We performed an observational cohort study in the Singapore military from 22 Jun 09 to 16 Jul 09. The Singapore military has a mix of regular employees and conscript personnel where all males are required to serve after high school. These personnel live in camps during the week and return home on weekends, resulting in a semi-closed community with exposures to the national community. The Singapore military identified its first imported case of 2009 Influenza A (H1N1) on 15 Jun 2009, and on 22 Jun 2009 identified its first outbreak cluster with local transmission.
In line with national protocols, cases of 2009 Influenza A (H1N1) were determined via laboratory confirmed infection by real-time reverse transcription polymerase chain reaction (RT-PCR) or viral culture . In addition to the national protocol of hospital or home isolation of cases during the early containment phase of the local epidemic , the Singapore military used the strategy of geographical oseltamivir ring chemoprophylaxis of affected military units with 10 days of oseltamivir 75 mg once a day, and cohorting of the entire units (as a form of social distancing) to prevent spread.
The study was performed among 252 personnel involved in 3 separate 2009 Influenza A (H1N1) outbreaks, whereby post-exposure oseltamivir ring chemoprophylaxis was administered. At the onset of each outbreak, a 10 day course of post-exposure oseltamivir chemoprophylaxis was given to each cohort and they continued to function in their normal capacity. The entire cohort was screened by RT-PCR using nasopharyngeal swabs three times a week, until no further positive cases were discovered for three days. All confirmed cases were given a minimum of 7 days home medical leave. The rest of the cohort continued their regular schedule, including staying in camp during weekdays and returning home during weekends.
In addition, three samples of 5 to 10 ml of venous blood were taken from each participant in for serological testing. The first baseline sample was taken at the start of outbreak. The second sample was taken between 2 to 3 weeks after the completion of oseltamivir prophylaxis. This timeframe allowed sufficient time for seroconversion from infections during prophylaxis, while reducing the likelihood of seroconversion from infections after prophylaxis . The third sample was taken 3 weeks after the peak of the pandemic in Singapore , between 4 to 6 weeks after the completion of prophylaxis, to assess for any additional seroconversion after prophyalxis. Questionnaires were administered to collect data on demographics, medical history, and clinical symptoms.
Written informed consent was obtained from participants, and the study was approved by the Singapore military's Joint Medical Committee (Research) and the Australian National University's ethics review board.
The nasopharyngeal swabs collected were resuspended in 3.0 ml of universal transport medium (Copan Diagnostics Inc., USA) and sent for laboratory testing. Total nucleic acid material was extracted using the DNA minikit (Qiagen, Inc, Valencia, CA, USA) according to manufacturer's instructions and subjected to real-time PCR testing for the presence of H1N1-2009 . Briefly, 5ul of nucleic extract was PCR-amplified with 0.8 uM of each of the forward (5'-GAC AAA ATA ACA AAC GAA GCA ACT GG - 3') and reverse primers (5'-GGG AGG CTG TTT ATA GCA CC-3') in the presence of 0.2 uM probe (5'-6-carboxyfluorescein-GCA TTC GCA AT(BHQ)G GAA AGA AAT GCT GG -3') using the Superscript III RT/Platinum Taq mix (Invitrogen Corporation, CA, USA) according to manufacturer's instructions. The reverse transcription (RT) was carried out at 50°C for 30 mins, the reaction denatured at 95°C for 2 mins, and PCR-amplified with 50 cycles consisting of 95°C for 15 sec and 55°C for 30 sec. The RT-PCR testing was carried out on a real-time PCR system (Applied Biosystems 7500, USA), A positive result is defined by a fluorescence growth curve that crosses the threshold line within 40 cycles. Sensitivity of this assay is about 100 copies of RNA genome equivalents per reaction (95% confidence level) .
For the blood samples, serum was extracted and tested by haemagglutination inhibition (HI) according to standard protocols (WHO CC, 1982) at the WHO Collaborating Center for Reference and Research for Influenza in Melbourne, Australia. The serum was pretreated with receptor destroying enzyme (RDE) (Deka Seiken Co. Ltd., Tokyo, Japan) at 1:4 (volume/volume), and the enzyme inactivated by addition of an equal volume of 1.6% tri-sodium citrate (Ajax Chemicals, Australia). Egg-grown A/California/7/2009 A(H1N1-2009) virus was purified by sucrose gradient, concentrated and inactivated with β-propiolactone, to create an influenza zonal pool preparation (a gift from CSL, Australia). 25 μL of Influenza Zonal Pool-A/California/7/2009 virus was incubated with an equal volume of RDE-treated serum, titrated in two-fold dilutions in phosphate buffer solution from 1:10 to 1:1280, and incubated for 1 hour. 25 μl 1% (v/v) turkey red blood cells was added to each well and read after 30 minutes. Controls for the HI assay were performed with positive ferret sera (sera collected from naive ferrets infected with A/California/7/2009 H1N1 pandemic virus and bled 14 days later), positive human sera from RT-PCR positive individuals collected in the convalescent phase, and negative human sera collected from non-infected individuals. Positive sera had high titres by both HI and MN assays against pandemic H1N1 viruses. Titres were expressed as the reciprocal of the highest dilution of serum where haemagglutination was prevented. Individual seroconversion was indicated by a four-fold or greater rise in titres between successive samples.
The data was analyzed using the Statistical Package for the Social Sciences (SPSS, version 16.0, Chicago, IL) with the level of significance set at 0.05. Categorical variables were summarized as percentages and continuous variables as means with standard error (SE); the Student's T-test was used to investigate the relationship between continuous variables.