the study population was the 1,307 students and 825 staff attending and working at the boarding school , and all were invited to participate in the study . however , because the investigation occurred during an examination period , not all students and staff were present , and the exact number staying at the school during this period is unknown . all students were boarders ; some staff members lived on the school grounds , and others lived outside . study participants were asked to complete an online questionnaire and provide a single serum sample . samples were collected from june 11 through june 26 , the last day of term . collection of data from the online questionnaire also began on june 11 and continued until october 15 . serologic testing by hemagglutination inhibition ( hi ) was carried out as previously described ( 1618 ) at the centre for infections , hpa , london , using egg - grown nibrg122 ( reverse genetics derivative of a / engl/195/2009 ) . serum specimens were pretreated with receptor - destroying enzyme ii ( denka seiken co. , ltd , tokyo , japan ) , 1:4 ( vol / vol ) , at 37c for 19 h , followed by heat inactivation at 56c for 1 h. the assay was performed by mixing 25 l of virus suspension ( containing 4 hemagglutinating units ) with an equal volume of receptor - destroying enzyme ii  treated serum , followed by 1 h incubation at room temperature , after which 25 l of 0.5% ( vol / vol ) turkey erythrocytes was added to each well . serum specimens were tested in a 2-fold serial dilution series with an initial dilution of 1:8 and ending at 1:1,024 . titers were expressed as a reciprocal of the highest serum dilution that fully prevented hemagglutination . serum specimens with no reactivity in the first dilution ( < 8 ; considered negative ) were assigned a titer of 4 ; serum specimens that showed titers > 1,024 were assigned a numerical value of 1,024 for statistical analysis . serologic samples were excluded from statistical analyses if a person had reported illness within 14 days of sample collection because previous data suggested that 1421 days is required for a measurable immune response ( 18 ) . the online questionnaire collected data on demographic characteristics : sex ; age ( age groups , years : 1315 , 1618 [ students ] ; 2049 , > 50 [ staff ] ) ; symptoms ; severity ( self - described as mild , moderate , severe ) ; self - reported use of antiviral drugs for treatment or prophylaxis ; and self - reported 200809 seasonal influenza vaccination . questionnaires were excluded if the person reported being away from the school during the outbreak or if symptom onset occurred after june 10 , 2009 . the outcomes of interest were seropositivity and clinical cases of acute respiratory infection ( ari ) . seropositivity was defined as having an hi titer > 32 , i.e. , a titer 4 the minimum detection limit . similar definitions have been used in population - based serosurveys in other countries ( 19,20 ) and have been shown to be specific in identifying recent infection in children ( 21 ) . for sensitivity analysis , we refitted the final logistic regression model ( below ) using an alternative cutoff value of 1:8 , the minimum detection limit ( 22 ) . a clinical case of ari was defined as a person reporting any one of the following respiratory symptoms ; runny / blocked nose , sore throat , or cough . those reporting ari were further subcategorized into a more specific case definition , i.e. , cases of ili , defined as a person reporting > 1 of the symptoms listed above and fever . exposures of interest were the use of antiviral drugs , prescribed for prophylaxis or treatment , and seasonal trivalent influenza vaccine in the previous year ( 200809 ) . we estimated the proportion of asymptomatic cases by determining the proportion of the population with positive serologic test results but no symptoms of ari . we also estimated the attack rate for those with ari , ili , and positive serologic test results and their distribution according to demographic variables . logistic regression models were constructed to estimate the independent association of antiviral drugs and seasonal influenza vaccine and the odds of being seropositive or having an ari . age was included in the model as a covariate ; other linear predictors were included if model fit was significantly improved ( likelihood - ratio [ l - r ] test p<0.05 ) . interaction between age group and antiviral agents for prophylaxis ; and seasonal influenza vaccine was tested to determine whether these associations between predictors and seropositive status and ari differed according to age group , and therefore according to student and staff categories . if interaction was observed ( i.e. , the model was improved by including the interaction term ) , students and staff would be reported separately . a further model was also fitted for staff to investigate whether staff role and sex were associated with a seropositive status or ari . data analysis was carried out by using stata version 11 ( statacorp lp , college station , tx , usa ) . informed consent was sought from all students and their parents or guardians if students were < 16 years old . because this was a field epidemiology study conducted during an emerging pandemic and involved a novel virus with unknown clinical effects , hpa did not require formal ethical approval since any information gained was essential in illuminating the effects of the infection and indicating possible control measures . in total , 746 questionnaires were completed online , of which 695 ( 93.2% ) met the inclusion criteria ( figure ) . this represented 35.9% of the 1,307 students and 27.4% of 825 staff who usually reside at the school . in total , 411 persons gave a serum sample and 353 ( 85.9% ) were matched to a valid questionnaire ( figure ) . of persons with a questionnaire and matched serologic test result , 216 were students and 137 staff , which accounts for 16.5% of the registered student population and 16.6% of the registered staff population ; these 353 persons composed our final cohort ( figure ) . number and proportion of boarding school staff and student populations who completed a questionnaire and had matched serologic test results , england , 2009 . the distribution of the study population by age , sex ( staff only ; all students were male ) , occupation ( staff only ) , self - reported illness , history of seasonal influenza vaccine in the previous year , and the use of antiviral agents is shown in table a1 . to determine whether our sample was representative of the total school population , we compared the final study population ( questionnaire and matched serologic results ) to those who completed only the questionnaire and to the whole school population . for staff , the proportion of men and women in the final study population was similar to the proportion that answered the questionnaire only ( 51.8% vs. 48.2% and 48.2% vs. 51.8% , respectively ;  p = 0.58 ) . the proportions of students in age groups 1315 years and 1618 years were similar for those completing a questionnaire only ( 46.7% vs. 53.3% ) compared to those in the matched study sample ( 41.7% vs. 58.3% ;  p = 0.25 ) ( table a1 ) , and similar to the school s student population . teaching staff made up 31.6% of those of the final study population and 38.6% of the questionnaire - only population (  p = 0.52 ) . for persons reporting ari , differences were significant between those who completed a questionnaire only and those who also gave a blood sample for students ( 37.3% vs. 48.3% ;  p = 0.020 ) and staff ( 10.2% vs. 30.8% ;  p<0.001 ) . this selection bias could have led to an overestimation of the infection attack rate . for ari , the attack rate was estimated at 35.9% , ( 237/661 , 95% confidence interval [ ci ] 32.2%39.6% ) or 16.6% ( 110/661 , 95% ci 13.9%19.7% ) by using the definition for ili ( table a1 ) . of those who reported ari and ili , serologic test results were negative for 64/141 ( 45.4% , 95% ci 37.0%54.0% ) and 19/63 ( 30.2% , 95% ci 19.2%43.0% ) persons , respectively . we found 143 seropositive persons , which gives an attack rate for infection of 40.5% ( 95% ci 35.3%45.8% ; table 1 ) . of these 143 persons , 4 students did not answer the question relating to their illness status , and of the remaining 139 persons for whom illness history was available , 62 ( 44.6% , 95% ci 36.2%53.3% ) did not report ari . * categories in which the response was missing or unknown are shown in table a1 . ci , confidence interval ; ari , acute respiratory infection ; ili , influenza - like illness ; pep , postexposure prophylaxis . 1 staff member was not included in the analysis because his occupation was unknown . .  excluding those who reported taking the treatment dose of antiviral drugs . in crude analyses , persons who reported ari were more likely to be seropositive ( 62/199 , 54.2% ; crude odds ratio [ or ] 2.66 , 95% ci 1.704.16 ) than were those without illness ( 77/141 , 29.9% ) . the odds of having serologic evidence of infection increased when the illness reported met the case definition for ili ( crude or 4.44 , 95% ci 2.458.02 ) ( table 1 ) . overall , those reporting moderate or severe illness were more likely to be seropositive than those reporting mild illness ( crude or 2.21 , 95% ci 1.104.41 ) . overall , fewer students reported antiviral drug use than did staff ( 59.4% vs. 80.0% , p<0.001 ) . most of those taking a treatment dose were students ( 16/20 , 80% ) ( table 2 ) . more students reported having had the 200809 trivalent seasonal influenza vaccine than staff ( 81.1% vs. 50.0% ; p<0.0001 in the matched sample ) . * categories in which the response was missing or unknown are shown in the table a1 . ari , acute respiratory infection ; ci , confidence interval ; pep , postexposure prophylaxis .  excluding those who reported taking the treatment dose of antiviral drugs . in logistic regression models for ari and serologic status ( table 3 ) , including age group , significantly improved the fit of the models ( both l - r tests p<0.001 compared models , including only antiviral drug use and vaccination status ) . no evidence of effect modification was found between age group and antiviral drugs or vaccination status for either outcome ( l - r test p = 0.87 and p = 0.77 , respectively ) . therefore , stratified models were not fitted for staff and students . * for ari , n = 654 who completed questionnaires ; for serology - confirmed infection , n = 333 who completed questionnaires and had a matched serology sample . ari , acute respiratory infection ; aor , adjusted odds ratio ; ci , confidence interval ; pep , postexposure prophylaxis . staff in the age groups 2049 years and > 50 years ( adjusted ors [ aors ] 0.30 [ 95% ci 0.120.73 ] and 0.20 [ 95% ci 0.080.53 ] , respectively ) were less likely to have positive serologic test results than students 1315 years of age ( table 3 ) . weak evidence suggests that those 1618 years were more likely to be seropositive and have ari than those 1315 years of age ( aor 1.85 [ 95% ci 0.953.60 ] and 1.57 [ 95% ci 0.982.53 ] , respectively ) . although odds of seropositivity did not increase significantly with receipt of 200809 seasonal influenza vaccine ( p = 0.10 ) , the point estimate was > 1 . likewise , the point estimate of the aor for the association between taking a prophylactic dose of antiviral drugs and seropositivity was < 1 ( p = 0.045 ) . in a similar model , with ari as the outcome of interest , having received a prophylactic dose significantly reduced the odds of ari ( aor 0.41 , 95% ci 0.270.61 ) . for the staff - only models ( table 4 ) , staff role improved the model that predicted serologic results and ari ( l - r test p < 0.001 and 0.01 , respectively ) . after staff role was taken into account , including sex as a factor did not improve the accuracy of either model and was therefore not included . for the multivariable logistic regression model , which included only staff , age groups , staff role , exposure to prophylactic dose of antiviral agents , and having received the 200809 seasonal influenza vaccine were considered . teachers were more likely be seropositive than other staff members ( aor 7.47 , 95% ci 2.3124.18 ) , and no association was found between seropositivity outcome and age , taking the prophylactic dose of antiviral drugs , or receiving the influenza vaccine ( table 4 ) . * ari , acute respiratory infection ; aor , adjusted odds ratio ; ci , confidence interval ; pep , postexposure prophylaxis . persons who reported taking the treatment dose of antiviral drugs were excluded . when the final logistic models were refitted by using the minimum detection limit ( > 1:8 ) to define seropositive status , this change made little difference to the point estimates of the ors for antiviral drug use , age group , or seasonal vaccine . for the staff - only model , using a cutoff value > 1:8 changed the point estimates for taking antiviral agents and age groups ( > 50 vs. 2049 years ) to > 1 ; however , neither linear predictor was significantly associated with the outcome with either cutoff value . in total , 746 questionnaires were completed online , of which 695 ( 93.2% ) met the inclusion criteria ( figure ) . this represented 35.9% of the 1,307 students and 27.4% of 825 staff who usually reside at the school . in total , 411 persons gave a serum sample and 353 ( 85.9% ) were matched to a valid questionnaire ( figure ) . of persons with a questionnaire and matched serologic test result , 216 were students and 137 staff , which accounts for 16.5% of the registered student population and 16.6% of the registered staff population ; these 353 persons composed our final cohort ( figure ) . number and proportion of boarding school staff and student populations who completed a questionnaire and had matched serologic test results , england , 2009 . the distribution of the study population by age , sex ( staff only ; all students were male ) , occupation ( staff only ) , self - reported illness , history of seasonal influenza vaccine in the previous year , and the use of antiviral agents is shown in table a1 . to determine whether our sample was representative of the total school population , we compared the final study population ( questionnaire and matched serologic results ) to those who completed only the questionnaire and to the whole school population . for staff , the proportion of men and women in the final study population was similar to the proportion that answered the questionnaire only ( 51.8% vs. 48.2% and 48.2% vs. 51.8% , respectively ;  p = 0.58 ) . the proportions of students in age groups 1315 years and 1618 years were similar for those completing a questionnaire only ( 46.7% vs. 53.3% ) compared to those in the matched study sample ( 41.7% vs. 58.3% ;  p = 0.25 ) ( table a1 ) , and similar to the school s student population . teaching staff made up 31.6% of those of the final study population and 38.6% of the questionnaire - only population (  p = 0.52 ) . for persons reporting ari , differences were significant between those who completed a questionnaire only and those who also gave a blood sample for students ( 37.3% vs. 48.3% ;  p = 0.020 ) and staff ( 10.2% vs. 30.8% ;  p<0.001 ) . for ari , the attack rate was estimated at 35.9% , ( 237/661 , 95% confidence interval [ ci ] 32.2%39.6% ) or 16.6% ( 110/661 , 95% ci 13.9%19.7% ) by using the definition for ili ( table a1 ) . of those who reported ari and ili , serologic test results were negative for 64/141 ( 45.4% , 95% ci 37.0%54.0% ) and 19/63 ( 30.2% , 95% ci 19.2%43.0% ) persons , respectively . we found 143 seropositive persons , which gives an attack rate for infection of 40.5% ( 95% ci 35.3%45.8% ; table 1 ) . of these 143 persons , 4 students did not answer the question relating to their illness status , and of the remaining 139 persons for whom illness history was available , 62 ( 44.6% , 95% ci 36.2%53.3% ) did not report ari . * categories in which the response was missing or unknown are shown in table a1 . ci , confidence interval ; ari , acute respiratory infection ; ili , influenza - like illness ; pep , postexposure prophylaxis . 1 staff member was not included in the analysis because his occupation was unknown . .  excluding those who reported taking the treatment dose of antiviral drugs . in crude analyses , persons who reported ari were more likely to be seropositive ( 62/199 , 54.2% ; crude odds ratio [ or ] 2.66 , 95% ci 1.704.16 ) than were those without illness ( 77/141 , 29.9% ) . the odds of having serologic evidence of infection increased when the illness reported met the case definition for ili ( crude or 4.44 , 95% ci 2.458.02 ) ( table 1 ) . overall , those reporting moderate or severe illness were more likely to be seropositive than those reporting mild illness ( crude or 2.21 , 95% ci 1.104.41 ) . overall , fewer students reported antiviral drug use than did staff ( 59.4% vs. 80.0% , p<0.001 ) . most of those taking a treatment dose were students ( 16/20 , 80% ) ( table 2 ) . more students reported having had the 200809 trivalent seasonal influenza vaccine than staff ( 81.1% vs. 50.0% ; p<0.0001 in the matched sample ) . * categories in which the response was missing or unknown are shown in the table a1 . ari , acute respiratory infection ; ci , confidence interval ; pep , postexposure prophylaxis .  excluding those who reported taking the treatment dose of antiviral drugs . in logistic regression models for ari and serologic status ( table 3 ) , including age group , significantly improved the fit of the models ( both l - r tests p<0.001 compared models , including only antiviral drug use and vaccination status ) . no evidence of effect modification was found between age group and antiviral drugs or vaccination status for either outcome ( l - r test p = 0.87 and p = 0.77 , respectively ) . therefore , stratified models were not fitted for staff and students . * for ari , n = 654 who completed questionnaires ; for serology - confirmed infection , n = 333 who completed questionnaires and had a matched serology sample . ari , acute respiratory infection ; aor , adjusted odds ratio ; ci , confidence interval ; pep , postexposure prophylaxis . staff in the age groups 2049 years and > 50 years ( adjusted ors [ aors ] 0.30 [ 95% ci 0.120.73 ] and 0.20 [ 95% ci 0.080.53 ] , respectively ) were less likely to have positive serologic test results than students 1315 years of age ( table 3 ) . weak evidence suggests that those 1618 years were more likely to be seropositive and have ari than those 1315 years of age ( aor 1.85 [ 95% ci 0.953.60 ] and 1.57 [ 95% ci 0.982.53 ] , respectively ) . although odds of seropositivity did not increase significantly with receipt of 200809 seasonal influenza vaccine ( p = 0.10 ) , the point estimate was > 1 . likewise , the point estimate of the aor for the association between taking a prophylactic dose of antiviral drugs and seropositivity was < 1 ( p = 0.045 ) . in a similar model , with ari as the outcome of interest , having received a prophylactic dose significantly reduced the odds of ari ( aor 0.41 , 95% ci 0.270.61 ) . for the staff - only models ( table 4 ) , staff role improved the model that predicted serologic results and ari ( l - r test p < 0.001 and 0.01 , respectively ) . after staff role was taken into account , including sex as a factor did not improve the accuracy of either model and was therefore not included . for the multivariable logistic regression model , which included only staff , age groups , staff role , exposure to prophylactic dose of antiviral agents , and having received the 200809 seasonal influenza vaccine were considered . teachers were more likely be seropositive than other staff members ( aor 7.47 , 95% ci 2.3124.18 ) , and no association was found between seropositivity outcome and age , taking the prophylactic dose of antiviral drugs , or receiving the influenza vaccine ( table 4 ) . * ari , acute respiratory infection ; aor , adjusted odds ratio ; ci , confidence interval ; pep , postexposure prophylaxis . persons who reported taking the treatment dose of antiviral drugs were excluded . when the final logistic models were refitted by using the minimum detection limit ( > 1:8 ) to define seropositive status , this change made little difference to the point estimates of the ors for antiviral drug use , age group , or seasonal vaccine . for the staff - only model , using a cutoff value > 1:8 changed the point estimates for taking antiviral agents and age groups ( > 50 vs. 2049 years ) to > 1 ; however , neither linear predictor was significantly associated with the outcome with either cutoff value . this study describes the seroprevalence of infection with the pandemic ( h1n1 ) 2009 virus in an enclosed institutional environment and provides evidence of widespread infection among both students and staff before the outbreak became evident to public health authorities . attack rates for infection were estimated as 40.5% by serologic testing and as 34.1% by clinical illness ( ari ) . an estimated 44.7% serology - positive persons did not report symptoms of ari , which agreed with previous findings ( 10,23 ) . no significant association was found between seropositivity and prophylaxis with antiviral drugs , although some evidence showed that it reduced the odds of ari . the point estimate of the aor indicated nonsignificant increased odds of infection ( indicated by serologic results ) for persons who had received the 200809 seasonal influenza vaccine , although it did not increase the odds for ari . the uncertainty regarding the associated illness of pandemic ( h1n1 ) 2009 at the time the study was initiated made the selection of a random sample not feasible . a pragmatic approach was therefore chosen to offer the entire school population the opportunity of being included in the study , resulting in 17% of the registered school population providing a serum sample . because the study was conducted during an examination period , and some students and staff were absent , the size of the population from which the study sample was drawn is not known . however , because this was definitely less than the registered population , our response rate estimation was conservative . the distribution of our study population was not significantly different from the school s population in age , school year , and sex ( among teachers ) . however , the subsample of persons who provided a serum sample likely were not representative of persons who answered the questionnaire . for example , persons who provided a serum sample were more likely to have reported an ari than persons who responded to the questionnaire only . selection bias in the serology study subsample is also evident in the ors for the 200809 seasonal influenza vaccine , for which the ors were different , according to whether a serologic or clinical outcome was used , because the effect would be expected to be in the same direction . in addition , vaccination status was self - reported and could not be validated against official records , and we did not collect dates that antiviral drugs were used from each person . we used a cutoff value of an hi titer > 32 to indicate recent seroconversion . a previous study ( 18 ) has indicated that cross - reactive antibody to pandemic ( h1n1 ) 2009 virus was prevalent in england s population before the pandemic and that seroprevalence was strongly associated with age . in addition , a high proportion of children at the school had been vaccinated and were therefore unlikely to be representative of children in england . conflicting evidence exists regarding the effect of prior trivalent influenza vaccination on cross - reactive titers for pandemic ( h1n1 ) 2009 virus in persons < 55 years of age ( 21,24 ) . therefore , some misclassification of cases ( persons who seroconverted as a result of exposure to pandemic [ h1n1 ] 2009 virus ) likely occurred , leading to possible overestimation of the proportion of asymptomatic patients . however , the proportion of misclassified seropositive persons is likely small , particularly among children . ideally , paired samples ( collected before and after the outbreak ) would have been able to measure seroconversion ; however , this opportunity was not available . these results highlight the fact that , depending on the virulence and transmissibility of an emerging influenza pandemic virus , extensive transmission may occur in a closed setting and thus by implication in the community over and above the observed clinical disease . this finding has notable implications for predicting the future course of a pandemic because the subsequent pool of those susceptible after initial transmission will diminish ( 18 ) . current policy in closed settings in the united kingdom is to isolate or place symptomatic persons in cohorts after diagnosis to minimize the risk for onward transmission . if a substantial proportion of mildly symptomatic or even possibly asymptomatic persons were able to transmit infection , current policy would be of limited value . first , infection may be widespread within an institution long before it becomes apparent to public health authorities ; second , a large number of persons may be infected but asymptomatic or mildly symptomatic when the first case is diagnosed . although conclusions can be drawn from this study that rapid transmission of influenza occurred in this environment and that infection may not always produce symptoms , the evidence of transmission of the influenza virus by asymptomatic persons remains scant ( 25 ) . our findings indicate that the use of prophylactic antiviral agents lowers the odds of an ari by 50% but has no effect on reducing the odds of serologic infection . several interpretations of these findings are possible ; for example , while prophylactic antiviral agents might not reduce the risk for infection , they could protect from clinical disease . published evidence from the occurrence of seasonal influenza has indicated that timely administration of prophylactic antiviral agents to close contacts of infected persons reduces the risk for disease ( 26 ) . first , the serology sample was a much smaller subset of the questionnaire survey respondents , and the results for a serologic outcome indicate a lack of power . thus , the effectiveness of antiviral prophylaxis would be underestimated . also , the low specificity of the case definition for aris could lead to an overestimation of the effect on clinical disease . the association between self - reported illness and severity of illness with an increasing likelihood of seropositivity suggests that even in facilities with a limited diagnostic capacity , simple definitions of ili may be a more specific indicator of the true presence of infection in communities in which a proven outbreak is under way . fever was an essential part of the clinical case criteria for testing in the united kingdom , in contrast to the united states , where the clinical criteria were either a  respiratory illness ( recent onset of > 2 of the following : rhinorrhea / nasal congestion , sore throat , cough , fever or feverishness ) or an ili ( fever > 37.8c [ 100f ] , plus cough or sore throat ) . this study has demonstrated evidence of widespread infection with pandemic ( h1n1 ) 2009 virus in a closed setting , with a substantial proportion of asymptomatic persons . although the study highlights the difficulties of obtaining a large representative sample from a boarding school population during a pandemic influenza outbreak , it also illustrates the value of such rapid field epidemiologic investigations in understanding an emergent threat . this was particularly relevant during the emergence of pandemic ( h1n1 ) 2009 virus at a time when its pathogenicity was uncertain and the benefit of using antiviral agents for postexposure prophylaxis was unclear . the results of this seroepidemiologic study in an outbreak setting during the pandemic of novel pandemic ( h1n1 ) 2009 highlight the need for health authorities to agree on protocols for similar investigations during future pandemics .
toc summary : prophylactic antiviral agents lower the odds of acute respiratory infection but not serologic infection.keywords : pandemic , influenza , a / h1n1 , pandemic ( h1n1 ) 2009 , seroepidemiology , outbreak , serology , asymptomatic , prophylaxis , antiviral agents , vaccine , viruses , research