs encode a hemagglutinin-neuraminidase (hn) glycoprotein, which interacts with gangliosides (sialic acid-containing oligosaccharides) on target host cells during infection. , seasonal and yearly prevalence of pivs have been shown to vary among the serotypes. piv- , - , and - have the highest prevalence in the autumn and winter months whereas piv- occurs mostly in the spring and summer. peaks in piv- prevalence have been shown to occur biennially. piv- prevalence exhibits some yearly variation and piv- is consistently detected each year. piv- is the least prevalent of all pivs. although each piv has the potential to cause respiratory illness in any age group, pivs to appear to be more associated with a particular clinical presentation at certain ages. piv- is associated with croup in children to years old, piv- with bronchiolitis in the age group younger than year, and piv- is more associated with viral pneumonia in individuals older than years. studies of asthmatic children have revealed that each piv type plays a different role in the production of asthma symptoms. in studies where the incidence of each individual piv is described, piv- is detected in . % to . %, piv- in . %, piv- in . % to %, and piv- in up to . % of wheezing illnesses. [ ] [ ] [ ] although pivs are largely considered as a single group in adult asthma studies, pivs are still detected in episodes of exacerbation. atmar and colleagues detected pivs to in of episodes of ari in asthmatic adults, with at least one infection each of piv- and piv- resulting in an ed visit, and nicholson and colleagues detected cases of piv - in asthmatic adults, of which had instances of a pefr mean decrease of greater than l/min. adenoviruses (advs) comprise a large group of dna viruses that are known to cause a wide variety of clinical syndromes including diarrhea, keratoconjunctivitis, and hemorrhagic cystitis. , however, advs are best known as a primary cause of ari and, particularly in young children, have been implicated in the production of more severe lrti. fifty-one adv serotypes have been identified, and as a group advs are associated with up to % of virus-related asthma exacerbations (see table ). , , , , advs are classified within the family adenoviridae, genus mastadenovirus, and divided into species a through g based on biochemical and biophysical properties, hemagglutination reaction, and sequence identity. the adv virion is a nonenveloped icosahedral capsid consisting of hexon and penton polyproteins or capsomers. a long fiber protein protrudes from each of the penton capsomers and contains a terminal "knob" domain, which interacts with host cell receptors. advs have been shown to employ a diverse variety of cell receptors, including coxsackie-adenovirus receptor (car), heparan sulfate glycosaminoglycans, cd , and an array of cell surface integrins. although each serotype exhibits some variation, advs are detected primarily during the winter and spring months. , of all the serotypes, adv- , - , - , and - are the viral diversity in asthma most common cause of ari. , however, similar to other respiratory viruses (eg, rsv subtype detections reported by lee and colleagues versus matthew and colleagues ) , location can affect the predominance of a given serotype, with adv- , - , and - being the most frequently detected in ari studies conducted in the united states. , as with most respiratory viruses, adv infections occur primarily in infants and children, and it is within these populations specifically that advs have been associated with more serious respiratory illness. , , [ ] [ ] [ ] in an -year study of children younger than years hospitalized with lrti, larranaga and colleagues detected adv at a rate of . % (sole detections), second only to rsv ( . %), and predominately in patients with pneumonia and wheezing bronchitis ( . %). the investigators also typed the adv isolates and determined that the species b and c were most frequently detected among their population and that serotype- h was particularly associated with a longer duration of hospitalization. although this study employed culture-based methods of detection, more recent studies employing pcr for adv detection describe comparable detection rates ( . %- %) in episodes of childhood wheeze and asthma exacerbations. , , , , an interesting aspect of the association of adv with asthma was described in a study of asymptomatic asthmatic children conducted by marin and colleagues. these investigators reported that adv dna was detected in . % of the asthmatic group but in only % of the nonasthmatic control group. this study also described hrv and rsv detection rates in the asthmatic group of . % and . %, respectively, despite none of the participants experiencing respiratory symptoms for the duration of the study ( weeks). however, a confounding aspect of this study is that the subjects were classified as having mild asthma that was well controlled (fluticasone - mg daily) in the months before the start of the study. nevertheless, the disparity in viral detection between the asthmatic and control groups may imply some mechanism of either persistence in asthmatics or an association with the long-term compliance of glucocorticoid therapy and inhibition of symptoms during respiratory virus infection. human bocaviruses (hbov) were discovered in by allander and colleagues in pooled nasopharyngeal aspirates obtained from children with lrti. further characterization in ari-focused studies has revealed that hbov is frequently associated with ari in both children and adults, with detection rates of . % to %. [ ] [ ] [ ] [ ] in addition, hbov has been associated with wheezing and gastrointestinal illness predominately in children younger than years. , - however, the exact role of hbov in respiratory and other illnesses remains ambiguous, with a high overall codetection rate with other respiratory viruses being described (median . %) and the detection of hbov dna in serum, urine, and lymph node samples. , , , , hbov is a parvovirus, a single-stranded dna virus of the family parvoviridae, genus bocavirus. the genome, which requires host cell dna polymerases for replication, encodes structural (vp and vp ) and nonstructural (ns and np ) proteins. although hbov has only recently been propagated in cultured cells and its host cell interactions remain to be determined, initial studies have found that hbov shares similar virion morphology with other parvoviruses, a nonenveloped icosahedral capsid consisting of copies of each structural protein. recently, viruses related to hbov have been identified, namely hbov- and hbov- , but it is unknown whether these are unique viral entities or closely related genotypes of the same virus. , while the epidemiology of hbov is still being determined, ari studies have shown that hbov infection occurs primarily during the winter, with a smaller peak in spring. , , several studies have indicated that hbov is associated with severe respiratory disease in children, particularly in those hospitalized with asthma and other wheezing illnesses. , a study conducted by vallet and colleagues of children aged to years hospitalized for asthma detected hbov in % of children with asthma exacerbations compared with only % in children with stable asthma, suggesting hbov plays a causative role in the development of exacerbations. similar findings were reported by nadji and colleagues, who detected hbov at a rate of % in children younger than years with asthma exacerbations, and garcia and colleagues, who reported that wheezing was seen in more than % of children in whom hbov was the sole virus detected. although hbov is more often detected in symptomatic than healthy individuals, high levels of codetection with other respiratory viruses, particularly picornaviruses and ifvs, confounds hbov's role in respiratory illness. , although a study of infants younger than months hospitalized with bronchiolitis found that dual infections of rsv and hbov were associated with higher clinical severity scores and longer length of hospitalization than infants with a single hbov or hrv and hbov dual infections, most studies have found that coinfection of hbov with another respiratory virus does not alter the severity or duration of associated illness. in , novel human polyomaviruses (pyvs) were described, the ki polyomavirus (kipyv) by allander and colleagues and the wu polyomavirus (wupyv) by gaynor and colleagues. initially identified in nasopharyngeal aspirates obtained from individuals with respiratory illness, further characterization has revealed kipyv and wupyv are detected in . % and . % of ari cases, respectively, suggesting these nivs have a causative role in respiratory illnesses. [ ] [ ] [ ] [ ] however, similar to hbov, a high rate of codetection (> %) with other respiratory viruses and detection in other tissue types currently confounds any direct association of kipyv and wupyv with ari and other illnesses. pyvs are classified in the single genus polyomavirus of the family polyomaviridae, and contain histone-associated circular dsdna genomes encoding structural (vp , vp , vp ) and nonstructural (large-t and small-t antigens) proteins. , as with some other nivs (eg, hrv-c, hcov-hku ), kipyv and wupyv cannot be grown using current cell culture systems. this drawback has postponed identification of host cell receptor interactions and determination of virion morphology, although the latter is believed to be similar to the nonenveloped icosahedral configuration of other pyvs. kipyv and wupyv characterization in retrospective ari-focused studies has determined that they exhibit a year-round prevalence, with the greatest peaks in detections occurring in the spring, autumn, and winter months. , [ ] [ ] [ ] although no studies have yet looked specifically for kipyv and wupyv during episodes of asthma exacerbations, wheezing illness and other more serious lrti have been reported in patients positive for kipyv and wupyv. bialasiewicz and colleagues showed that % of kipyv-positive and % of wupyv-positive patients had symptoms of bronchiolitis, and payungporn and colleagues found that of combined kipyv and wupyv sole detections originated in children younger than year with pneumonia. however, the overall high codetection rates ( %- %) described in these studies again indicates the need for further clarification of the role of kipyv and wupyv in respiratory illness. an interesting aspect of the previously described human pyvs is their ability to establish a form of latency (ie, low-level persistent infection) and undergo reactivation when an infected individual's immune system experiences stress (eg, infection, inflammation, or immune suppression). , while characterization of the host-virus interactions of kipyv and wupyv is still continuing, sharp and colleagues have proposed that they may also reactivate during illness, as evidenced by mutations in the transcriptional control region of kipyv and wupyv genomes detected in autopsy tissue from immunosuppressed individuals. given our incomplete knowledge about the role of kipyv and wupyv in respiratory illness, it may be plausible that the high codetection rates observed in ari studies are not the result of bona fide dual infections, but rather may be caused by infection with another respiratory virus evoking an immune response that reactivates latent kipyv and wupyv. although sharp and colleagues did not observe the high frequency of transcriptional control region mutations in kipyv and wupyv genomes detected from respiratory samples, a seroepidemiology study by nguyen and colleagues may provide evidence of kipyv and wupyv reactivation during ari. these investigators observed that the proportion of kipyv-and wupyv-seropositive individuals increased at around years of age, a finding that they speculated was the result of kipyv and wupyv exposure during school attendance. however, they conceded that the serum obtained for their study originated from tertiary referral hospitals and was likely from individuals with underlying medical conditions. given that ari represents the most common reason for hospital visits and admissions among school-aged children, and that the incidence of other respiratory virus infections also increases among this age group, the kipyv and wupyv antibody detected may not be that produced in response to initial exposure, but rather antibody produced during kipyv and wupyv reactivation evoked by infection with another ari-causing respiratory virus. indeed, nguyen and colleagues reported that a high proportion of adults were kipyv-and wupyv-seropositive which, considering the nature of the study cohort (ie, hospital-based), may not indicate preexisting immunity but again kipyv and wupyv reactivation during illness. future communitybased studies will no doubt determine when initial kipyv and wupyv exposure occurs and will better elucidate the relationships that exist between ari, viral codetections, and kipyv and wupyv reactivation. this review focuses on some of the less common respiratory viruses currently implicated in both childhood and adult asthma exacerbations. although the respiratory viruses covered here have a lesser, or in the case of nivs, an as yet uncharacterized involvement in the development of exacerbation compared with the "usual suspects" (eg, hrv, rsv, hmpv), they exemplify the diversity that exists in virus-related exacerbations. the implementation of more sensitive methods of virus detection and the identification and characterization of nivs in future asthma studies will further expand our understanding of viral diversity and the mechanisms underlying development of asthma exacerbations during 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infection presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection identification of the novel ki and wu polyomaviruses in human tonsils no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease philadelphia: lippincott-raven identification of wu polyomavirus from pediatric patients with acute respiratory infections in beijing clinical and epidemiologic characterization of wu polyomavirus infection prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in thailand the role of polyomaviruses in human disease reactivation and mutation of newly discovered wu, ki, and merkel cell carcinoma polyomaviruses in immunosuppressed individuals serologic evidence of frequent human infection with wu and ki polyomaviruses key: cord- -oob ktnz authors: proença-modena, josé luiz; gagliardi, talita bianca; escremim de paula, flávia; iwamoto, marisa akiko; criado, miriã ferreira; camara, ataíde a.; acrani, gustavo olszanski; cintra, otávio augusto leite; cervi, maria célia; de paula arruda, luisa karla; arruda, eurico title: detection of human bocavirus mrna in respiratory secretions correlates with high viral load and concurrent diarrhea date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: oob ktnz human bocavirus (hbov) is a parvovirus recently identified in association with acute respiratory infections (ari). despite its worldwide occurrence, little is known on the pathogenesis of hbov infections. in addition, few systematic studies of hbov in ari have been conducted in latin america. therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a -year retrospective hospital-based study of hbov in outpatients and inpatients with symptoms of acute respiratory infections (ari) in brazil. nasopharyngeal aspirates (npas) from patients with respiratory symptoms were tested for hbov dna by pcr. all samples positive for hbov were tested by pcr for all other respiratory viruses, had hbov viral loads determined by quantitative real time pcr and, when possible, were tested by rt-pcr for hbov vp mrna, as evidence of active viral replication. hbov was detected in . % of patients, with annual rates of . %, . % and . % in , and , respectively. the range of respiratory symptoms was similar between hbov-positive and hbov-negative ari patients. however, a higher rate of diarrhea was observed in hbov-positive patients. high hbov viral loads (> ( ) copies/ml) and diarrhea were significantly more frequent in patients with exclusive infection by hbov and in patients with detection of hbov vp mrna than in patients with viral co-infection, detected in . % of patients with hbov. in summary, our data demonstrated that active hbov replication was detected in a small percentage of patients with ari and was correlated with concurrent diarrhea and lack of other viral co-infections. the discovery of human bocavirus (hbov) was the result of a viral metagenomic study of respiratory secretions from swedish children with symptoms of acute respiratory infection (ari) reported in [ ] . hbov is classified in the family parvoviridae, which includes small non-enveloped, icosahedral viruses with . kb single-stranded dna genome containing three open reading frames (orfs). the first two sequential orfs encode non-structural proteins ns and np- , whereas the third downstream orf encodes two viral capsid proteins, vp and vp [ ] . hbov circulates worldwide [ ] and more recent phylogenetic analyses revealed that three novel hbov species, different from the respiratory hbov , named hbov , and , are frequently detected in stools from children with acute gastroenteritis [ , , , ] . hbov is often detected in patients with ari [ , , ] , including those with wheezing, croup, cough, rhinorrhea and fever [ , ] . the most frequent clinical diagnoses associated with respiratory hbov are general upper respiratory tract infections (urti), bronchiolitis, pneumonia, bronchitis and exacerbation of asthma [ ] . symptoms associated with hbov usually last - weeks [ , ] , but the agent has also been detected in association with prolonged fever [ ] . an association of hbov with respiratory disease has been based mostly on the significantly higher frequencies of detection of the agent in ari patients than in control subjects without respiratory symptoms [ , , ] . hbov is also detected in feces from children with diarrhea with frequencies ranging from . % to % [ , , ] . in addition, hbov dna has been detected in tonsil tissue in children with chronic tonsillitis undergoing surgical resection [ , ] . although hbov has been propagated in primary cultures of human respiratory cells [ ] , isolation of the agent from clinical samples has not been achieved in common cell lines. moreover, no experimental animal model of hbov infection has been developed, which hampers fulfillment of koch's postulates for a definitive association of hbov with disease. this scenario is further complicated by the possibility that hbov may persist in a way similar to other viruses of the family parvoviridae [ ] , with consequent prolonged detection in human secretions. in addition, detection of hbov dna by pcr is commonly associated with detection of genomes of other viruses whose roles in pathogenesis of human diseases is already established [ ] . this article reports a cross-sectional study of hbov in ari patients from ribeirão preto, brazil, in which the shedding of vp mrna in respiratory secretions was used as surrogate marker for active hbov replication, to look for correlations with viral load, and presence of particular clinical manifestations and simultaneous detection of other respiratory viruses. hbov genome was detected by pcr in out of ( . %) respiratory samples from ari patients. out of the hbov positive samples, ( . %) were from patients under or equal to years of age (# years old) and only ( . %) were from adult patients. hbov was detected in . % of adult patients enrolled in the study. the patient demographics and clinical information are summarized on figure ). it was noticeable that hbov circulated roughly along with hrsv ( figure ). in this year study period, there was a trend for an increase in the total number of respiratory samples from children submitted for viral testing, hrsv and hbov positivity, in association with dry season and declining average montly temperatures [ ] . it is noteworthy that hbov was not detected in stored samples obtained from patients without respiratory symptoms, seen at the emergency room for reasons unrelated to the respiratory tract from to [ ] . for the purpose of the analysis, the patients were divided into two groups: those with age equal or less than years of age ( males and females; median and mean age sd: and . . months), and those older than years ( males and females; . and . . months). in general, the clinical findings were more severe in patients younger than years (table s ). the most frequent respiratory symptoms/signs (present in . %) seen in hbov-positive patients were cough, fever, dyspnea and coryza, but wheezing, nasal obstruction and sneezing were also recorded. no significant differences were noted in the frequencies of specific respiratory symptoms/signs between hbov positive and negative patients (table ) . of studied patients, ( . %) were diagnosed with lrti and ( . %) with urti. the complications of aom and gerd were diagnosed in ( . %) and ( . %) patients, respectively (table s ) . over one third of the patients had illness of low or moderate severity and . % of them had an ics = or ,and only . % had ics = or (table ) . significantly higher frequencies of lrti ( . % vs. . %, p = . ), requirement for hospitalization ( . % vs. . %, p = . ), and diarrhea ( . % vs. . %, p, . ) were noted in the hbov-positive patients, in comparison with the hbov-negative ones (p, . ) ( table ) . no significant association was noted between hbov positivity and the presence of specific signs or symptoms (table ) . also, no significant differences were noted in length of hospital stay, rates of requirements for o and positive airway pressure, aom, and illness severity as measured by ics, between hbov positive and negative patients (table ) . while the frequency of gerd was roughly twice as high in hbov-positive, than in hbov-negative patients (p = . ), only hbov patients had gerd (table ) , a low number of cases that restricts the power of the comparison. logistic regression analyses of the relationship between hbov detection and disease, after adjustment for patient age, gender and concurrent hrsv infection, showed that the clinical features more significantly associated with hbov were diarrhea (odds ratio [ all samples positive for hbov by conventional pcr were tested for the other respiratory viruses, including hrsv, hrv, hmpv, flu, hpiv, hcov and hadv. of hbov-positive patients, ( . %) had at least one additional respiratory virus detected (table s ). the viruses most frequently detected simultaneously with hbov were hrsv in ( . %), hrv in ( . %), hmpv in ( . %), and hadv in ( . %) patients. the frequencies of co-detection of one, two, three and four additional viruses in patients with hbov were respectively . %, . %, . % and . % (table s ) . to assess whether this high frequency of co-detection of other respiratory viruses was a finding specific of hbov-infected patients, a subset of samples from hbov-negative ari patients, collected in the same season as those from hbovpositive ones, was randomly selected for testing by pcr for the other respiratory viruses, without knowing the result of the hrsv or influenza screening for which the samples had been originally submitted. such strategy was adopted since it would not be possible to test all samples for all respiratory viruses. a single respiratory virus different from hbov was detected in of ( . %) patients, and co-detections with more than one agent were found in an additional ( . %) (table s ) . there were no significant differences in frequencies of specific respiratory symptoms between the patients with ari in whose samples only hbov was detected, and those with hbov and codetection of other viruses. however, significantly lower frequencies of diarrhea (or = . ; % ci = . - . ; p, . ) and complication of gerd (or = . ; % ci = . - . ; p = . ) were noted in patients with co-detection of other viruses in addition to hbov (table s ) . absolute quantification of dna by qpcr revealed that the hbov viral load in hbov-positive patients varied very broadly, over a range that reached decimal orders of magnitude, with median value of . copies/ml ( figure and table s ). importantly, the median viral load in samples from patients in whom hbov was the only virus detected ( . copies/ml), was significantly higher than that in the patients with hbov in coinfection with other respiratory viruses ( . copies/ml) (p, . ) (figure a) . a comparison of demographics and clinical data between patients shedding very high hbov viral loads (. copies/ml of npa) and those shedding lower loads, evidenced significantly higher frequencies of diarrhea (p, . ) in patients shedding higher viral loads (table ) . remarkably, detection of other respiratory viruses simultaneously with hbov was significantly less frequent in samples from patients shedding very high loads of hbov (p, . ) ( table ) . logistic regression analyses confirmed that diarrhea (or = . ; % ci = . - . ; p = . ) were directly related, whereas simultaneous detection of other viruses in the npas was inversely related (or = . ; % ci = . - . ; p, . ) to the shedding of very high hbov loads. the association of shedding very high loads of hbov (. copies/ml) with lack of co-detection of other respiratory viruses suggested that such high viral loads may result from early acute hbov infections with active viral replication, as opposed to long term shedding unrelated to current clinical symptoms. in order to confirm this hypothesis, an assay was developed to detect hbov vp mrna in npas by real time rt-pcr done on total rna extracts previously treated with dnase i. as an internal control, real time rt-pcr was done for the housekeeping gene of b-actin. when simultaneously positive for both hbov vp and bactin mrnas, this assay was considered a marker of active hbov replication. positivity for hbov vp or b-actin by pcr done directly on dnase-treated material without previous reverse transcription were considered indicative of remaining genomic dna after incomplete dnase treatment, and samples were discarded. of the samples positive for hbov, had sufficient remaining volume left for testing. of those, only ( . %) tested positive for vp mrna and, remarkably, of these had very high hbov loads of greater than copies of dna/ml. the median hbov viral load in patients shedding vp mrna was . copies/ml, which was significantly higher than that in patients negative for vp hbov mrna, . copies/ml (p, . ) (figure b , table ). the association of high viral loads importantly, a significant inverse association was noted between shedding of hbov vp mrna and having another respiratory virus detected in the npa. four of ( . %) patients who shed hbov vp mrna also had another respiratory virus detected in their npas, whereas of ( %) of the patients in whose samples shedding of vp mrna was not present, had another respiratory virus detected in their npas (p, . ) ( table ) . such inverse association was confirmed by logistic regression analysis (or = . ; % ci = . - . ; p, . ). a comparison of clinical features between hbov patients with and without shedding of vp mrna revealed an association of active viral replication with diarrhea (or = . ; % ci = . - . ; p = . ) and a clinical diagnosis of urti (or = . ; % ci = . - . ; p = . ). the frequencies of diarrhea and diagnosis of urti in patients shedding vp mrna were %, while in patients without detectable vp mrna they were respectively % and . % (table ). the results of this cross-sectional study of hbov in ari patients from ribeirão preto, brazil, indicate that shedding of vp mrna in respiratory secretions, as a marker of hbov replication, correlates positively with high viral load, presence of diarrhea, and lack of co-infection by other respiratory viruses. during the last five or six years since its discovery, hbov has been detected in association with respiratory and gastrointestinal infections, mostly of children, in many countries in all continents [ , ] . rates of detection of hbov in ari studies have varied from . % to . %, depending on patient age, case definition, and geographic location [ , ] . in the present study, the overall detection rate of hbov in ari patients was . %, similar to rates in other regions of the world. however, it should be kept in mind that this study, like most others published on hbov, was a hospital-based surveillance, subject to selection bias. therefore, rates of hbov in ari in the community at large could be different. to the best of our knowledge, this has been the largest study of hbov in ari conducted in brazil and south america. a few previous studies conducted in the region reported hbov infection rates of . % to . % in patients with ari [ , , , ] and in . % of patients with gastroenteritis [ ] . importantly, the present study revealed a significantly higher rate of concurrent diarrhea in patients with ari positive for hbov as compared to those with ari but without detectable hbov, suggesting a causative role for hbov in gastrointestinal manifestations observed in ari patients. in general, parvoviruses replicate more efficiently in tissues with high cellular turnover, such as respiratory and digestive epithelia [ ] . several members of the family parvoviridae are known to cause gastroenteritis, such as canine parvovirus (cpv), feline panleukopenia virus (fpv) and canine minute virus (cnmv). in addition studies previously published by others have reported hbov detection in stools from patients with gastroenteritis [ ] . although the mechanisms of pathogenesis and routes of infection of hbov are not yet understood, some information is available for bovine parvovirus (bpv), another member of the genus bocavirus. bpv causes respiratory and enteric diseases in calves following initial replication in the tonsils and intestinal epithelium, from where it spreads into the blood, lymphoid tissues and respiratory epithelium. bpv induces atrophy of microvilli in the small intestine and death (by apoptosis and necrosis) of lymphoid cells and ciliated cells of the respiratory epithelium [ ] . it is interesting that novel species of hbov (hbov , and ) associated with gastrointestinal infections have recently been identified [ , , ] and two of those have been detected in stools from brazilian patients with acute diarrhea, at rates varying from . % for hbov to . % for hbov [ ] . however, a causative role for hbov in human diarrhea still awaits confirmation [ ] . interestingly, in this study a trend was noted for an increase in respiratory infections, positivity for hrsv and hbov, in association with drier and cooler months. while the reasons for this are not entirely understood, one possibility is that the increased respiratory virus activity in march and april could be favored by the initiation of school term one or two months before, when assembly of susceptible children could provide conditions for viral spread. the frequency of detection of hbov in was greater than three times that observed in the two following years, evidence that circulation of this agent in this region of brazil may vary greatly from year to year, in a way similar to that previously documented in italy [ ] . even though hbov has been regarded as an infectious agent present worldwide, its pathogenic role in respiratory illness is still debatable. this virus is frequently detected in co-infection with other respiratory viruses of well-established pathogenic role [ , ] . in addition, hbov is fastidious to propagate in conventional cell cultures and an experimental infection model is lacking, what limits advances in understanding pathogenesis. therefore, the search for an association of hbov infections with possible molecular markers of active viral replication is a useful approache to investigate pathogenesis of this emerging virus. in the present study, hbov dna was detected by pcr in samples from ari patients, but not in nasopharyngeal secretions collected from asymptomatic children approximately years before ( ) ( ) in the same hospital. the significance of this finding is limited by the relatively small number of control samples available for testing, and by the fact that they were collected years before. however, the lack of detection of hbov dna by a sensitive pcr assay in samples collected from children carefully scrutinized for the presence of respiratory symptoms, adds support for a pathogenic role of hbov in ari, in agreement with previous studies [ , , ] . high frequencies of co-infection by other respiratory viruses in patients with ari and hbov were confirmed in this study with a rate of . % ( of ) of co-infections, mostly caused by hrsv, hrv, hmpv and hadv, and frequently by more than one additional agent. this was similar to the % reported by allander et al., in sweden [ ] , and higher than the % reported by esposito et al., in italy [ ] . while viral co-infections have long been recognized in ari studies, the advent of highly sensitive pcr assays magnified their observation. in the particular case of hbov, frequencies of co-infections have consistently been reported to be higher than those observed for other respiratory infections, reaching rates similar to those reported for hadv [ ] , a tendency confirmed by the present study. yet unproven, this may suggest that long term shedding of hbov dna, unrelated to current illness may regularly occur, as it is known to happen with hadv [ ] . indeed, hbov dna was found by pcr in lymphocytes from palatine tonsils of % of children undergoing tonsillectomy [ ] , suggesting that this virus can establish latent or persistent infection in pharyngeal lymphoid tissue. another important piece of information befitting a systemic nature of hbov infections is the finding of viremia, reported by tozer et al. in children with ari [ ] . viremia was also reported by allander et al [ ] in hbov-exclusive infections, a feature strongly suggestive of a causal role for this agent in acute symptomatic infections. in addition, those authors reported that patients with hbov without concurrent infections by other respiratory viruses, tended to shed higher hbov loads [ ] . in this regard, an association of high viral loads in npas with hbov acute infections, as evidenced by igm seroconversion in wheezing children, was clearly documented in a study conducted in finland by söderlund-venermo [ ] . these results prompted us to determine hbov loads by qpcr in all hbov-positive npas of the study. the results revealed hbov viral loads to be significantly higher in patients without other viral co-infections than in those with multiple viruses detected. moreover, rates of diarrhea were significantly higher in patients with very high hbov viral loads in their npas. such finding, in the absence of respiratory viral coinfections, suggests a causal role for hbov in the genesis of the diseases presented by these patients. nevertheless, detection of a direct evidence of active replication of hbov in the secretions from patients was required to strengthen the causality link between the virus and disease. in order to do that, an assay to detect hbov vp mrna by real time rt-pcr in nasopharyngeal samples was developed. production of vp mrna is unequivocal evidence of hbov replication, but its shedding in nasopharyngeal secretions does not indicate the possible sources of virus production. because rna is very highly unstable in ribonuclease-rich respiratory secretions, one can speculate that vp mrna more likely originates from disruption of sloughed whole hbov-infected epithelial cells directly into the rnaseinhibiting extraction reagent. unfortunately, preparation of cytology slides that would have enabled detection of hbov on sloughed cells by in situ hybridization was not possible given the nature of the study. the presence of extremely high viral loads of hbov in secretions, argues in favor of abundant sources of virus, either on or adjoining the mucosal surfaces. this observation alone highlights the extremely productive replication levels reached by hbov in the permissive upper respiratory tract. however, in situ studies will be needed to provide definitive localization of viral replication sites. to the best of our knowledge this is the first report of an assay for mrna to confirm replication of hbov in clinical samples, and its application revealed that shedding of vp mrna in nasopharyngeal secretions was associated with high viral loads, presence of concurrent diarrhea, and lack of demonstrable coinfections by other respiratory viruses. conversely, in the absence of a marker of current viral replication, hbov was also often detected in nasopharyngeal secretions, but in much lower viral loads and frequently associated with shedding of other pathogenic respiratory viruses. it should be pointed out that vp mrna was found only in % of the hbov infected ari patients, with a significant part of whom with diarrhea. this may be interpreted to mean that only one quarter of the hbov respiratory infections are associated with genuinely acute viral replication, likely involving the gastrointestinal tract. conversely, in most cases of hbov detection in respiratory secretions, it may be the result of viral persistence/latency, with co-detection of another viral agent. a retrospective cross-sectional study was carried out with nasopharyngeal aspirates (npas) collected from patients with ari ( males and females) with month to years of age (median age of months). the samples were from outpatients and inpatients brought for health care at the hospital of the university of são paulo school of medicine, ribeirão preto, sp, brazil, from january through december . the study included all samples from patients with symptoms of ari seen at any of the hospital clinics and wards, submitted to the virology laboratory for testing. the vast majority of the samples had been originally sent to the laboratory for respiratory syncytial virus and influenza screening and no exclusion criteria were applied. clinical information obtained retrospectively from patient records upon discharge was entered on study forms. clinical data collected included the presence of wheezing, cough, fever, sneezing, dyspnea, coryza, nasal obstruction, diarrhea, requirement for oxygen therapy and length of hospitalization. npas were collected in saline solution from all patients [ ] and sent on ice to the virology laboratory within four hours. in addition to the samples from ari patients, a convenience set of control samples from patients without any respiratory symptoms who were participants in a previous study [ ] were used as negative controls. this group consisted of males, with median age of . months, mean of months (sd = months). after routine virology sample processing, remaining samples were split into backup aliquots, including two in trizol reagent (invitrogen carlsbad, ca), which were kept in storage at uc [ ] . this study was approved by the internal review board of the hospital das clínicas de ribeirão preto (hcfmrp-usp) under approval number / . all patients or the parents or guardians of the patients in the study signed an informed consent form. illnesses presented by all patients in this study were classified as lower respiratory tract infections (lrti) and upper respiratory tract infections (urti), with or without acute otitis media (aom) and gastro-esophageal reflux disease (gerd). patients with urti had only symptoms of the upper respiratory tract, such as coryza, sneezing, nasal obstruction and sore throat, without wheezing, and dyspnea, with or without fever. patients with lrti had diagnosis of bronchiolitis, bronchiolitis obliterans, bronchodysplasia or pneumonia. they had wheezing and/or dyspnea, with or without fever, cough, coryza, sneezing or nasal obstruction. all patients were assigned to a single clinical definition, and could not be allocated to more than one group. in order to assess disease severity, a numeric index of clinical severity (ics) was adapted from walsh et al ( ) [ ] , based on the presence of clear-cut criteria that indicate higher risk for severe disease: wheezing, requirement for hospitalization, oxygen therapy and positive airway pressure. for all patients the ics was obtained by attributing point each for the presence of wheezing, requirement for hospitalization, length of hospital stay longer than days, requirement for o , and use of o for longer than days; and extra points attributed in case of requirement for positive airway pressure, for a maximum ics equal to . dna and rna were extracted from ml of npa using, respectively, wizardh genomic dna purification kit (promegah, madison, wi, usa) and trizolh (invitrogenh, carlsbad, ca, usa), following manufacturers protocol. detection of hbov and other respiratory viruses was conducted by conventional pcr, as previously described [ ] . for detection of hbov and human adenovirus (hadv), ng of extracted dna was used, whereas for other respiratory viruses, including human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), human rhinovirus (hrv), human influenza virus (flu), human parainfluenza viruses (hpiv) and human coronaviruses (hcov), pcr was done with cdna generated by reverse transcription (rt) of mg of rna, primed with pmol of random hexamers, using improm-ii reverse transcriptase (promega, madison, wi) following manufacturer's instructions. prior to reverse transcription, rna was denatured at uc for minutes, then immediately placed on ice while reverse transcriptase was added and then incubated at uc for hours. pcr was done with ng of dna, pmol forward and reverse primers (table s ), mm mgcl , mm deoxynucleoside triphosphate (dntp), . ml of supplied x buffer, u of taq dna polymerase (invitrogen carlsbad, ca) and water to complete the volume of ml. the cycling conditions were denaturation at uc for min, followed by cycles of uc for min, uc to uc for min, uc for min, and then a final extension step of uc for min [ ] . all pcr products were analyzed by electrophoresis in ethidium bromide-stained . % agarose gels. pcr products of all tested viruses were cloned into the plasmid pgem-t easy (promega, madison, wisconsin) and used to determine detection limits for each assay. all clones were sequenced with bigdye terminator v . cycle sequencing (applied biosystems, foster city, ca) using the abi prism genetic analyzer. the detection limits for the pcr assays were determined by testing serial decimal dilutions of the clones, and results varied from to copies of plasmid dna. the limit of detection for hbov was determined to be copies. all applicable measures to prevent contamination of pcr reactions were taken in this study. the quantitative pcr (qpcr) for hbov was targeted to the viral np gene according to previously published procedures [ ] . briefly, the reaction was performed in triplicate in a final volume of ml, containing ng of template dna, ml of taqman universal pcr master mix (applied biosystems foster city, ca), nmol/l of each primer and nmol/l of probe (table s ). the amplification was performed on a real time pcr system (applied biosystems foster city, ca) with the following settings: uc for min, uc for min and cycles of uc for s and uc for min. a standard curve of amplification was produced using serial decimal dilutions of a plasmid (pgem-full hbov) in which a fragment of the hbov np gene ( nt to nt) was cloned. a positive qpcr for hbov was considered when the threshold was reached before the th cycle with fluorescence count higher than . . the detection limit was . copies of hbov plasmid, the slope and the r of the standard curve were , and , , respectively. all qpcr results were normalized by the amplification of the b-actin gene, included in duplicate in all tested batches, using nmol/l of each primer and nmol/l of the probe (table s ). with this approach, viral loads were determined as the number of copies of hbov dna per ml of npas. shedding of hbov vp mrna was tested by real-time rt-pcr in npas to ascertain the presence of active viral replication, as opposed to shedding of viral genome. to ensure target-specific amplification total rna extraction products were treated with dnase i (invitrogen carlsbad, ca) h prior to pcr. as a control, the same pcr was done in parallel directly with the rna extraction product without previous rt. samples were considered positive for hbov vp mrna only when they were simultaneously negative for amplification directly from the extracted rna without previous rt. rt was performed on mg of rna extracted by trizol using pmol of random primers, according to the protocol provided by the manufacturer. after rna denaturation at uc for min reverse transcriptase was added and incubation went on for h at uc. pcr was then carried out with ng of cdna, with nmol/l of each primer (vp -f and vp -r), nmol/l of the probe (vp -s, table s ), and ml of taqman universal pcr master mix (applied biosystems foster city, ca). amplification was done using the real time pcr system (applied biosystems foster city, ca) with the following settings: uc for min, uc for min and cycles of uc for s and uc for m. all samples, including all cdnas and rnas pre-treated with dnase, were tested by qpcr for b-actin mrna, following the protocol described above. samples found to be positive for b-actin by amplification directly from the rna not pre-treated with dnase, were considered to be false positives. as above mentioned, positive reactions were considered when the threshold was reached before the th cycle, with a fluorescence higher than . . the detection limit of this assay was found to be copies of dna, as determined using the decimal serial dilutions of viral cdna obtained from in vitro transcription of a plasmid containing thevp gene, according to manufactures' instructions (riboprobe in vitro transcription systems, promega madison, wi). correlation between positivity for hbov and clinical conditions was assessed by the chi-square test. statistical analysis was conducted using the bioestat . software (cnpq, brazil). logistic regression was done to examine the relationship of hbov shedding (including positivity for hbov dna, hbov loads, lack of other viral co-infections and detection of hbov vp mrna) and clinical data adjusted for age, gender and detection of hrsv in the same samples [ ] . to analyze the hbov loads obtained by qpcr, data in two unpaired groups of patients were compared with the mann-whitney test. these analyses were performed using the sash . software (sas institute inc, raleigh, nc). a p value of # . was chosen for significance. table s primers and probes used in conventional and real-time pcr for respiratory viruses and b-actin. 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realtime pcr for diagnosis of human bocavirus infections and phylogenetic analysis on the existence of maximum likelihood estimates in logistic regression models the authors thank the expert technical support of maria lucia silva, ariane matioli saranzo and luana sella delcaro. we thank also the staff responsible for the collection of the clinical samples on hcrp and the ''centro de métodos quantitativos'' (cemeq-fmrp), particularly mayara piani luna da silva sicchieri, for the statistical analysis and suggestions. key: cord- - m m authors: wishaupt, jérôme o.; versteegh, florens g.a.; hartwig, nico g. title: pcr testing for paediatric acute respiratory tract infections date: - - journal: paediatr respir rev doi: . /j.prrv. . . sha: doc_id: cord_uid: m m acute respiratory tract infection (ari) is a frequently occurring disease in children. it is a clinical diagnosis for which no internationally accepted diagnostic test is available. the majority of ari is viral in origin, though diagnostic tests for viruses were rarely performed in the past. in the past decades, new molecular techniques have been introduced in many hospitals. they are capable of generating a high yield of viral and bacterial diagnoses, but their impact upon clinical practices is still questionable. in this paper, we discuss the difficulties of diagnosing ari in children, the indications for conventional and new diagnostics and their implications. in developed countries, acute respiratory tract infection (ari) is the most common cause for hospitalization of young children [ ] . a rapid and accurate diagnosis is required to start adequate therapy, minimize hospital admissions and length of hospital stay and reduce unnecessary antibiotic use. clinical decision making in ari is based on interpretation of clinical signs and symptoms, often supported with results of laboratory parameters such as c-reactive protein and white blood count combined with conventional viral and bacterial tests. in addition, new molecular tests have been introduced in many hospitals. however the role (and impact) of these molecular tests in clinical decision making is not known. in this paper, we discuss the role of molecular diagnostics in relation to patient care in paediatric ari. the anatomic approach to ari creates categories: upper respiratory tract infection (urti) and lower respiratory tract infection (lrti). urti is confined to the ear, nose and throat region and is considered to be a rather harmless disease for which medical attention is generally restricted to family care. lrti on the contrary, which involves the bronchi, bronchiole and the lung tissue, is considered a more severe disease that should be seen by a paediatrician. pneumonia is thought to be more likely caused by bacteria for which prompt initiation of antibiotics is required. however, in paediatric patients this clear distinction between urti and lrti is difficult to make and appears not useful for clinical decision-making. in infants, classical lrti-symptoms like tachypnea or hypoxia are also common in paediatric urti due to nasal obstruction and mucus plugging of the anatomically small upper airways. general symptoms such as poor feeding, lowered alertness and low transcutaneous oxygen seem to influence the clinical course more strongly than the designated anatomical site of infection [ ] . based on pathophysiologic mechanisms, lrti can be subdivided in pneumonia and bronchiolitis. pneumonia reflects inflammation of lung tissue (bronchi, bronchioles and alveolar tissue) in response to infection by bacterial, viral or other pathogens, while bronchiolitis primarily affects the small airways (bronchioles) and is usually of viral origin only. the clinical presentation of pneumonia overlaps substantially with that of bronchiolitis and vice versa. pneumonia and bronchiolitis are clinical diagnoses without an internationally standardized test or definition [ , ] . though chest radiographs are often used in clinical settings, they cannot distinguish between these conditions [ ] . there have been attempts to distinguish viral from bacterial ari because of its therapeutic consequences. most paediatric ari are of viral origin and the risk of concurrent (or subsequent) bacterial infection has been reported to be low in children over three months of age [ ] . it is assumed that antibiotics are prescribed too often for these children [ ] . this is in part explained by a high level of similarity in clinical symptoms between viral and bacterial lrti. respiratory syncytial virus (rsv) pneumonia and serologically detected pneumococcal pneumonia did have considerably overlapping clinical signs and symptoms, whereas laboratory and chest radiography findings significantly differed [ ] . the majority of paediatric patients with ari are young and previously healthy children with no known risk factors for acquiring respiratory infection. assessment of disease severity in these children is based on patient's history, parental concern about the health of their child and objective findings by physical examination. different clinical scoring systems are in use, adapted to local circumstances and patient groups. examples are: the respiratory distress assessment instrument, based on wheezing and retractions [ ] , the (bedside) paediatric early warning sign scores [ ] [ ] [ ] , the preschool respiratory assessment measure, [ ] the clinical scoring system described by kristjansson [ ] , the silverman-anderson respiratory scale [ ] and the disease severity score by gern [ ] . however, none of the above scoring systems has been validated specifically for prediction of clinical outcome in paediatric ari. this diversity in scoring systems limits comparison of study data in the literature. assessment of respiratory illness in children with underlying conditions is often more difficult. known risk factors for severe bronchiolitis are best recognized for rsv. they include young age, prematurity, low birth weight, down syndrome, congenital heart disease, bronchopulmonary disease and immunodeficiency [ , ] . other underlying conditions that lead to higher hospitalisation rates are cerebral palsy, chronic lung diseases like cystic fibrosis, asthma and recurrent respiratory tract infections. use of viral diagnostics in these patient disease categories lies outside of the scope of this article. as stated above, diagnosing paediatric ari and assessment of disease severity is primarily based on clinical grounds. for borderline cases, laboratory results are often used in clinical decision making. since real time polymerase chain reaction (rt-pcr) assays have been widely introduced in many hospitals, we will discuss its impact and role in clinical decision making for paediatric ari. the pediatric infectious disease society (pids) and the infectious diseases society of america (idsa) recommend in their guideline 'community-acquired pneumonia (cap) in infants and children' the use of sensitive and specific tests for the rapid diagnosis of influenza virus and other respiratory viruses in the evaluation of children older than three months of age with cap [ ] . in the case of a positive test for influenza, they strongly recommend that no antibiotic therapy be employed in the absence of clinical, laboratory or radiographic findings suggestive for bacterial co-infection. no specific recommendation is given on antibiotic use when other viruses are detected. currently, there are no (p)idsa guidelines for paediatric ari in patients younger than three months. the american association of pediatrics (aap) recommends clinicians diagnose bronchiolitis and assess disease severity on the basis of history and physical examination without routinely ordering laboratory tests or radiography to make the diagnosis (evidence level b) [ ] . clinicians should assess risk factors for severe disease (age less than weeks, prematurity, underlying cardiopulmonary disease or immunodeficiency) (evidence level b). repeated observations over a short period of time will improve overall assessment and patient care. in a state of the art review in , this aap recommendation is supported by evidence of a low rate of bacterial co-infection in children younger than three months of age presenting with bronchiolitis [ ] . however, rates of rsv testing did not fall after publication of the guidelines [ ] . the authors suggests that hospitals continue to test for rsv in order to cohort patients after admission. the guidelines of the royal college of paediatrics and child health (rcpch) and the european society of paediatric infectious diseases (espid) recognize that rapid, sensitive and specific immunofluorescence viral tests are available and that rt-pcr is increasingly replacing immunofluorescence and serology, but they do not give recommendations when to use it and what the consequences are of the results when they become available [ ] . until the 's, viral testing was not routinely available in paediatric clinical practice. viral cultures were the gold standard, but they require specialized laboratory facilities and are timeconsuming. therefore, results are usually available too late to influence patient management. serology is more easy to implement, but requires two separate blood samples over time to show (preferably) a four-fold increase in antibody response. blood sampling is not a child-friendly procedure and the results are also not readily available. the use of serology seems restricted to epidemiologic studies in order to maximize etiologic diagnosis [ ] . rapid antigen tests like direct immunofluorescent antibody tests (dfa) provide more rapidly results, but are less sensitive than viral cultures [ , ] . they are available for rsv and influenza virus, but sensitivity ranges from . % to . % [ ] [ ] [ ] . according to earlier studies, rapid viral tests contribute to reduction in hospital stay and antibiotic use [ , [ ] [ ] [ ] . since the 's pcr techniques have become more widely available. conventional endpoint single-target pcrs were able to report positive and negative results of rna and dna viruses. later on, computerized quantitative rt-pcrs were able to correlate the amount of pcr product to a viral load. testing a panel of respiratory viruses within a time span of to hours has become routine practice in many hospitals. in paediatric populations, the sensitivity has shown to be higher than for viral culture [ ] . nowadays, qualitative and even quantitative multiplex pcrs are able to detect multiple respiratory viruses in one single sample simultaneously, however sensitivity in a multiplex pcr is generally lower than in single target rt-pcr [ ] . a disadvantage of commercially available multiplex pcrs is that the tests require external controls, which are difficult to perform since not all available platforms publish their targets [ ] . despite the availability of commercially quantitative multiplex pcrs, in-house quantitative single target rt-pcrs are used more frequently in current laboratory practice. in a recent review by jartti et al on new molecular virus detection methods, all nasopharyngeal sampling tests, including nasopharyngeal aspirates, washes, swabs or brush appear suitable for pcr analysis. viruses in the upper airways do reflect infections of the lower airways [ ] . sputum induction methods, trying to generate sputum samples from the lower respiratory tract are not recommended for routine use as the diagnostic yield will not significantly improve. introducing highly sensitive viral diagnostics has yielded a broad spectrum of viral diagnoses, but has not automatically lead to changes in patient management. in an adult population, implementation of rt-pcr in lrti increased the diagnostic yield, but did not reduce antibiotic use or costs [ ] . in a multicentre paediatric study, interviewing medical doctors on fictitious ari cases, rt-pcr decreased antibiotic use. however, in real life, the same physicians did not alter their antibiotic prescriptions based on the results of rt-pcr [ ] . in a cochrane review ( rct's) that evaluated the impact of rapid viral tests in children with ari in an emergency department (ed), rapid viral testing by immunofluorescence or pcr did not lead to changes in antibiotic use, length of ed visits, blood or urine testing, but did lower the rates of chest radiographs. the authors stated that routine viral testing in the ed is promising as a means to reduce antibiotic use, but there is yet insufficient evidence to support this. large trials are needed with a focus on patient management with respect to the results [ ] . in a recent retrospective study evaluating clinical differences between rsv and non-rsv patients, virological testing (enzyme linked immunoassay and / or pcr) did not help in management decisions and seemed insufficient to predict outcome at an individual level [ ] . in another recent retrospective study of children with ari in a general hospital, antibiotic management was not influenced after detecting a viral respiratory pathogen, although the authors state that routine testing of common respiratory pathogens could lead to a better understanding of their role in disease in children with respiratory symptoms [ ] . in a year prospective study of children with community acquired pneumonia (cap), designed to describe the frequency of respiratory viruses, antibiotics were prescribed less frequently in viral positive versus negative children, but only when they were > months old [ ] . our own data from a multicentre prospective controlled clinical trial of previously healthy children with respiratory symptoms showed a high diagnostic yield of rt-pcr, however the rapid communication (within hours) of results to the paediatrician did not change patient care versus those who received results later [ ] . these findings underlie the statement of the aap, not to recommend routine viral testing for standard ari cases, unless the physician is willing to change his patient management based on the results [ ] . at present, a panel of approximately respiratory rt-pcr assays is available in many hospitals. data suggest that rsv has the greatest disease burden both in hospitalized children and in outpatients, especially in children under five years of age [ ] . furthermore, human rhinovirus (hrv) is generally associated with the common cold, can cause severe ari as well and is the most common pathogen in ari in young children [ , ] . re-infections with hrv are commonly observed, and are usually caused by different virus strains. [ ] as reviewed by kim, recent studies suggest that hrv subtype c may be more virulent than other hrv [ ] , but not all rt-pcrs are capable of distinguishing between the different hrv strains. influenza virus (iv) is well known for its seasonal outbreaks of ari during a few weeks in winter, and also for a sepsis like syndrome without respiratory symptoms. human adenovirus (hadv) is common in respiratory disease with a severe disease course in children with underlying immunodeficiency [ ] . para-influenza viruses (piv) subtypes and are known to cause croup and subtype is known to cause bronchiolitis and pneumomia [ ] . piv subtype is has been reported to be a much less frequent cause of ari [ ] . the incidence of polyomaviruses wu [ ] and ki [ ] is low and their clinical relevance remain unclear and require further evaluation [ ] . the role of newly discovered viruses is of interest in many studies. some of these 'new' viruses are human metapneumovirus (hmpv), human bocavirus (hbov) and human coronavirus (hcov). hmpv was first detected in [ ] , and its clinical symptoms overlap with those of other respiratory viruses [ ] . disease severity seems less than for rsv [ ] . another 'new' virus is hbov, discovered in [ ] . in a review by brodzinsky, the incidence is found to be low ( . - . %) and rates of co-infections are high ( - %) [ ] . the spectrum of disease is similar to rsv and hmpv. the group of hcov's are heterogeneous. hcov e [ ] , oc , [ , ] nl [ , ] and hku [ ] are recognized frequently in young age and are correlated with less severe respiratory disease [ , ] . sars coronavirus and the novel mers coronavirus [ ] are not routinely incorporated in respiratory rt-pcr test panels. a recent swedish study compared viral pcr findings from children with ari versus asymptomatic matched controls. rsv, hmpv and pivs were highly overrepresented in symptomatic patients, suggesting that they are responsible for illness. asymptomatic controls showed high detection rates of hbov, hrv, hadv, hcov and enterovirus, suggesting that prolonged virus-shedding may occur and that pcr-results need to be interpreted with caution [ ] . the incidence of mixed viral infections is reported as high as - %, depending on different populations and test panels [ ] . in a multicenter study, involving children less than years of age, the incidence of mixed infection in children hospitalized for bronchiolitis was % [ ] . several circumstances can generate positive rt-pcr results. rt-pcr assays are very sensitive and can detect small amounts of viral nucleic acids, which are still present during a convalescence period. for each individual virus it is not known how long dna/rna shedding may continue during this convalescence period [ , ] . furthermore, children with a normal immune system can asymptomatically harbor viruses in their respiratory tract. usually, a primary viral infection leads to an adaptive immune response with induction of memory t-cells, so that a second hit with the same virus usually results in less serious or even absent symptoms [ ] . the relevance of finding only a single pathogen is also of interest. rt-pcr assays are sensitive tests, but detect only pathogens that are looked for. for each micro-organism a specific primer is used, therefore non-viral pathogens like bordetella pertussis, mycoplasma pneumonia and chlamydophyla pneumonia are not detected unless they are looked for. they can mimic viral disease as well, especially in young children [ ] . one could hypothesize that mixed infections may lead to a more severe disease. papers on this subject show somewhat contradictory results. there are a few reports suggest that there is no relation between mixed viral ari and disease severity [ ] [ ] [ ] [ ] , while others indicate a higher disease severity in children with a mixed respiratory infection [ , ] . further research as to how one should interpret multiple, positive rt-pcr results in one single sample is needed. quantitative studies with interpretation of viral load will possibly help to answer this question. epidemiologic surveillance programs benefit from viral rt-pcrs. examples include the worldwide search for sars and mers coronaviruses, well known for their febrile and atypical pneumonia and the dramatic course in some affected people [ , [ ] [ ] [ ] [ ] . other national and who-supported surveillance programs include seasonal influenza viruses. currently, the 'bird flu' influenza a subtype h n is of special interest for its infection of humans and possible easy transmission from wild birds to humans [ ] . prevention of nosocomial infections should be of high priority in any hospital and isolation or cohorting is a well-established policy. rt-pcr has shown to be a useful tool for epidemiologic studies. however, the epidemiology of many nosocomial respiratory infections is not well known. for rsv, the incidence of nosocomial spread seems low. although information about management of outbreaks is sparse and not well studied, normal prevention measurements appear to be rational [ ] . for cohorting, rapid testing for rsv has shown to be a safe, cost-effective and efficient way to improve bed management [ ] . although we do not doubt that isolation as strategy helps to prevent nosocomial infections, we still have concerns when cohorting is based on rt-pcr results of only a limited number of viruses. mixed infections occur in a substantial proportion of children and whether these children spread other non-detected viruses during cohorting is not yet clear. rapid antigen tests like dfa are limited useful for cohorting issues, since they may have a high proportion of false negatives. rt-pcr obviously is much more sensitive and specific, but the turnaround time is not sufficient to act in time for infection control measurements at admission. theoretically, one would expect that rt-pcr would be beneficial for infection control, however no large study has demonstrated a positive effect to date. the risk of cross-infection in children sharing a room was studied recently in a prospective observational cohort study of children with bronchiolitis. room sharing between rsv-positive and rsvnegative children on the first day of admission did not influence the risk of co-infection [ ] . children with respiratory failure induced by viral infection and admitted to paediatric intensive care units for mechanical ventilation frequently have concomitant bacterial infections [ ] . use of an extended rt-pcr panel of respiratory pathogens seems to be justified in those conditions to determine the causing micro-organisms. however, it is unclear what the exact relevance of the rt-pcr results is, except for those viruses for which treatment is available (like influenza and adenovirus) children with underlying immunodeficiencies are susceptible to severe complications of viral infections. indications for rt-pcr diagnostics in children with hematologic malignancies, hematopoietic stem cell transplantation, solid organ transplantation, premature infants and children with cystic fibrosis lies outside the scope of this article and was well reviewed by vallieres and renaud recently [ ] . an objective laboratory parameter, reflecting disease severity, would be helpful to estimate disease course in an early stage. viral load was thought to serve as such a parameter. viral load is the quantity or copy number of viral rna or dna detected per milliliter body fluid. in rt-pcr, the cycle threshold (ct) value is defined as the number of rt-pcr cycles required for a positive fluorescent amplification signal to cross the threshold. this is inversely correlated with the viral load. some authors indeed found a significant correlation between viral load and disease severity [ ] [ ] [ ] , thereby justifying broad use of rt-pcr. however viral load does not always correlate well [ ] [ ] [ ] [ ] [ ] . some viruses, like rsv, are short lived and others like hbov are long-lived, which is a complicating factor in demonstrating this relationship. the question how well disease severity is correlated with viral load is not answered yet. paediatric ari is a clinical diagnosis and most infections are caused by viruses. use of clinical scoring systems helps to assess disease severity for the individual patient and could be used for clinical management. thus far, an extended panel of rt-pcr assays contributes little to clinical decision making for the majority of children with ari; in fact rt-pcr for detecting respiratory infection is not routinely available in many hospitals. in today's paediatric practice, rt-pcr is used in patients with high-risk of complications or with an unexpected disease course. the panel of microorganisms should at least include pathogens for which a specific treatment is available, e.g. influenza virus and bordetella pertussis. the authors have no conflicts of interest to disclose. rt-pcr will help to explore the role of certain viruses in ari with mixed viral aetiology, to discover new viruses in future, and possibly to assess disease severity by repeated viral load quantification. rt-pcr is a powerful tool in public health surveillance. the role of rt-pcr in hospital hygiene is currently limited. infectious disease hospitalizations among infants in the united states clinical findings and severity of acute bronchiolitis a systematic review on the diagnosis of pediatric bacterial pneumonia: when gold is bronze the definition of pneumonia, the assessment of severity, and clinical standardization in the pneumonia etiology research for child health study evaluation of the utility of radiography in acute bronchiolitis rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital clinical profile of serologically diagnosed pneumococcal pneumonia wheezing in infants: the response to epinephrine the 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hospitalized infants: association between viral load, virus subgroup, and disease severity correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection prospective study of human metapneumovirus infection: diagnosis, typing and virus quantification in nasopharyngeal secretions from pediatric patients quantitation of respiratory viruses in relation to clinical course in children with acute respiratory tract infections illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus we thank p. goswami, md for critically reviewing the manuscript in the english language. key: cord- -kheek lx authors: carroll, kecia n.; gebretsadik, tebeb; minton, patricia; woodward, kimberly; liu, zhouwen; miller, e. kathryn; williams, john v.; dupont, william d.; hartert, tina v. title: influence of maternal asthma on the cause and severity of infant acute respiratory tract infections date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: kheek lx background: respiratory syncytial virus (rsv) and rhinovirus infections are the most common significant infant respiratory tract illnesses and are associated with increased but differential risks of childhood asthma. objective: we sought to determine whether maternal asthma is associated with higher odds of infant respiratory tract infection with rhinovirus versus rsv and increased infection severity. methods: mother-infant dyads were enrolled from - during an infant respiratory tract infection ( with rhinovirus and with rsv). mothers were classified into mutually exclusive groups (atopic asthma, nonatopic asthma, and no asthma). we determined viral cause using pcr and the severity of the infant’s respiratory tract infection using the bronchiolitis severity score. adjusted relative odds of maternal asthma with viral cause were calculated by using logistic regression. proportional odds models assessed the association of maternal asthma and infant infection severity. results: infants with a mother with atopic asthma compared with infants whose mothers did not have asthma were more likely to have rhinovirus versus rsv infection (adjusted odds ratio, . ; % ci, . - . ). similarly, among infants with rhinovirus, having a mother with atopic asthma was associated with increased infection severity (adjusted odds ratio, . ; % ci, . - . ). this relationship was not seen among infants with rsv. conclusions: clinically significant rhinovirus infection during infancy was more strongly associated with having a mother with atopic asthma than clinically significant rsv infection. having a mother with atopic asthma was associated with increased severity of infant rhinovirus but not rsv infections. infants with rhinovirus were more likely to have a familial atopic predisposition, which might partly explain the subsequent increased asthma risk. bronchiolitis, a lower respiratory tract infection (lrti) commonly caused by respiratory syncytial virus (rsv) and less commonly by human rhinovirus (hrv), affects an estimated % to % of children in the first year of life and is a leading cause of hospitalization during infancy. [ ] [ ] [ ] [ ] in addition to the acute morbidity seen with bronchiolitis, infants hospitalized with bronchiolitis and young children who experience virus-induced wheezing illnesses are at increased risk of recurrent wheezing and asthma in early childhood. [ ] [ ] [ ] [ ] [ ] the pathogenesis of the increased wheezing after viral bronchiolitis is not fully understood. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in efforts to learn whether children at risk of bronchiolitis are also at increased risk for asthma, studies have investigated whether a family history of asthma is associated with the severity or incidence of bronchiolitis during infancy, with some prior studies finding an association, whereas others did not. , , [ ] [ ] [ ] [ ] prospective birth cohorts in which all children have a familial predisposition to asthma, such as a cohort based in perth, australia, and the childhood origins of asthma (coast) cohort, have investigated the association of a viral cause of infections in early life and subsequent wheezing and asthma. , , in the coast cohort hrvinduced wheezing illnesses in early life were found to be stronger predictors of wheezing and asthma at age years than rsvinduced illnesses. , , however, it is not known whether infants with symptomatic hrv infections that lead to an unscheduled health care visit are more likely to have a familial predisposition to asthma than infants with symptomatic rsv infection. in this investigation that included mother-infant dyads enrolled in the tennessee children's respiratory initiative (tcri), we tested the hypothesis that a familial atopic predisposition was associated with viral cause and increased severity of viral acute respiratory tract infection (ari) during infancy. the tcri is a prospective cohort of term (> _ weeks), non-low-birth-weight (> _ g) infants and their mothers designed to investigate the association of characteristics of infant viral aris, such as severity and cause, and familial atopic predisposition on the development of early childhood asthma and atopy. this investigation included the mother-infant dyads in which the infant presented for an unscheduled clinic or emergency department visit or hospitalization and was determined to have a sole hrv-or rsv-induced ari. mother-infant dyads were enrolled at the time of an infant ari during viral respiratory seasons, september through may - . dyads were recruited in the inpatient, emergency department, and clinic settings at a single academic institution, and the children are currently being followed longitudinally through age years to ascertain asthma and atopy outcomes. each woman provided written informed consent for participation of herself and her infant. the vanderbilt university institutional review board approved the study. during study enrollment, research nurses administered an in-person structured questionnaire that included questions regarding demographics, the infant's home environment, the index infant's illness, previous medical history of the infant, and detailed family asthma and atopic disease history, including maternal responses to the international study of asthma and allergies in children questionnaire. at enrollment, research nurses obtained nasal and throat swabs from the infants for viral detection. through a structured medical chart review, information was abstracted regarding the infant's medical visit, including birth weight, room air pulse oximetry, requirement for supplemental oxygen, history of prior wheezing, and detailed medical information. final discharge diagnoses were obtained through chart review after discharge. self-reported maternal asthma status was defined as a positive response to the question ''have you ever had asthma?,'' which was asked as part of the international study of asthma and allergies in children questionnaire and/or to the question ''were you diagnosed with asthma as a child?'' , maternal atopy was determined based on skin prick test results or allergen-specific ige levels. preferentially, women underwent skin prick tests to saline, histamine, and aeroallergens: cat pelt, alternaria species, grass mix # , ragweed mix, oak mix, tricophyton species, mite mix, and cockroach mix (quintest extract tray; hollister-stier, spokane, wash). allergen-specific ige measurement (phadiatop; phadia, kalamazoo, mich) was performed on maternal blood samples for women who could not undergo skin prick tests or who had an inadequate skin test. multiallergen screens for specific ige (phadiatop) were measured with the immunocap (performed by the johns hopkins daci laboratory). a positive phadiatop result was defined as . ku a /l or greater by using the standards in place at the time the assays were performed. , among women for whom allergen sensitization status was determined based on skin prick test results or allergen-specific ige levels, we classified women by whether they reported a history of asthma into mutually exclusive categories: atopic asthma, nonatopic asthma, and no asthma. women with selfreported asthma and evidence of allergen sensitization (> _ positive skin prick test response or positive phadiatop result) were classified as having atopic asthma, women with self-reported asthma and without evidence of allergen sensitization were classified as having nonatopic asthma, and women who did not report self-reported asthma were in the no asthma group. nasal and throat swabs were obtained from infants at the time of enrollment, and the biospecimens were processed, placed in aliquots, and stored at c. the specimens were tested in batches for rsva and b, hrv, adenovirus, human metapneumovirus, coronaviruses, influenza a and b, and parainfluenza types , , and by using real-time rt-pcr with the cepheid smart cycler ii, as previously described. pcr results were used to identify infants with a sole rsv-or hrv-induced ari. infants included in this study had an hrv-or rsv-induced ari, either viral upper respiratory tract infections (urtis) or lrtis. children with a urti had a health care provider's diagnosis of a viral urti and/or symptoms, including fever, cough, congestion, hoarse cry, otitis media, and/or rhinorrhea without evidence of lower respiratory tract symptoms or respiratory distress. infants with a physician's diagnosis of bronchiolitis or wheezing, signs and symptoms consistent with bronchiolitis on chart review, or both were considered to have a viral lrti. , we determined the severity of the ari by using an ordinal bronchiolitis severity score with factors including respiratory rate, room air oxygen saturation, and the presence and extent of wheezing and flaring and retractions. scores range from to , with higher scores indicating more severe illness. , covariates other variables of interest obtained from the questionnaire administered at enrollment included self-reported maternal race/ethnicity, maternal education, secondhand smoke (shs) exposure, infant's insurance type (tennessee medicaid, private, or none), infant's birth weight (in grams), infant's sex, infant's age at enrollment (in weeks), and siblings. descriptions of demographics and characteristics of the infants with sole hrv or rsv infections are presented as frequencies and proportions for categorical variables and medians and interquartile ranges for continuous variables. univariate analyses were conducted to compare maternal asthma factors by the infant's hrv-or rsv-induced ari status by using the wilcoxon rank sum test for continuous variables or the pearson x test for categorical variables. in our analyses we first defined maternal asthma using self-reported asthma in the women. in addition, to examine the association of maternal asthma in combination with an objective measure of atopy, we repeated all analyses using a more detailed definition that classified women as having atopic asthma, nonatopic asthma, or no asthma by incorporating their skin prick test or allergen-specific ige findings. therefore we investigated whether ( ) having a mother with self-reported asthma and ( ) having a mother with atopic or nonatopic asthma was associated with an infant's ari with hrv or rsv. we assessed the association of measures of maternal asthma and virus type in the overall ari group (combined urti and lrti) and next among the lrti subgroup. we applied a logistic regression model with variable hrv( ) or rsv( ) as a binary outcome variable and maternal asthma as defined above, as our main factor along with covariates. because of our limited regression power determined by the hrv( ) group, we used propensity score adjustment to prevent overfitting because the propensity score analysis adjusts for many confounding factors simultaneously while preserving analytical power. variables in the propensity score model included a priori selected variables: maternal race/ethnicity, shs exposure, infant's insurance type, infant's birth weight, infant's sex, infant's age at enrollment, and number of siblings. in our next set of analyses we examined whether having a mother with asthma (first defined by maternal self-report and then using the atopic asthma and nonatopic asthma classifications) was associated with increased severity of the infant's hrv-or rsv-induced ari. we conducted a separate analysis for infants with sole hrv-or rsv-induced ari and in the subgroups with lrtis. we used the proportional odds model to evaluate the association of maternal asthma with infant hrvor rsv severity using the bronchiolitis severity score. for infants with rsv, a priori selected variables in the multivariable j allergy clin immunol volume , number models included maternal race/ethnicity, shs exposure, infant's insurance type, infant's birth weight, infant's sex, infant's age at enrollment, and number of siblings. analyses among hrv-induced lrtis were limited by small sample size for a full covariates model, and therefore we performed a propensity score-adjusted model that included infant's sex, age, and birth weight. we performed an interaction analysis to assess whether the association between maternal asthma and severity of the infant's ari was different depending on the infant's hrv or rsv status. the proportional odds model was used with a cross-product term of maternal history of asthma and virus positivity (rsv and hrv ) and adjustment for covariates. statistical analyses were performed with r version . . software. a total of infants with sole infection with either hrv or rsv were included in this study. table i highlights the demographics and characteristics of the cohort by infant ari cause, as determined by means of pcr: positive for hrv only (n ) or rsv only (n ). compared with infants with rsv, infants with hrv were more likely to be older ( vs weeks, p < . ), have medicaid insurance ( % vs %, p . ), have mothers who were african american ( % vs %, p < . ), have a urti versus an lrti ( % vs %, p < . ), and have a lower median bronchiolitis severity score ( vs . , p < . ). among infants with aris and the lrti subgroup, we determined the association of self-reported maternal asthma and infant hrv-or rsv-induced aris (both urtis and lrtis). infants with hrv were more likely to have a mother with self-reported asthma than infants with rsv ( % vs %, p . , table i ). in adjusted analyses, compared with infants whose mothers did not have asthma, infants with a mother with self-reported asthma had an increased relative odds of having hrv-induced than rsv-induced ari (adjusted odds ratio, . ; % ci, . - . ). in analyses limited to infants with lrtis, infants with hrv were more likely to have a mother with self-reported asthma than infants with rsv ( % vs %, p . ). in adjusted analyses infants with a mother with self-reported asthma had an increased relative odds of having hrv-induced than rsv-induced lrti (adjusted odds ratio, . ; % ci, . - . ). maternal allergen sensitization determined based on skin prick test results or allergen-specific ige levels was available for % of the mothers (n ) and was used to identify maternal atopic asthma. a larger percentage of infants with hrv-induced aris had a mother with atopic asthma than infants with rsv-induced aris ( % vs %, respectively), whereas the percentages of infants with a mother with nonatopic asthma were similar ( % vs %, respectively). having a mother with atopic asthma was associated with increased odds of an infant having hrv versus rsv infection when compared with having a mother without asthma (propensity score-adjusted odds ratio, . ; % ci, . - . ). having a mother with nonatopic asthma compared with no asthma was not associated with viral cause (adjusted odds ratio, . ; % ci, . - . ). a larger percentage of infants with hrv-induced lrtis had a mother with atopic asthma than infants with rsv-induced lrtis ( / [ %] vs / [ %]); however, the percentages of infants with a mother with nonatopic asthma were similar ( / [ %] vs / [ %]). in multivariable propensity score-adjusted analyses there was a statistically significant association with having a mother with atopic asthma compared with having a mother without asthma for infants with hrv-induced lrtis compared with rsv-induced lrtis (adjusted odds ratio, . ; % ci, . - . ). there was not a statistically significant difference between infants having a mother with nonatopic asthma compared with no asthma for infants with hrv-induced lrtis compared with those with rsv-induced lrtis (adjusted odds ratio, . ; % ci, . - . ) . in separate analyses for infants with either hrv or rsv infection, we examined whether the severity of the infant's respiratory tract infection was associated with having a mother with self-reported asthma. in infants with hrv, the association between self-reported maternal asthma and infant infection severity was not statistically significant in the ari group (both urtis and lrtis; adjusted odds ratio, . ; % ci, . - . ) or the lrti group (adjusted odds ratio, . ; % ci, . - . ; table ii ). in infants with rsv, the association between self-reported maternal asthma and infant infection severity was not significant in the total ari group (adjusted odds ratio, . ; % ci, . - . ) or the lrti group (adjusted odds ratio, . ; % ci, . - . ; table ii ). next, in separate analyses for infants with either hrv or rsv infections, we examined whether the severity of the infant's respiratory tract infection was associated with whether the infant's mother had atopic or nonatopic asthma. among infants with hrv-induced ari, having a mother with atopic asthma was associated with increased ari severity (fig ) , and in adjusted analyses there was a more than -fold increased relative odds of having more severe illness compared with infants whose mothers did not have asthma (adjusted odds ratio, . ; % ci, . - . ; table ii ). the interaction analysis investigating a differential effect of maternal atopic asthma by whether the infant had hrvor rsvon infection severity was also statistically significant (p . ). this relationship was not seen when limited to the subgroup of infants with hrv-induced lrtis; however, the number of infants with hrv-induced lrtis was very small (n ) and could be adjusted through propensity scores for only the infant's age, sex, and birth weight (table ii) . there was not an association between maternal atopic asthma and infection severity in infants with rsv-induced aris or the rsv-induced lrti subgroup (table ii) . rsv and hrv are the most common viruses associated with infant aris, and rsv-and hrv-induced lrtis are a leading cause of respiratory morbidity and hospitalizations in the first year of life. - viral lrtis during infancy and early childhood are also well established to be associated with an increased risk of asthma later in childhood. [ ] [ ] [ ] , because of the known differential risk of early childhood asthma after rsv-and hrv-induced infant infections, we were interested in studying whether a familial predisposition to asthma and allergies was associated with the viral cause of the infant's ari and the severity of the ari. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] several small studies have not found an association between familial predisposition and bronchiolitis; however, in our prior large, population-based cohort investigation of infants, we found that having a mother with asthma was associated with increased risk and severity of bronchiolitis during infancy. in the current investigation we sought to expand on previous findings by addressing the research questions of whether infants with hrvinduced aris or lrti subgroups are more likely to have a familial predisposition to asthma than those with rsv-induced infections and whether a familial predisposition to asthma is associated with more severe infant hrv-or rsv-induced aris. in our previous work we found that although, as a group, infants with a history of bronchiolitis had an increased risk of early childhood asthma, infants who had bronchiolitis during hrvpredominant months had a % increased risk of early childhood asthma compared with infants who had bronchiolitis during rsvpredominant months. furthermore, in the coast birth cohort, in which all children have a familial predisposition to asthma, , investigators found that hrv-induced wheezing illnesses in the first years of life were associated with a . -fold relative odds of asthma at years compared with a . -fold increase among children with rsv. we were able to assess whether maternal asthma and atopy were associated with the viral cause and the virus-specific severity of the infant's infection leading to an unscheduled health care visit because this cohort consisted of infants with and without a familial predisposition to asthma, and we used objective measures of maternal atopy and molecular techniques to determine the viral cause of the infant's respiratory tract infection. we found that infants with a mother with atopic asthma had an increased relative odds of having hrv-induced aris than rsv-induced aris compared with infants whose mothers did not have asthma. it is notable that although the prevalence of self-reported maternal asthma is higher in children with hrv ( %), the % prevalence of maternal asthma among infants with rsv-induced aris is higher than the asthma prevalence in the adult population in the united states. these study findings suggest that for infants with hrv-induced aris in particular, a familial predisposition to asthma might partly explain the increased risk of asthma seen later in life and/or that a familial predisposition to asthma predisposes infants to clinically significant hrvinduced aris during infancy, a viral infection that, unlike rsv, can often be isolated from young children without clinically obvious respiratory symptoms. we also found that infants with hrv who had a mother with atopic asthma had more than a -fold increased relative odds of having more severe illness than infants whose mothers did not have asthma. among those with rsv-induced aris, there was no relationship between maternal asthma and atopy and ari severity. the relationship between maternal atopic asthma and the infant's hrv infection severity is intriguing given the described altered host response to hrv, as has been found in vitro and among subjects with asthma. , [ ] [ ] [ ] [ ] [ ] these data support the notion of differential susceptibility to hrv among patients with atopic asthma. continued follow-up of these children until the age of years will further delineate whether it is the subset of infants with hrv-induced aris and a familial atopic predisposition who will have asthma and allergic diseases later in childhood or have more severe asthma or recurrent exacerbations. ultimately, this might help us to understand whether there is an altered host response or increased susceptibility to hrv among patients with atopic asthma. there are several limitations of this work. this study included a convenience sample of mother-infant dyads in which all infants presented for an unscheduled health care visit and not a cohort followed from birth. however, the study included participants recruited during viral seasons over years, which should serve to strengthen the generalizability of the findings. a single episode of rsv-or rhinovirus-induced ari was captured. it is likely that children had additional viral infections during infancy. the primary focus of this study was the association of a maternal atopic predisposition and the infant's ari severity, and therefore we were not able to investigate the outcome of bronchiolitis incidence as in our larger, population-based retrospective cohort of mother-infant dyads. in addition, maternal asthma was determined based on self-report and not based on objective criteria, such as airway reversibility testing. however, we used a validated instrument and self-reported asthma in young adults in whom there is little overlap with other diseases, and thus there is high specificity. furthermore, this study included objective measures of atopy. lastly, we cannot completely rule out the possible influence of unknown or unmeasured potential confounding factors, as with all observational studies. in summary, infant hrv infections requiring clinical care were more strongly associated with having a mother with atopic asthma than infant rsv infections. in addition, infants with hrv-induced aris who had a mother with atopic asthma had more than a -fold increased relative odds of having a more severe ari compared with that seen in infants with hrv whose mothers did not have asthma. this relationship was not seen in infants with rsv infections. these findings suggest that there is likely an underlying genetic basis for the risk of and response to respiratory tract infections during infancy and that the mechanisms underlying the increased asthma risk after hrv-and rsv-induced bronchiolitis might be different. future longitudinal investigations successful at preventing or modifying the host response to infant viral infections will provide insight into the relationship of infant viral infections and early childhood asthma, as will investigations that assess the atopic host and nonatopic host response to select respiratory pathogens. key messages d infants whose mothers had atopic asthma had increased relative odds of having a rhinovirus-induced ari than rsv infection compared with the odds in infants whose mothers did not have asthma. d in infants with rhinovirus, having a mother with atopic asthma was associated with -fold increased relative odds of more severe illness, a relationship not seen in infants with rsv. d for infants with rhinovirus-induced aris, a familial atopic predisposition might partly explain the subsequent increased risk of asthma and differential susceptibility to rhinovirus among asthmatic patients. bronchiolitis-associated hospitalizations among us children risk factors for respiratory syncytial virus-associated lower respiratory illnesses in the first year of life the increasing burden and risk factors for bronchiolitis-related medical visits in infants enrolled in a state healthcare insurance plan earlylife respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age the severity-dependent relationship of infant bronchiolitis on the risk and morbidity of early childhood asthma rhinovirus illnesses during infancy predict subsequent childhood wheezing the impact of respiratory viral infection on wheezing illnesses and asthma exacerbations atopy does not predispose to rsv bronchiolitis or postbronchiolitic wheezing wheezing, asthma, and pulmonary dysfunction years after infection with respiratory syncytial virus in infancy asthma and wheezing in the first six years of life. the group health medical associates respiratory syncytial virus in early life and risk of wheeze and allergy by age years viral infections, atopy, and asthma: is there a causal relationship? the childhood origins of asthma (coast) study childhood infections, the developing immune system, and the origins of asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease respiratory status and allergy nine to years after acute bronchiolitis family history of atopy and clinical course of rsv infection in ambulatory and hospitalized infants maternal asthma and maternal smoking are associated with increased risk of bronchiolitis during infancy host and viral factors associated with severity of human rhinovirusassociated infant respiratory tract illness wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the tennessee children's respiratory initiative: objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases relationship of maternal vitamin d level with maternal and infant respiratory disease international study of asthma and allergies in childhood (isaac): rationale and methods in vitro assays for the diagnosis of ige-mediated disorders in vitro diagnosis of atopic allergy in children. a comparison between total ige, conventional rast and a new multi rast (phadiatop) phadiatop compared to skin-prick test as a tool for diagnosing atopy in epidemiological studies in schoolchildren dexamethasone and salbutamol in the treatment of acute wheezing in infants prednisolone plus albuterol versus albuterol alone in mild to moderate bronchiolitis the central role of the propensity score in observational studies for causal effects r: a language and environment for statistical computing respiratory syncytial virus infection and recurrent wheeze/asthma in children under five years: an epidemiological survey community study of role of viral infections in exacerbations of asthma in - year old children the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings the september epidemic of asthma exacerbations in children: a search for etiology rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing allergic sensitization is associated with rhinovirus-, but not other virus-, induced wheezing in children season of infant bronchiolitis and estimates of subsequent risk and burden of early childhood asthma asthma prevalence, health care use, and mortality: united states asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma rhinoviruses in the pathogenesis of asthma: the bronchial epithelium as a major disease target rhinovirus infection induces expression of airway remodelling factors in vitro and in vivo validation of questionnaire and bronchial hyperresponsiveness against respiratory physician assessment in the diagnosis of asthma we conducted an analysis of mother-infant dyads enrolled in the tcri to investigate the association of a familial atopic predisposition with the viral key: cord- -ynh d authors: yoshihara, keisuke; le, minh nhat; toizumi, michiko; nguyen, hien anh; vo, hien minh; odagiri, takato; fujisaki, seiichiro; ariyoshi, koya; moriuchi, hiroyuki; hashizume, masahiro; dang, duc anh; yoshida, lay‐myint title: influenza b associated paediatric acute respiratory infection hospitalization in central vietnam date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: ynh d background: influenza b is one of the major etiologies for acute respiratory infections (ari) among children worldwide; however, its clinical‐epidemiological information is limited. we aimed to investigate the hospitalization incidence and clinical‐epidemiological characteristics of influenza b‐associated paediatric aris in central vietnam. methods: we collected clinical‐epidemiological information and nasopharyngeal swabs from ari children hospitalized at khanh hoa general hospital, nha trang, vietnam from february through june . nasopharyngeal samples were screened for respiratory viruses using multiplex‐pcrs. influenza b‐confirmed cases were genotyped by haemagglutinin gene sequencing. we analyzed the clinical‐epidemiological characteristics of influenza b lineages (victoria/yamagata) and who groups. results: in the pre‐a/h n pdm period, influenza b‐associated ari hospitalization incidence among children under five was low, ranging between . and . per population. the incidence increased to between . and in the post‐a/h n pdm . influenza b ari cases were slightly older with milder symptoms. both victoria and yamagata lineages were detected before the a/h n pdm outbreak; however, victoria lineage became predominant in ‐ ( % victoria vs % yamagata). victoria and yamagata lineages did not differ in demographic and clinical characteristics. in victoria lineage, group ari cases were clinically more severe compared to group , presenting a greater proportion of wheeze, tachypnea, and lower respiratory tract infection. conclusions: the current results highlight the increased incidence of influenza b‐related ari hospitalization among children in central vietnam in the post‐a/h n pdm era. furthermore, the difference in clinical severity between victoria lineage group and implies the importance of influenza b genetic variation on clinical presentation. influenza viruses belong to the family orthomyxoviridae, which possess a segmented negative-stranded rna genome. , among three influenza types, namely, type-a, b and c, influenza a and b often cause seasonal acute respiratory infections (ari) epidemics and impose a high socioeconomic burden, particularly among children and the elderly population. , in temperate climate regions, peaks of influenza-associated aris may appear in early autumn through winter, whereas year-round circulation with no apparent seasonal trend may be seen in tropical climate regions. [ ] [ ] [ ] [ ] influenza b differs from type-a in terms of genetic composition and its ability to infect only humans and seals. , two surface glycoproteins, haemagglutinin (ha) and neuraminidase (na), play pivotal roles during pathogenesis. unlike influenza a, influenza b does not possess multiple subtypes. instead, influenza b obtains its genetic variation mainly through genetic drift, including nucleotide substitutions, insertions, and deletions, which explains the slower molecular evolutionary rate and smaller capacity to cause seasonal ari outbreaks compared to influenza a. , genetic reassortment of influenza b genetic components was previously documented ; however, its frequency is not as high as that of influenza a due to its limited animal reservoir. despite the clinical importance of influenza b among ari cases, majority of previous literature focused on influenza a. therefore, clinical and molecular epidemiological information on influenza b is relatively limited worldwide. the first influenza b strain b/lee/ was isolated in . in the s, influenza b diverged into two genetically and antigenically distinct lineages, victoria-like (b/victoria/ / ) and yamagata-like (b/yamagata/ / ) lineages. , since then, the two lineages have been co-circulating in many regions of the world. previous studies from cambodia, china, india, and taiwan have reported the increase of influenza b-associated ari cases in the following seasons after the emergence of the pandemic a/ h n pdm strain. , , , , furthermore, some studies presented the circulation of victoria lineage as a dominant type in the post-a/h n pdm period, , , [ ] [ ] [ ] [ ] whereas others showed cocirculation of both lineages. , regardless of a few epidemiological studies on influenza b lineage-specific clinical presentation, , the clinical aspect of two genetically distinct lineages has not been clearly understood up to date. in this study, we investigated the incidence and clinicalepidemiological characteristics of paediatric hospitalized influenza b ari cases in vietnam. population-based prospective paediatric ari surveillance in khanh hoa province, nha trang, vietnam was established in february . all children from communes admitted to the paediatric ward of khanh hoa general hospital (khgh), which is the only hospital in the region, due to ari symptoms (cough and/or difficulty breathing) from february to june were enrolled in the current study. nha trang city in south central region of vietnam has a hot and dry season or tropical climate with a short rainy or wet season from september to december. the detailed characterization of the target population was described in the previous study. written informed consent from the parents or guardians of all the enrolled ari children were obtained. nasopharyngeal (np) swab, clinical-epidemiological information, chest radiograph, and laboratory test data were collected from each enrollee. currently, vaccination against influenza type a and b is not included in the nationwide immunization program in vietnam. its availability in our study site is limited and not commonly used among children. lower respiratory tract infection (lrti) was defined based on the modified world health organization (who) integrated management of childhood illnesses (imci) algorithms. the presence of tachypnea (respiratory rate > /min for children ≦ month, > /min for - months and > /min for - months of age) was categorized as mild lrtis. furthermore, children with a general danger sign (the situation in which children were either unable to drink, under convulsion, or lethargy), chest-wall indrawing, or stridor were categorized as severe lrtis. radiologically-confirmed pneumonia was defined as the presence of either substantial alveolar consolidation or pleural effusion in chest x-ray result following the standardized interpretation method established by who vaccine trial investigators group. ari cases with abnormal shadow but neither substantial alveolar consolidation nor pleural effusion were categorized into chest x-ray abnormality or other lower respiratory infections. adenovirus, and bocavirus as previously described. for the cur- confirmed that there was statistical evidence of an increase in influenza b-associated paediatric ari hospitalization in "post-a/ with regard to the demographic and clinical characterization of overall paediatric ari hospitalization cases enrolled in the current study (n = , ), , were male ( . %), and the median age (in months) was . (iqr: . - . ). other demographic information, including socioeconomic status, medical history, as well as detailed clinical information, was summarized in table . the demographic and clinical characteristics between influenza b (n = ) and non-influenza b ari groups (n = ) were compared ( table ). the male proportion was higher in the non-influenza b ari group ( . %, influenza b vs . %, non-influenza b, p = . ). regarding the median age (in month), the influenza b ari group was older ( . , influenza b vs . , non-influenza b, p < . ), and the greater proportion of ari children were years or older in the influenza b ari group. furthermore, family smoking was more frequent in the influenza b ari group (p = . ). with respect to the clinical information, the respiratory rate (per min) was faster in the non-influenza b ari group ( . , influenza b vs . , non-influenza b, p = . ) ( table ) . furthermore, ari children with wheeze (p = . ) and breathing difficulty (p = . ) were more common in the non-influenza b ari group. the proportion of paediatric ari hospitalizations with chest x-ray abnormal findings was also greater in the non-influenza b ari group ( furthermore, in the comparison between influenza a and b, influenza b-associated ari hospitalizations were slightly older with different age distribution pattern (p = . ) (table s ) . parental smoking was more common in influenza b ari group (p = . ). regarding the clinical information, influenza a group was associated with higher body temperature (p = . ) as well as frequent presence of chest x-ray abnormality (p < . ). (table s ) . on the contrary, the overall number of yamagata lineage-related ari hospitalizations was limited throughout the investigation period in both pre-and post-a/ h n pdm periods. in the demographic characteristics comparison between influenza b table s . firstly, demographic information among who groups ( , , and ) of victoria lineage was compared (table ). there were no significant differences in sex distribution and median age among the three groups. daycare attendance was more common in group com- (table s ) . with regard to demographic characterization, no statistically significant differences were detected between groups and . both group -and group -associated ari hospitalizations were commonly observed among children younger than years. the results of a comparison in clinical manifestations did not reveal any significant differences. interestingly, we also observed unusual split of rsv seasonal peak in as we previously described. further studies would be necessary to understand the effect of a/h n pdm on respiratory virus circulation trend. among all the paediatric ari hospitalization cases enrolled in the current study, were influenza b positive ( table ). the median age in influenza b ari group was older than non-influenza b aris, and influenza b group presented the higher proportion of family smoking, which is known to be one of the major risk factors for influenza morbidity and mortality. we also have to take the lineage-specific sero-epidemiological information into account to gain a better understanding of influenza b lineage circulation pattern. a study from taiwan suggested switching of dominantly circulating lineage may occur every - years. in the future study, we will continue monitoring the lineage-specific circulation trend of influenza b strains in order to investigate the lineagespecific viral transmissibility based on the viral genetic variation. regarding the demographic information of influenza b lineages, median age was slightly older in victoria lineage (table ) , which contradicted the reports from china and slovenia that presented victoria lineage aris were younger than yamagata lineage. , the difference in age of infection has been controversial due to the differences in inclusion criteria among studies. furthermore, clinical aspect of influenza b lineages is poorly understood. in the current study, the clinical data did not present a significant difference between lineages (table ) , which was in line with previous reports from china, france, serbia, and slovenia. , [ ] [ ] [ ] [ ] although the body temperature was slightly higher in yamagata lineage aris, it was probably due to the difference in age distribution. in our study, the hospitalization duration did not differ between lineages, which con- male sex (%) we would like to express our sincere gratitude to the medical doctors, nurses, and laboratory technicians at khanh hoa general hospital and staffs in khanh hoa health service for their kind support on clinical sample collection and ari clinical data management. we would like to express our special appreciation for administrative staff and professors in leading program at nagasaki university, graduate school of biomedical sciences for their kind support during the research. this study was conducted in part at the joint usage/research center on tropical disease, institute of tropical medicine, nagasaki university, japan. the authors have declared that no competing interests exist. lay-myint yoshida https://orcid.org/ - 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- journal: infection control and hospital epidemiology doi: . /ice. . sha: doc_id: cord_uid: rc a xs nan to the editor-since the emergence of coronavirus disease (covid- ) caused by severe acute respiratory coronavirus virus (sars-cov- ) in china, > , confirmed cases including > healthcare workers (hcws) have been reported in singapore. , healthcare workers (hcws) are at increased risk of nosocomial covid- infection. in , almost one-third of ward-based hcws at tan tock seng hospital (ttsh) in singapore were infected with the severe acute respiratory syndrome (sars) from an index patient. after the sars nosocomial outbreak, web-based staff sickness surveillance systems have been established at ttsh for the early detection of hcw clusters of acute respiratory infection (ari). [ ] [ ] [ ] additionally, a risk-based approach to the use of personal protective equipment (ppe) by hcws, with full ppe donned in high-risk areas and minimally surgical masks in low-risk areas were implemented. during the covid- pandemic, a team of public healthtrained personnel maintained close monitoring of staff sickness reporting to identify ari clusters among the , hcws working at the , -bed ttsh and its collocated -bed national centre for infectious diseases, the national referral centre for covid- response. we examined the epidemiology of ari clusters identified in hcws in the first weeks of , and we compared them with the ari clusters in . an ari cluster was defined as ≥ hcws or ≥ % of the total staff strength (whichever was higher) from the same work location reporting aris within consecutive days. each ari cluster was followed-up with active case findings and infection prevention measures, including enhanced hand hygiene and enforcement of adherence to appropriate ppe and referral of sick staff to the in-house occupational health clinic (ohc). nasal and throat swabs were taken from symptomatic hcws who worked at the ohc. samples were sent to the national public health laboratory for influenza polymerase chain reaction (pcr) and respiratory multiplex pcr (filmarray respiratory panel ) tests. sars-cov- pcr testing was implemented in at the start of the covid- pandemic. during the -week study period, the mean weekly number of staff absences due to ari was (standard deviation [sd], ). more than half of the ari clusters were identified among hcws who worked in inpatient wards (n = , %). other clinical areas (eg, pharmacy, physiotherapy, operating theatres, and outpatient clinics) accounted for more than one-quarter of the clusters (n = , %). the remaining clusters were identified among hcws who worked in nonclinical areas (n = , %) (fig. ) . in total, ari clusters were identified in the entire year of , and clusters were identified in first weeks of . compared to the first weeks of (n = ), the number of ari clusters identified among staff working in inpatient wards in (n = ) was significantly lower: % versus %, respectively (or, . ; % ci, . - . ; p = . ). median cluster sizes were slightly larger: (iqr, - ) for versus (iqr, - ) for (or, . ; % ci, . - . ; p = . ). median cluster duration was longer in than in : days (iqr, - ) versus days (iqr, - ), respectively (or, . ; % ci, . - . ; p = . ). among ari clusters, almost twice the number of clusters in had at least respiratory pathogen identified (n = , %) compared with : ( %) versus ( %), respectively (p = . ). rhinovirus was the most common viral pathogen detected in both years: clusters ( %) in and clusters ( %) in . this possibly reflects the most common circulating noninfluenza viral pathogen among ari episodes in the community. human coronaviruses e/hku /oc were detected in both years: clusters ( %) in and cluster ( %) in . adenovirus was identified in % of ari clusters in , although it was not detected in any of the ari clusters in . parainfluenza viruses ( %) were also detected during the first weeks of but not in . influenza viruses were detected in clusters in but in none of the clusters in . sars-cov- virus was not detected in any of the hcw ari clusters in . since start of the pandemic, despite an increase in ari clusters detected, sars-cov- has not been detected. this absence reflects the adequate protection of hcws from acquiring sars-cov- infection in the hospital. notably, no pathogen was identified in hcw ari clusters after epidemiological week in , and a downward trend of the weekly number of staff aris reported from epidemiological week . no staff ari cluster was identified after epidemiological week . these trends are likely the consequence of hospital-wide enhanced infection prevention measures (eg, safe distancing, having meals alone, and the donning of surgical masks at all times in all hospital areas) instituted since epidemiological week in (ie, the week ending april , ). close surveillance of staff absenteeism due to ari and epidemiological investigations of hcw ari clusters with screening for respiratory viruses and sars-cov- are crucial as covid- pandemic emergency responses relax, economic activities resume, and travel bans are lifted. because it is unlikely that covid- infections will taper off soon around the world, countries should consider having all hcws wear surgical masks at all times in healthcare settings. covid- cases among healthcare workers and support staff: gan kim yong. channel news asia website covid- situation report. ministry of health singapore website transmission of covid- to health care personnel during exposures to a hospitalized patient the outbreak of sars at tan tock seng hospital-relating epidemiology to control use of healthcare worker sickness absenteeism surveillance as a potential early warning system for influenza epidemics in acute care hospitals surgical masks for protection of healthcare personnel against pandemic novel swine-origin influenza a (h n )- : results from an observational study responding to the covid- outbreak in singapore: staff protection and staff temperature and sickness surveillance systems detection of viral respiratory pathogens in mild and severe acute respiratory infections in singapore acknowledgments. authors acknowledge members of surveillance team and infection control team who conduct staff sickness absenteeism surveillance throughout.financial support. no financial support was provided relevant to this manuscript. all authors report no conflicts of interest relevant to this manuscript. key: cord- -taj swwc authors: lu, guilan; peng, xiaomin; li, renqing; liu, yimeng; wu, zhanguo; wang, xifeng; zhang, daitao; zhao, jiachen; sun, ying; zhang, li; yang, peng; wang, quanyi title: an outbreak of acute respiratory infection at a training base in beijing, china due to human adenovirus type b date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: taj swwc background: twelve students experienced symptoms of acute respiratory infection (ari) at a training base in beijing from august to august , . we investigated the cause of this ari outbreak. methods: in partnership with the local center for disease control, we collected a total of twelve pharyngeal swab specimens as well as demographic information for the affected patients. we used multiplex real-time pcr to screen for sixteen common respiratory viruses in these samples. to isolate hadv, we inoculated hep- cells with the human adenovirus (hadv)-positive samples and then carried out sequencing and phylogenetic analysis of the hexon, fiber, and penton genes of the isolated adenoviruses. in addition, we analyzed the entire genome of one strain isolated from the index case to identify single-nucleotide substitutions. results: we identified ten hadv-positive students using multiplex real-time pcr. none of the students were co-infected with other viruses. we successfully isolated seven hadv strains from the pharyngeal swab specimens. the coding sequences of the hexon, fiber, and penton genes of these seven hadv strains were identical, suggesting that they represented seven strains from a single virus clone. one hadv isolate obtained from the index case, bjdx- - , was selected for whole genome analysis. from this isolate, we obtained a , -nucleotide sequence. the genome of bjdx- - clustered with hadv-b in phylogenetic analyses and had . % identity with human adenovirus isolate hadv-b/chn/bj / / (genbank accession no. jx ). conclusions: we identified hadv-b as the strain associated with the august ari outbreak at a training base in beijing. this was the first reported outbreak in beijing due to hadv-b . continuous surveillance of respiratory adenoviruses is urgently needed to understand the epidemiological and evolutionary features of hadv-b , and an epidemiological modeling approach may provide further insights into this emerging public health threat. furthermore, the clinical laboratory data from this outbreak provides important reference for the clinical diagnosis and may ultimately aid in informing the development of strategies to control and prevent respiratory tract infections caused by hadv-b . human adenoviruses (hadvs) cause a wide variety of clinical manifestations, including respiratory tract infection, gastroenteritis, kerato-conjunctivitis, acute hemorrhagic cystitis, nephritis, hepatitis, and encephalitis [ ] [ ] [ ] . hadvs are responsible for - % of all respiratory illnesses and for - % of pneumonias in children [ , ] . most hadv infections are mild, self-limiting, and indistinguishable from other viral infections. however, the illnesses caused by hadvs can be severe or even fatal and can result in substantial morbidity [ , ] . outbreaks of hadv-associated acute respiratory infection (ari) usually occur in healthy children or in adults in enclosed or crowded settings [ , ] . hadv was first reported as a viral pathogen in [ ] . since this initial identification, hadvs have been classified into seven species (a to g), and the human adenovirus working group has identified hadv types as of july (http://hadvwg.gmu.edu/) [ ] [ ] [ ] [ ] [ ] [ ] . adenoviruses are non-enveloped icosahedral particles that contain linear double-stranded dna genomes with sizes ranging from to kb. adenovirus genomes are characterized by inverted terminal repeat sequences (itrs) with sizes ranging from to over bp [ ] . the hadv viral capsid, which surrounds the genome, is composed of three major proteins: hexon, penton base, and fiber [ ] . different hadv species display various tissue tropisms that correlate with the different clinical symptoms of infection [ ] . hadv species a has often been associated with the gastrointestinal tract, whereas species b (hadv- , , , and ), c (hadv- , , , and ), and e (hadv- ) are known to cause respiratory tract infections. species d (hadv- , , and ) commonly causes adenoviral kerato-conjunctivitis. species f variants, including hadv-f and-f , and species g variant hadv-g are mainly associated with gastrointestinal tract infections [ ] . hadvs have been associated with previous outbreaks of ari. hadv-b and hadv-b cause frequent outbreaks in the united states [ ] . in asia, the prevalence of hadvs in patients with aris has ranged from . to . % [ ] [ ] [ ] [ ] [ ] . recently, guo et al. ( ) reported that hadv-b , hadv -b , hadv -b , and hadv -b were the most frequently detected virus strains among patients with acute adult adenovirus infections in beijing from may to july [ ] . in china, hadv-b and hadv-b , two hadv species b subtypes, are common causes of epidemic ari outbreaks [ ] [ ] [ ] [ ] . in , an ari outbreak occurred in qishan, shaanxi province, china. a re-emergent isolate of hadv-b (qs-dll), originally described as hadv- a and fully characterized in , was reported to be the cause of this outbreak [ ] . this re-emergent hadv-b was shown to have evolved from a hexon recombination between hadv-b and hadv-b [ , ] . since its characterization, hadv-b has been associated with several respiratory infection outbreaks and is known to be responsible for severe respiratory diseases [ , [ ] [ ] [ ] [ ] . here we describe an outbreak of ari caused by hadv-b at a training base in the daxing district of beijing, china. to help identify the causative pathogen, we collected pharyngeal swab specimens from the affected students and carried out molecular detection and typing, phylogenic analysis, and whole-genome sequencing. this is the first reported outbreak of ari in beijing due to hadv-b . the training base where the hadv-b outbreak took place on august , , local public health authorities were informed about an outbreak of ari among young students at a training base located in the daxing district of beijing. the training base consisted of two three-floor buildings for teaching and three three-floor dormitories with eight persons to a room. the training base recruits only male middle school graduates. approximately students majoring in mathematics, chinese, and english were enrolled in a total of sixty classes. the training base employs full-time staff members. on august , , one student reported symptoms of an ari and had a body temperature of . °c. by august , a total of ari patients from the same class were reported by the local hospital. for the purposes of our analysis, we defined ari cases as individuals with a body temperature over . °c and with at least one symptom of a respiratory tract infection, such as cough or sore throat. on august , the daxing district center for disease prevention and control (cdc) began an epidemiological investigation, collecting demographic, clinical and laboratory data. under the guidance of the cdc, the training base took precautionary measures, including quarantining the affected students, carrying out a routine cleaning and disinfection of living quarters, and morning body temperature checks. no further new cases were reported by september , . pharyngeal swab samples were obtained from each of the twelve students infected in this ari outbreak. the specimens were collected in ml vials containing viral transport medium and quickly transported on ice to the laboratory of the daxing district cdc. the specimens were stored at − °c until further use. patient information and laboratory results are shown in table . nucleic acids were extracted from μl of each of the clinical samples using qiaamp viral rna mini kits (qiagen, hilden, germany) according to the manufacturer's instructions. pharyngeal swab specimens were screened for common respiratory pathogens via realtime pcr multiplex assays using commercial kits (uninovo biological technology, zhenjiang, china) as described by shi w., et al. [ ] . the pathogens assayed using this approach were influenza virus a (h ), pandemic influenza virus a (h n ), influenza viruses a and b (flu a and b), parainfluenza viruses , , , and (piv , , , and ), human metapneumovirus (hmpv), human bocavirus (hbov), human coronavirus oc / nl , e/ hku , human respiratory syncytial virus (hrsv), human rhinovirus (hrhv), and hadv. hep- cells were inoculated with hadv pcr-positive specimens and cultured in high-glucose dulbecco's modified eagle medium (gibco, ny, usa) containing % fetal bovine serum (gibco, ny, usa), u/ml penicillin (gibco, ny, usa), and mg/ml streptomycin (gibco, ny, usa) at °c in a % co incubator for week following standard protocols [ ] . the cultured cells were checked regularly for cytopathic effects (cpe) and harvested when cytopathic effects (cpe) were observed. cultures with cpe were screened for specific hadvs as described by kim c et al. [ ] . molecular typing of hadvs was performed via conventional pcr using specific primers targeting the complete coding sequences of the hexon, fiber, and penton genes [ ] . viral dna was extracted from cultured medium using qiaamp rna mini kits (qiagen, hilden, germany) according to the manufacturer's instructions [ ] . conventional pcr was conducted using highfidelity dna polymerase (takara, dalian, china) according to the manufacturer's instructions. the hexon, fiber, and penton genes of hadv were amplified as described previously [ ] . for the hexon and penton genes, the pcr protocol was: °c for min., followed by cycles of s. at °c, s. at °c, min. at °c, and a final extension step of °c for min. the pcr protocol for amplification of fiber gene fragments was identical, except for the annealing temperature, which was °c instead of °c. the amplified pcr products were excised from agarose gels, purified using an axyprep dna gel extraction kit (axygen, hangzhou, china), and bidirectionally sequenced using the sanger sequencing method by invitrogen biotechnology co., ltd. (invitrogen, beijing, china) with an abi dna analyzer (applied biosystems, austin, tx, usa). to further analyze mutations in the genome sequences of the viruses isolated in this ari outbreak, we sequenced the whole genome of one isolate from the index case using the sanger method. targeted - kb segments that covered the entire genome with overlapping sequences of about bp were amplified by pcr. the ′ and ′ itrs of the genome were amplified and cloned into a plasmid t-vector and then sequenced. a set of pairs of primers was designed in-house to amplify the whole genome according to the reference sequence (genbank accession no. fj ) and then used for separate pcrs. primer sequences are available upon request. whole-genome sequencing segments were amplified using high-fidelity polymerase (takara, dalian, china) using . mm of each primer. pcrs were carried out using a biorad thermocycler (applied biosystems, austin, tx, usa) with the following protocol: °c for min., followed by cycles of s. at °c, s. at appropriate annealing temperature for separate primers, min. at °c, and a final extension step of °c for min. the amplified segments were purified and bi-directionally sequenced. gaps and ambiguous sequences were pcramplified using new primers and re-sequenced. dna sequence fragments were assembled using the seqman program implemented in dnastar lasergene . (dnas tar, inc. madison, wi) into a single contig. the genomic sequence determined in this study was deposited in gen-bank under accession number mk . nucleotide sequence homologies were identified using the basic local alignment search tool (blast, https://blast. ncbi.nlm.nih.gov/). multiple nucleotide sequence alignments were performed using the clustalw program implemented in bioedit. comparisons between the whole genome sequence of the bjdx- - virus strain and those of other types of hadvs were generated using clc genomics workbench (qiagen, hilden, germany). phylogenetic trees were constructed using the maximum likelihood method in the mega program (version . ). one thousand bootstrap replications were used to estimate distances. bootstrap values greater than % are shown for selected nodes in fig. (a-d) . wholegenome sequences and hexon, fiber, and penton gene sequences from other hadvs were downloaded from genbank on april , . on august , , a -year old male student developed a case of ari, with a peak body temperature of . °c. by august , a total of male students were infected. no females were infected. the mean age of the infected students was . years (median, years; range, - years). the distribution of daily cases is shown in fig. . reported students were from the same class but living in six different dormitory rooms. the outbreak lasted for days. the clinical symptoms of the infected students are described in table . all infected individuals had a fever; ( %) students had a sore throat, and ( . %) students had a headache. other symptoms reported by the patients were cough, body ache, stuffy nose, and diarrhea. all affected students had normal white blood counts. most of the infected students were treated in outpatient clinics; only one patient, who had non-severe pneumonia, was hospitalized. the index case (id no. ) was diagnosed as having an upper respiratory infection with a peak temperature of . °c, a neutrophil count of . % (normal range, - %), percentage of lymphocytes of . % (normal range, - %), and white blood cell count of . × / l (normal range, . - . × /l). the hospitalized student (id no. ) had a peak temperature of . °c accompanied by a cough, headache, sore throat, and diarrhea. he had a neutrophil count of . % (normal range, - %), a percentage of lymphocytes of . % (normal range, - %), and a white blood cell count of . × /l (normal range, . - . × /l). a chest x-ray showed patchy shadows on the right lung of the hospitalized student. a total of respiratory samples were obtained from the students. ten specimens were shown to be hadvpositive using multiplex pcr (uninovo biological technology, jiangshu, china). none of the hadv-positive patients were co-infected with other respiratory viruses. to isolate viruses, we inoculated hep- cells with the hadv-positive samples and isolated seven hadv virus strains. using typing primers, which allow for the determination of viral type, we sequenced the hexon, fiber, and penton genes in the seven hadv strains [ ] . the hexon ( bp), fiber ( bp), and penton ( bp) sequences from the seven hadv isolates were % identical, suggesting that this outbreak was caused by a single viral strain. we then compared the hexon, fiber, and penton sequences of one of the viral strains isolated from the index case (id no. ; referred to as bjdx- - ), with other hadv-b , hadv-b and hadv-b strains. based on blast analysis, the hexon, fiber, and penton genes were . , , and % identical to the genes of the hadv-b reference strain qs-dll in china (genbank accession number fj ), respectively,. the hexon, fiber, and penton genes were each %, identical to the genes of the isolate hadv-b/chn/bj / / (genbank accession no. jx ; table ), which was identified from a single patient in beijing with severe community-acquired pneumonia (cap) who was infected with hadv-b . one of the seven strains, bjdx- - , was selected for further whole-genome studies. we obtained and assembled the full genome of the index case isolate, bjdx- - . the sequence of this genome has been deposited in genbank (accession number mk ). the complete genome of bjdx- - was , nucleotides in length and had -bp inverted terminal repeat sequences in the ′-and ′untranslated regions ( ′-utr and ′-utr). to investigate the genetic relationships between isolate bjdx- - and other hadv strains, we constructed phylogenetic trees (fig. (a-d) ) using the maximum likelihood method based on the complete hexon, fiber, and penton gene sequences of strain bjdx- - and other hadv strains. all phylogenetic trees showed that bjdx- - , associated with this outbreak, clustered with hadv-b . the bjdx- - hexon gene also clustered with hadv-b (genbank accession number af ), while the fiber and penton genes and the full bjdx- - genome clustered with hadv-b (gen-bank accession number ay and jx ). comparisons of the whole genome sequence of the bjdx- - virus strain with other hadv-b , hadv-b and hadv-b strains are shown in fig. . fig. ). we then generated alignments between the genome sequence of strain bjdx- - and those of strains hadv-b , hadv-b , and hadv-b . most of the nucleotide differences in bjdx- - relative to hadv-b/chn/bj / / (genbank accession number jx ) were observed in the coding region of protein vi (fig. a) , where we found seven nucleotide changes and six amino acid substitutions. we also found different numbers of poly "t" and ploy "a" tracts (fig. b) . this study describes an ari outbreak with students infected at a training base in the daxing district of beijing, china, in august . based on epidemiological and laboratory investigation, we confirmed that the etiologic pathogen of this ari outbreak was hadv-b . hadv-b has received additional attention in recent years. hadv-b is a recombinant virus, and it is associated with more severe acute respiratory diseases than other types of hadvs [ ] . hadv-b was originally described as hadv-b a based on earlier putative, sporadic occurrences in spain ( ) [ ] , turkey ( ) fig. percent nucleotide identity and differences in the genome of bjdx- - relative to strains of hadv-b , hadv-b , and hadv-b . the numbers above the white grids represent the percent nucleotide identity, while those below the grids represent the nucleotide difference values calculated by clc genomic workbench fig. (a) alignment analysis on the coding sequence of protein vi among hadvs. '.' represents identical bases in the sequence alignment. (b) different numbers of poly "t" and poly "a" tracts were observed in the aligned genomes of the hadv-b , hadv-b , and hadv-b strains. '.' and '-' represent identical bases and deletions in the sequence alignment, respectively [ ] , and singapore ( ) [ ] . hadv-b was also identified as a re-emergent acute respiratory disease pathogen after a recent outbreak in qishan county, shaanxi province, china in [ , ] . subsequent analysis revealed that hadv-b consists of an hadv-b backbone and a partial hadv-b hexon gene. this re-emergent virus exhibited a neutralizing antigen epitope of hadv-b and the pathogenic properties of hadv- . therefore, this virus was renamed hadv-b [ , ] . in china, an increasing number of outbreaks of hadv-b have been reported since march [ ] . epidemic outbreaks of hadv-b have occurred in military camps, schools, and even hospitals in hebei province (february ) [ ] , tianjin city (january ) [ ] , beijing ( ; this study), guangzhou city ( ) [ ] , tibet, sichuan and yunnan provinces ( ) [ ] . in the hadv-b outbreak in china, a total of patients were infected, and one died [ ] . although the virus spread quickly in the outbreak reported here, only individuals were infected, including one student who developed pneumonia and was hospitalized. this suggests that the hadv-b strain associated with this outbreak is not as virulent as those reported previously [ , ] , yet the strain should still be considered an urgent public health threat that necessitates measures to contain or control it in order to prevent epidemics. unlike other outbreaks caused by hadvs in the northern regions of china [ , , ] that occurred in the winter or spring, this outbreak in beijing took place at the end of summer. studies by yu j., et al. and liu t., et al. have reported that, although infections of hadvs occur throughout the year, hadv outbreak prevalence often peaks in winter and spring in the north of china and in summer and spring in the south of china [ , ] . it is possible that hadv outbreaks differ in their seasonality based on relative humidity and temperature. the fact that the hadv outbreak reported here occurred in summer is thus significant because it could indicate this hadv-b virus strain is capable of circulating in the climate and environmental reservoir. the hexon, fiber, and penton genes of virus strain bjdx- - isolated in this study shared % sequence identity with those of the hadv-b strain hadv-b/chn/bj / / (genbank accession number jx ), which was isolated from a patient with severe cap in a previous study by bin cao et al. at beijing in [ , ] . the genome of bjdx- - is most similar to that of hadv-b/chn/bj / / . in our study, the strain of bjdx- - was obtained from the index case (id# ), who presented with an upper respiratory infection. furthermore most of the infected students were treated in outpatient clinics, and only one patient with non-severe pneumonia was hospitalized. the isolate of hadv-b/chn/bj / / was obtained from a patient with severe cap in a previous study by bin cao et al. at beijing [ ] , in which a total of cases with laboratory-confirmed adenovirus infections, including the mentioned case with hadv-b/ chn/bj / / , were detected in cap cases. furthermore, hadv-b was most frequently detected in the cases of adenovirus pneumonia ( / ), and six of the patients ultimately developed acute respiratory distress syndrome. based on the information of the two cases, two genetic viral samples, and the two independent studies that reported laboratory-confirmed aris with hadv-b in beijing, the isolate of bjdx- - led to milder aris. these findings imply that severe aris were not simply caused by hadv-b virus strains themselves but also depend on the conditions of the hosts, such as the individual's age, general state of health, and the presence of co-morbidities or additional infections. in this outbreak, we also found that for both the index case and the hospitalized case neutrophils increased and /or lymphocytes in the peripheral blood decreased. studies on other respiratory viruses, such as influenza virus, respiratory syncytial virus, or human rhinovirus, have shown that an increase in neutrophils has a significant role in limiting virus replication [ , ] . thus, the increase in neutrophils observed here may have limited and resulted in milder clinical symptoms in this outbreak. most of the mutations at the amino acid level in bjdx- - were observed in the coding regions of protein vi. during the replication of hadvs, protein vi functions as an adaptor to shuttle the hexon protein to the nucleus, where virus assembly occurs [ ] . whether these variations affect the virulence of bjdx- - and are responsible for the milder clinical symptoms observed in this study requires further investigation. we also found that bjdx- - has different numbers of poly "t" and poly "a" tracts compared with other hadv-b , hadv-b , and hadv-b strains. the role that poly "t" and poly "a" tracts play in the evolution of hadvs remains unclear and requires further research. the increasing frequency of ari outbreaks due to hadv-b suggests that this re-emergent virus poses a serious threat to public health. it is therefore urgent that the local cdc improve epidemiological and virological surveillance of hadv-b . we identified hadv-b as the cause of a recent localized ari outbreak. this incident was the first reported outbreak in beijing that can be attributed to this reemergent virus. our findings show that the risk of an hadv-b epidemic should be paid more attention in beijing, the capital of china, with a population of more than million. continuous surveillance of respiratory adenoviruses is an urgent need to understand the epidemiological and evolutionary features of hadv-b and could also find value in an epidemiological modeling approach. we also found that the clinical laboratory data from this outbreak provides important reference for clinical diagnosis and may ultimately aid in informing the development of strategies to control and prevent respiratory tract infections caused by hadv-b . abbreviations pcr: 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for their help in molecular detection and whole-genome analysis. they also thank the technicians of the daxing cdc of beijing for their excellent technical assistance with the epidemiological investigation and with the initial laboratory diagnosis. all authors made significant contributions to the data, analysis, and drafting of this manuscript and approved the final submitted version. gl wrote the manuscript. gl, xp and rl performed genomic analysis. yl, dz and jz conducted the diagnostic experiments. zw, xw, ys and lz participated in collecting information of this outbreak. py and qw helped review this manuscript. all authors have read and approved the manuscript. this study was supported by the research on early detection, genetic evolution and risk assessment for novel influenza virus by capital's funds for health improvement and research ( - - ). the datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request. this study was approved by the ethics committee of beijing cdc. written informed consent was obtained from all the participants or the guardians if participants were under years of age. not applicable. the authors declare no competing interests. key: cord- -rwf bly authors: sutrisna, b.; frerichs, r.r.; reingold, a.l. title: randomised, controlled trial of effectiveness of ampicillin in mild acute respiratory infections in indonesian children date: - - journal: lancet doi: . / - ( ) -y sha: doc_id: cord_uid: rwf bly the recommended treatment for mild acute respiratory infections (ari) in children is supportive care only, but many physicians, especially in developing countries, continue to prescribe antibiotic treatment because they believe it prevents progression to more severe ari. to find out whether ampicillin treatment conferred any benefit over supportive care alone, a randomised, controlled trial was carried out among children (under years) with mild ari in indonesia. were randomly allocated ampicillin ( - mg/kg body weight three times daily for days) plus supportive care (continued breastfeeding, clearing of the nose, and paracetamol to control fever); were allocated supportive care only. the treatment groups were almost identical after randomisation in terms of age, sex, level of parental education, history of measles immunisation, and fever. after week the percentages cured were nearly identical ( [ %] ampicillin; [ %] control), as were the percentages of cases progressing to moderate ari ( [ %] vs [ %]). the effect of treatment was not modified by age, sex, measles immunisation status, or the educational level of the parents. at the -week follow-up, the percentages cured were % ( ) in the ampicillin group and % ( ) in the control group; % of both groups had progressed to moderate ari; and % ( ) and % ( ), respectively, still had mild ari. none of the differences in outcome between the ampicillin and control groups was statistically significant. thus, ampicillin plus supportive care offers no benefit over supportive care alone for treatment of mild ari in young indonesian children. the findings obtained in necropsy studies. information collated from five independent necropsy studies indicated that mean prostate weight reaches g in men between the ages of and years and remains essentially constant at this weight with increasing age unless bph develops. o table ii summarises the prevalence rates of bph and prostatic weights of patients with bph in the survey in comparison with necropsy findings among men in similar age-groups with histologically confirmed bph. a surprising finding is that prostatic weights from these two sources are remarkably similar despite the differences in self-selection betwen the community survey and necropsy sources, the absence of prior knowledge of urinary dysfunction in the necropsy cases, and the absence of pathological confirmation of the diagnosis in the community survey. what is also of interest are the higher age-specific rates for bph in the necropsy than in the community survey. this difference suggests that a substantial reservoir of bph may exist below the "threshold" of signs and symptoms of urinary dysfunction that were used in the survey. the operational defmition adopted for this survey will probably change over time as knowledge of the natural history of bph increases, in the way that perception of what level of blood pressure constitutes hypertension has changed over the past several decades. whether the total urinary symptom score and qmax cut-off points used are ideal for enabling early cases of bph to be picked up through screening cannot be established from this survey. this issue can be determined only in a study that sets out to assess prostate size in a representative sample of men who have not been selected on the basis of their likelihood of having bph. the validity of symptom scores and voiding flow rate as preliminary screening criteria will eventually be required for identifying those men most likely to benefit from treatment should non-surgical therapy for bph be shown to be effective in the community. the recommended treatment for mild acute respiratory infections (ari) in children is supportive care only, but many physicians, especially in developing countries, continue to prescribe antibiotic treatment because they believe it prevents progression to more severe ari. to find out whether ampicillin treatment conferred any benefit over supportive care alone, a randomised, controlled trial was carried out among children (under years) with mild ari in indonesia. were randomly allocated ampicillin ( - mg/kg body weight three times daily for days) plus supportive care (continued breastfeeding, clearing of the nose, and paracetamol to control fever); were allocated supportive care only. the treatment groups were may start in the upper respiratory tract and progress to a more severe lower respiratory infection, such as pneumonia or bronchiolitis.l in less developed countries, pneumonia is a major cause of death in young children - antibiotics such as procaine penicillin, ampicillin, or co-trimoxazole are useful for treatment of pneumonia , , which is most commonly caused by bacterial agents, mainly streptococcus pneumoniae and haemophilus influenzae. , , , antibiotics are not recommended for treatment of upper respiratory infections which are believed to be caused mainly by viruses, most likely rhinoviruses and coronaviruses. the world health organisation (who) has promoted case-management as the appropriate approach to control of ari in children. , part of this recommendation is that supportive care by the mother rather than antibiotics be used to treat mild ari. inappropriate treatment of mild ari in developing countries wastes the resources of government-sponsored health services and is believed to increase the occurrence of drug-resistant bacterial strains in the population." when the who case-management programme was introduced in indonesia, many physicians were reluctant to stop giving antibiotics to children with mild ari, because they believed that antibiotic treatment prevents the progression of mild ari to moderate or severe ari. this belief is not supported by any objective evidence. we decided to test the effectiveness of antibiotics for treatment of mild ari in indonesian children. we now present the results from a randomised, controlled comparison of supportive care alone and ampicillin plus supportive care as treatment for mild ari in children under years of age. we chose ampicillin on the basis of our earlier findings that % of indonesian physicians who treated mild ari with antibiotics used ampicillin (unpublished). subjects and methods children under years of age with mild ari, who attended government health clinics in two regions of east jakarta, indonesia, were included in the trial. mild ari was defined by who criteria: mild upper respiratory signs such as cough or runny nose, and/or fever ( > &deg;c), and breathing at a rate less than breaths per minute. we excluded children with asthma or infections that required antibiotics, those who did not live in a defined service area, and those who had recently been treated by medical personnel in other locations. parental consent was obtained for all eligible children. through random allocation, children were offered ampicillin powder and supportive care and were offered only supportive care. parents in both groups were advised to provide supportive care at home including continuation of breastfeeding, clearing of the nose as needed, and control of mild fever by means of paracetamol, an inexpensive analgesic and antipyretic drug ( mg/kg body weight daily; doses per day for days). in addition to the supportive care, children in the ampicillin group received ampicillin powder (age-dependent treatment packets of - mg/kg body weight per dose every h [except midnight] for days). parents were asked to bring their child back to the health centre after days. home visits were made by nurses or midwives within days to children who did not appear at the clinic on the scheduled day. based on the clinic or home examination the health status was classified as: cured or recovered; static (no change); worse (moderate or severe ari); or dead. moderate and severe ari were defined according to who and ministry of health criteria. moderate ari was diagnosed if there was a respiratory rate greater than breaths per minute but no chest indrawing, and severe ari if the child had a respiratory rate greater than breaths per minute and chest indrawing, with or without cyanosis. children not cured after week were either referred to the health clinic for further medical care or sent home with instructions for additional supportive care. all children ( ampicillin, control) had stopped taking ampicillin or paracetamol because of side-effects (diarrhoea in [ ampicillin, control] and an allergic reaction in [control]). statistical analyses were done by means of 'epi info' and 'epi log plus', two microcomputer-based statistical analysis programs for epidemiology. these included standard chi-square tests (two sided) and confidence intervals for risk ratios (taylor series % confidence limits). the randomisation procedure successfully achieved two nearly identical groups in terms of the potential confounding variables of age, sex, level of parental education, history of measles immunisation, or fever at the time of enrolment (table ). after week, the percentages of children who were cured, still had mild ari, or had progressed to moderate ari in the two groups were almost identical (table n). there was no significant difference in the course of the disease between the ampicillin-treated and control groups at either -week follow-up (&khgr; = . , df; p= - ) or -week follow-up (x = - , df; p = - ) (table ii). after week the percentage cured was similar at all ages, in both sexes, at various education levels for fathers and mothers, and by measles immunisation status (table iii) . ampicillin appeared beneficial only among a few subgroups, but in each case the % confidence intervals included - , so the risk ratios are more likely to reflect variation inherent in the sampling process rather than a real benefit of ampicillin. the percentage of cases of mild ari that progressed to moderate ari at the -week follow-up was also similar in the various subgroups (table iv). in some subgroups the control cases were more likely to progress, whereas in others the ampicillin-treated cases were more likely to become worse. the width of the confidence intervals makes it unlikely that any of the group-specific values are truly different. after weeks, % of ampicillin-treated and % of control children still showed signs of mild ari (table ii) . % in each group had progressed to moderate ari and were referred to the health clinic for additional care. the remaining % of controls and % of ampicillin-treated children were cured of ari, and nearly three-quarters of them had recovered during the first week. the randomisation process in our study effectively created two groups with the same inherent risk of ari outcome independent of treatment. thus, we were able to assess the unconfounded effect of ampicillin plus supportive care compared with supportive care alone on mild ari. we conclude that there is no beneficial effect of ampicillin on the clinical course of mild acute respiratory infections among young indonesian children. for children breathing less than times per minute and showing minor signs such as a cough or runny nose, addition of ampicillin to simple supportive care supplemented with paracetamol conferred no benefit. since mild ari is primarily or entirely caused by viruses, our finding that ampicillin is of no benefit for such illnesses is hardly surprising, and readers might wonder why we undertook this study. in our previous work on ari in indonesia (unpublished), we observed that many children with mild ari were being treated with ampicillin by physicians at government clinics despite the ministry of health guidelines (which accord with who recommendations) that only supportive care is required. in our discussions with physicians, it became clear that many believed antibiotics were effective at preventing the progression of mild ari to pneumonia or other forms of severe ari, which are frequently bacterial in origin. when challenged to present data refuting this notion, we were unable to find support in the indonesian medical or public health literature. thus, we felt it necessary to assess in indonesia the effect of antimicrobial therapy on progression of mild ari to more severe forms. clinical studies of antibiotic effectiveness have been carried out in other nearby' countries-for example, thailand and australia. , these studies of children with upper (or mild) respiratory infections showed no therapeutic value for ampicillin, erythromycin, penicillin, or tetracycline. although our data clearly show that the use of ampicillin for mild ari had no beneficial effect, what is not evident is the potential harmful effects of inappropriate antibiotic use. first, ampicillin used for mild ari will not be available to treat moderate or severe ari. if there is a limited supply of antibiotics, as is the case in many government health clinics, the resultant shortage could lead to the use of less effective treatment and higher case-fatality for moderate and severe ari. even if antibiotics are widely available, use of ineffective treatment for many mild ari cases will substantially reduce the cost-effectiveness of ari treatments in general. second streptomyces, and arthrobacter genera, and more weakly related to mycobacteria. the biopsy specimen was estimated to contain around cells of the organism. the probable aetiological agent for our patient's illness has not been identified previously in a patient with whipple's disease. whipple's disease is a systemic infection associated with a small, largely intracellular bacillus of uncertain identity. the nature of the whipple's bacillus has not been established because efforts to culture the organism have been unsuccessful. we report the results of sequencing the ribosomal dna of the predominant bacterium associated with a biopsy taken from the small bowel of a woman with whipple's disease. comparison of s rrna sequences is a powerful approach to phylogenetic analysis, and had led to a new phylogenetic tree of all life forms. it was thus possible to place the whipple's-associated bacterial organism (wabo) phylogenetically by comparing its s rrna sequence with known sequences. a -year-old woman presented with a -year history of diarrhoea, a kg weight loss, and iron-deficiency anaemia. she complained of abdominal distension, arthralgia, fatigue, and myalgia. on physical examination the patient was cachectic with a distended abdomen and thickening of the metacarpals and wrists. x-ray examination of the small bowel suggested malabsorption, and an abdominal computed tomography scan revealed paracaval and periaortic lymphadenopathy. upper gastrointestinal endoscopy showed a bowel mucosa with a granular infiltrated pattern. histologically, the small bowel lamina propria was expanded by foamy macrophages and had prominent lymphatic dilatation. material in the macrophages stained periodic acid-shiff-positive and did not contain acid-fast organisms. electron microscopy showed large numbers of intracellular and extracellular bacilli characteristic of those found in whipple's disease. after informed consent, an endoscopic biopsy specimen of the proximal small bowel was taken and frozen rapidly in dry ice-ethanol. nucleic acids extracted from the biopsy specimen were amplified in a polymerase chain reaction (pcr) for cycles. the pcr primers were designed to amplify specifically a -base segment of bacterial s rdna (identical to an organism's rrna sequence). the resulting pcr product was sequenced directly. a computer search of the genbank and embi databases was done to find the rrna sequences most similar to the one we had isolated, and all sequences were aligned. the alignment was used to find a site at which the sequence of the wabo differed from the detailed diagnosis and procedures, national hospital discharge survey some clinical aspects of uroflowmetry in elderly males. a population study the development of benign prostatic hyperplasia among volunteers in the normative aging study are doctors able to assess prostatic size symptom status and quality of life following prostatectomy a technical and clinical evaluation of the disa uroflowmeter a simple uroflowmeter tester reproducibility of uroflowmetry variables in elderly males transabdominal ultrasound in the evaluation of prostate size the development of human benign prostatic hyperplasia with age natural history of benign prostatic hypertrophy acute respiratory infections in the developing world: strategies for prevention, treatment and control validation of postmortem interviews to ascertain selected causes of death in children acute lower respiratory infections: a major cause of death in children in bangladesh acute respiratory infections are the leading cause of death in children in developing countries antimicrobial susceptibility patterns of haemophilus isolates from children in eleven developing nations trial of co-trimoxazole versus procaine penicillin with ampicillin in treatment of community-acquired pneumonia in young gambian children world health organisation. clinical management of acute respiratory infections in children: a who memorandium aetiology of pneumonia in children in goroka hospital world health organisation. guidelines for research on acute respiratory infections: memorandum from a who meeting world health organisation. a program for controlling acute respiratory infections in children: memorandum from a who meeting report of a symposium on use and abuse of antibiotics worldwide epi info, version : a word processing, database, and statistics program for epidemiology on microcomputers epi log plus, statistical package for epidemiology and clinical trials ministry of health. health worker manual for ari case management and diarrhea among children under five communicable disease control and environmental health the value of antibiotics in minor respiratory illness in children-a controlled trial evaluation of orally administered antibiotics for treatment of upper respiratory infections in thai children we thank the consultant urologists and other staff of stirling royal infirmary, the general practitioners and their staff at bridge of allan health centre, the forth valley general practitioner research group, and all other members of the bph natural history study group for their advice, cooperation, and support. funding for this work was provided by merck, sharp & dohme. key: cord- -ts lyy p authors: pedersen, n.c; elliott, j.b; glasgow, a; poland, a; keel, k title: an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ts lyy p an isolated epizootic of a highly fatal feline calicivirus (fcv) infection, manifested in its severest form by a systemic hemorrhagic-like fever, occurred over a -month period among six cats owned by two different employees and a client of a private veterinary practice. the infection may have started with an unowned shelter kitten that was hospitalized during this same period for a severe atypical upper respiratory infection. the causative agent was isolated from blood and nasal swabs from two cats; the electron microscopic appearance was typical for fcv and capsid gene sequencing showed it to be genetically similar to other less pathogenic field strains. an identical disease syndrome was recreated in laboratory cats through oral inoculation with tissue culture grown virus. during the course of transmission studies in experimental cats, the agent was inadvertently spread by caretakers to an adjoining room containing a group of four normal adult cats. one of the four older cats was found dead and a second was moribund within – h in spite of symptomatic treatment; lesions in these animals were similar to those of the field cats but with the added feature of severe pancreatitis. the mortality in field cats, deliberately infected laboratory cats, and inadvertently infected laboratory cats ranged from – %. this new isolate of calicivirus, named fcv-ari, was neutralized at negligible to low titer by antiserum against the universal fcv-f vaccine strain. cats orally immunized with fcv-f , and then challenge-exposed shortly thereafter with fcv-ari, developed a milder self-limiting form of disease, indicating partial protection. however, all of the field cats, including the three that died, had been previously immunized with parenteral fcv-f vaccine. fcv-ari caused a disease that was reminiscent of rabbit hemorrhagic disease, a highly fatal calicivirus infection of older rabbits. feline calicivirus (fcv) is one of the most common viral pathogens of cats, especially in multi-cat environments such as shelters and catteries. fcv infection and disease occurs in acute and chronic forms (reviewed by studdert, ; reubel et al., ) . the manifesting signs of acute disease depends on the route (oral versus aerosol) and strain of virus. aerosol infection, which is commonly used in laboratory studies, produces a more serious disease than oral infection, with lesions extending further down the respiratory tract. hoover and kahn ( ) described the acute disease produced by ®eld strains of fcv. the disease differed only in severity, with the more virulent strains causing fever, depression, dyspnea, pneumonia, and vesicles/ulcers of the tongue, hard palate and nostrils. lower virulence strains were less likely to affect the lungs, although other signs were similar. transient mild conjunctivitis and rhinitis, has also been associated with acute fcv infection, but unlike pathogens such as feline herpesvirus, upper respiratory involvement is not the major feature of fcv infection. pedersen and colleagues ( ) added the feature of`limping' to the fcv disease syndrome. the lameness appears to be associated with acute viremia and localization of virus and/or immune complexes in the joints (bennett et al., ; dawson et al., ) . following recovery from acute disease, up to one-fourth of cats will shed the virus for a prolonged period of time from their oropharynx (bennett et al., ; harbour et al., ; dawson et al., a, b) . although most fcv carriers are asymptomatic, a small proportion will develop a distinct disease syndrome known as chronic plasmacytic/lymphocytic stomatitis or chronic ulceroproliferative stomatitis (reubel et al., ) . this chronic oral disease is progressive and extremely dif®cult to treat and is perhaps the single most important clinical manifestation of fcv infection today. it is fortunate for cats that fcv is not more virulent. related caliciviruses, such as rabbit hemorrhagic disease virus, have been associated with severe clinical disease and high mortality. although recognized strains of fcv have not been associated with signi®cant acute mortality, the calicivirus genome is highly mutable (johnson, ; pedersen and hawkins, ; geissler et al., ; kreutz et al., ; radford et al., ) and more highly virulent strains could arise at any time. the present report describes the sudden appearance and disappearance of a novel strain of fcv, which induced a severe systemic hemorrhagic-like fever and high mortality. the new strain, named fcv-ari, has weak to negligible cross-reactivity to current vaccine-induced immunity and would pose considerable risk to cats if it were to spread widely among either vaccinated or unvaccinated animals. speci®c pathogen free (spf) cats, of mixed gender, were obtained from the breeding colony of the veterinary nutrition laboratory, school of veterinary medicine, uc davis (compliments of dr. james morris and dr. quinton rogers). cats were from to weeks of age and housed in the experimental animal facilities of the center for companion animal health and cared for under the oversight of the animal resources services, uc davis. complete blood counts, parameter serum chemistries, and various clotting studies were done by either idexx veterinary services, western region, west sacramento, ca, or by the clinical pathology service of the veterinary medical teaching hospital (vmth), school of veterinary medicine, uc davis. gross and/or histopathologic studies were done by either idexx veterinary services or the pathology service of the vmth, uc davis. initial virus isolation were from cats vmth- (ari) and - (ian). swabs of nasal exudate, and herparinized whole blood, were taken from both animals on october . blood ( . ml) was diluted with an equal volume of hank's buffered salt solution (hbss) and centrifuged at Âg for min. the buffy coat was suspended in ml of hbss; the suspended cells were overlayered onto subcon¯uent monolayers of crandell feline kidney (crfk) and felis catus whole fetus- (fcwf- ) cells grown in cm ¯a sks. nasal swabs were placed in ml snap-top tubes containing ml of hbss with  gentamicin and penicillin/streptomycin and allowed to sit for at least h at room temperature. supernatant ( ml) was placed into cm ¯a sks containing subcon¯uent monolayers of crfk or fcwf- cells. cell cultures were observed every h for cytopathic effect (cpe). three -week old spf kittens were infected oronasally with . ml of tissue culturē uid containing approximately  tcid of fcv-ari. one half of the inoculum was instilled up the nostrils and one half placed in back of the throat. complete blood counts and serum chemistries were taken ± h prior to infection and every ± days thereafter. rectal temperatures were recorded starting days prior to infection and daily thereafter. cats were observed for clinical signs of depression, anorexia, limping, subcutaneous swelling and edema, skin lesions, nasal discharge/nasal congestion/ sneezing, diarrhea, and vesicles and/or ulcers of the palate, tongue, gums, cheeks, lips, or skin. for vaccine studies, four ± weeks old spf kittens were immunized oronasally, as described earlier, with ml of tissue culture¯uid containing approximately  tcid of the universal vaccine strain fcv-f (heska subisolate). two sibling kittens were kept strictly isolated and used as unimmunized controls. kittens were monitored for clinical signs of infection as outlined earlier. . . virologic studies . . . fcv strains a number of fcv strains, of vaccine and ®eld origin, were used in this study. the origin and serologic relationships of most of these strains has been previously reported (pedersen and hawkins, ) . vaccine strains, which were genetically identical to each other and to the original fcv-f , were re-isolated from several commercial brands of vaccine (pedersen and hawkins, ) . a nested rt-pcr reaction was used to detect fcv-rna in blood (buffy coat), oral swabs, and feces. forward and reverse primers amplifying a bp region of the capsid gene corresponding to nucleotides ± of the fcv genome were used, following the protocol of radford et al. ( ) . pcr products were run through microcon- columns (millipore, burlington, ma) to remove primers and salts and then submitted to a commercial sequencing laboratory (davis sequencing, davis, ca) for automated sequencing analysis. sequence data were analyzed on a vax computer using the university of wisconsin's genetics computer group (gcg) sequence analysis software package (gcg program manual for the wisconsin package, version , ). a dendrogram (cladogram) was generated by sequence comparison using the cantor-jukes correction, followed by distance calculations using the upgma algorithm. virus neutralizing (vn) antibodies to fcv were measured by a residual virus infectivity assay using constant amounts of virus and antiserum. a diluted tissue culturē uid stock of fcv-f or fcv-ari ( ml), containing approximately tcid , was incubated at c for h with either ml of a : dilution of tissue culture medium (negative control) or a : dilution in tissue culture medium of the cat serum to be tested. ninety-six well microtiter plates containing a monolayer of subcon¯uent crfk cells were used for the titration; each well contained ml of tissue culture¯uid. the virus/antiserum mixture of ml was added to the ®rst well, mixed well, and ml carried serially to each of the next wells in the same manner. this procedure was repeated in triplicate. a similar titration was done using the same virus stock and an equal volume of tissue culture medium alone. the plates were incubated for h and observed under an inverted microscope for typical fcv cpe; the last well containing any detectable cpe was read as endpoint. the vn antibody titer of the serum was calculated from the difference in the average virus titer with and without serum treatment, i.e. if the average virus titer of a sample of virusantiserum was : , and the titer of the corresponding virustissue culture¯uid : , , the difference was six wells ( / ) or a vn titer of : . fcv-ari was infected onto fresh crfk in a cm ¯a sk, and the cultures observed closely for cpe. about h after infection, when cytopathic effect was noticeable but most cells were still attached, the tissue culture medium was decanted and the monolayer overlayered with cold . % paraformaldehyde/ . % glutaraldehyde in . m sorenson's phosphate buffer, ph . . the¯asks were held for several hours at room temperature, and then placed into the refrigerator for several days. post-®xation was in % osmium tetroxide in . m sorenson's phosphate buffer at c. the samples were processed by the electron microscopy service of the dept. of pathology, school of medicine (with special assistance from mr. bob munn). grid staining was with % aqueous uranyl acetate and lead citrate. grids were examined and photographed using a philips em electron microscope. the ®rst tissue analyzed were three full-thickness skin biopsies from the face, nose and ear of the cat named ria. the samples were ®xed in formol-saline and hematoxylin and eosin stained tissue sections prepared by a private laboratory (idexx veterinary services, inc., west sacramento, ca ). subsequent necropsies and gross and microscopic pathologic analyses were done by the veterinary pathology service of the vmth, department of pathology, school of veterinary medicine, university of california, davis, ca . the index case in this outbreak of atypical fcv infection was probably an orphaned, fully vaccinated, -month old, female domestic kitten from a local animal shelter. one of the authors (jbe), noticed that the kitten had a severe upper respiratory infection. therefore, the kitten was brought from the shelter into the clinic on september for hospitalization and treatment. the kitten subsequently developed crusty, ulcerative lesions on her face and oral vesicles on the margins of the tongue and soft palate. the kitten was intensively treated for days before being well enough to be adopted. the kitten intussuscepted week later and died. the second cat in the outbreak was ria, a -year old, spayed female, domestic, strictlyindoors cat owned by a veterinary assistant in the same veterinary clinic. the cat received her last feline panleukopenia-/herpes-/calici-virus (fphcv) immunization on april . the only exposure outside the home was a brief hospitalization for dental prophylaxis on october . ria presented on october with a primary complaint of h of lethargy, anorexia, and fever. physical examination was uninformative except for fever. tests for felv and fiv infections were negative and cbc and blood chemistry analyses were unremarkable. a slight swelling of the dorsal muzzle, extending from one canthus to the other, was observed that afternoon. shortly thereafter, hair over the right side of the muzzle epilated, revealing a mm diameter erythematous patch with a dark necrotic center. ria was sent home for observation, but returned on october still lethargic, anorectic and febrile. additional lesions, with focal crusting, erythema and epilation, were present on the right and left medial canthi, commissures of the mouth, and on the margins of both pinnae. the patient also demonstrated diffuse cutaneous edema of the face and submandibular area and extending down the limbs. biopsies were taken from the muzzle and pinnae lesions and swabs of several sores were collected for routine aerobic bacterial culture. ria was still febrile on october , but did not appear to be in discomfort and was beginning to eat and groom. skin biopsies taken on october revealed multifocal moderate to severe neutrophilic/lymphoplasmacytic perivascular and periadnexal dermatitis, super®cial pyoderma, and extensive ulceration and super®cial dermal necrosis with underlying vasculitis. no infectious organisms were observed in routine or special stained tissues sections and no aerobic bacteria grew in culture. ria was treated with prednisolone and antibiotics; within the next days she was eating well and actively grooming, the cutaneous edema was resolving, and the skin lesions were healing. the third affected cat, indy, was a -month old, domestic, indoor/outdoor cat. he was brought to the hospital on october for castration and his ®nal parenteral fphcv immunization. indy returned to the hospital on october with depression and fever. abnormalities on a standard blood chemistry panel included a mild increase in creatinine phosphokinase (cpk) and total bilirubin. felv and fiv tests were negative and a coronavirus ifa antibody titer was : . indy was treated with iv electrolyte solution and antibiotics. the fever was resolved by october , but ulcerative lesions appeared on the margins of both pinnae on october. the cat made an uneventful recovery. the fourth cat, garth, was a -year old, castrated male, domestic, indoor/outdoor cat and the housemate of indy. the cat had received the recommended series of parenteral fphcv immunizations as a kitten and yearly boosters. garth was seen on october for mild conjunctivitis and squinting and sent home on ophthalmic antibiotic ointment. the cat appeared to make an uneventful recovery, but weeks later the owner noticed focal areas of alopecia and encrustation on the muzzle, base of the right pinna, and bilaterally on both¯anks, compatible with old healing ulcers. the ®fth cat in this focal epidemic was aristotle (ari), a . -year old, neutered male, domestic, indoors-only cat and the son of ria. there were no signi®cant past health problems; he tested negative for felv and fiv infections and had been parenterally vaccinated against rabies and fphcv on april ari presented on october with a history of an acute onset of lethargy and anorexia of days duration. a slight lameness in the right foreleg was noted days prior but had spontaneously resolved. the patient had vomited a bile tinged¯uid with¯ecks of red blood. physical examination revealed fever and a small crusty lesion on the lower lip. ari returned to the hospital on october afebrile and alert, but moderately dehydrated and anorectic. diffuse facial edema with central ulceration were noted, primarily over the muzzle and left pinna; focal crusty and erythematous lesions were also present on the upper and lower lips. intravenous¯uids, metoclopramide and antibiotic therapy were instituted. ari was afebrile on october but anorectic. the facial swelling was mildly increased but the central ulcerated areas seemed to be less exudative. the patient's right forepaw was slightly edematous. ari was still afebrile on october but remained anorectic; the edema of the right forepaw remained unchanged. the patient was transported to the uc davis vmth for additional testing, including blood and nasal swabs for virus isolation and blood for chemistry and coagulation pro®les. blood chemistries revealed moderate elevation of cpk, mild elevation of total bilirubin, and moderate hypoproteinemia. there was evidence of a coagulopathy with moderately increased prothrombin and partial thromboplastin times and a slightly prolonged activated clotting time (act). a fcv was isolated in cell cultures exposed to both blood and nasal exudate. ari was still euthermic on october ; however, he was anorectic and his entire face was edematous. the facial lesions had coalesced to form large irregular crusts on both sides of the muzzle (fig. ) , and the mucus membranes were slightly icteric. a nasoesophogeal feeding tube was positioned and oral hyperalimentation instituted; this therapy that would be continued until his death. ari seemed more responsive on october , although still anorectic and hypocoagulable. the patient appeared comfortable on october ; there was no more vomiting, but the subcutaneous edema of the right foreleg was persisting. ari was transfused with one unit of whole blood and low level heparin therapy was instituted to counteract a suspected disseminated intravascular coagulopathy (dic). dextran was given initially as an iv bolus and then maintained as a continuous infusion. laboratory tests taken in the evening of october indicated a persisting coagulopathy, hypoproteinemia, and hyperbilibubinemia. ari was alert, responsive, and afebrile on the morning of october; however, he was diffusely edematous, had mild watery diarrhea and exhibited a slightly increased respiratory rate and effort. laboratory work revealed a decreased hematocrit, hypoproteinemia and greatly elevated act. the patient was then given a second unit of cross matched whole blood. the respiratory rate continued to increase throughout the day and a thoracocentesis was performed late that evening and a yellowish-transudate withdrawn. ari was alert and responsive on october , but still anorectic. the patient received a third unit of whole blood at midday and more pleural¯uid was removed. on october , ari was quiet, responsive and vocal when handled, but polypneic and totally anorectic. a chest tube was placed to facilitate thoracic drainage and the dextran replaced with hetastarch. ari was still anorectic and lethargic on the morning of october; he was diffusely edematous with small areas of miliary crusting noted in areas of greatest swelling, especially on the distal limbs. despite treatment, ari's condition slowly deteriorated and he died on november from complications of pneumothorax, shock and cardiac arrest. a fcv, genetically indistinguishable from the earlier isolate, was recovered from blood and tissues at postmortem examination. ian, a . -year old, neutered male, domestic, indoors-only cat, also a son of ria, was the sixth cat to be affected. on october , the owner observed that ian was febrile and manifesting signs similar to those shown by his mother. previous clinical history included a negative felv/fiv test on august and routine parenteral vaccination for rabies and fphcv on june . ian became febrile on october and was lethargic and sneezing occassionally. physical examination revealed mild edema of his muzzle with patchy erythematous macules and miliary crusts on margins of both pinnae. ian was transported to uc davis, vmth for consultations with one of the authors (ncp) and a veterinary dermatolgist (dr. peter ihrke). blood and nasal swabs were collected and the cat returned home. a fcv, which was genetically identical to the isolate from ari, was obtained in tissue cultures from both blood and nasal exudate. on october , ian was still febrile at home but was eating and drinking. the facial and pinna swellings were increasing and new crusts were present on his pinnae and around his nares. ian continued to do well at home on october, although the owner reported sneezing and nasal congestion. ian was still sneezing and congested on october and the nasal and ear lesions had begun to coalesce. his appetite and attitude remained very good and he made an uneventful recovery. the seventh cat in the outbreak was emma, a . -year old spayed female, domestic, who was kept strictly indoors. emma tested negative for felv and fiv as a kitten and was last vaccinated for felv and fphcv on june . emma was owned by a second technician who worked at the same practice. emma presented on november with a history of days of lethargy and anorexia. her physical examination was unremarkable except for fever, moderate dehydration and possible abdominal tenderness. the patient was hospitalized and started on intravenous¯uids and antibiotics. presenting lab work revealed a slightly elevated total blood bilirubin and glucose. felv and fiv tests were negative. on november , emma was depressed, anorectic, febrile and hyperirritable. the fever increased by evening and her left hind paw appeared swollen. emma was still anorectic on november but was much more alert; rectal temperatures continued to be elevated and her right hind paw was swollen. emma remained anorectic on november , her hind paws remained swollen and slight swelling was observed on the muzzle. small areas of ecchymosis were noted on the right caudal abdomen. a naso-esophageal feeding tube was inserted and the patient was started on a full strength hyperalimentation solution. the next morning emma became restless and mildly dyspneic, with pronounced decreases in the hematocrit and total plasma proteins. emma was started on vigorous treatment for a developing hypoproteinemia and dic; therapy included intravenous hetastarch, subcutaneous heparin, intravenous antibiotics, and nasoesophageal tube feeding. the patient's respiratory rate continued to increase throughout the day even though her fever abated. a chest tube was placed but only small amounts of a transudate were withdrawn. she became progressively more dypneic and depressed throughout the day and was hypothermic by midnight. results of laboratory tests taken in the evening revealed a moderate hypoproteinemia, hyperbilirubinemia and thrombocytopenia. blood tests taken on november showed an elevated glucose, decreased hematocrit and total protein, and a prolonged act. emma's condition continued to decline throughout the day, with increasing respiratory dif®culty. lasix and dobutamine were administered by intravenous drip and intravenous hetastarch and nasoesophageal tube feeding discontinued. on november , emma's condition continued to decline overnight and the owner elected to have the cat euthanatized. affected cats eight through ( - , - , - and - ) were . ± . years old, neutered male, moderately obese, laboratory cats that were housed two rooms away from kittens that had been experimentally infected with an fcv isolate obtained from the blood of ari (see section . ). the four cats had not been exposed to other agents and were quarantined from other cats through separate ante-rooms, and were cared for by different caretakers. the caretakers took extra precautions in terms of disposable overalls, gloves, boots, and foot baths, as well as separate food and litter sources, to prevent inadvertent infections from the outside. on the morning of november , one of the four cats ( - ) in this group was found dead and the other three animals were depressed and febrile. gross necropsy ®ndings showed a reddened and somewhat swollen pancreas with areas of hemorrhage and saponi®cation in peripancreatic mesenteric fat. several large areas of yellow-tinged subcutaneous edema were observed in the groin and trunks. the lungs appeared grossly congested with many areas of collapse, and there was a small amount of free¯uid in the abdomen and chest cavities. the condition of the remaining three cats rapidly deteriorated, in spite of antibiotic,¯uid and whole blood therapy. cat - was in shock on november and euthanatized. the remaining two cats ( - and - ) were still febrile on november but were becoming rapidly more depressed; a decision was made to euthanatize them rather than to continue symptomatic treatment. blood chemistry panels taken near the time of death from all four animals revealed mild to moderate elevations in blood glucose, moderately decreased serum total proteins, and mild to moderate hyperbilirubinemia. gross necropsy ®ndings in the three latter cats were identical to those observed in the ®rst cat. all three cats had reddened and swollenappearing pancreases with hemorrhage and saponi®cation of peripancreatic fat. in addition to these ®ndings, cat - had an edematous omentum, ulceration of the skin on the phalanges of the right hindleg, sloughing of the claw on second digit of the right hindleg, an ulcer of the skin under the jaw, a yellowish edema under the tongue and icteric appearing nictitating membranes. cat - , in addition to pancreatic lesions, had congestion and consolidation of the right lung lobes, an edematous omentum, and splenomegaly. cat - had identical lesions on the pancreas and surrounding tissues, but also had subcutaneous edema with yellowing of the tissues of the hind limbs and edema of all four paws. crfk and fcwf- cell cultures of blood and pancreatic tissue from all four cats yielded fcv. genetic sequencing of fcv isolates from all four cats showed them to be identical to fcv-ari (data not shown). maximum quarantine was initiated at the time of the secondary outbreak; the virus was only handled under the strictest of quarantine conditions, with special isolation facilities, separate caretakers, complete changes of disposable coveralls, gloves, and foot coverings. no additional cases were observed in either the clinic of origin or in the experimental animal facilities after this time. cytopathic effect, typical of fcv, was evident within h on both crfk and fcwf- cells cultures exposed to blood and nasal secretions of ari. transmission electron micrographs of the cells demonstrated characteristic fcv capsids, ± nm in diameter, within paracrystalline viral arrays in the nuclei (not shown) and parallel linear viral arrays in the cytoplasm (fig. ) . rna was extracted from infected cultures, reverse transcribed, and subjected to pcr. the pcr product was sequenced (table ) and proved to be identical, within the range of strain variation, of a number of vaccine-and ®eld-origin fcvs (fig. ) . the virus isolate was designated fcv-ari. three -week old spf kittens, - , - , and - , were infected oronasally with fcv-ari on october . the kittens were febrile by h post-inoculation. the fever continued unabated for the course of the study, while other signs appeared in rapid progression. evidence of nasal congestion with occasional sneezing and slight ocular discharge were noticeable by the second day. nasal congestion was associated in all three cats with subcutaneous edema over the bridge or side of the nose. in one cat, a shallow ulcer appeared over the site of edema. the feet and lower limbs of the kittens also became edematous. all of the infected cats were anorectic after day and required subcutaneous uid therapy to maintain hydration. cat - was in shock on november and was given ml of fresh whole blood intravenously. he failed to rally and was euthanatized on november . the remaining two cats, - and - , began to show progressive improvement from november onward and were essentially normal by november . signi®cant abnormalities, including hypoproteinemia with hypoalbuminemia and hypoglobulinemia, hyperglucosemia, and hyperbilibubinemia were seen at the peak of illness in cat - on november . hyperbilibubinemia was the only signi®cant ®nding on the remaining two animals. virus was readily isolated from both blood and nasal secretions of the experimentally infected cats. the genetic sequence of pcr products from the two isolates that were tested was identical to fcv-ari isolates obtained from the ®eld cases. detailed necropsy and histopathologic studies were done on ari, four laboratory cats inadvertently infected with fcv-ari, and on four laboratory cats deliberately infected with fcv-ari. gross lesions present on ari included alopecia of the rear limbs, inguinal and perineal regions and the dorsal aspects of the forelimbs, with dozens of ulcers ranging from . ± cm in diameter within alopecic foci. a small vesicle was present on the left carpus and all of the digital pads were ulcerated. small ulcers were also present on the lateral aspects of the gum and medial buccal mucosa. subcutaneous edema was prominent on the lateral body wall, and numerous irregular chalky, white foci were widely disseminated in the subcutaneous fat. the lungs were reddened and atelectatic and ml of a pinkish milky¯uid was present in the pleural cavity. fifty or more miliary grey foci were randomly distributed on the surface of all lung lobes. the cranial mediastinum was thickened and a thrombus was present in the right jugular vein. the abdomen contained about ml of serosanguinous¯uid with ®brin clots and ®brin tags adherent to the surface of the viscera. a . cm . cm ulcer was present in the gastric cardia, and smaller ulcer was also observed on the mucosa of the trigone of the bladder. histopathologic examination showed a severe multifocal epidermal necrosis with ulceration. there was a chronic suppurative necrotizing bronchopneumonia with squamous metaplasia and intralesional fungal hyphae consistent with aspergillus sp. the pulmonary aspergillosis was presumed to be a terminal sequelae of the persistent calicivirus infection, debilitation of the immune system, and prolonged antibiotic therapy. the lung tissue was severely atelectatic, with intraalveolar histiocytic exudate and multifocal vascular thrombosis. a moderately severe and chronic pleuritis was present. tracheobronchial lymph nodes manifested widespread ®brinous sinus thrombi and moderate lymphoid depletion. a severe diffuse and chronic thrombosis was evident in subcutaneous vessels with associated fat necrosis. the spleen showed marked red-pulp histiocytosis. cat - , which was representative of the four laboratory animals that were inadvertently infected with fcv-ari, demonstrated several large areas of subcutaneous yellowish edema, diffuse consolidation of the lungs, swelling and reddening of the pancreas, and numerous chalk-like plaques with surrounding hemorrhage were present in the peripancreatic fat. histopathologic ®ndings included a moderate to severe multifocal pancreatitis with a mixed in¯ammatory in®ltrate, peripancreatic fat necrosis, mild to moderately severe multifocal crypt necrosis in the duodenum and colon, moderately severe and diffuse individual hepatocellular necrosis with mild periportal lymphocytic/ histiocytic in®ltrate, and acute multifocal interstitial pneumonia. gross ®ndings in laboratory cats ( - , - , - , - ) , which were purposefully infected with fcv-ari, were similar, and included patchy to diffuse consolidation of the lungs, small amounts of free pleural¯uid, and areas of subcutaneous edema, especially around the face. intestinal crypt lesions were apparent in all four animals; crypts were lined with large pleomorphic, or¯attened, epithelial cells and necrotic cells and cellular debris often ®lled the crypt lumens. peyer's patches were hyperplastic, and the sinuses of lymph nodes were expanded by numerous foamy macrophages and subcapsular neutrophilic in®ltrate. paracortical lymphoid hyperplasia with abundant apoptotic cells were also evident. the medullary centers of the thymus were often expanded by ®brin and eosinophilic cellular debris. a foci of pancreatic necrosis with in¯ammatory in®ltrate was noted in one animal. pulmonary changes included a mild multifocal atelectasis with a few neutrophils and lymphocytes present in the interstitium of capillaries and small airways. rare in¯ammatory cells were present in the lumen of pulmonary capillaries and bronchioles. multiple randomly situated foci of necrotic hepatocytes, with or without a small number of neutrophils, were observed. marked diffuse edema and epidermal necrosis were present in sections of diseased appearing skin. four -week old spf cats ( - , - , - and - ) were immunized orally with fcv-f and serum collected weeks later. these sera were then tested in a virus neutralization assay against fcv-f and fcv-ari (table ). there was low to negligible cross-reactivity between fcv-f immune sera and fcv-ari. serum collected from cat - weeks following fcv-ari infection was titrated for virus neutralizing activity against a number of vaccine and ®eld strains of fcv (table ) . fcv-ari antiserum neutralized all four vaccine strains (three containing fcv-f ), but failed to neutralize / ®eld isolates. six spf kittens were divided into vaccine-(cats - , - , - and - ) and control-groups (cats - , - ) . the vaccine group was inoculated orally on january with . ml of a tissue culture supernatant containing approximately  table virus neutralizing titer of serum from fcv-ari recovered cat - against several vaccine and ®eld strains of table ). the four recently vaccinated cats, along with two additional unvaccinated animals, were challenge-exposed to fcv-ari on february , which was weeks postvaccination. the route and dosage were identical to the initial experimental infection study. both vaccinated and unvaccinated cats became febrile within h, and the fever persisted for ± days (fig. ) . the febrile responses were slightly lower and about day shorter in duration in vaccinated versus unvaccinated cats. clinical signs were similar in both groups, but noticeably less severe in vaccinates than nonvaccinates. clinical signs included transient anorexia, dehydration, lameness, slight ocular and nasal discharge, mild sneezing, swelling of the face with occasional ulceration, edema of the pinnae with ulceration or crusting of the edges, and variable swelling of the lower limbs. there table the oral shedding (culture/pcr) of feline calicivirus by fcv-f vaccinated cats, following vaccination and after challenge exposure to fcv-ari at week post-immunization a weeks post oral vaccination with fcv-f appeared to be no relationship between whether the cats were still shedding fcv-f vaccine virus at the time of challenge-exposure and subsequent disease severity; cat - , a vaccine virus carrier, was almost as symptomatic as the nonvaccinates. both vaccinated and unvaccinated cats were treated with subcutaneous lactated ringers solution for several days, but all animals made a rapid uncomplicated recovery starting at days ± post-infection. virus shedding, as measured by both tissue culture isolation and pcr, from the oral cavity was observed in both nonvaccinates following fcv-ari challengeexposure and in / vaccinates (table ). virus shedding lasted from ± weeks; no cats were still shedding calicivirus by culture or pcr after week . virus shed at the time of fcv-ari challenge-exposure and earlier was of the f strain, while virus shed after challenge was entirely of the fcv-ari strain as determined by sequence analysis (data not shown). fcv is endemic in most catteries, shelters and large multiple cat households, where up to one-fourth of cats may be orally shedding the virus at any given time (bennett et al., ; harbour et al., ; dawson et al., a, b) . fcv exists in numerous overlapping serotypes but is basically a single species (gillespie and scott, ) . different strains vary somewhat in the main types of disease signs that they cause pedersen et al., ; pedersen and hawkins, ) , but these differences are hard to discern from genetic or serologic comparisons (dawson et al., a, b; pedersen and hawkins, ; geissler et al., ; teewee et al., ) . although original descriptions of fcv infection suggested that it might be a major cause of mortality in young cats, this has not been borne out by subsequent experimental studies. so-called highly virulent strains of fcv, such as fcv- (gillespie and scott, ) , can exist in breeding populations of cats without signi®cant disease; kittens being infected from ± weeks of age when they still have signi®cant levels of passive immunity (johnson and povey, ) . clinical disease can be of acute or chronic form. acute disease, when it is seen, is generally systemic with vesicular involvement of the oral cavity (ulcers on palate, tongue, in¯ammation of gums and oral fauces), arthropathy (limping) and mild interstitial pneumonia pedersen et al., ; pedersen and hawkins, ; teewee et al., ) . upper respiratory disease is relatively uncommon, associated with certain strains much more than others, and manifested as a mild and transient bilateral conjunctivitis and rhinitis (compare fcv- and fcv-cook in pedersen and hawkins, ) . a viremia is detectable during the ®rst week of infection (pedersen et al., ) , and oral shedding occurs for several weeks to months (reviewed by pedersen and hawkins, ) . the source of viral carriage and shedding appears to be the tonsils and surrounding mucosa (dick et al., ) . there seemed to be little doubt that the infection in the ®rst six client-owned cats was nosocomial. however, catteries and shelters are more common sources of infection than veterinary clinics, an observation which may have been borne out by the theorized index case in this outbreak, a -month old female domestic kitten that had been brought into the hospital from a local shelter and treated for a severe atypical viral infection during the period of september to october . the dates that this particular animal was in the clinic both preceded and overlapped the dates when client-owned cats were exposed. this kitten was receiving almost constant medical attention and contamination of hands, clothes, instruments, etc., would have been dif®cult to prevent under the strictest of conditions. the disease caused by fcv-ari in both natural and experimental infections appeared to target blood vessels, as evidenced by the severe edema (sometimes with hemorrhage) in subcutaneous tissues and lungs and local necrosis of skin and adipose tissues. although vasculitis was dif®cult to discern as a distinct lesion in tissues of laboratory infected cats, it was de®nitely a feature of the early skin lesions that were ®rst observed on the cat ria. loss of vascular integrity was also the best explanation for the signi®cant drop in serum proteins, icteric serum (from breakdown of extravasated red blood cells), variable thrombocytopenia, and coagulopathies. elevations in cpk also indicated myonecrosis. the noteworthy feature of the infection was its persistence in the blood of those cases that went on to die; virus was still present in the blood of ari at the time of his death, almost weeks after ®rst clinical presentation. persistence of viremia may also have explained the death of the index kitten after a -week course of illness. the type of disease caused by fcv-ari is highly reminiscent of rabbit hemorrhagic disease, also caused by a calicivirus and ®rst reported in china (liu et al., ) . similarities included the high mortality, acute nature, the tendency to cause more severe disease in older animals, the ease of spread, hepatocyte tropism, and widespread vascular disease. following its apperance in china, rhd spread throughout europe, where it caused severe disease in rabbits but mild to unapparent infections in hares (mitro and krauss, ) . a closely related calicivirus, the european brown hare syndrome virus (ebhs virus) (bascun Äana et al., ) , affects both wild and farmed hares, and was also ®rst reported in the s in europe. both viruses cause severe necrotizing hepatitis and widespread hemorrhaging. the disease caused by the rhdv is particularly severe, with mortalities approaching % in rabbits older than weeks of age (nowotny et al., (nowotny et al., , . the high species speci®city and virulence of rhdv led the australian government to experiment on the virus as a means to control wild rabbits (kovaliski, ) . experiments conducted on an offshore island showed the potential of the virus as a biologic agent, but before a ®nal decision for controlled release could be made, the rhdv appeared in wild rabbits on the mainland. the virus spread rapidly, with over % mortality on wild rabbit populations recorded for one region of south australia (mutze et al., ) . reasons for the high mortality associated with fcv-ari were undetermined. the virus is de®nitely more virulent than any fcv strain yet tested by one of the authors (ncp) or reported in literature. it was noteworthy that the cats that became the most ill were also among the oldest. rhdv causes a mild self-limiting infection in rabbits less than weeks of age, while the mortality approaches % in older animals (mutze et al., ) . inherent resistance factors also seemed to play a role; some individual cats developed mild self-limiting disease, while others were devastated by the infection. the close genetic relationship of ria, ari and ian could have contributed to the severity of their illness. at least one of the cats (ari) was also treated with prednisolone early in the course of his infection, in the belief that the disease was some sort of immune vasculitis. however, another cat (emma) in the initial outbreak, and several laboratory cats, were not treated with prednisolone and had a similar fatal disease course. even though many ®eld strains of fcv do not cross-react with the almost universal fcv-f vaccine strain (pedersen et al., ; knowles et al., ; pedersen and hawkins, ; laruritzen et al., ; hohdatsu et al., ) , none of these strains have been of this virulence and the consequences have therefore been small. antibodies against the universal fcv-f vaccine strain also did not signi®cantly cross-react with fcv-ari, and laboratory cats immunized by the oral route with fcv-f weeks prior to challenge-exposure possessed only a small measure of immunity. two ®eld cats that died of the infection (ari and emma) had been previously immunized for fcv also indicating that current fcv vaccines, given by prescribed parenteral routes, will not protect against fcv-ari. why such a virulent and obviously contagious strain of fcv would appear and disappear so suddenly is unknown. the virus was spread readily from one group of cats to another both within the practice of origin and in the laboratory setting with ease, and it is fortunate that this spread was contained in both areas. unfortunately, highly virulent, vaccine resistant, viruses like fcv-ari may arise again in the future and vigilance is required. detection and differentiation of rabbit hemorrhagic disease and european brown hare syndrome viruses by ampli®cation of vp genomic sequences from fresh and ®xed tissue specimens detection of feline calicivirus antigens in the joints of infected cats typing of feline calicivirus isolates from different clinical groups by virus neutralization tests investigation of vaccine reactions and breakdowns after feline calicivirus vaccination acute arthritis of cats associated with feline calicivirus infection sites of persistence of feline calicivirus genetic and antigenic heterogeneity among feline calicivirus isolates from distinct disease manifestations feline viral infections isolation of feline calicivirus and feline herpesvirus from domestic cats to neutralizing feature of commercially available feline calicivirus (fcv) vaccine immune sera against fcv ®eld isolates experimentally induced feline calicivirus infection: clinical signs and lesions antigenic change in feline calicivirus during presistent infection feline calicivirus infection in kittens born by cats persistently infected with the virus induction of immunity to feline caliciviral disease neutralisation patterns among recent british and north american feline calicivirus isolates from different clinical origins phenotypic and genotypic variation of feline calicivirus during persistent infection of cats monitoring the spread of rabbit hemorrhagic disease virus as a new biological agent for control of wild european rabbits in australia serological analysis of feline calicivirus isolates from the united states and united kingdom a new viral disease in rabbits rabbit hemorrhagic disease: a review with special reference to its epizootiology the initial impact of rabbit hemorrhagic disease on european rabbit populations in south australia zum auftreten der rabbit hemorrhagic disease (rhd) in o È sterreich. i. pathomophologische und virologische untersuchungen mechanisms for persistence of acute and chronic feline calicivirus infections in the face of vaccination a transient febrile limping syndrome of kittens caused by two different strains of feline calicivirus quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats acute and chronic faucitis of domestic cats. a feline calicivirus-induced disease comparison of the primary signs induced by experimental exposure to either a pneumotrophic or a limping strain of feline calicivirus cat # group . b - vaccinate /À c / / À/À À/ /À À/À / À/ À/À - vaccinate / /À / / /À À/À / À/ À/ À/À - vaccinate / / À/ À/ /À / / À/ À/ À/À - vaccinate / / /À /À À/À À/ À/ À/ À/ À/À - nonvaccinate À/À À/À À/À À/À À/À À/À / À/ / À/À - nonvaccinate À/À À/À À/À À/À À/À À/À / À/ À/ À/À a fcv isolates prior to challenge-exposure were uniformly of the fcv-f strain by sequence analysis, while isolates after challenge-exposure were identical to fcv-ari.b challenge-exposed with fcv-ari immediately after oral swabbing. c culture/pcr. fig. . rectal temperatures of previously fcv-f immunized (^ÐÐÐÐ^) and nonimmunized (&ÐÐÐÐ&) cats that were challenge-exposed to fcv-ari. the febrile responses of vaccinates developed more slowly and were somewhat less severe than those of the unvaccinated cats. key: cord- -mfta emi authors: jolliffe, d.; camargo, c. a.; sluyter, j.; aglipay, m.; aloia, j.; bergman, p.; damsgaard, c.; dubnov-raz, g.; esposito, s.; ganmaa, d.; gilham, c.; ginde, a.; grant, c.; griffiths, c.; hibbs, a. m.; janssens, w.; khadilkar, a. v.; laaksi, i.; lee, m. t.; loeb, m.; maguire, j.; mauger, d. t.; majak, p.; manaseki-holland, s.; murdoch, d.; nakashima, a.; neale, r. e.; rake, c.; rees, j.; rosendahl, j.; scragg, r.; shah, d.; shimizu, y.; simpson-yap, s.; trilok kumar, g.; urashima, m.; martineau, a. r. title: vitamin d supplementation to prevent acute respiratory infections: systematic review and meta-analysis of aggregate data from randomised controlled trials date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mfta emi objectives: to assess the overall effect of vitamin d supplementation on risk of acute respiratory infection (ari), and to identify factors modifying this effect. design: we conducted a systematic review and meta-analysis of data from randomised controlled trials (rcts) of vitamin d for ari prevention using a random effects model. pre-specified sub-group analyses were done to determine whether effects of vitamin d on risk of ari varied according to baseline -hydroxyvitamin d ( [oh]d) concentration or dosing regimen. data sources: medline, embase, the cochrane central register of controlled trials (central), web of science, clinicaltrials.gov and the international standard rct number (isrctn) registry from inception to may . eligibility criteria for selecting studies: double-blind rcts of supplementation with vitamin d or calcidiol, of any duration, were eligible if they were approved by a research ethics committee and if ari incidence was collected prospectively and pre-specified as an efficacy outcome. results: we identified eligible rcts (total , participants, aged to years). data were obtained for , ( . %) of , participants in studies. for the primary comparison of vitamin d supplementation vs. placebo, the intervention reduced risk of ari overall (odds ratio [or] . , % ci . to . ; p for heterogeneity . ). no statistically significant effect of vitamin d was seen for any of the sub-groups defined by baseline (oh)d concentration. however, protective effects were seen for trials in which vitamin d was given using a daily dosing regimen (or . , % ci . to . ); at daily dose equivalents of - iu (or . , % ci . to . ); and for a duration of [≤] months (or . , % ci . to . ). vitamin d did not influence the proportion of participants experiencing at least one serious adverse event (or . , % ci . to . ). risk of bias within individual studies was assessed as being low for all but two trials. a funnel plot showed asymmetry, suggesting that small trials showing non-protective effects of vitamin d may have been omitted from the meta-analysis. conclusions: vitamin d supplementation was safe and reduced risk of ari, despite evidence of significant heterogeneity across trials. the overall effect size may have been over-estimated due to publication bias. protection was associated with administration of daily doses of - iu vitamin d for up to months. the relevance of these findings to covid- is not known and requires investigation. conclusions: vitamin d supplementation was safe and reduced risk of ari, despite evidence of significant heterogeneity across trials. the overall effect size may have been over-estimated due to publication bias. protection was associated with administration of daily doses of - iu vitamin d for up to months. the relevance of these findings to covid- is not known and requires investigation. . cc-by-nc-nd . 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 july , . . https://doi.org/ . / . . . doi: medrxiv preprint • a previous individual participant data meta-analysis from • sub-group analysis revealed most benefit in those with the lowest vitamin d status at baseline and not receiving bolus doses. • we updated this meta-analysis with trial-level data from an additional placebo-controlled rcts published since december (i.e. new total of studies with , participants). • an overall protective effect of vitamin d supplementation against ari was seen (nnt= ). • a funnel plot revealed evidence of publication bias, which could have led to an over-estimate of the protective effect. • no statistically significant effect of vitamin d was seen for any of the sub-groups defined by baseline (oh)d concentration. • strongest protective effects were associated with administration of daily doses of - iu vitamin d for ≤ months (nnt= ). . cc-by-nc-nd . 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) interest in the potential for vitamin d supplementation to reduce risk of acute respiratory infections (ari) has increased since the emergence of the covid - pandemic. this stems from findings of laboratory studies, showing that vitamin d metabolites support innate immune responses to respiratory viruses, together with observational studies reporting independent associations between low circulating levels of -hydroxyvitamin d ( [oh]d, the widely accepted biomarker of vitamin d status) and increased risk of ari caused by other pathogens. randomised controlled trials (rcts) of vitamin d for the prevention of ari have produced heterogeneous results, with some showing protection, and others reporting null findings. we previously meta-analysed individual participant data from rcts and showed a protective overall effect that was stronger in those with lower baseline (oh)d levels, and in trials where vitamin d was administered daily or weekly rather than in more widely spaced bolus doses. since the date of the final literature search performed for that study (december ) , fifteen rcts with , participants fulfilling the same eligibility criteria have been completed and analysed. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we therefore sought data from these more recent studies for inclusion in an updated metaanalysis of aggregate (trial-level) data to determine whether vitamin d reduced ari risk overall, and to evaluate whether effects of vitamin d on ari risk varied according to baseline (oh)d concentration and/or dosing regimen (frequency, dose size, and trial duration). . cc-by-nc-nd . 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 july , . . https://doi.org/ . / . . . doi: medrxiv preprint methods were pre-specified in a protocol that was registered with the prospero ppi representatives were not involved in the conduct of this study. randomised, double-blind, trials of supplementation with vitamin d , vitamin d or (oh)d of any duration, with a placebo or low-dose vitamin d control, were eligible for inclusion if they had been approved by a research ethics committee and if data on incidence of ari were collected prospectively and pre-specified as an efficacy outcome. the latter requirement was imposed in order to minimise misclassification bias (prospectively designed instruments to capture ari events were deemed more likely to be sensitive and specific for this outcome). studies reporting results of longterm follow-up of primary rcts were excluded. . cc-by-nc-nd . 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 july , . summary data from trials which contributed to our previous meta-analysis of individual participant data were extracted from our central database, with permission from the principal investigators. summary data relating to the primary outcome (overall and by sub-group) and secondary outcomes (overall only) from newly identified trials were requested from principal investigators. on receipt, they were assessed for consistency with associated publications. study authors were contacted to provide missing data and to resolve any queries arising from these consistency checks. once queries had been resolved, clean summary data were uploaded to the study database, which was held in stata ic v . (statacorp, college station, tx). data relating to study characteristics were extracted for the following variables: study setting, eligibility criteria, (oh)d assay and levels, details of intervention and control regimens, trial duration, case definitions for ari and number entering primary analysis (after randomisation). follow-up summary data were requested for the proportions of participants experiencing one or more ari during the trial, both overall and stratified by baseline serum (oh)d concentration, where this was available. we also requested summary data on the proportions of participants who experienced one or more of the following events during the trial: upper respiratory infection (uri); lower respiratory infection (lri); emergency department attendance and/or hospital admission for ari; death due to ari or respiratory failure; use of antibiotics to treat an ari; absence from work or school due to ari; a serious adverse event; death due to any cause; and potential adverse reactions to vitamin d (hypercalcaemia and renal stones). . cc-by-nc-nd . 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 july , . . we used the cochrane collaboration risk of bias tool to assess the following variables: sequence generation, allocation concealment, blinding of participants, personnel and outcome assessors, completeness of outcome data, evidence of selective outcome reporting and other potential threats to validity. study quality was assessed independently by two investigators (arm and daj), except for the five trials for which daj and/or arm were investigators, which were assessed by cac. discrepancies were resolved by consensus. the primary outcome of the meta-analysis was the proportion of participants experiencing one or more aris, with the definition of ari encompassing events classified as uri, lri and ari of unclassified location (i.e. infection of the upper and/or lower respiratory tract). secondary outcomes were incidence of uri and lri, analysed separately; incidence of emergency department attendance and/or hospital admission for ari; death due to ari or respiratory failure; use of antibiotics to treat an ari; absence from work or school due to ari; incidence of serious adverse events; death due to any cause; and incidence of potential adverse reactions to vitamin d (hypercalcaemia and renal stones). data were analysed by daj; results were checked and verified by jds. our metaanalysis approach followed published guidelines. the primary comparison was of participants randomised to vitamin d vs. placebo: this was performed for all of the outcomes listed above. for trials that included higher-dose, lower-dose and placebo arms, data from higher-dose and lower-dose arms were pooled for analysis of the primary comparison. a secondary comparison of participants randomised to higher vs. lower doses of vitamin d was performed for the primary outcome only. a log odds ratio and its standard error was calculated for each outcome within each trial from the proportion of participants experiencing one or more events in the intervention vs. . cc-by-nc-nd . 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 july , . . control arm. these were meta-analysed in a random effects model using the metan package within stata ic v . to obtain a pooled odds ratio with a % confidence interval and a measure of heterogeneity summarized by the i statistic and its corresponding p value. the number needed to treat for an additional beneficial outcome (nnt) was calculated using the visual rx nnt calculator (http://www.nntonline.net/visualrx/) where meta-analysis of dichotomous outcomes revealed a statistically significant beneficial effect of allocation to vitamin d vs. placebo. to explore reasons for heterogeneity of effect of the intervention between trials we sub-optimal vitamin d status (so-called 'vitamin d insufficiency', - . nmol/l). an exploratory analysis restricted to studies with optimal frequency, dose size and duration was also performed. to investigate factors associated with heterogeneity of effect between subgroups of trials, we performed multivariable meta-regression analysis on trial-level characteristics, namely, dose frequency, dose size and trial duration, to produce an adjusted odds ratio, a % confidence interval and a p value for interaction for each factor. independent variables were dichotomised to create a more parsimonious model the meta-regression analysis excluded data from one trial that included higher-dose, lower-dose and placebo arms, since the higher-dose and lower-dose arms spanned the , iu/day cut-off, rendering it unclassifiable for the purposes of this analysis. . cc-by-nc-nd . 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 july , . . for the primary analysis, the likelihood of publication bias was investigated through the construction of a contour-enhanced funnel plot. we used the five grade considerations (study limitations, consistency of effect, imprecision, indirectness and publication bias) to assess the quality of the body of evidence contributing to analyses of the primary efficacy outcome and major secondary outcomes of our metaanalysis. we conducted two exploratory sensitivity analyses for the primary comparison of the primary outcome: one excluded rcts where risk of bias was assessed as being unclear; the other excluded rcts in which incidence of ari was not the primary or coprimary outcome. this study was conducted without external funding. . cc-by-nc-nd . 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 july , . . the study selection process is illustrated in figure compared effects of higher-dose, lower-dose and placebo arms, and compared effects of higher-vs. lower-dose regimens of vitamin d only. aggregate data were sought and obtained for all but study. table s . characteristics of the studies contributing data to this meta-analysis and their participants are presented in table . trials were conducted in different countries on continents, and enrolled participants of both sexes from birth to years of age. baseline serum (oh)d concentrations were determined in of trials: mean baseline (oh)d concentration ranged from . to . nmol/l (to convert to ng/ml, divide by . ). thirty-eight studies administered oral vitamin d to participants in the intervention arm, while study administered oral (oh)d. vitamin d was given as monthly to -monthly bolus doses in studies; as weekly doses in studies; as daily doses in studies; and as a combination of bolus and daily doses in studies. trial duration ranged from weeks to years. incidence of ari was primary or co-primary outcome for studies, and a secondary outcome for studies. details of the risk of bias assessment are provided in supplementary table s . two trials were assessed as being at unclear risk of bias due to high loss to follow-up. in the trial by laaksi and colleagues, % of randomised participants were lost to follow-. cc-by-nc-nd . 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 july , . . up. in the trial by dubnov-raz and colleagues, % of participants did not complete all symptom questionnaires. all other trials were assessed as being at low risk of bias for all seven aspects assessed. table ; cates plot, figure s ). heterogeneity of effect was moderate (i . %, p for heterogeneity . ). the associated nnt was ( % ci to ). for the secondary comparison of higher-vs. lower-dose vitamin d, we observed no statistically significant difference in the proportion of participants with at least one ari (or . , % ci . to . ; , participants in studies; i . %, p for heterogeneity . ; figure s ). to investigate reasons for the observed heterogeneity of effect for the primary comparison of vitamin d vs. placebo control, we stratified this analysis by one participant-level factor (baseline vitamin d status) and by three trial-level factors (dose frequency, dose size, and trial duration). results are presented in table figure s ). with regard to dosing frequency, a statistically significant protective effect was seen for trials where vitamin d was given daily (or . , % ci . to . ; , participants in studies), but not for trials in which it was given weekly (or . , % ci . to . ; , participants in studies), or monthly to -monthly (or . , % ci . to . ; , participants in studies; figure . cc-by-nc-nd . 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 july , . . s ). statistically significant protective effects of the intervention were also seen in trials where vitamin d was administered at daily equivalent doses of - iu ( figure s ). statistically significant protective effects were also seen for trials with a duration of ≤ months (or . , % ci . to . ; , participants in studies) but not in those lasting > months (or . , % ci . to . ; , participants in studies; figure s ). an exploratory analysis restricted to placebo-controlled trials investigating effects of daily dosing at doses of - iu/day with duration ≤ months showed a statistically significant reduction in the proportion of participants experiencing at least one ari (or . , % ci . to . ; , participants in studies; figure s ; cates plot, figure s ). heterogeneity of effect was low (i . %, p for heterogeneity . ). the associated nnt was ( % ci to ). multivariable meta-regression analysis of trial-level sub-groups did not identify a statistically significant interaction between allocation to vitamin d vs. placebo and dose frequency, size or trial duration (table s ) . meta-analysis of secondary outcomes was performed for results of placebo-controlled trials only; results are presented in table . overall, without consideration of participant-or trial-level factors, vitamin d supplementation did not have a statistically significant effect on the proportion of participants with one or more uri, lri, courses of antimicrobials for ari, work/school absences due to ari, hospitalisations or emergency department attendances for ari, serious adverse events of any cause, death due to ari or respiratory failure, death due to any cause, or episodes of hypercalcaemia or renal stones. . cc-by-nc-nd . 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 july , . . https://doi.org/ . / . . . doi: medrxiv preprint a funnel plot for the proportion of participants experiencing at least one ari showed left-sided asymmetry, raising the possibility that small trials showing non-protective effects of vitamin d may not have been included in the meta-analysis ( figure s ). an egger's regression test for publication bias confirmed asymmetry (p= . ). accordingly, the quality of the body of evidence contributing to analyses of the primary efficacy outcome and major secondary outcomes was downgraded to moderate (table s ). results of exploratory sensitivity analyses are presented in table s . meta-analysis of the proportion of participants in placebo-controlled trials experiencing at least one ari, excluding studies assessed as being at unclear risk of bias, pandemic, we used a trial-level approach for this update, which includes data from a total of , participants in trials. overall, we report a modest statistically significant protective effect of vitamin d supplementation, as compared with placebo (or . , % ci . to . ). as expected, there was significant heterogeneity (p= . ) across trials, which might have led to an under-estimate of the protective effect. on the other hand, a funnel plot revealed evidence of publication bias, which might have led to an over-estimate of the protective effect. in contrast to findings of our . cc-by-nc-nd . 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 july , . . previous meta-analysis, we did not observed enhanced protection in those with the lowest (oh)d levels at baseline. however, there was evidence that efficacy of vitamin d supplementation varied according to dosing regimen and trial duration, with protective effects associated with daily administration of doses of - iu vitamin d given for ≤ months. an exploratory analysis restricted to data from trials fulfilling these design criteria revealed a larger protective effect (or . , % ci . to . ) without significant heterogeneity across trials (p for heterogeneity . ). the magnitude of the overall protective effect seen in the current analysis (or . , % ci . to . ) is similar to the value reported in our previous meta-analysis of individual participant data (adjusted or . , % ci . to . ). in keeping with our previous study, the point estimate for this effect was lower among those with baseline (oh)d < nmol/l than in those with higher baseline vitamin d status. however, in contrast to our previous finding, a statistically significant protective effect of vitamin d was not seen in those with the lowest (oh)d concentrations. this difference reflects the inclusion of null data from three new rcts in which vitamin d was given in relatively high doses at weekly or monthly intervals over - years. null results of these studies contrast with protective effects reported from earlier trials in which smaller daily doses of vitamin d were given over shorter periods. these differing findings suggest that the frequency, amount and duration of vitamin d supplementation may be key determinants of its protective efficacy. in keeping with this hypothesis, statistically significant protective effects of vitamin d were seen for meta-analysis of trials where vitamin d was given daily; where it was given at doses of - iu/day; and where it was given for months or less. when results of trials that investigated daily administration of - iu over ≤ months were pooled in an exploratory meta-analysis, a protective effect was seen (or . , % ci . to . ) with low heterogeneity (i . %, p for heterogeneity . ) and a nnt of ( % ci to ). the current study has several strengths: it contains the very latest rct data available in this fast-moving field, including findings from a soon-to-be published very large trial conducted using directly-observed, higher-dose, weekly vitamin d supplementation in very deficient children. the inclusion of additional studies allowed us to analyse results of placebo-controlled studies vs. high-dose / low-dose studies separately, and . cc-by-nc-nd . 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 july , . . https://doi.org/ . / . . . doi: medrxiv preprint gave us the power to investigate reasons for heterogeneity of effect observed across trials. for example, we could distinguish the effects of daily vs. weekly dosing, which were previously pooled. our work also has limitations. given the need to generate a rapid update of our previous work in the context of the covid- pandemic, we meta-analysed aggregate (trial-level) data, rather than individual participant data; this allowed us to proceed rapidly, without the delays introduced by the need to establish multiple data sharing agreements. however, we did contact authors to get unpublished estimates of effect that were stratified by pre-defined baseline (oh)d levels, harmonised across studies: thus, we were able to provide accurate data for the major participant-level effect-modifier of interest. despite the large number of trials overall, relatively few compared effects of lower-vs. higher-dose vitamin d: our power for this secondary comparison was therefore limited. we lacked the individual participant data to investigate race/ethnicity and obesity as potential effect-modifiers. we also could not account for other factors that might influence the efficacy of vitamin d supplements for ari prevention (e.g., taking the supplement with or without food) or secular trends that would influence trials, such as the increased societal use of vitamin d supplements; concurrent use of standard dose vitamin d supplements or multivitamins in the "placebo" group would effectively render these as high-vs. low-dose trials and potentially drive results toward the null. a final limitation relates to the funnel plot, which suggests that the overall effect size may have been over-estimated due to publication bias. in summary, this updated meta-analysis of data from rcts of vitamin d for the prevention of ari showed a statistically significant overall protective effect of the intervention. the number needed to treat to prevent one ari was . the protective effect was heterogenous across trials; it also may have been over-estimated due to publication bias. in contrast to findings of our previous meta-analysis of individual participant data, we did not see a protective effect of vitamin d supplementation among those with the lowest baseline vitamin d status. the vitamin d dosing regimen of most benefit was daily and used standard doses (e.g., to iu) for up to months. the relevance of these findings to covid- is not known and requires investigation. . cc-by-nc-nd . 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 july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . 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 july , . . this study was conducted without external funding. daj is supported by a barts students. the views expressed are those of the authors and not necessarily those of barts charity or the office for students. sources of support for individual trials are detailed in supplementary material. we thank dr emma c goodall (glaxosmithkline plc) for contributing data. we also thank all the people who participated in primary randomised controlled trials, and the teams who conducted them. daj and arm wrote the study protocol and designed statistical analyses. daj, cac and arm assessed eligibility of studies for inclusion and performed risk of bias assessments. statistical analyses were done by daj; results were checked and verified by jds. daj and arm wrote the first draft of the report. all authors revised it critically for important intellectual content, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved. all authors have completed the icmje uniform disclosure form. no author has had any financial relationship with any organisations that might have an interest in the submitted work in the previous three years. no author has had any other relationship, or undertaken any activity, that could appear to have influenced the submitted work. daj and arm are the manuscript's guarantors and they affirm that this is an honest, accurate, and transparent account of the study being reported and that no important aspects of the study have been omitted. all analyses were pre-specified in the study protocol, other than the exploratory analyses whose results are presented in table s and figure s . data sharing: the study dataset is available from d.a.jolliffe@qmul.ac.uk. . cc-by-nc-nd . 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 july , . . 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 july , . . 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 july , . 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 july , . 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 july , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : flow chart of study selection . cc-by-nc-nd . 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 july , . . . cc-by-nc-nd . 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 july , . . vitamin d and sars-cov- virus/covid- disease modulation of the immune response to respiratory viruses by vitamin d vitamin d in the prevention of acute respiratory infection: systematic review of clinical studies acute respiratory tract infection and -hydroxyvitamin d concentration: a systematic review and meta-analysis a randomized controlled trial of vitamin d supplementation for the prevention of symptomatic upper respiratory tract infections randomized trial of vitamin d supplementation to prevent seasonal influenza a in schoolchildren effects of vitamin d supplementation to children diagnosed with pneumonia in kabul: a randomised controlled trial vitamin d supplementation for the prevention of acute respiratory tract infection: a randomized, double-blinded trial among young finnish men vitamin d supplementation in children may prevent asthma exacerbation triggered by acute respiratory infection effect of weekly vitamin d supplements on mortality, morbidity, and growth of low birthweight term infants in india up to age months: randomised controlled trial high doses of vitamin d to reduce exacerbations in chronic obstructive pulmonary disease: a randomized trial effect on the incidence of pneumonia of vitamin d supplementation by quarterly bolus dose to infants in kabul: a randomised controlled superiority trial randomized trial of vitamin d supplementation and risk of acute respiratory infection in mongolia effect of vitamin d supplementation on upper respiratory tract infections in healthy adults: the vidaris randomized controlled trial vitamin d supplementation in patients with frequent respiratory tract infections: a randomised and double-blind intervention study vitamin d supplementation reduces the risk of acute otitis media in otitis-prone children vitamin d supplementation and upper respiratory tract infections in a randomized, controlled trial effect of vitamin d supplementation on antibiotic use: a randomized controlled trial vitamin d and gargling for the prevention of upper respiratory tract infections: a randomized controlled trial effects of vitamin d supplements on influenza a illness during the h n pandemic: a randomized controlled trial reduced primary care respiratory infection visits following pregnancy and infancy vitamin d supplementation: a randomised controlled trial vitamin d supplementation in patients with chronic obstructive pulmonary disease (vidico): a multicentre, double-blind, randomised controlled trial. the lancet respiratory medicine double-blind randomised placebocontrolled trial of bolus-dose vitamin d supplementation in adults with asthma (vidias) double-blind randomised controlled trial of vitamin d supplementation for the prevention of acute respiratory infection in older adults and their carers (vidiflu) weekly cholecalciferol supplementation results in significant reductions in infection risk among the vitamin d deficient: results from the cipris pilot rct vitamin d supplementation and upper respiratory tract infections in adolescent swimmers: a randomized controlled trial vitamin d supplementation and the risk of colds in patients with asthma improved control of childhood asthma with low-dose, short-term vitamin d supplementation: a randomized, double-blind, placebo-controlled trial high-dose monthly vitamin d for prevention of acute respiratory infection in older long-term care residents: a randomized clinical trial vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data vitamin d supplementation for treatment and prevention of pneumonia in under-five children: a randomized double-blind placebo controlled trial effect of high-dose vs standard-dose wintertime vitamin d supplementation on viral upper respiratory tract infections in young healthy children randomized trial of vitamin d supplementation to prevent seasonal influenza and upper respiratory infection in patients with inflammatory bowel disease effect of vitamin d supplementation on recurrent wheezing in black infants who were born preterm: the d-wheeze randomized clinical trial randomized phase trial of monthly vitamin d to prevent respiratory complications in children with sickle cell disease effect of vitamin d supplementation to reduce respiratory infections in children and adolescents in vietnam: a randomized controlled trial. influenza other respir viruses effect of higher vs standard dosage of vitamin d supplementation on bone strength and infection in healthy infants: a randomized clinical trial intake of -hydroxyvitamin d reduces duration and severity of upper respiratory tract infection: a randomized, double-blind, placebo-controlled, parallel group comparison study vitamin d and acute respiratory infections-the poda trial effect of monthly high-dose vitamin d supplementation on acute respiratory infections in older adults: a randomized controlled trial winter cholecalciferol supplementation at degrees n has little effect on markers of innate immune defense in healthy children aged - years: a secondary analysis from a randomized controlled trial vitamin d supplements and prevention of tuberculosis infection and disease occurrence of infections in schoolchildren subsequent to supplementation with vitamin d-calcium or zinc: a randomized, doubleblind, placebo-controlled trial high-dose oral vitamin d supplementation and mortality in people aged - years: the vidal cluster feasibility rct of open versus doubleblind individual randomisation impact of two oral doses of , iu of vitamin d in preschoolers with viral-induced asthma: a pilot randomised controlled trial the cochrane collaboration's tool for assessing risk of bias in randomised trials cochrane handbook for systematic reviews of interventions version metan: stata module for fixed and random effects meta-analysis, revised diagnosis and management of vitamin d deficiency contour-enhanced meta-analysis funnel plots help distinguish publication bias from other causes of asymmetry grade: an emerging consensus on rating quality of evidence and strength of recommendations bias in meta-analysis detected by a simple, graphical test longitudinal and secular trends in dietary supplement use: nurses' health study and health professionals follow-up study pharmacokinetics of oral vitamin d( ) and calcifediol key: cord- - n ej f authors: masse, shirley; capai, lisandru; villechenaud, natacha; blanchon, thierry; charrel, rémi; falchi, alessandra title: epidemiology and clinical symptoms related to seasonal coronavirus identified in patients with acute respiratory infections consulting in primary care over six influenza seasons ( – ) in france date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: n ej f there is currently debate about human coronavirus (hcov) seasonality and pathogenicity, as epidemiological data are scarce. here, we provide epidemiological and clinical features of hcov patients with acute respiratory infection (ari) examined in primary care general practice. we also describe hcov seasonality over six influenza surveillance seasons (week to of each season) from the period / to / in corsica (france). a sample of patients of all ages presenting for consultation for influenza-like illness (ili) or ari was included by physicians of the french sentinelles network during this period. nasopharyngeal samples were tested for the presence of respiratory pathogens by real-time rt-pcr. among the ili/ari patients, were positive for at least one hcov ( . %). on an annual basis, hcovs circulated from week (november) to weeks – (may) and peaked in week (february). overall, among the hcov-positive patients detected in this study, hcov-oc was the most commonly detected virus, followed by hcov-nl , hcov-hku , and hcov- e. the hcov detection rates varied significantly with age (p = . ), with the age group – years accounting for . % (n = ) of hcov-positive patients. fever and malaise were less frequent in hcov patients than in influenza patients, while sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more associated with hcov positivity. in conclusion, this study demonstrates that hcov subtypes appear in ari/ili patients seen in general practice, with characteristic outbreak patterns primarily in winter. this study also identified symptoms associated with hcovs in patients with ari/ili. further studies with representative samples should be conducted to provide additional insights into the epidemiology and clinical features of hcovs. coronaviruses (covs) are an enveloped, single positive-strand rna species of viruses belonging to the coronaviridae family, which infect birds and mammals. in animals, covs cause respiratory, enteric, cardio-vascular, and neurological disorders [ ] . in humans, these viruses result in respiratory and gastro-intestinal symptoms, ranging from cold symptoms to severe diseases [ , ] . covs recognized to infect humans belong to the genera alphacoronavirus and betacoronavirus [ ] . seven cov species are known to cause human infection, of which four hcovs (namely hcov e, nl , oc , and hku ) are known as non-severe acute respiratory syndrome (sars)-like covs. hcov- e and hcov-nl belong to the genus alphacoronavirus. the genus betacoronavirus includes hcov-hku , hcov-oc , sars-cov- [ ] , the middle east respiratory syndrome (mers) coronavirus (mers-cov) [ ] , and the sars-cov- , which is currently associated with a global outbreak [ ] . the four non-sars/mers species circulate widely in humans and infect individuals of all ages [ , ] . hcov- e and hcov-oc were identified in and were primarily associated with mild upper respiratory tract infections [ , ] . hcov-nl was identified in from a -month-old child suffering from bronchiolitis and conjunctivitis [ ] and hcov-hku was discovered in in hong kong and isolated from patients with pneumonia [ ] . in general, the four common circulating hcovs mostly infect humans during the winter season (december-april) [ ] , whereas circulation of hcov-hku has been observed during the spring-summer period [ ] . the world health organization has highlighted the need to improve epidemiological surveillance and knowledge of the health burden imposed by non-influenza respiratory viruses [ ] . hcovs are generally associated with mild upper respiratory tract infections [ ] , but severe infections with hcov- e, hcov-nl , and hcov-oc have been reported [ ] [ ] [ ] . in the context of the spread of sars-cov- in the community, a better understanding of the seasonality and clinical features of patients with confirmed hcovs could be useful for mathematical modelling and clinical diagnosis. there is currently a debate about hcov seasonality and pathogenicity, as epidemiological data are scarce and mostly from hospitalized populations. here, we document the epidemiological and clinical features of hcov patients with acute respiratory infection (ari) observed in general practice. we also describe hcov seasonality over six influenza surveillance seasons ( / to / ) in corsica, france. nasopharyngeal samples were collected: i) as part of the community influenza surveillance conducted in collaboration with the french sentinelles network from patients seen in general practice, consulting for influenza-like illness (ili) or ari (for patients aged > years old) during six influenza seasons (week to of each season) from to in corsica, france; and ii) from ari patients enrolled throughout mainland france by general practitioners (gps) of the french sentinelles network, during an epidemiological study of the risk factors for seasonal influenza (iriis study; - influenza seasons (week to )) [ ] . notably, to ensure that the selection of ili/ari patients remained random, each gp was required to include, each week, the first two patients unrelated to one another, consulting within < h since symptom onset and consenting to provide a nasopharyngeal specimen. each patient could be included only once a year (table and figure ). the surveillance uses a specific definition of ili for patient recruitment: sudden onset of fever > • c with myalgia and respiratory signs, diagnosed by the physician. the case definition of ari was "any person with a sudden onset of symptoms and 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, or shortness of breath." . this rrt-pcr did not provide details for hcovs and hadv, hbov, and hpiv were not analysed. thus, samples still available were retrospectively analysed for cov- e, cov-oc , and cov-nl by ftd. all nasopharyngeal samples collected by the french sentinelles network during the / season were analysed for sars-cov- . rdrp-ip and rdrp quantitative rtrt-pcr was used for detection of sars-cov- . the rdrp rtrt-pcr corresponds to the charité protocol [ ] . when a sample was positive for rdrp-ip , quantification of the number of rna copies was performed according to a scale ranging from to copies per µl [ ] . all analyses were conducted by the laboratory of virology, university of corsica. the hcov seasonality in corsica from / to / was studied using samples collected by gps of the corsican sentinelles network, as constant and homogeneous monitoring of this population of ili/ari patients has been conducted throughout six influenza seasons (table and figure ). to study the weekly number of hcovs detected among ili/ari patients seen in general practice during the six influenza seasons, we gathered all samples collected by gps for influenza surveillance and for the iriis study (table and figure ). the samples were obtained as part of influenza surveillance. the protocol was conducted in agreement with the helsinki declaration. we obtained authorization from the french data protection agency (cnil# ). the iriis study was approved by the ethics committee (cpp sud mediterranee v, / / ; ref. number . ). differences according to viral infection and epidemiological data (sex, age, clinical symptoms, and risk factors) were analysed and tested using fisher's exact test or chi-square test. results were considered statistically significant when the p value was lower than . . all statistical analyses were performed using r software version . . (r foundation, vienna, austria). during the six-year study, among the ili/ari patients enrolled by the corsican sentinelles network, . % (n = ) were positive for at least one of the respiratory viruses analysed ( table ) . hcovs were the third most frequently detected viruses ( . %; n = ) after influenza viruses subtypes a ( . %; n = ) and b ( . %; n = ) ( table ). the number of hcov infections ranged from . % the seasonal distribution per week of hcov, influenza viruses (a and b), rsv, and hrv was studied in ili/ari patients (table and figure ). among the patients, . % (n = ) were positive for at least one respiratory virus, of which . % (n = ) were positive for influenza a virus, . % (n = ) for influenza b virus, and . % (n = ) for hcovs. hcovs were detected during the entire winter season (weeks - ), with the highest number of hcovs detected in week ( figure ) . notably, week had a decrease in influenza a virus detection (figure ). since the number of hcovs detected by species per season was low, the seasonality of hcovs was established from all viruses detected per week over the six influenza seasons. the hcov peak trend detection among week to was clearly observed during three influenza seasons ( / ; / ; / ). among the hcov cases, . % (n = ) were typed. of these . % (n = ) of cases were infected with hcov-oc , . % (n = ) with hcov-nl , . % (n = ) with hcov-hku , and . % (n = ) with hcov- e ( table ). the weekly number of hcov types detected among the ili/ari patients is illustrated in figure . a degree of synchrony was observed during all winter seasons and in the timing of the peak (week ) for hcov-hku , nl , and e, whereas hcov-oc infections occurred earlier with a peak in weeks - ( figure ). among the ili/ari patients, ( . %) were positive for at least one hcov. among them, ( . %) were infected with one hcov, while ( . %) were infected with at least two different respiratory viruses (including two co-infections with three viruses) ( table ) . of these, the most frequently observed co-pathogens were influenza viruses (flu a: n = / ; . %, and flu b: n = / ; . %) ( table ) . two co-infection combinations were detected: hcov- e/nl and hcov-hku /oc . the demographic data and clinical characteristics of the confirmed hcov-positive patients are summarized in table . the age of patients infected with hcov varied from month to years with mean and median ages of years. the male to female ratio of hcov-infected patients was . ( / ). hcov infections were observed in all age groups (table and figure ). however, the detection rates in these different age groups varied significantly (p = . ) with the age group of - years accounting for . % (n = ) of hcov-positive patients. in the remaining groups, detection rates were observed of . % (n = ) in the - age group, % (n = ) in the - age group, . % (n = ) in the - age group, . % (n = ) in the - age group, and . % (n = ) in the group aged ≥ years old. * overall hcov infections also include strains without species determination (n = ). hcov-oc and hcov-hku were detected in patients of all ages (table and figure ). the oldest patients (≥ years old) were infected by hcov-oc and hcov-hku only ( figure a) , and hcov-hku was the most prevalent among patients aged ≥ years old ( figure b ), but a significant difference in the age-group distribution of hcov species was not observed (table ) . the epidemiological and clinical features of patients were compared according to their viral infection: influenza a cases, influenza b cases, and hcov cases (single or co-infection). fever and malaise were less frequent in hcov patients than in influenza patients, while sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more frequently associated with hcov positivity (table ) . sore throat and dyspnoea were more frequently detected in patients with hcov single infection ( . %, / ) than in patients with hcov co-infection ( . %, / ; p = . ) (table ) . notably, oseltamivir was prescribed more frequently to influenza a patients (table ) . lastly, when comparing epidemiological and clinical features of patients according to the hcov strain detected, there was a significant effect on the prevalence of sex (p = . ), with the hcov- e strain detected more frequently in male patients ( . %) ( table ). a significant difference in the prevalence of malaise (p = . ) among hcov strain patients was also identified, as this symptom was exclusively related to hcov-hku patients ( . %, / ) (table ). among the ari patient samples collected during the / influenza season and tested retrospectively for sars-cov- from week to week , four ( . %) were positive for sars-cov- . these four infections were detected during weeks (n = ), (n = ), and (n = ). fever, cough, and headache were the most commonly declared symptoms, while anosmia was reported for one patient. the mean age of the four patients was . years and the sex ratio was : (two women and two men). data from continuous surveillance are very important for identifying the pattern of hcov epidemiology. this report is the first to describe patterns of circulation of the four common hcov strains in french ili/ari patients seen in general practice over six influenza seasons. hcov was the third most detected respiratory virus in ari/ili patients after the influenza a and b viruses. similar to their susceptibility to other respiratory viruses, young children aged < years old had the highest risk of hcov infection. sore throat, dyspnoea, and conjunctivitis were more frequently described among hcov cases than among influenza cases. similar to other countries in the northern hemisphere, we reported a winter seasonal prevalence pattern for the four hcov strains [ ] , with the highest number of cases presenting in february [ , ] . in our study, the peak in february was attributable to the e, nl , and hku strains, whereas the oc strain had peaks in december. overall, among the hcov-positive patients in this study, the oc strain was the most commonly detected virus, followed by the nl , hku , and e strains, in agreement with previous studies [ , , ] . hcov-oc is routinely associated with~ % of acute respiratory infections and was the most commonly detected virus in hcov patients [ ] [ ] [ ] . a seroconversion study in hospitalized children reported that hcov-oc and hcov-nl might induce cross-immunity, and therefore reduce the subsequent number of clinically identified hcov-hku and hcov- e infections [ ] . we confirmed that, similar to other respiratory pathogens, hcovs were widespread in all age groups [ ] . a significant difference in the age distribution of hcov patients was noted, with the youngest ( - years old) patients displaying a threefold higher level of infection than those aged - years old and a sevenfold higher infection level than patients aged ≥ years old. among hcov patients aged < years old, the most frequently detected viruses were hcov-oc and hcov-nl , while among patients aged ≥ years old, hcov-hku was the most frequently detected. this trend of hcovs infection has already been reported in other studies [ , , , ] . a recent surveillance study on the occurrence and hospitalization rates in children with respiratory infections over years reported that hcovs were involved in . % of episodes [ ] . to date, in contrast to that observed for hcovs, few sars-cov- have been observed in children. official national statistics in italy reported that % of total cases diagnosed country-wide were below years [ ] . it could be possible that pre-existing cross-immunity provides protection and/or reduces the severity of covid- , thereby reducing the number of children who are tested/hospitalized [ ] . the fewer number of hcov infections in older than younger patients may result from higher antibody levels or other immune mechanisms of protection [ ] . the lower number of hcov infections in older than younger patients may result from higher antibody levels or other immune mechanisms of protection [ ] . it is well known that hcovs co-circulate endemically with other common respiratory viruses and co-infections are frequently observed. in our study, six other common respiratory viruses were detected simultaneously and . % of the hcov-positive patients were co-infected by at least one of the other respiratory viruses. the occurrence of co-infection of hcov, including other hcovs, rsv, influenza a and b viruses, hadv, and hmpv has been reported [ , ] . similar to other studies, we observed that influenza and rsv were the most common respiratory viruses co-infected with hcov [ , ] . we did not observe co-infection of sars-cov- and another respiratory virus among the four covid- patients detected. a recent co-infection of sars-cov- and hcov-hku has been reported [ ] . in this study, we compared the demographic data and clinical characteristics of hcov patients with those of influenza a and b patients, respectively (table ). sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more often observed in hcov cases than in influenza cases. while dyspnoea was reported as not common in seasonal influenza viruses [ ] , studies of hospitalized patients reported the role of hcov in causing lower respiratory illness [ , ] . fever was more often observed in influenza cases, as reported [ ] . these observations may be of importance, especially when hcovs and influenza virus co-circulate. the clinical impact of hcovs in co-detection is not fully understood, with previous studies observing unchanged morbidity or increased illness severity [ ] . when we compared the clinical presentations of patients with a single hcov infection with those with co-infection, sore throat and dyspnoea were more commonly reported in cases with a single infection. a growing number of studies are currently describing the clinical features due to sars-cov- infection [ ] [ ] [ ] [ ] [ ] . although most of the covid- studies have been conducted on hospitalized patients some comparisons with data of the present study might be useful. fever and cough were the most common symptoms reported for sars-cov- and hcov patients [ ] . compared to symptoms observed in hcov patients included in the present study, sars-cov- patients seem to show less headache ( . %- . % vs. . %), rhinorrhea ( % vs. . %) and myalgia ( . - . % vs. . %) [ ] . sore throat, which was one of the most detected symptoms among hcovs patients, was less common among covid- patients [ ] . about half of covid- patients had an underlying disease [ ] , whereas in our study less than a quarter of the hcov patients suffered from an underlying disease. this study has some limitations. first, we used two ili/ari patient samples, which can introduce bias, even if the methodology of enrolment was very similar. second, the number of hcov patients included did not allow the identification of meaningful associations by subanalyses. third, we studied hcov circulation during influenza season and not on annual basis. as previously described, hcovs co-circulate and showed marked winter seasonality, and are not detected in the summer period [ ] . lastly, we did not identify the species of hcov cases because relevant samples were not available. in conclusion, this study demonstrates that hcov subtypes appear in ari/ili patients seen in general practice, with characteristic outbreak patterns primarily in winter. hcovs were detected at a significantly higher rate in children aged < years 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characteristics of hospitalized patients with novel coronavirus-infected pneumonia in clinical findings in a group of patients infected with the novel coronavirus (sars-cov- ) outside of wuhan, china: retrospective case series clinical characteristics of coronavirus disease in china the epidemiology and clinical information about covid- we thank all general practitioners and paediatricians participating in the french sentinelles network. the authors declare no conflict of interest. the funder had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit this article for publication. all researchers' decisions have been entirely independent from funders. key: cord- -ch prsf authors: moran, elizabeth; kubale, john; noppert, grace; malosh, ryan e; zelner, jon l title: inequality in acute respiratory infection outcomes in the united states: a review of the literature and its implications for public health policy and practice. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ch prsf seasonal and pandemic respiratory viruses such as influenza and the novel coronavirus (sars-cov- ) currently sweeping the globe have often been described as 'equal opportunity infectors', implying little socioeconomic disparity in susceptibility. however, early data from the covid- pandemic has underscored that the burden of respiratory viruses actually reflect and magnify existing socioeconomic inequalities. we review the literature on socioeconomic and racial disparities in acute respiratory infection (ari), as well as ari-associated hospitalization and mortality. our goal is to identify key principles of the relationship between socioeconomic inequality and ari outcomes, as well as highlighting poorly understood areas that need to be addressed by research and policy in the wake of the covid- pandemic. we find that there has been descriptive work in this area, but that there is a distinct lack of cohesive methodology in the literature exploring social determinants and ari. we propose the fundamental cause theory is a useful framework for guiding future research of disparities in ari and for the design of interventions to alleviate these disparities. acute respiratory infections (ari) cause substantial morbidity and mortality worldwide , both as the result of seasonal epidemics and pandemics, such as covid- . respiratory viruses such as influenza and respiratory syncytial virus (rsv), have often been described as 'equal opportunity infectors', implying little socioeconomic disparity in susceptibility. however, early data from the covid- pandemic has underscored that the burden of respiratory viruses actually reflect and magnify existing socioeconomic inequalities . these trends have been made clear in the united states by the alarming disparities in the toll of severe disease and mortality experienced by african-americans . at the population level, viral and immune factors are necessary pre-conditions for the emergence and transmission of pathogens causing seasonal and pandemic ari . at the same time, socioeconomic inequalities are clearly key drivers of exposure, severe disease, and mortality. the rapid pace of transmission and mortality resulting from the covid- pandemic has shone a bright light on these disparities. but they are no less acute in the context of other causes of seasonal and pandemic ari, despite attracting less attention. in this paper, we review the literature on socioeconomic and racial disparities in ari infection, hospitalization, and mortality in the united states. by focusing on the existing literature on influenza, rsv, and all-cause ari we hope to identify key principles of the relationship between socioeconomic inequality and ari outcomes, as well as highlighting poorly understood areas that need to be addressed by research and policy in the wake of the covid- pandemic. disparities in ari outcomes may result from ) differential rates of exposure owing to occupation, housing, and other factors, ) differential susceptibility to infection upon exposure, owing to both social and medical factors, e.g. access to vaccination where it is available, and ) differential ari-towards a more broadly protective influenza vaccine, increased use of antivirals to improve outcomes, and advances in genomics and mathematical modeling to understand transmission, can lead to significant strides against influenza and other endemic and emerging viral aris. however, such advances in population-level protection are often accompanied by a widening of disparities in outcomes, as new preventive measures and therapies become available first to the well-off. as recent discussions of the ability to practice social distancing, and whether a covid- vaccine will become broadly available shows, these issues remain woefully unaddressed, and have significant implications for public health. we searched pubmed for articles documenting socioeconomic disparities in ari infection/disease risk, severe outcomes, and mortality. first, we attempted to capture articles investigating disparities in all-cause ari, influenza-like illness (ili), and rsv. then, we identified articles documenting disparities in influenza infection risk and outcomes. we separated these searches to ensure each was sensitive to key differences in the literature, most notably around access to and uptake of influenza vaccination. we used the pubmed special query for health disparities in conjunction with search terms relating to all-cause ari, rsv, and influenza ( figure ). the health disparities query identifies articles evaluating disparities in health outcomes and healthcare access with inequities in dimensions of race/ethnicity, ses, gender identity and sexual orientation, insurance status, and other populations described as "vulnerable." we then screened studies using title and abstract review to identify articles with a specific focus on all-cause ari, ili, rsv, and influenza. additional articles and reviews were added to our results upon recommendation from topic-area experts. articles were not excluded based on year of publication. full search queries are available in the supplementary materials. . cc-by-nc . 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 our all-cause ari and rsv search returned papers, of which studies met the inclusion criteria. of these, only of the retained articles used data from the us. the influenza search returned results, of which were deemed relevant following title and abstract review. of these, addressed seasonal influenza in the us, with the remaining focused on the h n influenza pandemic. all selected articles, organized by etiology, dimension of disparity assessed, and measured outcome (incidence, severity, death, etc.) are listed in table . a wide range of ses-related disparities in all-cause ari were identified in studies from the us , , , uk , , , and argentina . two studies reported increased ari incidence , among those with lower self-reported social status and ses . race/ethnicity was also found to be associated with increased severity, as indicated by two studies reporting increased ari-related hospitalization in a us study of children under using data from new vaccine surveillance network (nvsn) and primary care seeking in uk using sentinel surveillance data . ses disparities were also evident in all-cause ari-related complications: two studies reported increased hospital admission for ari and bronchiolitis and increased rates of medical intervention due to bronchiolitis townsend deprivation score, index used to quantify material deprivation based on car and house ownership, household overcrowding, and employment, with a higher score showing more material deprivation , . finally, one study reported increased under- mortality due to ari among those with proxy measures for poverty including having an adolescent mother, household crowding, and lacking running water in home . . cc-by-nc . 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 incidence: in a multi-state cohort study, female healthcare workers from texas, washington, and oregon, with lower self-reported social status reported higher rates of ari symptoms . in an experimental study, higher subjective ses was protective against the development of cold symptoms after exposure to rhinovirus or influenza virus . severity: disparities in severity of ari were indicated using hospitalization and care seeking rates in a variety of study designs. using population-based surveillance data, the nvsn study found increased hospitalization rates for all-cause ari among black children as compared to white children; however, these results were not adjusted for ses . additional surveillance data from the royal college of general practitioners research and surveillance centre in england found racial and ethnic minorities were more likely to seek care for cold/flu symptoms than whites . in the uk, a case-control study matching children under year old admitted to sheffield hospital with clinical suspicion of bronchiolitis to healthy community controls identified from birth records found that children living in the two most deprived electoral wards, as defined using the townsend index, were . times as likely to be admitted to the hospital and . times as likely to require a medical intervention than children living in other parts of the city . an ecological study in the west midlands region of the uk found neighborhoods with higher townsend scores of deprivation experienced higher rates of childhood hospital admissions due to ari and pneumonia . mortality: a case-control study in buenos aires found death due to ari in children under was associated with living in a crowded household, having an adolescent mother, and lacking running water in the home . no other papers discussed mortality due to all-cause ari. . cc-by-nc . 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 , . studies of disparities in seasonal influenza outcomes have focused primarily on the relationship between neighborhood ses and rates of hospitalization for severe disease in [ ] [ ] [ ] [ ] [ ] with fewer studies of individual-level outcomes . in addition, studies have investigated inequalities in influenza-related healthcare utilization (e.g. visits to outpatient clinics) , , incidence , and exposure as they relate to ses, race/ethnicity , , , and socioemotional stress . ses and exposure indicators utilized in these studies include percentage of households living in poverty , - , female headed households , , household crowding , , and neighborhood population density . incidence: two papers described disparities in rates of influenza susceptibility and exposure in older african americans and college students with higher perceived stress . black americans in nursing homes were less likely to be vaccinated against seasonal influenza, and were less likely to have vaccinated contacts, due to racial and socioeconomic segregation in nursing home care. the authors of this study argue that the results from nursing homes may provide clues to residential segregation's effects on seasonal influenza risk at the population level . in a cohort study of u.s. college students, an increase in perceived stress score was associated with a % greater rate of self-reported ili, after adjustment for demographic factors, behaviors and flu vaccination . severity: hospitalization was the most frequently studied dimension of severity used to characterize influenza disparities, followed by overall healthcare utilization , . area-level poverty and race/ethnicity were used as predictors of risk in a number of studies examining disparities in rates of influenza-related hospitalization. two ecological studies found that living in neighborhoods with a high percentage of residents below the poverty line and in female-headed households experienced higher rates of influenza . cc-by-nc . 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 , . , . similarly, census-tract rates of influenza-associated hospitalization surrounding nashville, tennessee, increased with population density and the percentage living in poverty, female headed/crowded households . in new haven, connecticut, the annual incidence of pediatric influenzaassociated hospitalizations in high-crowding census tracts (defined as > % of households with > one occupant per bedroom), and high-poverty census tracts (defined as > % of households below the poverty line), was > times higher than in low-crowding/low-poverty census tracts , . this disparity remained even after adjustment for comorbid medical conditions and influenza vaccination , . in a study of individual-level outcomes using data from fluserv-net, across multiple us counties and influenza seasons, african american and latino adults had increased odds of influenza-related hospitalization than whites . one study using population-based surveillance data found significantly higher hospitalization rates for laboratory-confirmed influenza for black children than for white children . viral and bacterial pneumonias are common ari-related complications that reflect ses-based disparities in baseline health status as well as access to care. several studies identified by our search focused specifically on disparities in hospitalization , , , and mortality associated with diagnosis of pneumonia and influenza (p&i). severity: in a study examining socio-demographic factors increasing risk of p&i hospitalization in elderly populations using medicare data from - , counties with high proportions of live-in grandparent caregivers and lower median income experienced higher rates of pneumonia-related hospitalization . in a study of alaska natives using hospitalization records from - , regions . cc-by-nc . 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 with a lower proportion of households with in-home water service, indicating higher poverty rates, also had higher hospitalization rates for p&i . mortality: there is some evidence that disparities in p&i associated mortality have decreased over time. one study using age-adjusted mortality rates for p&i show a decreasing ratio and absolute difference between black and white americans from to . the authors attribute this reduction in disparity due to widely available treatment to guard against p&i complications and mortality . literature examining rsv-specific disparities was limited to hospitalization/severity. disparities were identified in dimensions of race/ethnicity , , , household socioeconomic status , , high population density , maternal unemployment , younger maternal age , and household crowding . a study using population-based surveillance data found that hospitalization rates for laboratory confirmed rsv were similar for black and white children under months of age, but for children > months, hospitalization rates for lab-confirmed rsv were higher for black children than for white children . a study utilizing surveillance and hospital admittance data in southwestern alaska identified villages with lower proportion of houses plumbed water, higher proportion of household crowding, and higher proportion of families living below the poverty line as risk factors for hospital admission for rsv . similar risk factors were also identified in non-us studies . pandemics of influenza and other viral respiratory pathogens, such as covid- , are characterized by higher overall attack rates, elevated morbidity and mortality, unevenly adopted countermeasures, and increased strain on health systems. all of which may magnify inequalities in patterns of disease, despite a complete or near-complete lack of protective immunity. because of this lack of acquired immunity in the population, the age distribution of infection is likely to be different than seasonal epidemics, resulting in differential patterns of healthcare utilization and mortality risk. nevertheless, in a pattern echoing findings for seasonal flu, rsv and all-cause ari, inequalities in the most severe ari pandemic outcomes have consistently been documented. for example, historical data from chicago illustrated disparities in influenza pandemic mortality by neighborhood ses and racial composition , , with some of this disparity explained by differential rates of transmission in low-ses, overcrowded neighborhoods . in , a number of studies documented social race/ethnic outcome disparities [ ] [ ] [ ] [ ] [ ] [ ] , with lower ses associated with increased exposure risk , as well as overall incidence , hospitalization , , , complications , and death , due to pandemic h n . incidence: a nationally representative survey from the u.s. measured risk factors for exposure during the pandemic, including living in an apartment building, relying on public transportation, and difficulty finding day care that was separate from other children. this study found that these risks were significantly more common among non-white participants . another us study identified a lack of access to sick-leave and increased number of children in the household as risks for higher ili incidence during the h n pandemic . the authors determined that this increased risk was associated with hispanic ethnicity even after controlling for education and income , suggesting that additional, unmeasured ses-related risk factors contributed to this disparity. severity: in a study using surveillance data from illinois, hospitalization rates were twice as high for hispanics and blacks compared to whites during the h n pandemic . an analysis using data from multiple nationally representative surveillance networks investigated disparities in hospitalization . cc-by-nc . 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 and death found substantial differences in morbidity and mortality between race/ethnic groups . ageadjusted hospitalization rates for minorities were two times higher than those for non-hispanic whites. mortality: one study using nationally representative surveillance networks found non-hispanic black and hispanic children had disproportionately higher mortality than non-hispanic whites . during the h n pandemic, american indians and alaskan natives had a mortality rate times higher than all other racial/ethnic groups . conversely, a study using hospital discharge and census data found the living in a higher poverty census tract (> % below poverty line) and hispanic ethnicity was associated with lower risk of icu admission for h n related hospitalizations in massachusetts , although it is unclear if this result reflects differential severity or disparities in access to care. outside the united states: research outside of the united states has identified similar relationships between inequality and pandemic ari outcomes. for example, in a case-control study in ontario, canada of persons hospitalized for ari during the h n pandemic, the odds of h n infection were higher among adults identifying as east/southeast asian, south asian, and black compared to whites . in spain, individuals belonging to an ethnic minority group were more than twice as likely than whites to be admitted to the hospital, and individuals with a secondary or higher education were . times less likely to be admitted to the hospital as individuals with less education . in england, the h n pandemic caused . and . times higher mortality rates in the two most socio-economic deprived quintiles compared to the least deprived quintile, and this disparity persisted after adjustment for underlying medical conditions and age . . cc-by-nc . 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 , . the covid- pandemic has shown in stark relief the ways in which ses and racism structure ari outcomes at every level from exposure to disease severity and mortality. however, despite the large toll of mortality exacted by seasonal and pandemic ari and the clear evidence of disparities, the number of studies investigating disparities in ari outcomes and incidence is too limited to help chart a way towards more equitable outcomes. notably, most of the analyses discussed in this review come from data collected by studies in which identifying disparities was not a primary goal, and thus was not explicitly incorporated into study design and the enrollment of participants. further, much of the previous work has documented disparities, for example by race/ethnicity, but has not explored the history of policies and practices that specifically marginalized these racial/ethnic minorities and led to disparate outcomes . this echoes earlier findings that studies focusing on identifying and addressing social determinants are underrepresented relative to the importance of social factors in structuring infectious disease risk . the lack of studies dedicated to identifying and ameliorating ari disparities guarantees that the analyses reviewed here provide a decidedly incomplete picture of the drivers of disparities in arirelated outcomes. for example, many of the studies identified in our search is that they characterized disparities among the population of cases presenting at a point of care, ranging from routine medical visits to hospitalization. this reliance on clinical data is likely to under-count risk in populations without access to care, who are disproportionately likely to have low ses, and/or come from marginalized groups, e.g. undocumented immigrants, for whom real and perceived overlaps between medical and legal systems may serve as a disincentive to care-seeking. finally, most ari cohort studies, which allow for granular examination of individual-level risk and protective factors, drew largely on geographically concentrated study populations that are largely homogeneous in terms of ses and race/ethnicity. . cc-by-nc . 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 , . it is evident from our review that the lack of a coherent theoretical framework for understanding relationships between social inequality and ari incidence, severity, and mortality is a significant factor in the dearth of research in this area. the evidence turned up by our review, as well as the issues of racially and socioeconomic disparate exposure risks, coupled with unequal access to testing and treatment made clear by the covid- pandemic, demonstrate that the principles underlying link & phelan's theory of ses and race as fundamental causes of health and illness apply to aris as much as many non-communicable diseases as well as infections more classically understood has having social antecedents, such as tuberculosis and diarrheal disease. fundamental cause theory (fct) posits ses drives disparities through its impact on the financial and social power to marshal the material and social resources that are protective of health . fct is a mainstay of the literature on ses-related disparities in non-communicable diseases, but has enjoyed very limited application to infectious diseases . a key tenet of fct is that these disparities are persistent and are likely increase even as population-level risk goes down. for example, medical innovation is likely to increase disparities because new interventions typically become available first to the well-off and well-insured. this strongly suggests that progress in the treatment and prevention of aris using antivirals, monoclonal antibodies , influenza vaccination, and molecular genotyping to inform surveillance and control, are likely to result in increased inequality in both ari incidence and severe outcomes. these issues have once again been front-and-center during the covid- pandemic, as well-off individuals in the united states enjoyed preferential access to testing in the early days of the pandemic. similarly, the ability to participate in 'social distancing' by working from home, having food and other essential delivered to one's home, and engage other protective measures are clearly a function of power and material resources. in the absence of effective vaccines and antivirals, this is the key technology available to protect oneself from an emerging . cc-by-nc . 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 infection. as preventive and therapeutic interventions against covid- become available, we are likely to see these also distributed preferentially to those with greater means, thereby only exacerbating the yawning disparities in exposure, severity, and mortality that are already evident. surveillance and large network studies can estimate burden of disease and influenza vaccine effectiveness (ve), but may under enroll low-ses and non-white populations and are by design limited to those with access to care since participants are enrolled at the point of care. ideally, large longitudinal cohort studies with study samples that are representative of the us population would be available to estimate differences in incidence and risk and ve. a meta-analysis found that % of observational studies on influenza severity were cohort studies, but very few of these studies collected information on race/ethnicity and the authors do not report on measures of ses in their analysis . to capture the components of risk associated with social disparities, these studies would ideally span multiple seasons and account for known factors associated with risk such as age, influenza infection and vaccination history, household composition, crowding, and other potential exposure and susceptibility related factors. in the absence of such studies, greater attention is needed to increase the socioeconomic diversity of the populations that studies of seasonal ari risk and ve draw upon, as well as the potential impact of ses on infection outcomes and vaccine effectiveness. where such data are not available, careful statistical modeling is essential. for example, approaches such as multiple regression with poststratification (mrp), which can be used to make unbiased population-level predictions from nonrepresentative data, may help address some of these questions while we wait for more detailed data . . cc-by-nc . 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 fct highlights why identifying and intervening only on mediators of the relationship between ses and disease is unlikely to be effective: risk of ari infection, severe disease, and mortality cannot be reduced to the impact of a single variable, e.g. vaccination. instead, it reflects a spectrum beginning with comorbid health conditions, the ability to avoid exposure, access to preventive and therapeutic treatment, and other factors. these issues are arguably even more acute for infectious diseases than for non-communicable ones, as the risks of our friends and family directly impact our own risks via transmission. an fct perspective suggests that effective policies must address inequality in access to prevention and treatment, as well as inequity in the ability to avoid exposure. for example, policies limiting or eliminating paid sick leave have been shown to increase rates of influenza transmission in the workplace, and to disproportionately affect racial/ethnic minorities , with similar patterns evident in the covid- pandemic. in order to be able to reduce transmission, symptomatic persons should be treated with antivirals (when applicable) and practice home isolation . this cannot be achieved without policies that support rather than penalize such behavior. the lack of supportive workplace policies has been suggested to result in a population-attributable risk of million additional cases of ili in the us with a disproportionate burden on hispanic americans . the lack of a health system ensuring equal access to prevention and care is clear a key factor in exacerbating ses-related disparities in seasonal ari incidence, as well as undermining pandemic preparedness. however, in the absence of sweeping changes that would address the root causes of these disparities , targeted vaccination for influenza , and rsv based on age specific contact and transmission patterns has been theorized to improve both the efficacy of vaccines. identifying high priority groups for vaccination has been shown to be effective in reducing disease burden when target groups are those with high-risk of infection or high transmission potential for both rsv and influenza [ ] [ ] [ ] [ ] . the expansion of targeted programs to improve access to . cc-by-nc . 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 , . vaccination among those of lower ses could be beneficial in reducing disparities. when identifying groups for targeted vaccination, especially those of disadvantaged backgrounds, it is important to address past abuses that may lead to mistrust of these programs. finding ways to engage with organizations that serve low ses and racial minority communities to address concern and reduce vaccine hesitancy will be paramount to the success of these programs in sum, our findings highlight wide disparities in ari outcomes by socioeconomic status and race/ethnicity in the u.s. though the covid- pandemic has brought many of these concerns to the forefront of the public and scientific consciousness, it has also shown how poorly developed our set of tools for addressing both the root and proximal causes of these disparities is. this study represents a first step towards the development a coherent framework for identifying disparities in ari and their causal antecedents. this will hopefully increase clarity around when and where socially focused interventions (i.e. healthcare and workplace policies) should be prioritized, as well as how biomedical innovations (e.g. vaccination, antivirals) should be distributed to shrink group-level disparities at the same time that they minimize population risk. . cc-by-nc . 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 to accomplish this, a sustained effort to build studies and create standardized measures purposively aimed at understanding and curbing ses-related disparities in aris is urgently needed. however, even with this lack of consensus, the body of evidence suggests there are disparities in ari incidence, severity, and mortality. with potential new technologies on the horizon to prevent and better treat aris from rsv to influenza as well as emerging infections with pandemic potential, such as covid- , the time to make a plan for how to ensure the equitable distribution of benefits from these and other technologies is now. . cc-by-nc . 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-nc . 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-nc . 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-nc . 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 , . . cc-by-nc . 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 , . 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 , . regions with lower proportion (< %) of inhome water service had a . times higher rate of hospitalization due to pneumonia and influenza and a . cc-by-nc . 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-nc . 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 , . māori or pacific ethnicity was associated with over times the risk of . cc-by-nc . 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-nc . 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 , . living in a census tract with higher poverty levels ( . - . % and % or more vs. - . % households living below poverty) and hispanic ethnicity was associated with lower risk of icu admission in h n related hospitalizations. when stratified by race, living in a census tract with higher poverty levels ( . - . % vs. - . % households living below poverty) was associated with lower risk of icu admission . cc-by-nc . 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-nc . 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. (which was not certified by peer review) 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. (which was not certified by peer review) 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. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint global, regional, and national causes of under- mortality in - : an updated systematic analysis with implications for the sustainable development goals why inequality could spread covid- african americans are at higher risk of death from coronavirus. the washington post drivers of airborne human-to-human pathogen transmission global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis reasons for the decline of mortality in england and wales during the nineteenth century influenza-related hospitalizations and poverty levels -united states social determinants of influenza hospitalization in the united states. influenza other respi viruses socioeconomic disparities and influenza hospitalization neighborhood socioeconomic status and influenza hospitalizations among children influenza-related hospitalization of adults associated with low census tract socioeconomic status and female sex the relationship between in-home water service and the risk of respiratory tract , skin , and gastrointestinal tract infections among rural alaska natives subjective social status predicts wintertime febrile acute respiratory illness among women healthcare personnel objective and subjective socioeconomic status and susceptibility to the common cold deprivation and bronchiolitis social deprivation and hospital admission for respiratory infection: ecological study mortality associated with acute respiratory infections among children at home disparities between black and white children in hospitalizations associated with acute respiratory illness and laboratory-confirmed influenza and respiratory syncytial virus in us counties - - rcgp research and surveillance centre annual report - : disparities in presentations to primary care socio-economic disparities in the burden of seasonal influenza: the effect of social and material deprivation on rates of influenza infection increased influenza-related healthcare utilization by residents of an urban aboriginal community stress, adherence to preventive measures for reducing influenza transmission and influenza-like illness health care segregation and race disparities in infectious disease a spatial analysis of the determinants of pneumonia and influenza hospitalizations in ontario ( - ) disparities in influenza mortality and transmission related to sociodemographic factors within chicago in the pandemic of pandemic influenza and socioeconomic disparities: lessons from racial disparities in exposure, susceptibility, and access to health care in the us h n influenza pandemic the impact of workplace policies and other social factors on self-reported influenza-like illness incidence during the h n pandemic disparities among pandemic influenza a (h n ) hospital admissions: a mixed methods analysis -illinois racial and ethnic disparities in hospitalizations and deaths associated with pandemic influenza a (h n ) virus infections in the united states deaths related to pandemic influenza a (h n ) among american indian/alaska natives states effect of race/ethnicity and socioeconomic status on pandemic h n -related outcomes in massachusetts ethnic disparities in acquiring pandemic h n influenza: a case-control study social factors related to the clinical severity of influenza cases in spain during the a (h n ) virus pandemic socio-economic disparities in mortality due to pandemic influenza in england neighborhood-level mass incarceration and future preterm birth risk among african american women analyses of infectious disease patterns and drivers largely lack insights from social epidemiology: contemporary patterns and future opportunities social conditions as fundamental causes of health inequalities: theory, evidence, and policy implications contemporary social disparities in tb infection and disease in the usa: a review cost-effectiveness of palivizumab for respiratory syncytial virus: a systematic review results from the us hospitalized adult influenza vaccine effectiveness network (haiven) measurement of vaccine direct effects under the test-negative design measurement of vaccine direct effects under the test-negative design influenza vaccination coverage --united states interim estimates of - seasonal influenza vaccine effectiveness -united states interim estimates of - seasonal influenza vaccine effectiveness -united states populations at risk for severe or complicated influenza illness: a systematic review and meta-analysis struggles with survey weighting and regression modeling the impact of illness on social networks: implications for transmission and control of influenza on the relative role of different age groups in influenza epidemics or "medically underserved area"[mesh terms] or minority group[tiab] or "minority groups or alaska natives[tiab] or people of color[tiab] or "poverty areas"[mesh terms] or poverty area[tiab] or poverty areas[tiab] or "rural health"[mesh terms] or rural health[tiab] or "rural health services"[mesh terms] or "rural population"[mesh terms] or rural population[tiab] or rural populations[tiab] or slum[tiab] or slums[tiab] or "urban health"[mesh terms] or "urban health services"[mesh terms] or "urban population or "minority health and ((((human influenza[mesh terms]) or human influenzas[mesh terms]) or human influenza[title/abstract]) or human influenzas key: cord- -p f pl y authors: masoud, khaldoun; hanna-wakim, rima; zaraket, hassan; kharroubi, samer; araj, george f; matar, ghassan m; dbaibo, ghassan title: viral etiology of acute respiratory infections in pediatric patients in lebanon date: - - journal: mediterr j hematol infect dis doi: . /mjhid. . sha: doc_id: cord_uid: p f pl y background: acute respiratory infections (ari) are the leading cause of death worldwide, especially among children. the majority of these infections in children are of viral etiology. in this study, we evaluated the incidence of viral ari among children in lebanon. patients and methods: children presenting with symptoms of ari were prospectively recruited between september to february . nasopharyngeal aspirates were obtained from patients and screened for respiratory viruses using a multiplex luminex-based pcr assay. results: two hundred twenty-one patients were recruited with a median age of year (iqr: – ). out of patients, ( . %) were positive for at least one virus, the majority ( / ; . %) of which were in children under -year of age. overall, viruses were detected. rhinovirus (rhv) was the most common virus detected in ( . %) patients followed by coxsackie virus and echovirus (cvev) which were detected as one target in the panel in ( . %), and parainfluenza viruses (piv types: , , , ) in ( . %) patients. coinfection with more than one virus was detected in ( . %) patients. rhv and cvev were the most common viruses associated with co-infections and higher risk of rhinorrhea. conclusions: viral pathogens account for at least half of the aris in lebanon, with a high frequency of co-infections being detected. introduction. acute respiratory tract infections (aris) are among the most common reasons for primary care consultations. the world health organization (who) ranks aris as the fourth major killer after cardiovascular diseases, general infections and parasitic diseases, and cancer. aris cause million death globally. the burden is especially high in children where aris are responsible for - % of deaths. aris can lead to severe complications requiring hospitalizations and can have fatal outcomes. viruses are the most common etiology of aris in children. , these include rhinovirus (rhv), respiratory www.mjhid.org mediterr j hematol infect dis ; ; e pag. / syncytial virus (rsv), influenza (ifn), parainfluenza virus (piv), coronavirus (cov), human metapneumovirus (hmpv), enteroviruses (ev), adenovirus (adv), and human bocavirus (hbov). , , each of these viruses poses a significant health burden. nair et al. estimated that deaths in children < years were attributable to influenza-associated lower respiratory tract infections (lri) in , the vast majority of which occurred in developing countries. rsv was estimated to have caused . million lri episode in children under five, of which . million were severe causing up to deaths. in addition to the health burden of viral respiratory tract infections (rtis), the economic impact is also high if we account for health care costs (direct cost) and loss of productivity (indirect cost). a study by fendrik et al. estimated the total economic impact of non-influenzarelated viral rtis in the united states at $ billion annually. the advancements that have been achieved in developing antiviral drugs, some of which have already been approved, against respiratory viruses allow for targeted therapy of viral aris. [ ] [ ] [ ] this possibility calls for better and faster diagnosis of the etiologic agents in ari patients to benefit from the full potential of these drugs. furthermore, aris are associated with the greatest amount of excess use of antibiotics that has led to unprecedented increase in antimicrobial drug resistance; , therefore, proper and timely diagnosis of viral infections can help reduce unnecessary antibiotic prescriptions. , in lebanon, studies investigating the viral etiologies of aris are very scarce. in this study, we determined the viral etiologies among ari patients at a tertiary care hospital that serves an ethnically and socioeconomically diverse patient population. patients and samples collection. infants and children younger than years of age with symptoms of ari disease presenting to the emergency department or the departments of pediatrics of the american university of beirut medical center (aubmc), beirut, lebanon were prospectively recruited between september to february . an ari was defined as an acute infection of the upper and lower respiratory airways. recruited patients had one or more of the following symptoms: fever, cough, sore throat, rhinorrhea, headache, conjunctivitis, wheezing, dyspnea, and vomiting. medical history and demographic data were obtained from the patients and their medical records. a respiratory sample was collected and stored at - °c for viral assessment. the study was approved by the institutional review board (irb) of the aubmc, and written informed consent was obtained from all parents. nucleic acid extraction and viral detection. nucleic acid was extracted from clinical specimens by using the qiaamp minelute virus spin kit (qiagen) according to the manufacturer's protocol. a µl aliquot of each specimen was used for nucleic extraction. specimens were then analyzed by the resplex ii panel (qiagen) using the manufacturer's protocol. the resplex ii panel can detect viral targets: rsva, rsvb, infa, infb, piv , piv , piv , piv , hmpv, cvev (coxsackievirus and echovirus), and rhv. briefly, µl of each specimen were added to µl reverse transcription-pcr (resplex ii) master mix, including the supplied primers. targets were detected by mixing µl portions of amplification products with resplex ii bead in hybridization buffer at °c for min. streptavidin-phycoerythrin conjugate was added, and mixtures were incubated at °c for a further min before the addition of stop buffer. the samples were then analyzed on a luminex bio-rad bioplex system (bio-rad laboratories) using bio-rad bioplex manager software. the cutoff value for each target was determined, as previously described by lia et al. statistical analysis. the data were checked for completeness, and responses were coded and entered into the statistical package for the social sciences (spss) software version for windows, which was later used for statistical analyses. descriptive statistics were presented to summarize the study variables of interest as counts and percentages for the categorical variables and as medians and interquartile range (iqr) for the continuous ones. the chi-square test was used to calculate the association between two categorical variables. pearson's chi-square analysis with bonferroni-holm p-value correction was used for multiple comparisons to assess infectivity enhancing correlations. univariate and multivariate logistic regression analyses were applied to determine which factors are associated with rhinorrhea. in the regression model, rhinorrhea was used as the dependent variable. odds ratios and their respective % confidence intervals were calculated. for all analysis done, a pvalue of less than . was considered statistically significant. patient characteristics. a total of specimens were collected from children presenting with symptoms of ari between september and february ( table ) . the socio-demographic characteristics of the study patients are presented in table . overall, the study consisted of males ( . %) and females ( . %) with patients' median age of (iqr: - ) years. seventy-four patients ( . %) were children under one year of age, ( . %) were between to years old, ( . %) were to years old, and ( . %) were to years old. sixty-seven ( . %) of the children had an underlying disease (asthma, immune-deficiency, allergic rhinitis, or cystic fibrosis). at the time of diagnosis ( . %) patients were receiving chemotherapy, and ( %) had received an antibiotic. virological characterization. samples were screened for virus targets included in the resplexii respiratory panel. of the ari episodes, ( . %) were confirmed to be of viral etiology being positive for at least one of the virus targets tests ( table ). the majority (n= ; %) of viral ari episodes were observed in children under -year of age (chisquare, p< . ). figure shows the frequency of each of the viruses detected in the study population. overall viruses were detected. rhinovirus (rhv) was the most common virus detected in ( . %) patients followed cystic fibrosis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) . a significant majority (n= ; . %; chi-square p-value< . ) of coinfections occurred in children under six years of age. the most frequent viral co-infections involved two viruses (n= ), cases had a triple infection, had four viruses detected, and one case had five viruses. almost half cases of rhinovirus ( . %) were positive for at least another virus in the panel. cvev positive cases also had a high rate of coinfection ( %). moreover, hmpv and infb were detected in samples, and all were co-infected. table summarizes the correlation of different viruses among our patients. several correlations enhancing infectivity were evident in our analysis. of note, rhv was the most frequently detected virus in co-infections and was significantly associated with rsvb, infb, piv , hmpv, and cvev. underlying conditions and clinical presentation. we next analyzed the correlation between each of the viral etiologies or co-infection with the underlying conditions ( table ) . to simplify the analysis, we treated subtypes or genotypes of a virus as a single group (e.g. rsv for rsva and rsvb, etc.). having an underlying condition or receiving chemotherapy or a course of antibiotics were not found to be a risk factor for having a viral etiology or co-infection ( table ) . additionally, we investigated the seasonal variation of viruses. rhinovirus infections were detected throughout the year however the peak rate occurred during the main rainy months (november, december), likely for coxsackie/echovirus and rsv. on the other hand,influenza a virus infections had a peak in the fall (september, october); (figure ). in terms of clinical symptoms, fever, cough and rhinorrhea were major symptoms observed in most of the patients infected with one or more respiratory virus ( table ) . chisquare analysis revealed a significant correlation between rhinorrhea and inf, cvev, and rhv and coinfection ( table ) . bivariate logistic regression was then performed to determine the risk associated with these infections. our analysis revealed that rhv or a multivariate logistic regression model was used to examine the correlates of rhinorrhea in the study patients. variables were put in the model in order of strength of their correlation with rhinorrhea as per the bivariate analysis. the effect of each variable on the model was assessed, and the variable was kept if it significantly contributed to a better fit of the model. the final model included the following variables: rhv and cvev. the results of the multivariate model showed that cvev was independently associated with rhinorrhea (or: . ; ci: . - . ). cvev infected patients were . times more likely to have rhinorrhea compared to none-cvev patients controlling for rhv. unlike the bivariate analysis, rhv was not significantly associated with rhinorrhea (or: . ; ci: . - . ). rhv infected patients were . times more likely to have rhinorrhea compared to none-rhv patients controlling for cvev; however, this was not statistically significant. discussion. we demonstrated that viral infections are responsible for at least half of the aris in children in lebanon. rhinovirus infection was the most common etiology of ari consistent with other studies from lebanon and other countries. , , in neighboring jordan and egypt rhinovirus incidence was second to rsv, but the population captured in these studies was younger than that included in our study. , the overall viral ari incidence ( . %) in our study lower than that recently reported by finianos et al. ( %) in lebanon. both studies targeted children; however, finianos et al. screened their specimens for more viral targets than those included in our analysis. in our study we did not test for hcov, adv, ev, and hbov which collectively accounted for % of viral ari in the study by finianos et al. the coinfection rate in our study ( . %) was higher than that previously reported in lebanon ( %), qatar ( . %), and egypt ( . %). , , this incongruence could be because cvev, which was frequently detected with other viruses in our study, was not screened in the previous studies from the region. cvev infections are not commonly reported in studies investigating respiratory infections. in our study, cvev infection constituted . % of all viral ari cases and was independently associated with rhinorrhea. this incidence is much higher than that reported in other countries. a recent study in latin america reported that cvev was associated with % of the ari cases. in central america, cvev was even much lower ( . %). the very low prevalence of cvev in other regions might have discouraged its testing. given the high prevalence of cvev in lebanon, we recommend testing for enteroviruses, including (cvev). co-infections were found to be more common younger children in lebanon, and that is similar to a previous study done in mexican children showing that the majority of coinfections occur in children < months of age. younger children are likely to be more prone to infections due to their lack or still weak immunity to respiratory viruses. the effect of coinfections on disease outcomes is not well understood. patients coinfected with pandemic h n influenza and rhinovirus tended to have milder clinical severity when compared with non-rhinovirus coinfections; while the patients coinfected with hmpv and rsv were prone to a higher risk of severe bronchiolitis. additionally, the prevalence and severity of obstructive airway disease were higher in patients with coinfections. in our study, coinfection was associated with higher risk of rhinorrhea but not with more severe symptoms like dyspnea. in contrast, some studies showed no correlation between coinfection status and clinical severity. , the complexity of viral coinfections and the large number of respiratory viruses involved make challenging to study the effect of coinfection on disease outcome in a clinical setting. therefore, there is a need for developing in vitro or in vivo models to allow a better understanding of coinfections. for example, dual infection with inf was shown to suppress rsv growth in vitro. the suppression of rsv by inf was suggested to be due to competition for protein synthesis and budding from the cell surface. further studies are warranted to investigate the interactions among respiratory viruses during coinfection and their effect on the host. our study had a couple of limitations. first, we have not screened for hbov, and hcov which are not included in resplex ii kit and thus the prevalence of viral ari is expected to be higher than %. another limitation was our inability to rule out bacterial etiologies which was not tested for in the current study. in conclusion, viral etiologies contribute to a large proportion of aris many of which involve more than one viral agent. the burden of acute respiratory infections in crisis-affected populations: a systematic review who | the global burden of disease estimates of world-wide distribution of child deaths from acute respiratory infections respiratory viruses other than influenza virus: impact and therapeutic advances detection of respiratory viruses by molecular methods epidemiology and prevention of pediatric viral respiratory infections in health-care institutions identification of new respiratory viruses in the new millennium global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. the lancet the economic burden of non-influenza-related viral respiratory tract infection in the united states recent advances in diagnosis, prevention, and treatment of human respiratory syncytial virus developing new antiviral agents for influenza treatment: what does the future hold? advances in antivirals for non-influenza respiratory virus infections. influenza other respir viruses use of antibiotics for adult upper respiratory infections in outpatient settings: a national ambulatory network study excessive antibiotic use for acute respiratory infections in the united states viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections etiology, seasonality and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon viral and atypical bacterial aetiologies of infection in hospitalised patients admitted with clinical suspicion of influenza in thailand, vietnam and indonesia. influenza other respir viruses viral aetiology of acute respiratory infections with cough in infancy: a community-based birth cohort study human metapneumovirus in hospitalized children in viral etiologies of lower respiratory tract infections among egyptian children under five years of age the role of human metapneumovirus in pediatric respiratory tract infection in qatar human rhinoviruses and enteroviruses in influenza-like illness in latin america influenza and other respiratory viruses in three central american countries. influenza other respir viruses viral coinfection in acute respiratory infection in mexican children treated by the emergency service: a cross-sectional study respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology rate and influence of respiratory virus co-infection on pandemic (h n ) influenza disease dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections viral and atypical bacterial detection in acute respiratory infection in children under five years in vitro growth profiles of respiratory syncytial virus in the presence of influenza virus key: cord- -vfagxsdz authors: althouse, benjamin m; flasche, stefan; minh, le nhat; thiem, vu dinh; hashizume, masahiro; ariyoshi, koya; anh, dang duc; rodgers, gail l.; klugman, keith p.; hu, hao; yoshida, lay-myint title: seasonality of respiratory viruses causing hospitalizations for acute respiratory infections in children in nha trang, vietnam date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: vfagxsdz background: acute respiratory infections (aris) are the most common causes of death in children under years of age. while the etiology of most pneumonia and ari episodes is undiagnosed, a broad range of ari-causing viruses circulate widely in south east asia. however, the patterns and drivers of the seasonal transmission dynamics are largely unknown. here we identify the seasonal patterns of multiple circulating viruses associated with hospitalizations for aris in nha trang, vietnam. methods: hospital based enhanced surveillance of childhood ari is ongoing at khanh hoa general hospital in nha trang. rt-pcr was performed to detect respiratory viruses in nasopharyngeal samples from enrolled patients. seasonal patterns of childhood ari hospital admissions of various viruses were assessed, as well as their association with rainfall, temperature, and dew point. results: respiratory syncytial virus peaks in the late summer months, and influenza a in april to june. we find significant associations between detection of human parainfluenza and human rhinovirus with the month's mean dew point. using a cross-wavelet transform we find a significant out-of-phase relationship between human parainfluenza and temperature and dew point. conclusions: our results are important for understanding the temporal risk associated with circulating pathogens in southern central vietnam. specifically, our results can inform timing of routing seasonal influenza vaccination and for when observed respiratory illness is likely viral, leading to judicious use of antibiotics in the region. acute respiratory infections (aris) in south east asia cause substantial morbidity and mortality, especially in children under years of age (walker et al., ) . pneumonia continues to be the number one cause of under death despite effective treatments (wardlaw et al., ) . a substantial contributor to this is the largely unknown etiology of most pneumoniae, with both viral and bacterial origin. the patterns of pneumonia and other ari hospitalizations serves as a proxy for determining the transmission dynamics of viruses and bacteria contributing to these hospitalizations, and is of key importance in understanding and limiting burden of this childhood killer. in addition to informing on the relative likelihood of the potential viral etiology of a pneumonia case based on seasonally circulating pathogens, knowledge of seasonal influenza epidemiological dynamics can aid in informing optimal timing for vaccination efforts (saha et al., ; lambach et al., ) and judicious use of antivirals tanaka et al., ) . identification of low incidence seasons would provide a target window for vaccination, with the hope of maximizing population immunity before the onset of the influenza season. similarly, when respiratory syncytial virus (rsv) vaccination becomes available, knowledge of its seasonality will be useful to maximize benefit (drysdale et al., ; anderson et al., ) , and in the potential administration of passive immunoprophylaxis with palivizumab to reduce the number of severe outcomes associated with rsv infection among high-risk infants (committee on infectious diseases, ). finally, knowledge of seasonal patterns of virus circulation can inform the clinical use of antibiotics, again limiting use when viral circulation is traditionally high to minimize antibiotic resistance ( van nguyen et al., ; laxminarayan et al., laxminarayan et al., , van boeckel et al., ) . previous work has identified the potential etiology of pneumonia and other aris in southern central vietnam (do et al., ; yoshida et al., ) . adenovirus (aov), bocavirus (hbov), coronavirus (cov), human metapneumovirus (hmpv), human parainfluenza - viruses (hpiv - ), human rhinovirus (hrv), influenza a and b, and rsv all contribute to the disease burden and circulate widely. information on viral transmission dynamics across seasons in vietnam is relatively under-explored. do et al. found seasonality of rsv infections and slight seasonality of hmpv infections in ho chi minh city, but no seasonality of influenza (do et al., ) . several studies have similarly found high variability in influenza a & b incidence over the year (nguyen et al., ; le et al., ; thai et al., ) . yoshida et al. found rsv occurring in the hot months, influenza a in the cool months, and year-round detection of hrv in nha trang (yoshida et al., ) . few other studies have examined seasonality of these common viruses across south east asia. here we examine the seasonal trends of hospitalizations and circulation of multiple viruses in nha trang, vietnam. using enhanced hospital based surveillance of childhood ari we identify seasonal patterns in hospitalizations as a proxy for transmission and explore the relationship of hospitalizations associated with virus detection with rainfall, temperature, and dew point, to try and identify contributing factors to observed seasonality. the study site is nha trang, central vietnam, where the study population has been described previously (yoshida et al., (yoshida et al., , flasche et al., ) . the hospital based enhanced surveillance of childhood ari is ongoing. we analyze data from january , to april , which is the only tertiary care facility located in khanh hoa province. according to the field site census survey in july , the study catchment area encompassing the non-touristic of the communes in nha trang city, had , residents including , children less than years of age. an ari case was defined as any child presenting to khgh with cough or/and difficulty in breathing. before study enrollment, informed consent was obtained from parents of children who presented with ari and lived in the study catchment area. clinical and demographic information, chest radiographs (cxr), laboratory data, and nasopharyngeal (np) samples were collected from all enrolled patients. khgh is the only hospital in nha trang, khanh hoa province and the only one accessible for residents of the catchment area. hence for incidence calculations we assume that all children with ari are eligible to be hospitalized and enrolled into the study and use the population of the catchment area as denominator. acute respiratory infection patients with normal cxr were categorized as upper respiratory tract infection (urti). patients with abnormal cxr were categorized as lower respiratory tract infection (lrti). np samples were collected at the time of admission and viral nucleic acid was extracted using qia viral rna minikit (qiagen inc., valencia, ca). four multiplex-pcr assays ( : influenza a, influenza b, rsv, hmpv; : piv- , - , - , and - ; : rhinovirus, coronavirus e, coronavirus oc ; : adenovirus and bocavirus) were performed to detect respiratory viruses in each np sample. a second confirmatory-pcr was performed for samples positive on the initial pcr test. samples positive for both pcr assays were defined as positive. reverse transcription-pcr (rt-pcr) assays were performed using one-step rt-pcr kit from qiagen. for the multiplex pcr and hemi-nested pcr assays, taqdna polymerase (promega, san luis obispo, ca) was used as previously described (yoshida et al., ) . positive templates were used in each assay for quality control. three weather variablesrainfall (inches), temperature (f ), and dew point (the temperature to which air must be cooled in order to reach saturation with water)were collected from the nha trang station (id: ) reported by the us national oceanic and atmospheric administration (national oceanic and atmospheric administration, ). we considered monthly averages of all weather variables as well as both the weather variable on the day of admission (t ) as well as averaged over the previous days (t À to t À ) assuming up to a week incubation period for the viral infections (see supplementary material) (lessler et al., ) . the weather in nha trang central vietnam is warm throughout the year (between and c ). in terms of temperature, december to february months are cooler (referred to here as the winter months) while june to august months are hottest months (referred to here as the summer months). september, october, november are the wettest months. for each pcr+ for virus a series of log-link poisson models were fit to assess respective seasonality with calendar month as the main predictor, log-commune population size as an offset term, monthly averaged rainfall, temperature, and dew point, and calendar year as adjusting variables. the outcome was monthly aggregate cases, with resulting coefficients as incidence rate ratios as compared to january. we excluded hospitalizations with more than one virus detected in the nasopharynx to adjust for a potential bias through inclusion of cases who by virtue of being co-infected may otherwise have been asymptomatic (althouse and scarpino, ) . this approach underestimates the true incidence of np carriage among ari cases but allows estimation of seasonal patterns that are not biased by other circulating viruses; although co-circulation of bacteria was not accounted for. we assessed the numbers and variety of viruses in ari hospitalizations using binomial proportion tests for each virus. to examine the relationship between monthly average rain, temperature, and dew point and incidence hospitalized childhood ari infections, we estimated the cross-wavelet transform between the z-standardized time series (we subtracted the mean of the time series and divided by the standard deviation) of weather and viral detections (cazelles et al., ) . the cross-wavelet transform identifies regions of high power in phase-space and identifies the relative phases of each time series, i.e., in-phase or out-of-phase (grinsted et al., ) . the wavelet transform can be thought of a fourier transformation over time that can identify what is the dominant frequency composing a time series as the signal changes in time. the cross-wavelet transform allows us to compare how two time signals co-vary: we can identify if the presence of a particular frequency at a given time in the time series of hospitalizations corresponds to the presence of that same frequency at the same time in a weather covariate (chaves and pascual, ) . additionally, we can identify the magnitude by which weather precedes or follows hospitalizations through the phase angle of the two time series. finally, we can identify the statistical significance of the identified constituent frequencies over time by comparing the observed frequencies to a red-noise process. sensitivity analyses, presented in the supplementary materials, were performed as follows: ) case counts of less than per virus over the whole study period were deemed too low for robust statistical inference; ) alternative poisson regression models where the reference category is july; ) logistic regression models were formulated as an alternative to the poisson regressions above with detection of a virus by pcr (yes/no) as the outcome, with month as the main predictor, adjusted for weather, commune of residence, age, sex, smoking indoors, socioeconomic status (ses), and calendar year, with weather variables on the day of admission (t ) as well as averaged over the previous days (t À to t À ); and ) additional wavelet analyses of viral isolations not presented in the main text. the study enrolled children between to . among those, ( %) had multiple viruses detected in their np swabs, for presence of viruses in the np was not determined, and were excluded from the analyses, thus the total study population was . among all cases with a virus detected, hrv, rsv, and influenza a were the most frequently detected viruses, with ( . % of all viral detections), ( . %), and ( . %) detections, respectively (table and figure ). counts of bocavirus (hbov), coronavirus (cov), and human parainfluenza , , and viruses (hpiv - ) were less than and are reported in the supplementary material. strong seasonality, as defined by at least three consecutive months with a consistently higher or lower incidence than expected (irr or or greater or less than , respectively) and at least one of those statistically significantly different from the baseline, was observed for influenza a, rsv, and the presence of any virus (figures and ) . rsv peaked in july through november, with august seeing a . ( % confidence interval [ci]: . , . ) times higher risk of identifying rsv as the sole viral agent from the nasopharynx of a childhood case as compared to january. influenza a peaked in may with an irr of . ( % ci: . , . ) as compared to january. estimates of odds ratios from the supplemental logistic regression are qualitatively similar to the poisson regression, save for hpiv , which exhibits a consistent yet nonsignificant peak in the cool months in the primary analysis which becomes significant in the supplemental analysis (see supplemental material). weather patterns over the study period were similar to patterns before and after the study period (supplementary material, figure s ). monthly average rainfall (in inches), temperature ( f), and dew point ( f) correlated with the seasonality of some of our endpoints. figure shows the incidence rate ratios for the three weather effects from the seasonally-adjusted models. overall hospitalizations for ari were negatively associated with temperature (irr . per degree increase, % ci: . , . , p = . ) and positively associated with dew point (irr . per degree increase, % ci: . , . , p < . ). of the few other significant effects, influenza a and hrv had a negative associations with temperature (irr . , % ci: . , . , p = . , and irr . , % ci: . , . , p < . , respectively), and hpiv and hrv were positively associated with dew point, with irrs . ( % ci: . , . , p = . ) and . ( % ci: . , . , p = . ), respectively. logistic regression found that rsv was positively associated with the previous week's rain, with an odds ratio of of . ( % ci: . , . , p = . ). previous week's temperature was marginally associated with rsv (or: . ( % ci: . , . , p = . ) (see supplementary material). figure shows the cross-wavelet transform of all hospitalizations and the three weather variables in the month of admission. significant bands of high power around year can be seen for temperature and dew point. this indicates that hospitalizations and temperature and dew point share variability at yearly frequencies over the study period, and the phase relationship indicates that changes in weather slightly precede changes in hospitalizations (arrows point about down). figure shows the cross-wavelet transform of rsv and the three weather variables with similar significance bands around year for temperature and dew point. the phase indicates temperature and dew point lead rsv incidence. while not significant, rsv was found to be leading rainfall by ( months) at the one year period band (see supplementary material). significant bands of power were seen between temperature and dew point with hpiv with an indicated phase relationship of nearly completely out-of-phase (figure ) . similar patterns were seen ( year significant bands between temperature and dew point and virus) for adv, hbov, cov, hpiv , & , hrv, and influenza a, though the phase differences varied across these viruses; hmpv and influenza b had bands lying outside the cone of influence (ie, not statistically significant; see supplementary material). these results in general indicate strong associations between weather covariates and viral hospitalizations at a yearly timescale. here we have identified seasonal trends of several common respiratory viruses in hospitalized children in nha trang, vietnam. by fitting a series of statistical models to the observed data, we allow the data to identify salient features contributing to the seasonality of these viruses. we evaluated seasonal patterns and associations with weather of hospitalizations for several respiratory viruses using three lines of evidence: ) poisson regression examining the relative incidence across months of virus detections adjusted for weather covariates, ) cross-wavelet transforms of hospitalizations with viral detections, and ) a sensitivity analysis with a logistic regression model finding odds ratio of hospitalizations with viral detections and weather variables. any viral detection showed distinct seasonality with peaks in may through september, a negative association with temperature, positive association with dew point, and crosswavelets indicating temperature and dew point leading viral detection. of commonly detected viruses, rsv, influenza a, and hpiv had significant seasonality. rsv peaked in july through december, was positively associated with the week's previous average rainfall. cross-wavelets showed temperature and dew point to lead rsv, rain was found to non-significantly fall behind rsv at year frequencies, and precede rsv at shorter (< day) frequencies. finally, hpiv while not significant, had peaks in january and february, was positively associated with dew point, and was completely out of phase with temperature and dew points in cross-wavelet analyses. these results contribute to the growing body of knowledge on the epidemiology of respiratory pathogens in south east asia and southern central vietnam. using a cross-wavelet transform we evaluated the timedependence of virus hospitalizations with the weather covariates. we found strong yearly associations with rsv over the study period with temperature and dew point in phase with hospitalizations. this seasonality is opposite to observed seasonality in temperate climates, where rsv typically peaks in winter (tang and loh, ; shek and lee, ) . recent reviews of rsv seasonality in tropical regions highlights the uncertainty in the effects of weather on rsv transmission. studies in brazil (straliotto et al., ) , hawaii (reese and marchette, ) , india (agrawal et al., ) , kenya (hazlett et al., ) , and malaysia (chew et al., ; chan et al., ; khor et al., ) , have shown negative associations between temperature and rsv, while studies in hong kong (chan et al., ) , mexico (yusuf et al., ) , singapore (chew et al., ) , and taiwan (huang et al., ) have shown positive associations. we find strongly positive associations between temperature and rsv hospitalizations with a slight lead of temperature on rsv. this is evident both from the cross-wavelet transform and the regression results indicating a positive effect of the previous week's temperature on rsv. there is less uncertainty in the role of rainfall on rsv transmission in tropical areas, where the majority of work indicates that rsv generally occurs during rainy seasons (shek and lee, ) . colombia (bedoya et al., ) , the gambia (weber et al., ) , hong kong (chan et al., ) , kenya (hazlett et al., ) , malaysia (chan et al., ) , and papua new guinea (hierholzer et al., ) all show positive rsv associations with rainfall. omer et al. (omer et al., ) showed significant positive associations between rainfall and temperature in the previous days and rsv incidence in lombok, indonesia. we find similar associations, with the mean rainfall and temperature over the previous days having odds ratios for rsv of . and . , respectively. this association is plausible as the incubation period of rsv is estimated to be between and days (lessler et al., ) . detailed contact tracing studies, coupled with climatological data could refine this association. somewhat surprisingly, the cross-wavelet transform of rsv and rain showed no significant association, though areas of high power were observed in the -year and -month bands, with phase indicating rsv leading weather. however, none of the viruses studied here showed appreciable associations with rain in the cross-wavelet transform, possibly indicating the dominance of other weather effects (temperature and dew point) on virus hospitalizations. as with previous studies examining hpiv incidence, we found a predominance of hpiv ( detections) compared to , , and for hpiv , , and , respectively. typical seasonality for hpiv is the spring and early summer months in the temperate regions (fry et al., ; hall, ) , and has little to no observed seasonality in the tropics and subtropics (chew et al., ; branche and falsey, ) . we find evidence for winter peaks in hpiv hospitalizations when employing both the poisson regression model as well as the logistic regression model to estimate odds ratios, though the peaks were not statistically significant when controlling for weather. the cross-wavelet transforms reveals hpiv to vary significantly at year periodicity with temperature and dew point across the study. it also shows that hpiv hospitalizations are nearly completely out of phase with temperature and dew point throughout the study period. hpiv has been associated with low temperature and low relative humidity (miller and artenstein, ) though there is in general a paucity of data on the transmission routes of hpiv (pica and bouvier, ; meissner et al., ) . future work could explore in more depth the epidemiological relationship between hpiv and weather variables. this study is not without limitations. first, while we have nearly years of data, this is still a relatively short period to assess longterm seasonal trends, or to increase confidence in the estimates of seasonal patterns. however, the length of the analyzed time series is similar to other studies examining seasonal trends in viral respiratory pathogens in the tropics (chew et al., ; hall, ) , and gives indications for areas of future study. second, we excluded individuals with more than one virus detected. examination of changes in the seasonality of other viruses and coinfection over this period is worthy of study and is outside the scope of this paper. third, this study used hospital-based surveillance, necessarily presenting the most ill children. we take as an assumption that hospitalizations are a fraction of all transmission and severity of illness is not related to weather. finally, this study examines the influence of weather on viral hospitalizations and does not address other drivers of seasonal patterns of transmission, such as school closures (fine and clarkson, ) , differences in other social behavior such as contact rates (cook et al., ; dushoff et al., ) , susceptible recruitment through births (metcalf et al., ) , or possible seasonal changes in host immune responses (dowell et al., ) . future work examining these drivers in this setting is necessary. limitations aside, our study adds to the body of literature on seasonality of common respiratory patterns in tropical regions and will be of use when consideration of the epidemiology of these pathogens is necessary. for example the timing of influenza peaks in the mid-spring months (april, may, june) would indicate routine vaccination in the winter months would be of biggest impact. similarly, 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to rsv seasonality seasonality of absolute humidity explains seasonality of influenza-like illness in vietnam global antibiotic consumption to : an analysis of national pharmaceutical sales data antibiotic use and resistance in emerging economies: a situation analysis for viet nam global burden of childhood pneumonia and diarrhoea pneumonia: the leading killer of children the clinical spectrum of respiratory syncytial virus disease in the gambia viral pathogens associated with acute respiratory infections in central vietnamese children population based cohort study for pediatric infectious diseases research in vietnam the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus study design: bma, sf, hh, lmy; data collection: lmy, lnm, vdt; data analysis: bma, sf; writing first draft: bma, writing subsequent drafts: all authors; contributed intellectually: all authors the study was approved by institutional review boards in the national institute of hygiene and epidemiology, vietnam and the institute of tropical medicine, nagasaki university, japan. bma and hh were supported by bill & melinda gates through the global good fund. sf and lmy were partially supported by bill & melinda gates foundation (bmgf opp ). the pediatric ari surveillance study in nha trang, vietnam was supported by the japan initiative for global research network on infectious diseases (j-grid) from ministry of education, culture, sport, science & technology in japan, and japan agency for medical research and development (amed). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.ijid. . . . key: cord- -wbb h zu authors: walker, gregory j.; stelzer‐braid, sacha; shorter, caroline; honeywill, claire; wynn, matthew; willenborg, christiana; barnes, phillipa; kang, janice; pierse, nevil; crane, julian; howden‐chapman, philippa; rawlinson, william d. title: viruses associated with acute respiratory infection in a community‐based cohort of healthy new zealand children date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: wbb h zu acute respiratory infections (aris) are a major cause of morbidity among children. respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. the rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. children otherwise healthy aged to years were prospectively enrolled in the study during two consecutive winters, when aris peak in new zealand. parents completed a daily symptom diary for weeks, during which time they collected a nasal swab from the child for each clinical ari episode. a further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. all samples were tested for respiratory viruses commonly causing ari using molecular multiplex polymerase chain reaction assays. there were aris identified from children completing the study, at a rate of . per child‐month. swabs collected during an ari were positive for a respiratory virus in . % ( of ), compared with . % ( of ) of swabs collected during asymptomatic periods. the most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. all of these were significantly more likely to be detected during aris than asymptomatic periods. parent‐administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses. metapneumovirus (hmpv), human enterovirus (ev), and adenovirus (adv), are the most common viral aetiological agents associated with aris. although most respiratory viral infections are mild, they can potentially cause life-threatening illness, particularly in high-risk groups such neonates, the elderly, and those with chronic respiratory disease. advances in molecular diagnosis have made it possible to easily and quickly identify viruses in people with aris, with quantitative polymerase chain reaction (pcr) now considered by most to be the reference standard for detection of respiratory viruses over traditional methods such as viral culture. however, the clinical significance of virus detection is not always clear, as respiratory viruses are also detected in asymptomatic subjects. , further understanding of the role of viral detection in aris may improve clinical decision-making around infection control and therapy. direct comparisons between studies of respiratory virus epidemiology can be difficult for a number of reasons. climatic and social factors affect the survival and transmission of respiratory viruses, as well as host mobility and susceptibility. , this results in many viruses having characteristic seasonal patterns that vary by geographical location. reported epidemiology is also affected by study design. only one-fifth of aris result in a health care visit, were recruited for the study. this was made up of children from a previous study, while the remaining participants replied to advertising via staff email for the local district health boards, via posters and through referral from family and friends. there were of participants deemed ineligible for various reasons including that they slept in more than one bedroom, the bedroom was already heated, or they were going away for more than weeks during the study period. before commencement of the study of participants withdrew, while of participants commenced the program but were later excluded after failing to comply with study conditions. the remaining cohort for analysis was children, pcr, respectively, as previously described. sequences were compared to the ncbi nonredundant nucleotide (nr/nt) database using the basic local alignment search tool (blast) on the ncbi website (https://blast.ncbi.nlm.nih.gov/blast.cgi) and genotype assigned according to the closest identifying sequence. details of primers used in detection of respiratory viruses are included in the supplementary material (table s ). any period in which participants had a multi-day cumulative symptom score ≥ , with a single day symptom score ≥ was defined as an ari episode. the end of an ari episode was marked by three consecutive symptom-free days. a symptom score correction (daily baseline score of − ) was applied to participants who were considered to have background rhinitis. this included children who had (i) rhinitis symptoms (runny nose or sneezing) for ≥ of the study days, (ii) no more than days with no symptoms, or (iii) or more days of only rhinitis symptoms. in cases where multiple swabs were taken during a single ari episode, only the first swab taken was included in analysis. the exception to this was if a new virus was detected in subsequent swabs, or if swabs were taken > days apart. risk ratio (rr) was calculated as the proportion of samples positive for a virus with ari symptoms divided by the proportion of samples negative for a respiratory virus with ari symptoms. there were child-days of symptom observed and a total of nasal swabs collected from the participants. there were aris identified (table ) . nasal swabs were received from of of these episodes ( . %). in addition, swabs were taken when children were asymptomatic, most of which were performed during the clinical visit by researchers at the conclusion of the study. due to inclusion of children from a previous study of wheezing, those with asthma were highly represented within our cohort ( . %). overall, children with asthma displayed a small increase in the rate of aris per child-month ( . vs . ) compared with nonasthmatics (table s ), but this increase was not significant (p = . ). of the swabs taken during an ari, ( . %) were positive for a virus. during an ari, detection of respiratory viruses in nasal swabs was highest amongst the youngest age group ( year old), and decreased to age ( table ). during aris, multiple viruses were codetected in ( . %) swabs. the most commonly detected viruses in samples collected during ari were hrv ( . %), hcov ( . %), parainfluenza virus (pif) ( . %), ifv ( . %), rsv ( . %), and hmpv ( . %). these were all significantly more likely to be associated with ari episodes than asymptomatic detection (table ). adv was detected with similar frequency in both ari ( . %) and asymptomatic samples ( . %), while detection of hbov was relatively low in both groups. detection of any virus and codetection of viruses were both significantly associated with swabs collected during ari episodes. sequencing was performed on of a possible picornavirus positive samples ( . %). rhinovirus strains hrv-a and hrv-c were significantly associated with ari episodes (table ) . overall, detection of individual viruses was similar between asthmatic and nonasthmatic children in swabs taken during an ari (table s ) . hrv-b (p = . ) and rsv (p = . ) were significantly over represented in asthmatics during aris. aris were most frequent in may ( . ), which was also the month in which the least data was collected. this was followed by june ( . ) august ( . ), and july ( . ). the rate of aris steadily decreased following the winter season ( figure ). hrv was the predominant virus detected in each month, and was most prevalent in late-winter and spring (figure ). hcov and rsv had peaks during early winter, while adv peaked in spring. ifv and hmpv were most prevalent in july, but were also detected in other months. pif was detected consistently throughout study months, although not in may. there is a need for improved population data regarding respiratory viruses in children, particularly in the southern hemisphere. this is the first study assessing the epidemiology of an extended range of respiratory viruses in a community cohort of healthy children in new zealand. infection with respiratory viruses was common in both symptomatic and asymptomatic children, and this study demonstrated these relatively high frequencies were true of children with asthma ( . ) and those without asthma ( . ). the proportion of aris associated with a virus-positive swab in this population declined with age. children year of age were positive for a respiratory virus in . % of samples taken during an ari, compared to . % in participants aged greater than or equal to years age. a similarly designed household surveillance study showed that weekly detections of viruses in young (aged < ) and older (aged - ) children were significantly more frequent than in adults. in the current study swabs were taken at the onset of illness, when viral load reportedly peaks for some respiratory viruses. it is possible that this method is more likely to detect viruses associated with ari than other studies obtaining weekly swabs. sample sizes of each virus detected here were too small to compare individual viruses between age groups. however, a multi-country community surveillance study has previously shown that with the exception of influenza, prevalence of individual respiratory viruses was highest in younger children. the rates of ari per child-month were similar between age groups, suggesting that other factors such as development of allergy may play a more significant role than age in acute respiratory health. the study was conducted during the winter/spring seasons of consecutive years. consistent with previous reports in the southern hemisphere rsv peaked in this pediatric population in early winter. , interestingly, we found a similar pattern for detection of hcov. it has recently been reported in china that while overall hcov infection is most prevalent in winter, hcov subtypes display different epidemic patterns. as only a small number of hcovs were detected (n = ), we did not distinguish between subtypes when reporting our results. influenza was detected from june to october and peaked in july. a limitation was that no data were collected for the summer and autumn periods, capturing only the times of highest respiratory virus activity. this may account for the low detection of adv, hbov, and ev in comparison to other year-round epidemiological studies. a yearround analyses may also allow for investigation of phenomena such as the february back to school hrv peaks. our study differs from a previous nz-based analysis of viruses in hospitalized children with lrti, where relatively high rates of rsv ( %) and hmpv ( %) were detected. in our community-based study, incidence of these viruses was considerably lower. previous groups have reported that rsv and hmpv rarely occur in asymptomatic subjects, , , suggesting that detection of these viruses is almost always of clinical relevance. ifv may be present in people with few or no symptoms. however, the duration and copy number of viral shedding is considerably lower in these cases, and further research is needed to understand the contribution of asymptomatic detection to community transmission. in the present study, we found these viruses in very few samples of asymptomatic subjects, and demonstrated these three viruses (and others) to be significantly associated with aris. respiratory viruses were detected in a large proportion of swabs taken during asymptomatic periods ( . %), with the vast majority of these detections caused by hrv. this is unsurprising as hrvs were the most frequently detected virus overall, and have been commonly detected in asymptomatic children before. it has previously been unclear whether subtypes of hrv have varying clinical impact on their hosts, with studies of healthy, and asthmatic children reporting no differences in symptom risk between hrv subtypes. our data, along with another recent study suggest that hrv-a and hrv-c subtypes are significantly more likely to cause symptoms related to ari. in our study hrv-b, adv, and hbov were detected similarly in samples from both ari episodes and asymptomatic periods. while their corresponding risk ratios are not considered significant, the number of detections of these viruses is relatively small, and a larger analysis would be required to rule out the clinical significance of detecting hrv-b, adv and hbov in aris. the effect of viral coinfection on respiratory disease severity in children has not been well established. a number of studies have found increased hospitalizations, longer length of stay in hospital, and higher mortality when two or more respiratory viruses were detected. [ ] [ ] [ ] however, others report no association between viral coinfection and clinical outcomes , including a systematic review of such publications. in this study we found a codetection was observed in . % of total virus detections during aris, which is similar to rates reported previously. we also codetected viruses in a small number of swabs collected during asymptomatic periods, and show that codetections are significantly more likely to be associated with aris. our sample size for codetection of viruses is relatively small, and larger studies have demonstrated a more significant association. respiratory viruses cause significant morbidity, particularly for disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis respiratory viruses and influenza-like illness: epidemiology 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- - key: cord- -i xv o authors: bishop-williams, katherine e.; sargeant, jan m.; berrang-ford, lea; edge, victoria l.; cunsolo, ashlee; harper, sherilee l. title: a protocol for a systematic literature review: comparing the impact of seasonal and meteorological parameters on acute respiratory infections in indigenous and non-indigenous peoples date: - - journal: syst rev doi: . /s - - -x sha: doc_id: cord_uid: i xv o background: acute respiratory infections (ari) are a leading cause of morbidity and mortality globally, and are often linked to seasonal and/or meteorological conditions. globally, indigenous peoples may experience a different burden of ari compared to non-indigenous peoples. this protocol outlines our process for conducting a systematic review to investigate whether associations between ari and seasonal or meteorological parameters differ between indigenous and non-indigenous groups residing in the same geographical region. methodology: a search string will be used to search pubmed(®), cab abstracts/cab direct(©), and science citation index(®) aggregator databases. articles will be screened using inclusion/exclusion criteria applied first at the title and abstract level, and then at the full article level by two independent reviewers. articles maintained after full article screening will undergo risk of bias assessment and data will be extracted. heterogeneity tests, meta-analysis, and forest and funnel plots will be used to synthesize the results of eligible studies. discussion and registration: this protocol paper describes our systematic review methods to identify and analyze relevant ari, season, and meteorological literature with robust reporting. the results are intended to improve our understanding of potential associations between seasonal and meteorological parameters and ari and, if identified, whether this association varies by place, population, or other characteristics. the protocol is registered in the prospero database (# ). electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. acute respiratory infections (ari) contribute to a substantial global burden of morbidity and mortality [ ] [ ] [ ] . an estimated . million children were hospitalized for ari in , of which , died [ ] . defined as an acute infection with coughing as a symptom [ ] , ari is often associated with meteorological parameters [ ] and commonly varies by season [ ] . for instance, ari is commonly associated with temperature parameters, with increasing incidence during cold periods as a result of an individual's exposure, susceptibility, and the infection type [ ] . furthermore, seasonal associations with ari also have been identified; in some cases, these seasonal associations have been attributed to varying meteorological parameters and in other cases attributed to the pathogen's own rhythmicity [ ] . the associations between ari and meteorological parameters and season may be modified by social gradients of health [ ] [ ] [ ] and affected by type of livelihood [ , ] . for instance, among indigenous peoples, a strong connection to the land [ ] [ ] [ ] , resource-based livelihoods [ , ] , interacting social determinants of health (i.e., housing, education) [ , , ] , and the legacies of colonization [ ] may modify the association between infections and meteorological and seasonal parameters [ ] . therefore, it is possible that there are differences in the association between weather variables and ari among indigenous and non-indigenous communities. it is important to know if these associations differ between indigenous and non-indigenous communities to aid in better planning, resource allocation, and intervention strategies. this paper outlines a protocol for conducting a systematic review to investigate whether associations between ari and seasonal or meteorological parameters differ between indigenous and non-indigenous groups residing in the same geographical region. this protocol, which outlines methods for the proposed systematic review, was designed in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) guidelines [ ] . the protocol is registered in the prospero database (# ). the items of this protocol are presented in accordance with the prisma-p checklist (additional file ) [ ] . this systematic review protocol outlines the procedures for a systematic literature review that is intended to answer the question: is the association between seasonal or meteorological parameters and ari the same in indigenous and non-indigenous peoples who live in the same geographical region? the components of population, exposure, comparator, and outcome (peco) are as follows: population: communities with indigenous and non-indigenous community members; exposure: indigeneity; comparator: non-indigenous; and outcome: association between seasonal or meteorological parameters and ari. all primary epidemiological observational study designs (i.e., cross-sectional, cohort, case-control studies) are eligible for inclusion (table ) . ecological studies will be eligible, as the population in a singular location should be equally exposed to seasonal or meteorological parameters. experimental studies (i.e., intervention studies) will not be eligible, as the exposure (i.e., indigeneity) cannot be assigned. further, reviews, commentaries, editorials, mathematical models, or other non-primary research articles will not be eligible, as these studies are not comparable with observational results. eligible study populations are those in which a portion of the population living in the same region is explicitly defined as indigenous and a portion of the population is explicitly defined as non-indigenous. this research builds from the united nations declaration of the rights of indigenous peoples [ ] understanding of the term indigenous peoples, which states that an indigenous person self-identifies as indigenous; has historical continuity table inclusion and exclusion criteria for a systematic literature review investigating the impact of seasonal and meteorological parameters on acute respiratory infection (ari) in indigenous and non-indigenous peoples the study is a primary epidemiological observational study design (cross-sectional, case-control, cohort, or ecological studies) the study is an experiment (i.e., interventional), review, commentary, editorial, mathematical model, or other non-primary research the research report exceeds words the research report is less than words the study's population includes both indigenous and non-indigenous peoples the study's population includes only indigenous or only non-indigenous peoples the study's outcome is the association between ari and seasonal or meteorological parameters the study's outcome is something other than the association between ari and seasonal or meteorological parameters the study provides sufficient epidemiological data to investigate the question of interest (i.e., two models representing the indigenous and non-indigenous strata separately with the same seasonal or meteorological parameter in both models, producing two measures of association for which the ratio of odds ratios (ror) can be calculated, or (ii) a model for an ari outcome that includes indigeneity and seasonal or meteorological parameter(s) as independent variables from which further calculations can be made) the study provides insufficient information to investigate the question of interest the study presents unique results which have not been previously published, or is the most recent and comprehensive analysis of the data. the study which duplicates the results of a previous study ari acute respiratory infection with pre-colonial society; has a strong link to territory and natural resources; has a distinct social, economic, or political system; has a distinct language, culture, and/or belief system; forms a non-dominant societal group; and/or resolves to maintain and reproduce their ancestral environments and systems as distinctive peoples and societies. indigeneity is the only eligible exposure. the comparator group is a non-indigenous population living in the same region as the indigenous population. all eligible studies will present the association between ari and weather variables. eligible studies must investigate this association among both indigenous and non-indigenous peoples living in the same region. the results can be presented in one of two eligible ways: (i) two models representing the indigenous and non-indigenous strata separately with the same seasonal or meteorological parameter in both models, producing two measures of association for which the ratio of odds ratios (ror) can be calculated (i.e., ari-exposure model for indigenous peoples and ari-exposure model for non-indigenous peoples); or (ii) a model for an ari outcome that includes indigeneity and seasonal or meteorological parameter(s) as independent variables from which further calculations can be made (fig. ) . the case definition for ari is an acute infection (i.e., less than days duration, if duration is stated) with coughing as a primary symptom, with or without any accompanying symptoms. if duration is not provided in the case definition, the word "acute" will be sufficient for inclusion. the majority of ari outcomes are anticipated to be respiratory syncytial virus, influenza, or pneumonia; however, studies reporting any ari outcome are eligible. a diagnosis of ari may include any symptomatic description that meets the case definition, a positive biological sample (e.g., swab), clinical diagnosis from a practitioner, or self-reported illness. studies of non-infectious respiratory outcomes (e.g., asthma) are not eligible. seasonal variables refers to a pattern (i.e., season) in meteorological parameters related to a predictable trend such as temperature, hours of sunlight, or total precipitation that is repeated annually [ ] . meteorological parameters are defined as observable weather events, at a fig. options for presentation of results in eligible studies, where option represents a study presenting two models and option represents a study presenting one model. e+ study exposure positive, estudy exposure negative, se standard error, β coefficient point in time, primarily consisting of temperature, precipitation, barometric pressure, humidity, sunlight, and the interactions and variability of these parameters [ ] . in this review, any study that has seasonal or meteorological parameters measured at two or more points in time, and where the points are meteorologically or seasonally contrasting (i.e., measured in two different dates or in two different seasons), will be eligible. the search strategy comprises three main components: indigenous communities (population and exposure terms); and association between seasonal or meteorological parameters and ari (outcome terms; table ). terms that will be used to identify indigenous peoples globally are based on a series of umbrella terms for indigenous used globally and throughout time, adapted from bartlett et al. [ ] . individual group names were added to the umbrella terms from two sources. first, the international work group for indigenous affairs (iwgia; www.iwgia.org) registry provides a continental directory of indigenous peoples, further sorted as a country-bycountry list of recognized indigenous groups. these terms may be at the greater population level, rather than individual group level if the category can be expected to represent and include all of the unique peoples groups within it (i.e., the name "maori" was included in the search terms, but individual maori group names, such as ngati kuri, ngati maru, and to arawa, were not included). secondly, the united nations refugee agency (unhcr) provides a country-by-country database of minority and indigenous peoples (www.refworld.org). since this list provides both indigenous and non-indigenous peoples, only those groups explicitly listed as indigenous were collected. the names of all identified groups defined as indigenous were added to the search. when the two lists were complete, the lists were merged into one, alphabetized, and de-duplicated. the list of terms for indigenous peoples is comprehensive to the best of the ability of this strategy. searches of mesh terms for "season," "meteorology," and "weather" were performed in pubmed ® and used to compile terms for the search strategy. terms that will be used to identify ari outcomes in the literature include any pathogen known to primarily cause ari (i.e., enteric pathogens that rarely cause ari are not included) based on the medical microbiology th edition, chapter , online version [ ] . any terms used for ari by the lung disease alphabetical listing generated by the american lung association (www.lung.org) were added to the search strategy. a search of mesh terms for "respiratory" and "lung infection" was performed in pubmed ® and any additional terms were added to the search strategy. it is difficult to develop a search strategy that is robust enough to represent all nuances of the terms indigenous, seasonal or meteorological parameters, and ari, and thus, the use of multiple databases is intended to increase sensitivity of the search. this review will search the following databases: pubmed ® (via ovidsp ® ), cab abstracts/cab direct © , and science citation index ® (via web of knowledge™). the search string will be appropriately adapted for each of the selected databases (tables , , and ). a university librarian was consulted in preparation of the search strategy for pubmed ® . the search will not be limited by language, date, or study design. search terms will be in english, although the names of indigenous groups are commonly stated in their own languages (i.e., non-english names in the roman alphabet syntax will be used). an english search string should identify all english articles and any non-english articles with an english title and abstract. if a non-english citation is collected by the search, google translate © will be used to translate the title and abstract for initial screening [ ] , and if maintained for full article screening, google translate © will be used for full text screening. this will allow calculation of the total number of eligible articles to generate appropriate denominators. if after full article screening a non-english article is eligible, the article will be formally translated by a paid service, if funding is available. if it is not possible to translate non-english articles, they will be excluded from data extraction and risk of bias (i.e., after full-text screening). to minimize the risk of exclusion of relevant citations, the citation list of each included study will be searched (i.e., a snowball search). additionally, google scholar © will be used to complete a citation search on eligible studies, to identify studies that have referenced these studies. studies identified by either the hand-searches or citation searches will be screened for relevance. published and unpublished literature will be eligible. published literature can be collected by all of the proposed databases. unpublished literature can be collected by cab abstract/cab direct © and science citation index ® . unpublished primary research that exceeds words in length will be eligible if it meets one of the three following criteria: (i) governmental report (i.e., produced by a regional or national government ministry); (ii) non-governmental report (i.e., produced by nongovernmental organizations); or (iii) graduate or honor undergraduate thesis or dissertation. reports that are fewer than words will be excluded. if the initial search identifies any relevant government or non-governmental reports, or theses, a search will be conducted in pubmed ® to identify any relevant journal publications by the first author of the citation. this pubmed ® search will include the first author's last name "acute chest syndrome" or "acute respiratory distress syndrome" or "ards" or "bacterial pneumonia" or "black lung disease" or bronchitis or bronchiectasis or bronchiolitis or "bronchiolitis obliterans organizing pneumonia" or "boop" or "bronchopulmonary dysplasia" or bronchopneumonia or byssinosis or "chest infection*" or congest* or coccidioidomycosis or cocci or cough* or "cryptogenic organizing pneumonia" or "flu" or "hantavirus pulmonary syndrome" or histoplasmosis or "human metapneumovirus" or "hypersensitivity pneumonitis" or influenza* or "lower respiratory tract infection" or "lrti" or "lrtis" or "lung inflammation" or "mers" or "middle eastern respiratory syndrome" or "mycoplasma" or "non-tuberculosis mycobacterium" or pertussis or pleuropneumonia or pneumonconiosis or pneumocystis or and first initial, institutional affiliation, and one to three keywords from the abstract (i.e., author identified key words if available, or reviewer key words if not available). to keep the review current, if more than months pass from the date the search was conducted to completion of data extraction and analysis, an update search will be conducted. if conducted, the second search will use the same search strategy as the first and will not be restricted by date. the search will be conducted in all of the original databases. thus, a recall strategy is employed [ ] , which should identify all of the initial studies and all studies published since the previous search. a hand-matching method will be used to identify whether all of the original citations are included. all search result citations will be loaded into and managed in endnote™ bibliographic software and deduplicated automatically. then, citations will be uploaded from endnote™ into distillersr ® , which will be used for form generation, screening, and management of relevant screening level statistics. screening will be completed in two stages. screening processes will be piloted and tested by the reviewers on a subset of studies ( % of studies if n > , % of studies if n ≤ ). first, title and abstract screening will be conducted on all citations identified. two reviewers with graduate-level training in epidemiology and systematic literature review processes will screen articles independently, using five evaluation questions ( table ). all questions will be answered as "yes," "no," or "unsure." in this screening phase, questions will be hidden. a hidden question will not be answered if the article is excluded based on previous screening questions. articles will be excluded if both reviewers answer "no" to any of the five questions. if both reviewers answer "yes" and/or "unsure" to all questions it will be maintained for full article screening. any disagreements will be resolved by consensus. when consensus cannot be reached, a third reviewer will arbitrate. second, full article screening will be conducted on all citations remaining after title and abstract screening. two reviewers will screen articles independently, using a second form in distillersr ® . reviewers will use seven evaluation questions (table ). in full article screening, reviewers will identify any studies using a duplicate dataset and will maintain the research that is most comprehensive. duplicate results will be removed. questions will not be hidden in full article screening, allowing for analysis of the reason for exclusion. studies will be excluded if both reviewers answer "no" to any question. disagreements between reviewers will be resolved by consensus and, when consensus cannot be reached, a third reviewer will arbitrate. data extraction will include study identifiers and study design; participant, exposure, and outcome information; and information about analytical methods. missing information will be noted. extracted study identifiers will be the authors' names; study title; publication type; publication date; journal, volume, issue, and page numbers of publication; place of publication (i.e., first author's institutional address); and digital object identifier. study design, time frame of study, climate zone of interest, location of study (i.e., country), and region of study (localized when reported) will also be extracted. data extracted about participants will include the definition of the target and source populations, size of the target population, and size of the source population. relevant demographic information (e.g., age, sex) at the study population level will be extracted when reported. exposure related data extracted will include the name of the indigenous population, the size of the indigenous source population, and the size of the indigenous study population. for the comparator, the size of the non-indigenous source population, and the size of the non-indigenous study population will be extracted. for all studies, the definition provided for indigenous peoples will be extracted. further, if causal mechanisms for differences in seasonal or meteorological effects on ari are provided, these will be extracted. information related to the seasonal or meteorological parameter(s) of interest, as well as the ari outcome(s) of interest will be extracted. in addition, the association(s) between ari and season or meteorological parameters will be extracted. for season and meteorological parameters, extracted information will include the name of each parameter (e.g., rainfall) and its related measure (e.g., millimeters); type of temporal pneumon* or pneumophila or "pneumocystis carnii" or pulmonary or respiratory or "respiratory distress" or "respiratory distress syndrome" or "respiratory tract infection*" or "rti" or "rsv" or "respiratory syncytial virus" or sarcoidosis or "severe acute respiratory syndrome" or streptococcus or "tuberculosis" or "tb" or "upper respiratory tract infection*" or "urti" or "urtis" or wheez* or "viral pneumonia" all terms searched as "all fields" terms. collected citations on september , exposure "atmospheric pressure" or barometric or cloud* or cold or "dew point" or heat* or humidity or meteorolog* or precipit* or rain* or season* or snow* or storm* or sunshine or temperature* or "uv" or "uv index" or "ultraviolet radiation" or vapor or warming or weather or wind or winds or windy cycle (e.g., daily, seasonal, annual); and number of cycles completed (e.g., years). additionally, the source of data used to evaluate the season or meteorological parameter will be collected (e.g., meteorological stations). the extracted information for each ari outcome will be the specific ari outcome (i.e., case definition), measurement of the ari outcome (e.g., self-reported), group-level metric for each population group (e.g., prevalence) and the effect size (i.e., beta) comparing the indigenous and non-indigenous peoples or strata (e.g., odds ratio). where two models are presented (e.g., ari-exposure models for indigenous and non-indigenous peoples), odds ratios will be extracted for each strata (fig. ). where one model is presented (e.g., a model with indigeneity and weather parameter(s) as fixed effects), the regression coefficients for the season or meteorological parameter and indigeneity will be extracted. in the case that a study presents results for both options (i.e., two strata models and a single model with fixed effects), data will be extracted for both options. for each association, the measure(s) of precision (e.g., standard error of the mean, standard deviation, and/or confidence intervals) will be extracted when provided. if only the p value and sample size are reported, these data will be extracted and a measure of precision will be calculated from the available data for each association. finally, information will be extracted on the type of modeling or statistical approach (e.g., linear regression) used, and if and which confounders were considered. confounders considered will be extracted and a list will be generated for various ari outcomes. additionally, the unit of analysis (e.g., individual, household, or community) and spatial resolution of the climate data used for modeling will be extracted. a data extraction form will be created in distillersr ® . the extraction form will be piloted and tested by the data extractors on a subset of studies ( % of studies if n > , % of studies if n ≤ ). following pilot testing, the form will be adapted as recommended by the extractors to improve usability and completeness. the first author and one additional extractor who each have training in epidemiology and systematic literature review processes will complete data extraction. data extraction will be completed independently and the extractors will compare the data for consensus. if the extractors cannot answer a question, consensus will confirm that the data are unavailable to answer the question. in the event that the data presented in a study are unclear, missing, or presented in a non-extractable or "acute chest syndrome" or "acute respiratory distress syndrome" or "ards" or "bacterial pneumonia" or "black lung disease" or bronchitis or bronchiectasis or bronchiolitis or "bronchiolitis obliterans organizing pneumonia" or "boop" or "bronchopulmonary dysplasia" or bronchopneumonia or byssinosis or "chest infection*" or congest* or coccidioidomycosis or cocci or cough* or "cryptogenic organizing pneumonia" or "flu" or "hantavirus pulmonary syndrome" or histoplasmosis or "human metapneumovirus" or "hypersensitivity pneumonitis" or influenza* or "lower respiratory tract infection" or "lrti" or "lrtis" or "lung inflammation" or "mers" or "middle eastern respiratory syndrome" or "mycoplasma" or "non-tuberculosis mycobacterium" or pertussis or pleuropneumonia or pneumonconiosis or pneumocystis or pneumon* or pneumophila or "pneumocystis carnii" or pulmonary or respiratory or "respiratory distress" or "respiratory distress syndrome" or "respiratory tract infection*" or "rti" or "rsv" or "respiratory syncytial virus" or sarcoidosis or "severe acute respiratory syndrome" or streptococcus or "tuberculosis" or "tb" or "upper respiratory tract infection*" or "urti" or "urtis" or wheez* or "viral pneumonia" all terms searched as "topic" terms. collected citations on september , bronchitis or bronchiectasis or bronchiolitis or "chest infection*" or congest* or cough* or "flu" or influenza* or "lung inflammation" or "middle eastern respiratory syndrome" or "mycoplasma" or pertussis or pneumon* or pulmonary or respiratory or "respiratory syncytial virus" or streptococcus or wheez* removed all acronyms, maintained symptoms and key diseases (more generic names only) all terms searched as "title" or "abstract" or "subject." collected citations on september , unusable form, authors of studies published in the last years (since january , ) will be contacted for clarification. authors will be contacted via email, and a follow-up email will be sent weeks later. authors will be provided weeks from the initial contact to respond. if data from older studies are unclear, missing, or presented in a non-extractable or unusable form, authors will not be contacted. missing data will be noted in the report. the risk of bias (rob) assessment was adjusted from existing tools (i.e., risk of bias in non-randomized studies of interventions) [ ] . in particular, adjustments were needed to account for ecological studies and repeated measures. one question was added to investigate rob due to ecological studies and one question was added to investigate rob due to repeated measures. questions related to experimental unclear: it is unclear if the study design is primary research from the title and abstract. publication type: does the title and abstract come from a published study, government report, nongovernmental report, or postsecondary institutional thesis (exceeding words in length)? yes: the study is a published study, or government report, nongovernmental report, or postsecondary institutional thesis (exceeding words in length). no: the study is not a published study, government report, nongovernmental report, or postsecondary institutional thesis or is fewer than words in length. unclear: it is unclear if the study is a published study, or government report, non-governmental report, or post-secondary institutional thesis (exceeding words in length) from the title and abstract. population: does the population of interest include both an indigenous population and non-indigenous population living in the same geographical region? yes: the study population describes both indigenous and non-indigenous peoples living in the same geographical region. no: the population of interest is entirely indigenous or non-indigenous or the populations are not within the same region. unclear: it is unclear if the population of interest is both indigenous and non-indigenous from the title and abstract. outcome: does the title and/or abstract describe research on the association between ari and any exposure? yes: one or all of the outcomes of interest in the study are ari, defined by the case definition for this review (i.e. acute infection (less than days duration, if duration is stated) with coughing as symptom, with or without any accompanying symptoms). no: there is no ari-related health outcome measured in the study. unclear: it is unclear if the health outcome of interest is ari from the title and abstract. exposure: is the study's exposure of interest a seasonal or meteorological factor (defined as observable weather events, primarily consisting of temperature, precipitation, barometric pressure, humidity, sunlight, and the interactions and temporal variability of these parameters)? yes: the exposure of interest is a seasonal or meteorological factor. no: the exposure of interest is not a seasonal or meteorological factor. unclear: it is unclear if the exposure of interest is a seasonal or meteorological factor. yes: the research compares associations between seasonal or meteorological parameters and ari in the context of indigeneity. no: the research does not compare associations between seasonal or meteorological parameters and ari in the context of indigeneity. duplicates: does the research use a new dataset for analysis (i.e., the dataset was not previously analyzed in another included study). yes: the research uses a new dataset for analysis. no: the research uses a dataset that has been previously analyzed by another included study. (note: when identifying the study to be included, the most comprehensive study will be used.) full article screening questions will include all screening domains and questions from title and abstract screening as well as two additional questions interventions were removed. two reviewers will conduct the rob assessment independently, in conjunction with data extraction, at the study level (i.e., one rob analysis will be conducted per study). both reviewers conducting the rob assessment will have advanced graduate training in epidemiology and bias assessment. in total, nine domains of bias will be tested according to predetermined criteria for high, low, or unclear rob. the rob will be conducted using a form in distillersr ® with a textbox to record the rationale for selecting the level of rob for each domain. since this review will focus on the association between weather parameters and ari outcomes, confounders in this study are those that affect the association between these weather parameters and ari in indigenous versus non-indigenous peoples. important confounders that could affect all or most studies are: (i) gender, (ii) age, and (iii) local wealth (e.g., regional or national). the review will analyze information for both associations (systematic review and meta-analyses) and context (e.g., via descriptive statistics, narrative, and descriptive spatial analyses). this approach is necessary to avoid comparing unlike populations, exposures, or outcomes. analyses will be completed using stata © version . and revman © version . prior to beginning meta-analyses, descriptive statistics will be conducted on extracted data. frequencies, proportions, and missing data will be considered for each extracted variable. the descriptive data will serve to describe the literature available on this topic and to represent the dataset under study. meta-analyses will be conducted for each ari association (i.e., each ari outcome and weather parameter identified) that has at least two studies providing data (i.e., two studies presenting the association between the same ari outcome and same weather parameter). the outcome used for meta-analyses will be the ror representing the relative effects of weather parameters on ari between indigenous and non-indigenous peoples (fig. ) . for studies presenting two models (i.e., ari associations for each strata), odds ratios for each eligible study for each group will be extracted (i.e., indigenous, non-indigenous) to calculate a ror. in studies presenting a single model, regression coefficients will be used directly to calculate odds ratios and solve for the ror (fig. ) . the standard error of the ror will be calculated according to golder et al. [ ] for both study types. meta-analyses will be conducted using random effects models. between-study heterogeneity will be explored using the i statistic (i < . considered homogeneous). if heterogeneity exists, sources of heterogeneity will be explored by sub-group analyses. for eligible studies, we propose to categorize the studies by (i) population, (ii) outcome, (iii) exposure, and (iv) location. studies will be categorized by groups of indigenous peoples (e.g., all studies of maori peoples) for population; as upper respiratory, lower respiratory, or unclear/both for outcome; as seasonal or meteorological parameters for exposure; and as a high-, middle-, or low-income country and by climate zone for location. when there are at least two effect sizes for each category, we will calculate a summary effect size. descriptive statistics will be conducted on each of the domains of the rob. if enough data are available and rob profiles vary, heterogeneity will be explored using sub-group analyses on each domain as an independent variable. publication bias is the concept that significant results are more likely to be published than non-significant research results [ ] . an evaluation of publication bias will be conducted using a funnel plot (if n > studies). ratio measures of association will be plotted on the logarithmic scale to increase symmetry. a % confidence interval will be plotted, and different symbols will be used if heterogeneity is present. interpretation of the funnel plot will be done visually. a test of significance for publication bias will be conducted using egger's test [ ] . finally, to summarize data about region of study and climate zone, descriptive spatial analyses will be performed. specifically, point maps will be generated. the point map will illustrate specific study locations. when point locations are not provided in text with latitudinal and longitudinal coordinates, study sites will be geolocated using google maps © and the best information available in the study. the point map will use open source shape files and will be built in r spatial © and r maptools © statistical packages. the final search strategy for each database and all ancillary searches conducted will be provided in the additional file of the final report. a flow chart, following the prisma guidelines [ ] , will be used to illustrate where citations were eliminated during screening and ancillary searches, including information about the rationale for exclusion in full article screening. to illustrate the potential for publication bias and small study effects, a funnel plot will plot the effect estimates (horizontal axes) against the standard error (vertical axes) for each meta-analysis with n > studies [ ] . the results of this review will be provided via text and characteristics of studies tables in a published journal article. the tables will describe the seasonal or meteorological exposure, the outcome(s) measured, the direction and magnitude of association, and the period of study. descriptive statistics (i.e., frequencies, proportions, missing data) will be provided as extensions of this table when appropriate or in narrative (i.e., proportion of studies with each ari outcome). individual study results will be presented in forest plots [ ] . if heterogeneity exists, separate forest plots will be used to illustrate results by strata. if the data are too limited (i.e., fewer than two studies with the same population, exposure, outcome, and region) or are heterogeneous, the results will be presented in a forest plot without a summary effect size. a summary of findings table for key outcomes will be generated based on the grading of recommendations assessment, development and evaluation [ ] . a priori key outcomes will include prevalence of upper ari and prevalence of lower ari. additional key outcomes identified in the systematic literature review will be documented as protocol amendments. a table will be provided to summarize the findings of the rob assessment. this table will follow the rob presentation suggested in the prisma guidelines [ ] . an additional column will highlight the rationale for the study's rob level. maps indicating the specific location of studies (point map) will be generated. climate zones will be indicated on each map (e.g., tropical, temperate, or arctic). this research does not involve working directly with indigenous and non-indigenous communities, but rather with previous research conducted with these communities. in conducting this research, ethical principles will still be at the forefront, and will involve considerations for small population sizes and framing of the findings. this systematic review protocol presents the method for the synthesis of current evidence related to differences in seasonal or meteorological association with ari between indigenous and non-indigenous peoples living in the same region. this proposed review will likely be the first to summarize the potentially different associations between ari and weather parameters between indigenous and non-indigenous peoples. the results of the meta-analysis will examine whether indigenous peoples are equally susceptible to associations between weather parameters and ari, and whether this relationship varies by place, population, or other characteristics. a deeper understanding of this relationship will advance the academic literature and potentially lead to intervention strategies as climate change progresses. further, an understanding of the differences between indigenous and non-indigenous communities can aid in planning, resource allocation, and determination of appropriate interventions. global burden of acute respiratory infections in children: implications for interventions global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in : a systematic analysis the respiratory health of urban indigenous children aged less than years: study protocol for a prospective cohort study what is the seasonal distribution of community acquired pneumonia over time? a systematic review are meteorological parameters associated with acute respiratory tract infections? urban as a determinant of health social status and susceptibility to respiratory infections indigenous health and climate change seasonal pattern of pneumonia mortality among under-five children in nairobi's informal settlements the land enriches the soul: on climatic and environmental change, affect, and emotional health and well-being in rigolet from this place and of this place:" climate change, sense of place, and health in nunatsiavut united nations: indigenous peoples, indigenous voices factsheet. edited by issues upfoi vulnerability of indigenous health to climate change: a case study of uganda's batwa pygmies health of indigenous people in africa social determinants of health inequalities preferred reporting items for systematic reviews and meta-analyses: the prisma statement preferred reporting items for systematic review and metaanalysis protocols (prisma-p) statement seasonality in six enterically transmitted diseases and ambient temperature descriptive meteorology identifying indigenous peoples for health research in a global context: a review of perspectives and challenges infections of the respiratory system international burden of chronic kidney disease and secondary hyperparathyroidism: a systematic review of the literature and available data precision and recall of search strategies for identifying studies on return-to-work in medline the risk of bias in nonrandomized studies -of interventions (robins-i) assessment tool meta-analyses of adverse effects data derived from randomised controlled trials as compared to observational studies: methodological overview trim and fill: a simple funnel-plot-based method of testing and adjusting for publication bias in meta-analysis bias in meta-analysis detected by a simple, graphical test the cochrane collaboration's tool for assessing risk of bias in randomised trials grading quality of evidence and strength of recommendations the authors wish to thank ali versluis, university of guelph library, for her time and expertise contributed to this protocol. funding for this protocol was provided through scholarships from the ontario veterinary college (k. bishop-williams) and the ontario graduate scholarship program (k. bishop-williams).availability of data and materials not applicable.authors' contributions kbw will serve as the first author of the protocol and review paper. she led all stages of protocol development, including development of the research question and objectives, search strategy, and extraction and analysis plans. jms, lbf, vle, ac, and slh supervised and contributed to the entirety of the development of plans for searching, screening, extracting, and writing phases. all authors have graduate training in epidemiology and/or health studies. all authors read and approved the final manuscript. the authors declare that they have no competing interests. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -hcg rrhl authors: feikin, daniel r.; njenga, m. kariuki; bigogo, godfrey; aura, barrack; aol, george; audi, allan; jagero, geoffrey; muluare, peter ochieng; gikunju, stella; nderitu, leonard; balish, amanda; winchell, jonas; schneider, eileen; erdman, dean; oberste, m. steven; katz, mark a.; breiman, robert f. title: etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya, – date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: hcg rrhl background: few comprehensive data exist on disease incidence for specific etiologies of acute respiratory illness (ari) in older children and adults in africa. methodology/principal findings: from march , , to february , , among a surveillance population of , persons > years old in rural western kenya, we collected blood for culture and malaria smears, nasopharyngeal and oropharyngeal swabs for quantitative real-time pcr for ten viruses and three atypical bacteria, and urine for pneumococcal antigen testing on outpatients and inpatients meeting a ari case definition (cough or difficulty breathing or chest pain and temperature > . °c or oxygen saturation < % or hospitalization). we also collected swabs from asymptomatic controls, from which we calculated pathogen-attributable fractions, adjusting for age, season, and hiv-status, in logistic regression. we calculated incidence by pathogen, adjusting for health-seeking for ari and pathogen-attributable fractions. among , ari patients > years old (adjusted annual incidence . per person-years), influenza a virus was the most common virus ( % overall; % inpatients, % outpatients) and streptococcus pneumoniae was the most common bacteria ( % overall; % inpatients, % outpatients), yielding annual incidences of . and . episodes per person-years, respectively. influenza a virus, influenza b virus, respiratory syncytial virus (rsv) and human metapneumovirus were more prevalent in swabs among cases ( %, %, % and %, respectively) than controls. adenovirus, parainfluenza viruses, rhinovirus/enterovirus, parechovirus, and mycoplasma pneumoniae were not more prevalent among cases than controls. pneumococcus and non-typhi salmonella were more prevalent among hiv-infected adults, but prevalence of viruses was similar among hiv-infected and hiv-negative individuals. ari incidence was highest during peak malaria season. conclusions/signficance: vaccination against influenza and pneumococcus (by potential herd immunity from childhood vaccination or of hiv-infected adults) might prevent much of the substantial ari incidence among persons > years old in similar rural african settings. compared with other regions, the mortality rate among older children and adults remains several-fold higher in sub-saharan africa, where acute respiratory infections (ari) are a leading cause of this high mortality, as well as associated morbidity [ ] . however, data on etiologies and rates of ari among persons $ years old in africa have been principally focused on few geographic areas and pathogens (e.g., pneumococcus, tuberculosis). few studies have comprehensively examined the etiologies of ari among africans $ years of age. more importantly, there is even less available data on disease incidence for specific etiologies, which can inform public health policies regarding treatment and prevention. from population-based surveillance in rural western kenya undertaken from - , we report bacterial and viral etiologies of ari by age group, hospitalization status, hivinfection status and season. we also provide incidence by etiology, adjusted for health-care seeking and presence of pathogens in asymptomatic controls. written informed consent was obtained for data and specimen collection at the clinics and households. for children , years of age, written informed consent was obtained from parents or guardians for specimen collection. for minors aged - years of age, written informed consent was obtained from parents or guardians and written assent from the minor him/herself for specimen collection. the protocol and consent forms were reviewed and approved by the institutional review boards of kemri (# ) and cdc (# ). cdc's international emerging infections program and the kenya medical research institute (kemri) have conducted population-based, infectious disease surveillance since late in asembo, nyanza province, in rural western kenya [ , ] . all households in villages within kilometers of the referral facility, st. elizabeth lwak mission hospital (lwak hospital), were offered enrollment - % were enrolled. the surveillance population on july , , included , persons $ years old, who had resided permanently in the area for at least calendar months [ , ] . enrollment was continuous since the project's beginning. malaria transmission was holoendemic [ , ] . hiv prevalence was high ( % in adults $ years in ) [ ] . from march , , to february , , all enrolled participants received free medical care by kemri/cdc-trained nurses and clinical officers at lwak hospital for acute illnesses [ , ] . lwak hospital has inpatient beds and manages most hospitalizations, only referring complicated patients. no chest radiographs were taken during the study period. ari was defined, using a variation on the definition of severe acute respiratory illness suggested for influenza surveillance, as cough or difficulty breathing or chest pain and documented axillary temperature $ . o c or oxygen saturation , % or hospitalization [ , ] . for ari patients, blood, nasopharyngeal and oropharyngeal specimens using polyester-tipped swabs, and urine were collected. giemsa-stained malaria blood smears were performed on patients with history of fever or documented temperature $ . uc. community interviewers visited enrolled households every two weeks and questioned participants using a standardized questionnaire, in the local language, about all illness episodes in the past two weeks, including symptoms and health-seeking [ ] . for this analysis, we defined ari from the household visits as cough, difficulty breathing or chest pain and reported fever. the ari cases from the household visit were only used to assess healthseeking patterns in the area and not directly included in the incidence calculations. no specimens were collected at household visits. from january , , asymptomatic controls were enrolled from lwak hospital. eligible controls were those who presented with non-severe illness (i.e., not requiring hospitalization), for immunizations, or for medicine refills. eligible controls could not have had fever, any respiratory symptoms or diarrhea in the past two weeks. each month we attempted to enroll a targeted number of controls, frequency-matched to cases by age and hiv status. the monthly target was controls aged $ years old (six hivinfected), yielding power to detect a significant difference in detection of a pathogen between % of cases and % of controls. nasopharyngeal and oropharyngeal swabs were collected on controls. clinic nurses attempted to collect five to ten ml of blood for culture, which was inoculated into commercially-produced blood culture bottles and bacterial growth identified using standard methodology, which we have described previously (bactec tm aerobic plus tm , becton dickinson, belgium) [ , ] . naso-and oropharyngeal swabs were placed together in ml viral transport media without antibiotics and transported the same day at uc- uc to kemri/cdc laboratories near kisumu, approximately km from lwak hospital, where each specimen was divided into four aliquots and stored at uc. in monthly batches, a frozen aliquot was transported on dry ice to the kemri/cdc laboratory in nairobi, where lab technicians, blinded to case-control status, tested it after one freeze-thaw cycle, using quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr). nucleic acid was extracted from ml aliquots of each sample using qiagen's qiaamp viral rna minikit (qiagen inc, california), according to manufacturer's instructions. prior to august , taqmanh universal pcr master mix (applied biosystems, california) was used for qrt-pcr; later qrt-pcr was carried out using agpath-id tm one- step rt-pcr reagents (applied biosystems). samples were tested for the presence of adenovirus, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), influenza a and b viruses, and parainfluenza virus (piv) types - using qrt-pcr assays using previously published assays [ , ] . each clinical specimen was also tested for the human ribonuclease p gene to measure nucleic acid integrity and to confirm sample adequacy. a qrt-pcr test result was considered positive if an exponential fluorescence curve was produced that crossed the assigned threshold at c t , . [ ] . between january , , and february , , enhanced testing was done, which included testing respiratory swabs for three additional viruses (rhinovirus, enterovirus, and parechovirus) and atypical bacteria, and testing of asymptomatic controls. for testing of swabs for these additional pathogens, total nucleic acid extracts were prepared from ml of specimen per aliquot using magmax viral rna isolation kit (applied biosystems) in nairobi and transported to the viral respiratory diseases laboratory at cdc atlanta on dry ice. singleplex qrt-pcr for rhinovirus, enterovirus and parechovirus were performed using previously published methodologies [ , , ] . the rhino-and enterovirus assays targeted the noncoding region of an area of high sequence similarity between human rhinovirus and enterovirus and exhibit some cross-reactivity (s. oberste, personal communication). therefore, positive rhinovirus and/or enterovirus qrt-pcr were reported together as rhino/enterovirus positive. for atypical bacteria, multiplex qrt-pcr was performed at kemri/cdc laboratories in nairobi for mycoplasma pneumoniae, chlamydia pneumoniae and pan-legionella species using published assays [ , ] . urine specimens were tested for pneumococcal urine antigen using the binaxnowh kit (binax inc., maine) starting in may ; kits were not consistently available throughout the entire study period. sputum specimens were not collected by protocol, but only based on the clinician's suspicion of pulmonary tuberculosis, and microscopy was done at lwak hospital using ziehl-neelsen staining. hiv testing was performed as part of a home-based testing initiative during when all persons $ years in the surveillance area were offered hiv testing (two parallel rapid hiv tests), as described previously [ ] . seventy-eight percent of eligible adults agreed to be tested. we assumed that a person's hiv status during home-based testing was the same throughout the study period. hiv-testing was not performed routinely in the clinic on most patients during this period. structured questionnaires on scannable paper forms detailing the current illness were completed for all sick visits at lwak hospital. (teleformh software, cardiff tm , vista, ca). at the household visits, data were collected using personal digital assistants (pdas) [ ] . analysis was performed using sas (version . , cary, nc). proportions were compared using chi-square test or fisher's exact test, and medians by wilcoxon rank sum test. rate ratios and % confidence intervals were calculated using fisher's method (computer programs for epidemiologists, pepi, version . x) for crude rates and the delta method for adjusted rates [ ] . correlation between monthly rates of ari and percent positive for each pathogen were tested by pearson's or spearman's correlation coefficient, depending on normality of the data. we compared detection of each virus by qrt-pcr between cases and asymptomatic controls. odds ratios (or) and % confidence intervals were calculated using unconditional logistic regression, adjusting for age group, season when the swab was taken (december-february, hot and dry; march-may, long rains; june-august, cooler and dry; september-november, short rains) and hiv status (positive, negative, unknown). we used the or to calculate pathogen-attributable fractions, which estimated the proportion of cases positive for each virus in which the virus was the likely cause [ , ] . the pathogen-attributable fractions were calculated as (or- )/or; pathogen-attributable fractions were only calculated for viruses with ors that were statistically significant (p, . ). for purposes of this analysis, we assumed the pathogen-attributable fraction was . for s. pneumoniae based on the negligible probability of detection of s. pneumoniae in blood or urine of asymptomatic controls [ , ] . ari incidence was calculated as the number of ari clinic visits per person-years of observation. revisits for the same illness were not counted as separate episodes. permanent residence status in the surveillance area was used to determine person-time contribution. adjusted rates of clinic visitation were calculated accounting for the percentage of all clinic visits made for ari that went to lwak hospital as opposed to other area clinics, as determined from the household visits [ ] . etiology-specific incidence was calculated by applying the proportions of each etiology to the adjusted incidence of ari ( figure ). inpatients were over-represented among ari patients tested at lwak hospital ( % inpatients), compared to ari patients who visited a health facility in the community ( % inpatients), as determined by the household surveillance. therefore, the inpatient and outpatient etiologic results from lwak hospital for each pathogen were weighted using a : outpatient:inpatient ratio. after adjusting for the outpatient/inpatient distribution, the pathogenattributable fraction for each pathogen was applied to adjust the etiology-specific proportions. for those pathogens found to have an or that included . , no incidences were calculated because the role of the pathogen as a cause of ari was not supported by our data. during the study period, , ari patients $ years old were seen at lwak hospital, of whom , ( %) were outpatients and , ( %) were hospitalized. among ari patients, , ( %) had cough, ( %) had difficulty breathing and , ( %) had chest pain; , ( %) had documented temperature $ . uc, ( %) had oxygen saturation , % without fever (among a total of with oxygen saturation , %), and ( %) were hospitalized without documented fever or low oxygen saturation. the majority ( %) was aged - years old and only % were $ years old. fifty-eight percent were female. overall, ( %) ari patients died within days of their clinic visit ( . % among outpatients, % among inpatients). among ari patients $ years old, ( %) had hiv test results available, of whom ( %) were hiv-infected, compared to % hiv-positivity among the entire surveillance population $ years old [ ] . among , ari patients, . % reported receiving one or more antibiotics before presentation ( . % cotrimoxazole, . % penicillin or amoxicillin, . % other). epidemiologic characteristics were similar between patients who did (n = , , %) and did not (n = , , %) have blood cultures taken ( table ). the median blood volume for culture was . ml. four percent of blood cultures grew contaminants ( table ). among uncontaminated cultures, the most common bacteria isolated were streptococcus pneumoniae ( %) and non-typhi salmonella ( %). the percentage of patients with s. pneumoniae identified increased to % when adding urine antigen results to blood cultures ( % inpatients versus % outpatients, p = . ). among the patients who had s. pneumoniae detected, ( %) were positive by blood culture only, ( %) by urine antigen only, and ( %) by both tests. the case-fatality ratio was higher among those with positive blood cultures for s. pneumoniae ( %) compared with those with only urine antigen detected ( . %, p = . ). s. pneumoniae were detected more frequently among hiv-positive patients ( %) than hiv-negative patients ( %, p = . ). mycoplasma pneumonia was detected in . % of patients; no legionella or chlamydia pneumoniae were detected (table ) . among sputum samples tested by smear for mycobacterium tuberculosis, ( %) were read as positive. epidemiologic characteristics were similar between patients who did (n = , , %) and did not (n = , , %) have naso/ oropharyngeal swabs taken (table ) . overall during the time period of full viral testing, % of swabs were positive for at least virus, detection being higher among outpatients ( %) than inpatients ( %, p = . , table ). the detection of at least one virus decreased with increasing age (p, . ). the most commonly detected virus was rhino/enterovirus ( %) followed by influenza a virus ( %). detection of other viruses ranged from , % to %. influenza a virus was more often detected in outpatients ( %) than inpatients ( %, p, . ) and among those , years old ( %) than those $ years old ( %, p, . ). influenza b virus was also more common among outpatients ( %) than inpatients ( %, p, . ). all other viruses were similarly detected among inpatients and outpatients. there were no significant differences in viral detection between hivpositive and hiv-negative patients. of patients with full pathogen testing, % had . pathogen identified (table ). detecting . pathogen was more common among hiv-positive patients ( %) than hiv-negative patients ( %, p = . ), but in similar proportions among inpatients ( %) and outpatients ( %, p = . ). the coinfection prevalence among those who had influenza ( %) was lower than that among those with other pathogens ( %, p, . ). when limited to only those pathogens significantly more common in cases than controls, % of ari patients had . pathogen identified; still no differences between inpatients ( %) and outpatients ( %) existed for coinfection (p = . ). cases (n = ) had a younger age distribution than controls (n = ), with % and % aged - years old, respectively (p, . , table ). more cases ( %) than controls ( %) were enrolled in the hot, dry season (p, . ). among those with hivtesting results available, % of cases and % of controls were hiv-positive (p = . ). after adjustment for age, season, and hiv status, detection of influenza a virus, influenza b virus, rsv and hmpv in the naso/ oropharynx was associated with being a case (table ). influenza a virus and hmpv were more strongly associated with outpatients than inpatients, although the confidence intervals overlapped. in contrast, rsv was associated with being an inpatient, but not an outpatient. piv and piv were also more common among cases, but did not reach statistical significance. rhino/enteroviruses and adenovirus were equally common among cases ( % and %, respectively) and controls ( % and %, respectively). the number of ari cases tended to peak in june and july each year, with a smaller peak in october-december ( figure ). there was a trend towards increasing ari cases during the three year period (p = . , linear regression). the number of ari cases was highest in january-february when pandemic h n influenza a virus circulated widely in asembo [ ] . of the pathogens associated with case status in the case-control study, the monthly rate of ari was correlated with the percentage positive for influenza a virus (p = . ), influenza b virus (p = . ), and hmpv (p = . ), but not for s. pneumoniae and rsv. of note, when we limited the analysis to the months prior to november , when pandemic h n virus first appeared, the monthly correlation between ari case rates and viral respiratory pathogens was no longer significant. the percentage of malaria blood smears positive among ari patients ranged from ( ) ( ) ( ) ( ) - ( ) ( ) ( ) ( ) cfr a for ari cases ( ) ( ) ( ) - ( ) ( . ) ( ) ( ) blood cultures ( ) ( ) ( ) ( ) - ( ) ( ) ( ) ( ) without contaminant ( ) ( ) ( ) ( ) - ( ) ( ) ( ) ( ) s. pneumoniae ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) positive urine pneumococcal antigen c ( ) ( ) ( ) ( ) ( . ) ( ) ( ) ( ) ( ) s. pneumoniae by blood culture or urine antigen c ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) h. influenzae ( ) ( ) ( ) ( ) other pathogenic bacteria d ( . ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) viruses and atypical bacteria naso/orophangeal specimens ( ) ( ) ( ) ( ) - ( ) ( ) ( ) ( ) influenza a virus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) influenza b virus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) rsv ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) adenovirus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) parainfluenza ( ) ( ) ( ) ( ) ( ) ( ) ( ) parainfluenza virus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) human metapneumo ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) rhino/enterovirus e ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to % by month, and tended to be higher in the rainy months ( figure ). the monthly ari rate was correlated with the malaria prevalence throughout the months (p = . ), as well as in the months prior to pandemic h n (p = . ). the adjusted overall incidence of ari resulting in a clinic visit was . per person-years for persons $ years of age and . per person-years for persons $ years of age (table ) . among hiv-positive persons $ years old, the rate of ari was . per person-years, compared to . for hiv-negative persons (rr . , % ci . - . ). the highest pathogen-specific incidence among persons $ years old was influenza a virus at . per person-years, followed by pneumococcus at . per person-years ( table ). rates of all pathogens were higher among hiv-positive than hiv-negative persons. our study was unique in several respects. first, we compared etiologies in inpatient and outpatient settings, whereas most previous adult etiology studies in africa have focused on severely ill, hospitalized patients [ , , , ] . we demonstrated different etiologic predominance for the same broadly defined ari syndrome based on hospitalization status, with pneumococcus more common among inpatients and influenza viruses and hmpv among outpatients. second, we enrolled a group of asymptomatic controls. as has been noted elsewhere, a positive result from pcr is not necessarily conclusive as to etiology because of its high analytic sensitivity; pcr may detect small amounts of nucleic acid due to past or asymptomatic infections [ , , , ] . by enrolling asymptomatic controls, we were able to calculate a pathogenattributable risk, which suggested that several viruses commonly found among ari cases (i.e., rhino/enterovirus and adenovirus), were not associated with illness. third, because we embedded our etiologic study in ongoing population-based surveillance, we were able to calculate incidence of etiology-specific ari. the accuracy of our rate calculation was likely improved by adjusting for several factors that are often neglected, namely health-seeking patterns in the population and the attributable fraction of illness for each pathogen. using the calculated incidence, we estimated that approximately % and % of adults in nyanza province (approximately . million persons $ years, ), where lwak is located, will have an episode of ari from influenza viruses and pneumococcus each year, respectively, which translates to approximately , and , illnesses, respectively, in the province [ ] . as expected, pneumococcus was the most common bacterial cause of serious ari in this population, and was particularly prevalent among hiv-infected persons [ , , , , , , ] . there was more than a -fold increase in the frequency of positive results for pneumococcus when incorporating the urine antigen test, supporting previous evidence that most pneumococcal pneumonia is non-bacteremic [ , ] . non-typhi salmonella (nts) was the second most common bacterium identified in ari patients. nts bacteremia commonly presents with respiratory symptoms in children, although its role in causing pneumonia based on lung aspiration studies is doubtful [ , ] . alternatively, patients could have a mixed infection where nts bacteremia is accompanied by another pathogen causing ari, particularly among hiv-infected persons who are at increased risk for both [ , , , ] . the role of atypical bacteria as a cause of ari in african adults is unclear. in south africa, c. pneumoniae and legionella pneumophila table . coinfection among ari cases aged $ years of age, limited to those that had full testing -nasopharyngeal/oropharyngeal specimens, blood culture and urine antigen testing for pneumococcus. were detected by seroconversion in % and % of adults with pneumonia [ ] . in botswana, % of mostly hiv-infected adults had evidence for m. pneumoniae infection [ ] . apart from these studies, other studies of adults in less developed african countries showed low prevalence of atypical bacteria, a finding supported by our study [ , , , ] . influenza a virus was the most commonly detected virus. a prominent role of influenza viruses as a cause of ari in africa is becoming clear as more surveillance data becomes available [ , ] . we found influenza viruses more often in outpatients than inpatients. we have previously speculated that even when detected among inpatients with ari, influenza viruses might not be the primary reason for the admission [ ] . several other viruses besides influenza virus were associated with ari. we found rsv associated with ari among inpatients, but not outpatients. rsv has been shown to be the leading cause of hospitalized ari in african children [ ] . rsv has a less clear etiologic role among african adults. one study among south african adults did not find an excess in hospitalizations or mortality during rsv seasons [ ] . in the u.s., several studies have postulated that rsv can cause pneumonia in adults, especially those with underlying disease or the institutionalized elderly, although none of these studies evaluated asymptomatic controls [ , ] . in contrast to rsv, we found hmpv associated with ari among outpatients, but not inpatients. the only study of hmpv among adults on the african continent was in egypt and found hmpv in % of hospitalized adults with lower respiratory tract infections, but no asymptomatic controls were included [ ] . coinfection was common, with almost one-third of ari patients having . pathogen identified. coinfection could represent a true biologic phenomenon where infectious cofactors are necessary for pathogenesis. alternatively, the frequency of coinfection could reflect the fact that certain pathogens, such as rhino/enteroviruses and adenoviruses, are commonly detected in the upper airways, with unclear clinical significance. of note, ari patients with influenza viruses detected had less coinfection, which might be related to the high pathogen attributable risk of influenza viruses and the lack of need for a coinfection to cause illness. our study cannot be considered a comprehensive assessment of the etiologies of ari. additional specimens likely would have increased the yield of pathogens identified. lung aspirates have been shown to have high diagnostic yield in african children, and one study in african adults, as they sample material directly from the affected sections of the lung [ , , ] . however, lung aspirates are rarely performed in african adults with pneumonia due to the potential risk of pneumothorax in populations with high risk of pneumocystis jiroveci pneumonia (pcp). bronchoscopy with bronchoalveolar lavage has been used in a few hospitals in africa, and has improved diagnostic yield, although its use is usually reserved for severely ill, hypoxic patients in tertiary hospital settings [ , , , , ] . while good-quality sputum specimens might have a role in diagnosis of some etiologies, if strict criteria for assigning a predominant organism are used, sputum is generally considered too prone to contamination with upper respiratory tract secretions to be useful diagnostically [ , , , ] . lack of these additional specimens limited our detection of pneumocystis jiroveci and mycobacteria tuberculosis, two important causes of severe respiratory infection in high hiv prevalence populations in africa [ , , , , , , , ] . moreover, we did not test for some viruses, such as coronaviruses and bocavirus, which might play a role in pneumonia, although their pathogenicity, particularly in adults, is still debated [ , , , ] . while we did not observe discrete seasonality of ari in western kenya as previously shown in temperate climates, there tended to be yearly peaks coincident with the peak malaria season, which usually occur - months after the rainy season begins. there could be several explanations for this. malaria infection could augment the risk for ari. an association between malaria and bacteremia in african children has been shown, although no direct associations between malaria and pneumonia in children or adults has been proven [ ] . second, similar climactic conditions could favor both malaria and pneumonia. we have shown previously that influenza incidence tends to be highest in kenya during a broad wave that mostly corresponds with the southern hemisphere winter, from june to october, a time period that also encompasses peak malaria season [ , ] . lastly, malaria might cause symptoms that meet the nonspecific ari case definition we used. the overlap in symptoms between malaria and pneumonia in children is well-documented [ ] . although similar evidence does not exist for older persons, the non-specificity of the ari definition we used suggests that symptomatic malaria could have resulted in an illness that met the ari case definition. our study had several other limitations. first, not all ari patients were sampled. reasons for not collecting swabs included high patient volume, after hours clinic attendance, and rare refusals (, %). while we showed similar demographic characteristics and case-fatality ratios between sampled and non-sampled patients, undetected sampling bias might still have occurred that could influence the pathogens detected. second, we used a broad ari definition that likely captured a range of illnesses from influenza-like illness to pneumonia. without further diagnostic procedures, such as chest radiographs, we could not confirm the anatomic location of the respiratory infection. third, although the controls were by definition asymptomatic in the two weeks prior to enrollment, they might have been in the incubation period of a viral infection and subsequently developed symptoms. if this were true, a small number of controls might have been inaccurately classified. fourth, unlike in developed countries, we found lower rates of ari among the elderly, which we speculate is due to lower healthcare utilization by the elderly in rural kenya [ , , ] . fifth, we assumed that a person's hiv status during home-based testing in was unchanged throughout the study period, which might have led to some misclassification of hiv status. however, we feel that the number of misclassified individuals is small because the annual incidence of hiv among adults in the area has been estimated at % (kemri/cdc unpublished data), which at the most would have resulted in individuals among the , ari patients with hiv results having misclassified hiv status for some period of the three year surveillance period. lastly, despite the lack of a statistical association with ari at the population level for some viruses, an etiologic role of a virus detected in any given individual case cannot be ruled out. a study in thailand showed that rhinovirus was associated with hospitalized respiratory illness among adults, particularly among the elderly [ ] . outbreaks of rhinovirus-associated pneumonia have been suspected among the elderly in developed countries [ ] . adenovirus has on occasion been implicated as a cause of pneumonia in african adults, but without a control group for comparison [ ] . our study demonstrated several opportunities to make an impact on the incidence of ari among older children and adults in africa. the high incidence among hiv-infected persons suggests that expanded testing and access to cotrimoxazole prophylaxis and anti-retroviral drugs will likely decrease the number of ari cases, as has been shown before for pneumococcal infections [ , ] . vaccines could also decrease the incidence. pneumococcal conjugate vaccine was introduced for infants in kenya in early and if herd immunity occurs, as it has in developed countries, then decreases in adult pneumococcal disease incidence are expected [ ] . moreover, targeted vaccination among high-risk groups, such as hiv-infected adults, might be applicable for both influenza and pneumococcus [ , ] . participation with kemri/cdc. we thank brett whitaker for assistance in virologic testing at cdc, atlanta. we thank sonja olsen for her critical review of the manuscript. this paper is published with the approval of the director of kemri. the findings and conclusions are those of the authors and do not necessarily represent the views of the centers for disease control and prevention. the pathogen-attributable fraction (paf) is calculated as (or- )/or using viral prevalence data from cases and controls. for pneumococcus, afe was assumed to be . b the percentage of ari was adjusted for the paf and then adjusted for the prevalence among inpatients and outpatients for each pathogen at lwak hospital and the percentage of ari cases in the community who were seen in clinics as inpatients and outpatients (see methods). c only persons with complete testing for s. pneumoniae including blood culture and urine antigen testing were used to calculate the percent of ari due to s. pneumoniae. doi: . /journal.pone. .t high rate of pneumococcal bacteremia in a prospective cohort of older children and adults in an area of high hiv prevalence in rural western kenya the burden of common infectious disease syndromes at the clinic and household level from population-based surveillance in rural and urban kenya health and demographic surveillance in rural western kenya: a platform for evaluating interventions to reduce morbidity and mortality from infectious diseases a reversal in reductions of 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human bocavirus infections in hospitalized children and adults relation between falciparum malaria and bacteraemia in kenyan children: a populationbased, case-control study and a longitudinal study distinguishing malaria from severe pneumonia among hospitalized children who fulfilled integrated management of childhood illness criteria for both diseases: a hospital-based study in mozambique the epidemiology of hospitalized pneumonia in rural kenya: the potential of surveillance data in setting public health priorities healthcareseeking behaviour for common infectious disease-related illnesses in rural kenya: a community-based house-to-house survey human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus respiratory viruses in hospital patients on the witwatersrand. a -year study the impact of antiretroviral treatment on the burden of invasive pneumococcal disease in south african children: a time series analysis changes in invasive pneumococcal disease among hiv-infected adults living in the era of childhood pneumococcal immunization sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine we thank oliver morgan for assistance with control enrollment, joe oundo for establishing blood cultures, and joy chebet for graphical assistance. we thank the management of st. elizabeth lwak mission hospital and the people of asembo for their continued support and august | volume | issue | e key: cord- -v wovcqd authors: akmatov, manas k.; gatzemeier, anja; schughart, klaus; pessler, frank title: equivalence of self- and staff-collected nasal swabs for the detection of viral respiratory pathogens date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: v wovcqd background: the need for the timely collection of diagnostic biosamples during symptomatic episodes represents a major obstacle to large-scale studies on acute respiratory infection (ari) epidemiology. this may be circumvented by having the participants collect their own nasal swabs. we compared self- and staff-collected swabs in terms of swabbing quality and detection of viral respiratory pathogens. methodology/principal findings: we conducted a prospective study among employees of our institution during the ari season / (december-march). weekly emails were sent to the participants (n = ), reminding them to come to the study center in case of new symptoms. the participants self-collected an anterior nasal swab from one nostril, and trained study personnel collected one from the other nostril. the participants self-collected another two swabs (one from each nostril) on a subsequent day. human β-actin dna concentration was determined in the swabs as a quality control. viral respiratory pathogens were detected by multiplex rt-pcr (seeplex rv kit, seegene, eschborn, germany). of participants, ( %) reported at least one ari episode, participants two, and one participant three. self-swabbing was highly accepted by the participants. the amount of β-actin dna per swab was higher in the self- than in the staff-collected swabs (p = . ). β-actin concentration was lower in the self-swabs collected on day than in those collected on a subsequent day (p< . ). a respiratory viral pathogen was detected in % ( / ) of staff- and in % ( / ) of self-collected swabs (p = . ). with both approaches, the most frequently identified pathogens were human rhinoviruses a/b/c ( / swabs, %) and human coronavirus oc ( / swabs, %). there was almost perfect agreement between self- and staff-collected swabs in terms of pathogen detection (agreement = %, kappa = . , p< . ). conclusions/significance: nasal self-swabbing for identification of viral ari pathogens proved to be equivalent to staff-swabbing in this population in terms of acceptance and pathogen detection. the need for the timely collection of diagnostic biosamples (such as nasal swabs) during acute symptomatic episodes represents a major obstacle to large-scale studies on acute respiratory infection (ari) epidemiology. this may be circumvented by having the participants collect their own nasal swabs (''selfcollected nasal swabs'' or ''self-swabbing''). this method has been shown to be highly acceptable and feasible in various populations, e.g. among parents who collected nasal swabs from their children [ ] or adults who collected swabs from themselves [ ] (also reviewed in [ ] ). while the mere feasibility of nasal self-swabbing has thus been amply demonstrated, efforts to validate the diagnostic equivalence of self-swabbing compared to staff-swabbing are still ongoing. in a sample of individuals with ari symptoms, larios et al. compared self-collected midturbinate swabs with staff-collected nasopharyngeal swabs (gold standard) that were collected the same day. the self-collected swabs had a sensitivity of % for the detection of respiratory pathogens compared to the gold standard [ ] . luinstra et al. found similar detection rates for respiratory pathogens between self-and staffcollected midturbinate swabs when one staff-collected and one selfcollected swab were taken from opposite nostrils during the same visit to a campus health center [ ] . ip et al. investigated the validity of self-collected nasal (posterior nares) and pharyngeal swabs to detect influenza virus infection and came to the conclusion that self-swabs may be a good alternative [ ] . while these results do provide substantial evidence for the validity of selfcollected midturbinate swabs, anterior nasal swabs have not been evaluated in this respect. moreover, self-collected nasal swabs collected on separate days have not been compared with each other. in the present study, we thus compared quality of swabbing and efficiency of viral detection of anterior nasal swabs in the following scenarios: ) self-vs. staff-collected swabs that were taken during the same visit to the study center (day ), and ) self-and staff-collected swabs from day vs. self-collected swabs that were obtained at home on a later day. a prospective study was conducted during the / ari season among a convenience sample of employees of our institution, the helmholtz centre for infection research in braunschweig, germany. in december , employees ( to years old) were sent messages through the internal e-mail system inviting them to participate in the study. this invitation contained a link to the institutional intranet where information about the study was made available in english and german. individuals planning to leave braunschweig during the study period and staff members of the departments of epidemiology and infection genetics were not eligible to participate, the latter due to concern over a potential conflict of interest. at the baseline visit (december -january ), the study aims were explained to the participants and informed consent was obtained. a self-administered questionnaire was used to collect basic sociodemographic data. at the end of the ari season (april/ may ) the study participants completed a short acceptance questionnaire. all participants received a remuneration of j. the study was approved by the ethics committee of the state board of physicians of the german federal state of lower saxony. during january-march weekly e-mail messages were sent to the participants reminding them to come to the study center within days of onset of at least one of the following symptoms: sudden onset of stuffy or runny nose, cough, sore throat, headache, malaise, chills, or fever, defined as body temperature . uc. in the study center, a trained staff member (a.g.) obtained a nasal swab (regular flocked swab, copan, brescia, italy, product number c) from the participant's left nostril and instructed him/her how to perform a self-swab. the participants also received written and visual instructions for nasal self-swabbing. the participants then self-collected a swab from the right nostril. briefly, the swab was to be inserted into the nostril to the point where the basal edge of the flocked tip had just entered the nostril, corresponding to a depth of insertion of approx. cm. the swab was then rotated three times, being careful to swab all u of the anterior nasal lining and to include the superior recess (''little's area'') and the latero-inferior recess. the swab was then placed into ml universal viral transport medium (copan). a swabbing kit containing written and visual self-swabbing instructions, two nasal swabs and two vials of ml transport medium was then given to the participants with the request to self-collect two nasal swabs (one from each nostril) at home the next day, to place each swab in ml transport medium and to return the swabs to the study center as soon as possible. the swabs were stored at uc until analysis. the timeline of the study is shown in fig. . detection of human b-actin coding sequences was used as a measure of sample adequacy, assuming that the amount of b-actin gene dna can serve as proxy for the presence of human epithelial cells [ , ] , which contains these b-actin gene sequences in a pcdna . backbone, were analyzed in parallel to obtain the standard curve. b-actin dna concentration was determined in all four swabs. the values of the two swabs that were self-collected at home were pooled. the technician performing the laboratory analyses was not blinded as to whether a swab was staff-or selfcollected. detection of viral respiratory pathogens. rna was extracted from ml aliquots of transport medium (utm kit, copan, brescia, italy) with the qiaamp minelute virus spin kit (qiagen gmbh, hilden, germany). cdna was synthesized with the transcriptor first strand cdna synthesis kit (roche diagnostics gmbh, mannheim, germany) and tested by multiplex pcr (seeplex rv ace detection kit, seegene germany, eschborn, germany) for the presence of any of human viral respiratory pathogens (adenovirus a/b/c/d/e, human metapneumovirus, enterovirus, bocavirus / / / , human coronavirus e/nl and oc , parainfluenza virus , , and , influenza virus a and b, respiratory syncytial virus a and b, and rhinovirus a/b/c), following the manufacturer's recommendations except that ml instead of ml cdna was used as input for the pcr reaction. satisfaction and acceptability were assessed using a nine-item questionnaire. participants rated each item on a five-point likert scale with indicating strong disagreement, disagreement, neither disagreement nor agreement, agreement, and strong agreement. some of these items were reverse-phrased to reduce response bias. data were described by percentage for categorical variables and median with interquartile range (iqr) for continuous variables. a staff-collected and a self-collected swab were obtained on day from separate nostrils. the participants were instructed to collect a self-swab from each nostril the next day, but the actual day of self-swabbing ranged from day to day , as indicated by the triangle. doi: . /journal.pone. .g table . acceptability of nasal swabbing. the collection of the nasal swab by the study personnel was acceptable (n = )* collecting the nasal swab myself was acceptable (n = )* i felt comfortable when the study personnel collected the swab (n = )* ( - ) i felt comfortable when taking the swab myself (n = )* i would prefer taking a nasal swab myself and not having it taken by study personnel (n = )* ( - ) nasal self-swabbing was easy to perform (n = )* the instructions how to take the self-swab were understandable (n = )* i would participate in a study where nasal swabs are to be taken by study personnel (n = )** i would participate in a study where nasal swabs are to be taken by myself (n = )** *only those participants who collected at least one nasal swab. eighty-four participants responded to the invitation e-mail, corresponding to a response rate of %. seventy-two participants ( %) were women, the median age was years (range, - ). about half of the respondents had a university degree (including university of applied sciences) and about % were born outside germany. overall, both staff-and self-collected nasal swabs were highly accepted ( table ). most participants reported that instructions how to collect the swab were easy to understand and that the nasal self-swab was easy to perform. the participants did not make a preference regarding self-or staff-collected swabs ( table ) . fifty-six of ( %) participants reported at least one ari episode, ( %) participants reported two episodes, and one participant reported three, resulting in a total of ari episodes for the final analysis. the number of ari symptoms reported in a single ari episode ranged from one to seven (median, . ). thus, matched pairs of staff-and self-collected nasal swabs, performed on the same day, were available. about % of these were taken on the day of symptom onset (day ), % on day , % on day , % on day , and % on or after day . the additional set of self-collected swabs, which was to be collected at home the next day, was obtained in the majority of ari episodes ( / , %), for / ( %) of which swabs from both nostrils were returned, and from only one side in the remaining four. sixty-two percent of these swabs were indeed collected the next day (day ) and the latest one on day ( fig. ) . when comparing the staff-and self-swabs collected on day , bactin dna concentration was higher in the self-collected swabs (p = . ) (fig. a) . the b-actin dna concentration was higher in the self-swabs collected on or after day than in the self-swabs from day (p, . ). interquartile distance was smallest in the staff-collected swabs and greatest in the swabs that were selfcollected at home, indicating lower variability in the staff-collected than in the self-collected swabs. b-actin dna levels did not correlate with the duration of symptoms preceding the day of swab collection (fig. b) . a respiratory viral pathogen was detected in % ( / ) of staff-and in % ( / ) of self-collected swabs collected on day (p = . , mcnemar test). in both, the most frequently identified pathogens were human rhinoviruses a, b or c ( / positive swabs, %), human coronavirus oc ( / swabs, %) and parainfluenza viruses , , or ( / , %). table contains the complete list of detected pathogens, expressed as percentages of all swab pairs collected on day in which a pathogen was detected in at least one of the two swabs. there was nearly perfect agreement between the staff-and self-swabs collected on day in terms of pathogen detection (percent agreement = %, k = . , fig. ). the overall sensitivity of self-collected swabs to detect a respiratory pathogen was %, compared to the staff-collected swabs (table ). this very good agreement between the staff-and self-collected swabs from day is also evident in the flow diagram shown in fig. . analyzing the results obtained with the self-swabs from day or later, a viral pathogen was detected in % ( / ) of the swab pairs when the results of both nostrils were combined. when the swabs from each side were considered separately, a pathogen was detected in % ( / ) of the self-collected swab from the right and in % ( / ) of swabs from the left nostril. however, this apparent difference was not significant (p = . , mcnemar test). comparing viral detection of these self-swabs with that of the staffcollected swabs from day revealed substantial agreement table . sensitivity and specificity of self-collected swabs, obtained in the study center, to detect viral respiratory pathogens (compared to staff-collected swabs)*. (percent agreement = %, k = . ). the detection of a viral pathogen was independent of the amount of b-actin dna in both staff-and self-swabs collected on day (fig. ) . likewise, there was no association between viral positivity status and b-actin dna levels across all samples (p for trend = . ). this prospective study comparing staff-and self-collected nasal swabs for the detection of ari pathogens clearly demonstrated the validity of self-swabbing; specifically, self-swabbing was not inferior in terms of acceptance, satisfaction, sample adequacy, and viral detection rate. of note, we observed excellent agreement in viral detection between self-and staff-collected swabs collected the same day. the agreement between the swabs that were self-collected at home and the staff-collected swabs was somewhat less pronounced, but still was classified as substantial (k = . ). this lower agreement may be explained by the time lag between collection of the staff-swabs and the self-swabs at home, which spanned up to days and resulted in five additional positive self-swabs, but only two additional negative ones. thus, taken together, the results strongly indicate equivalence of self-collected and staff-collected swabs in this study population. noteworthy differences were detected in b-actin dna concentration, which was used to quantify the amount of host cells and thus served as a measure of sample adequacy [ ] . median b-actin dna concentration was significantly higher in the self-collected than in staff-collected swabs, but also in the swabs that were selfcollected at home than in the self-collected swabs from day . taken together, these results suggest ( ) that the participants applied higher swabbing pressure than the trained staff member, likely due to high confidence in the self-swabbing procedure, and ( ) that a training effect resulted in yet greater confidence and more vigorous swabbing when self-swabbing was repeated at home $ day later. in support of this notion, in a study comparing staffand self-collected swabs that were obtained the same day smieja et al. observed a higher number of epithelial cells and a tendency toward a higher b-actin dna level in a second self-swab that was collected immediately after the first one [ ] . alternatively, the presence of leukocytes in purulent secretions may have contributed to b-actin levels in some swabs. however, we do not consider this to be a major contributing factor since most participants did not have purulent secretions. another theoretical reason for the higher b-actin dna levels in the swabs that were self-collected at home might have been the longer disease duration, leading to higher shedding of epithelial cells or higher leukocyte numbers in the secretions. however, as shown in fig. b , there was no association between duration of symptoms and b-actin concentration in the swabs, thus ruling out this possibility in the present study. of note, more thorough sampling (as reflected by higher b-actin dna levels) did not improve viral detection in our study, suggesting that the amount of host cells on the swabs was near the optimum in most cases. alternatively, the sampling of host cells may not be a major determinant for the detection of ari viruses due to the presence of sufficient amounts of viral particles in nasal secretions. a criticism of studies comparing staff-and self-collected swabs from the same day has been that the participants might feel more confident in the presence of study personnel [ ] and that selfswabs might actually be inferior when collected in the absence of study personnel. we tested this hypothesis by including two additional nasal swabs which were self-collected at home $ day later. comparison of these swabs with staff-collected swabs collected on day revealed good agreement in terms of viral detection and even better sample adequacy, thus demonstrating the diagnostic equivalence of self-swabbing in an unsupervised setting, provided that the participants have been trained adequately. it should be kept in mind that only two individuals with pcrproven influenza virus infection were detected. therefore, anterior nasal self-swabbing for the detection of influenza virus infection still needs to be validated in dedicated studies. recently, a flocked mid-turbinate swab was developed and turned out to be superior to the gold standard nasopharyngeal swab in terms of ari virus detection [ ] . the anterior nasal swab used in the present study has not been compared to this midturbinate swab. this should be done in a future study. our findings are also limited by the fact that the study was conducted in a selected study population recruited within a research institution. however, the population included a mixture of scientific, clerical and support staff. notably, education level spanned a broad range and did not influence viral detection or b-actin dna levels. nonetheless, our findings need to be validated in future studies using random samples drawn from the general population. due to much lower expenses for personnel and travel, selfcollection would be a highly cost-efficient way to obtain diagnostic nasal swabs in medium and large scale population-based studies on ari epidemiology [ ] . the presented study adds significantly to the growing body of evidence demonstrating its diagnostic equivalence to staff-collection. nasal self-swabbing proved to be an acceptable, feasible and valid method to identify viral respiratory pathogens in this selected adult population. its applicability in the general population should be tested in future studies. parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research e-mail-based symptomatic surveillance combined with self-collection of nasal swabs: a new tool for acute respiratory infection epidemiology self-collected nasal swabs to detect infection and colonization: a useful tool for population-based epidemiological studies? self-collected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses validation of self-swab for virologic confirmation of influenza virus infections in a community setting evidence for altered regulation of i kappa b alpha degradation in human colonic epithelial cells development and evaluation of a flocked nasal mid-turbinate swab for selfcollected respiratory virus diagnostic testing the measurement of observer agreement for categorical data collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood we would like to thank the participants for their kind participation in the study. we also thank gérard krause (helmholtz centre for infection research, braunschweig, and robert koch institute, berlin) and aparna schweitzer (helmholtz centre for infection research, braunschweig) for helpful comments and a critical reading of the manuscript. key: cord- -nalulzfo authors: bastien, nathalie; anderson, kelly; hart, laura; caeseele, paul van; brandt, ken; milley, doug; hatchette, todd; weiss, elise c.; li, yan title: human coronavirus nl infection in canada date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: nalulzfo the isolation of human coronavirus nl (hcov-nl ) in the netherlands raised questions about its contribution to respiratory illness. in this study, a total of respiratory specimens, collected in canada primarily during the winter months of – , were tested for hcov-nl ; tested positive for hcov-nl , demonstrating virus activity during january–march . patients with hcov-nl were month- years old (median age, years). the main clinical presentations were fever ( / ), sore throat ( / ), and cough ( / ), and patients were hospitalized. these results provide evidence for the worldwide distribution of hcov-nl . hcov-oc , are responsible for up to % of the commoncold syndrome [ ] . however, several studies have found that these viruses are associated with more-severe respiratory infections [ ] [ ] [ ] [ ] . a novel coronavirus, hcov-sars, has been associated with severe atypical pneumonia and caused deaths worldwide during november -july [ ] . coronaviruses are divided into serotypes, on the basis of both their host range and their genome sequence. group and viruses are mammalian coronaviruses and include hcov- and hcov-oc , respectively, whereas group consists of avian coronaviruses. hcov-sars does not closely resemble viruses in any of the known groups of coronaviruses. analyses of the complete genome sequence of hcov-nl have revealed that the virus is a new group coronavirus that is closely related to hcov- [ ] . the relative importance of hcov-nl in viral respiratory-tract illnesses is still not known. in the present study, we retrospectively looked for the presence of hcov-nl in canadian patients suffering from acute respiratory-tract infection (ari) during the winter months of - , to assess the impact that hcov-nl infections have on ari and to describe the presenting signs and symptoms of the ari caused by this virus. materials and methods. primers used for amplification and sequencing were based on the published hcov-nl genome sequence [ ] . two nested sets of primers, described by van der hoek et al. [ ] , were used in this study. the primer set based on the b gene-primers repsz- f-( ) ( -gtgatgcata-tgctaatttg- ), repsz- r-( ) ( -ctcttgcaggtat-aatccta- ), repsz- f-( ) ( -ttggtaaacaaaagat-aact- ), and repsz- r-( ) ( -tcaatgctataaacag-tcat- )-were used for diagnosis; the primer set based on the a gene-primers ss - pf-( ) ( -cttttgataacggt-cactatg- ), p g m- - pr-( ) ( -ctcattacataaaa-catcaaacgg- ), p e - pf-( ) ( -ggtcactatgtag-tttatgatg- ), and ss - pr-( ) ( -ggtcactatgt-agtttatgatg- )-were used for confirmatory purposes and sequence analysis. the primers used for amplification of the rnase p housekeeping gene were based on the published sequences (genbank accession number nm_ ) -agatt-tggacctgcgagcg- (forward primer) and -gagcgg-ctgtctccacaagt- (reverse primer). viral rna was extracted from ml of either original samples or tissue-culture fluid, by use of the rneasy mini kit (qiagen). viral rna was amplified by use of a -step reverse transcription-polymerase chain reaction (rt-pcr) kit (qiagen), according to the manufacturer's recommendations. in brief, ml of rna was added to the rt-pcr mixture, which the dna sequences were assembled and analyzed with the seqman, editseq, and megalign programs in the lasergene suite (dnastar). phylogenetic trees were generated by the neighbor-joining method using the mega program [ ] . to monitor the efficiency of nucleic-acid extraction, each sample was also tested for the presence of the rnase p housekeeping gene by use of a -step rt-pcr kit under the following conditions: min at Њc, for reverse transcription; min at Њc, for the activation of the hotstart dna polymerase; cycles of s at Њc, s at Њc, and s at Њc; and an extension for min at Њc. of the specimens tested, all were positive for the rnase p gene. the hcov-nl sequences described in the present study have been deposited in genbank, under accession numbers ay -ay . results. the national microbiology laboratory received a total of specimens collected from patients with ari, from provincial public health laboratories in manitoba ( specimens), saskatchewan ( specimens), and nova scotia ( specimens). these representative specimens were selected to be negative for ( ) swabs, from throat washes, and other, miscellaneous specimens from the respiratory system. of the specimens tested, ( . %) were positive, by rt-pcr, for hcov-nl , and they were from all provinces: ( %) from manitoba, ( %) from saskatchewan, and ( %) from nova scotia. hcov-nl activity was found during january ( specimens [ %]), february ( specimens [ %]), and march ( specimens [ %]) and subsided during late spring; in contrast, no specimens positive for hcov-nl were retrieved for the same period during . the sex distribution of these specimens was % ( ) male and % ( ) female (table ) . patients with hcov-nl were month- years old (median age, years). the proportion of positive specimens was significantly higher in the - -year-old age group than in the -year-old age group (table ) ( / vs. / ; ). p p . seven patients from the -year-old age group were involved in an outbreak of ari in a personal-care home in manitoba (table ) . hcov-nl was found in of the patients from this outbreak whose samples were analyzed in this study. (table ) . other observed symptoms included bronchiolitis ( patients), rhinitis ( patient), and lymphadenitis ( patient), and patients died- because of unrelated causes (this patient, a male infant, was smothered) (table ). of the patients ( %) with hcov-nl who were hospitalized, ( %) were in the - -year-old age group, and was in the -year-old age group (table ) . for of the specimens positive for hcov-nl , nucleotide sequences were determined for nucleotides - of the a gene. comparison of these sequences with those published for dutch isolates of hcov-nl showed that the a genes were relatively well conserved, with . %- % nucleicacid identity between specimens. the phylogenetic tree of the hcov-nl isolates showed the existence of major groups or clusters that contained both canadian and dutch strains of hcov-nl (figure ). similar canadian hcov-nl strains were isolated from adults and children in all provinces and during different outbreaks. strains isolated from the outbreak of ari in a personal-care home in manitoba were grouped together into cluster . discussion. although, in a study like this one, the inclusion of a control group of healthy individuals is necessary to clearly demonstrate a causal relationship, the detection of hcov-nl in respiratory-tract specimens from patients suffering from ari of unknown causes strongly suggests that it is associated with respiratory illness. this finding supports the association, found by van der hoek et al. [ ] , of hcov-nl with ari. it also demonstrates for the first time that hcov-nl was present in several canadian provinces during the winter season, suggesting that hcov-nl may be circulating worldwide. we detected the presence of hcov-nl in ( . %) of the analyzed specimens that were negative for ( ) influenza viruses a and b; ( ) piv , , and ; ( ) adenovirus; ( ) rsv; and ( ) hmpv; and these results provide further evidence of the contribution of hcov-nl to ari-and of the significant burden that it therefore may present to health-care systems. for the time period that was analyzed in the present study, peak activity of hcov-nl was found to occur during january-march . although sampling occurred only during the winter season (october-april), our results are consistent with van der hoek et al.'s [ ] finding that hcov-nl appears to be transmitted predominantly during the winter season. other hcovs, as well as influenza and rsv, which are involved in a substantial number of hospitalizations for ari, were also shown to be present in patients with ari during that time of the year [ ] . the clinical symptoms associated with hcov-nl infection are also comparable to those observed to be associated with influenza, rsv, and other hcovs, making it impossible, at least on the basis of seasonality and clinical manifestations, to differentiate between these viral infections. previous estimates of the contributions of rsv and other etiological agents to ari based on these parameters may have been biased because the involvement of other new respiratory viruses such as hmpv and hcov-nl were overlooked [ ] . thus, the systematic detection of hcov-nl in respiratory specimens may improve the understanding of the etiology of ari; however, the possibility of dual infection cannot be excluded, because the present study utilized samples from patients with ari who were tested-and found to be negative-for only ( ) influenza viruses a and b; ( ) piv , , and ; ( ) adenovirus; ( ) rsv; and ( ) hmpv. nor can it be ruled out that hcov-nl can exist asymptomatically in an individual, because samples from healthy individuals were not included. in addition, the present study provides evidence that hcov-nl may have been associated with an outbreak of ari in a personal-care home. all hospitalizations occurred in the - -year-old and -year-old age groups, which suggests that, like the other hcovs and rsv, hcov-nl may cause more-severe ari in frail patients, such as infants and the elderly, than in other patients [ , , [ ] [ ] [ ] . phylogenetic analysis based on the a gene confirms previously published results and shows the presence of viruses with different molecular markers [ ] . the increased number of isolates detected provides further evidence of genetic diversity and the presence of genetic clusters. close clustering of hcov-nl isolates recovered from different canadian provinces and from the netherlands suggests that the evolutionary pattern of hcov-nl does not presently correlate with geographic location. in summary, our data suggest that hcov-nl may play a significant role in ari, especially in young children and the elderly. more-comprehensive studies, which would include data on prevalence, risk factors, and use of health services, are necessary to determine both the importance of hcov-nl in ari and its impact on the health-care system. editorial board of respiratory disease in canada. respiratory disease in canada identification of a new human coronavirus fields virology spectrum of clinical illness in hospitalized patients with "common cold" virus infections rhinovirus and coronavirus infection-associated hospitalizations among older adults impact of respiratory virus infections on persons with chronic underlying conditions an outbreak of coronavirus oc respiratory infection in normandy, france world health organization, department of communicable disease surveillance and response. summary of probable sars cases with onset of illness from mega : molecular evolutionary genetics analysis software contribution of rsv to bronchiolitis and pneumonia-associated hospitalizations in english children respiratory syncytial virus coronavirus infection in acute lower respiratory tract disease of infants coronavirus e-related pneumonia in immunocompromised patients