Procedures

At the time of study initiation, influenza virus circulation in India was poorly understood.¹¹ Furthermore, only influenza vaccines with a northern hemisphere formulation were available in India; therefore, we used northern hemisphere vaccine formulations for the study. Enrolled children were vaccinated in the following periods: year 1 (Nov 24, 2009, to Jan 17, 2010), year 2 (Oct 13, 2010, to Dec 13, 2010), and year 3 (Oct 7, 2011, to Dec 18, 2011). Children were re-vaccinated in years 2 and 3 if they remained in the eligible study age range. Intramuscular, northern hemisphere seasonal inactivated split-virion IIV3 (Vaxigrip Junior [0·25 mL dose, 7·5 μg of each haemagglutinin antigen for children aged 6–35 months] or Vaxigrip [0·5 mL dose, 15 μg of each haemagglutinin antigen for children aged 3–10 years]), and IPV (Imovax Polio, 0·5 mL dose for all children) were purchased from Sanofi Pasteur (India).¹⁰ Two doses of the study vaccines were planned for year 1 (2009–10) and one dose in subsequent years for patients aged between 6 months and 8 years, and only one dose yearly for those aged 9–10 years. However, in response to the 2009 emergence of influenza A/California/7/2009 (H1N1pdm09) virus, two doses of vaccine (for both IIV3 and IPV groups) were administered to children between 6 months and 8 years of age in year 2 (2010), even if vaccinated in year 1.¹¹ In year 1 (2009–10), vaccine comprised influenza A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2), and B/Brisbane/60/2008 (Victoria lineage) strains, and in years 2 and 3, influenza A/California/7/2009 (H1N1pdm09), A/Perth/16/2009 (H3N2), and B/Brisbane/60/2008 (Victoria lineage).²

Adverse events (both expected and unexpected), including serious events, were monitored and recorded. At time of vaccination, children were observed for immediate adverse events for 30 min and received follow-up visits by study staff at days 1, 7, and 30 after vaccination. Any reports of a child’s admission to hospital or death during the entire study period were evaluated and assessed for a relationship with vaccination.

Year-round active surveillance for FARI was done via weekly household visits from November, 2009, until April, 2012.¹¹ Year 3 included only a partial year of surveillance (ended on May 1, 2012), because of the initiation of a second phase of this study with pre-monsoon (June) vaccination beginning in 2012.¹¹ FARI was defined as reported fever and any respiratory complaint (cough, sore throat, nasal congestion, runny nose, earache, or difficulty breathing) with onset in the previous week; measurement of temperature was not required. An episode of FARI was considered a new event if 2 weeks had passed from the onset of a previous episode. Throat and nasal swabs were collected (nasal swabs alone in infants), from all participants with FARI, placed in transport medium, and maintained at 4°C for up to 24 h until transported to the laboratory.

Specimens were tested by real-time RT-PCR for influenza A and B viruses,¹² and, if positive, the subtype was determined with the same method. A subset of influenza B viruses were classified into Victoria and Yamagata lineages by real-time RT-PCR as previously described.¹² A third of the real-time RT-PCR-positive specimens were inoculated into Madin-Darby Canine Kidney cells for virus isolation; isolates were subtyped by use of WHO kits for haemagglutination inhibition and guinea pig red blood cells.¹³

In year 1 and 2, a subset of children aged 3–6 years were enrolled in a post-hoc analysis (ie, not included in the protocol, but decided before starting the study) immunogenicity study (appendix). The first 531 age-eligible children in the three villages whose parents consented to blood draw were selected to participate in this substudy and 1–2 mL of venous blood was collected before vaccination. Approximately half of the children provided a second blood sample 4 weeks after the first dose, and the other half 4 weeks after the second dose of the vaccine. Serum samples were tested for influenza antibodies by use of a haemagglutination inhibition assay with turkey red blood cells and vaccine-matched antigens following WHO protocols.¹³ For influenza B virus, ether-treated antigen was used. Seroprotection was defined as a titre of 1:40 or more, seroconversion as a prevaccination titre of less than 1:10, and a post-vaccination titre of 1:40 or more; or a prevaccination titre of 1:10 or more and at least a 4-fold rise in post-vaccination antibody titre.¹⁴