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.

Most of the outbreak cases occurred in May 2009. 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 (16–18) 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 37°C for 19 h, followed by heat inactivation at 56°C for 1 h. The assay was performed by mixing 25 μL 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 μL 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 14–21 days is required for a measurable immune response (18).

The online questionnaire collected data on demographic characteristics: sex; age (age groups, years: 13–15, 16–18 [students]; 20–49, >50 [staff]); symptoms; severity (self-described as mild, moderate, severe); self-reported use of antiviral drugs for treatment or prophylaxis; and self-reported 2008–09 seasonal influenza vaccination. Results from these questionnaires were subsequently linked to the serology results. 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 (2008–09).

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.

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 2008–09 seasonal influenza vaccine, although it did not increase the odds for ARI.

Our study has some limitations, however. 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. This resulted in the overestimation of attack rates. Selection bias in the serology study subsample is also evident in the ORs for the 2008–09 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). However, our results should be viewed with caution. 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 >37.8°C [100°F], 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.