Seroepidemiology of pandemic influenza A (H1N1) 2009 virus infections in Pune, India

The first pandemic influenza A (H1N1) 2009 case in India was reported in Hyderabad city on 16^th May 2009 [1]. Pune city reported the first pandemic influenza A (H1N1) 2009 case on 22^nd June 2009. The first pandemic death in Pune on 3^rd August 2009 caused panic in the general public. Subsequently, widespread transmission was reported in community [2].

The critical need of population-based serology has been advocated to determine the extent of infection and age-specific infection rates [3]. Wide geographical variations are expected in the incidence of infection in different populations. Therefore, large serosurveys covering different areas and age groups at different times are necessary to understand the extent of the infection in community. Further, seropositivity in population may provide appropriate denominator for pandemic severity estimates and the data for delineation of risk populations for priority in vaccination [4].

Several studies have been conducted to address the issues of cross-reactivity or pre-existing immunity using sera from the archives or collections from the routine diagnostic or screening programmes [5–10]. Some studies were done involving hospital staff [11], blood donors and patients without acute respiratory illness [12].

Pune is one of the cities in India reporting higher number of cases and deaths during this pandemic [2]. We report results of serosurveys undertaken in Pune in the risk groups, general population and household contacts of the PCR-confirmed cases. We also tried to detect the change in seroprevalence over time by resurveys in the selected risk groups.

This is the first report of population-based large-scale serological surveys undertaken during the continuing influenza A (H1N1) 2009 pandemic in India. We also report the repeat surveys among adults in the selected risk groups for new infections over 5-13 weeks period and secondary infections among household contacts of the PCR-confirmed cases. We have surveyed a large number of school-aged children in the study. A recent report from New Zealand investigated 521 pre-pandemic sera and surveyed 1156 subjects (including 361 children) from community and 540 healthcare workers at the end of first wave [16]. Another study from Singapore investigated 727 adult community participants and 537 hospital staff [17]. A review of seroepidemiological studies clearly indicated the need of more studies using standardized methods to be able to accurately estimate global infection rate [18].

Seropositivity in the pre-pandemic period was negligible (0.9%) in our study. Other studies reported seropositivity in pre-pandemic sera at different levels [5–10, 19]. A recent study from Singapore observed no or minimal cross-reactivity [17, 20]. Indian population has experienced co-circulation of seasonal and pandemic viruses with almost equal contribution during the study period [2]. Also, seasonal influenza vaccination uptake is negligible. Seropositivity in the PCR-confirmed cases was 73.8% considering a cut off of 1:40 and 81.5% considering 1:10. Similar seroconversion of 89.1% in PCR-confirmed cases was reported using a titre cut-off of 1:32 in England [9].

The highest seropositivity was observed in the school children and the staff. Seropositivity was higher among students in the divisions with PCR-confirmed cases than in other divisions. Repeat serosurvey in school staff indicated significant increase in seropositivity after 5 weeks. The academic school calendar starts from June and continues through March. Seropositivity of 52% among 415 school children and staff in a residential school in Panchgani, India using a cut off titre of 1:10 [21] was higher than 31.7% in our study. Similar observations on incidence of influenza disease were reported during the school outbreaks in London [22] and New York [23]. In the UK, schools were identified as the most important source of infection [24]. Higher seropositivity in schools than households and communities could be attributed to the sustained close contact favouring transmission among the susceptible age groups [16].

In August, seropositivity among general practitioners (4.9%) was higher than the hospital staff (2.8%). During the repeat serosurvey, seropositivity was almost three times higher among general practitioners after 13 weeks. Among hospital staff, seropositivity was two times higher after 9 weeks. The higher seropositivity among general practitioners may be attributed to the higher possibility of contact with the patients as general practitioners are the first point of contact for influenza-like illness. Lack of infection-control practices may be another important reason. A study in Taiwan [11] reported 20% seropositivity among 295 hospital staff. Seropositivity of 29.6% in hospital staff and 25.3% in primary healthcare staff at the end of first wave in New Zealand indicated no additional risk to the healthcare workers as compared to general community (26.7%) [16]. The lower seropositivity among hospital staff in our study may be due to the widespread use of both therapeutic and prophylactic antiviral therapy and other infection-control measures.

In our study, higher seropositivity was noted in 15-19 years age group in both schools (42.2%) and in general population (20.3%). Seropositivity was lower in elderly population indicating low infection rates (Fig. 1). The incidence, severity and mortality of pandemic H1N1 disease was also lower in elderly than in other age groups in the study area [2]. The similar lower incidence of infection was reported in elderly in New Zealand [16]. This is also similar to the studies in some other countries [5–10]. It indicates that the pre-existing immunity and cross-reactivity levels vary in populations and age groups [20]. Seropositivity among elderly in China has been reported to be 9.4% in pre-pandemic and 42.5% in post-pandemic sera [25]. The likely hypotheses being forwarded include the differential exposures [26], the role of cell-mediated immunity [27] and immune epitopes or genetic differences [6].

Seropositivity of 25.6% in household contacts of the PCR-confirmed cases and influenza-like illness in 14% of those seropositive is noteworthy. The incidence of influenza-like illness among household contacts in our study was similar to the secondary attack rate of 13% detected in the USA [27]. The transmission of the pandemic H1N1 2009 virus is reported to be less-efficient in households as compared to the school settings [23]. It is also likely that the infections were mild, unnoticed or not remembered by the household members [23]. The widespread use of oseltamivir therapy in suspected cases and their household contacts, hospital or home isolation and the other prevention practices might have contributed to the lower infections in the household contacts. Post-exposure oseltamivir prophylaxis has been reported to reduce the rate of infection during outbreaks [28].

In our study, symptomatic infection was around 10%. Incidence of influenza-like illness among those identified with the serological infections in schools, hospital staff and general practitioners was similar to that reported in France [29], New Zealand [16, 30] and the USA [31]. The 20-40% infections were reported to be symptomatic in the 1957 and 1968 pandemics [32, 33]. The percentage of asymptomatically infected subjects was estimated at 2-10 times of the clinical cases during the current pandemic [9, 34]. The asymptomatic infections seem to play an important role in transmission of the pandemic H1N1 virus as evidenced recently by RNA detection in persons with subclinical infection [35].

The possibility of underestimation of seropositivity may be due to the specificity and the threshold titre of HI assay [16]. This is evident from the data presented with a minimum detectable titre of 1:10 as compared to 1:40. The sera treated with turkey red blood cells were few and randomly distributed. We do not consider that these led to biases in the study. The pandemic H1N1 infections are widespread and mostly subclinical or mild. Asymptomatic or mild infections are reported to have low antibody titres as compared to clinically manifested patients [11]. There is under-representation of some age groups in general population. Information about underlying health conditions of 222 subjects sampled during pre-pandemic period could not be made available. Comparison of serology data with the incidence of clinical disease was not possible due to the non-availability of baseline seropositivity in representative pre-pandemic sera, non-reliability of disease incidence estimates due to changing policies of sampling and testing of throat swab specimens at different time periods, and co-circulation of seasonal and pandemic viruses in the community. We have certain limitations in estimation of incidence of infection as we could study only the adults by repeat surveys and limited number of household contacts of PCR-confirmed cases. Different levels of morbidity and mortality have been reported in different cities of India. Further serological surveys in different cities in India may help us understand infection rates and the factors responsible for such variation.

We conclude that the schools played an important role for transmission of pandemic influenza A (H1N1) 2009 virus infections. General practitioners were at higher risk than the hospital staff. There is a need to provide health education regarding infection-control practices to these groups. The study may also help in prioritization for vaccination.