Seroprevalence of Antibodies to Avian Influenza Virus A (H5N1) among Residents of Villages with Human Cases, Thailand, 2005

No evidence of influenza virus A (H5N1) neutralizing antibodies was found in residents of 4 villages where human cases had occurred the previous year.

T hree apparent waves of highly pathogenic avian infl uenza virus A (H5N1) infection in humans occurred in Thailand from early 2004 through 2006; these waves, which corresponded to infl uenza (H5N1) outbreaks in poultry, resulted in 25 confi rmed human cases and 17 deaths (1)(2)(3)(4). However, the frequency of asymptomatic and clinically mild cases of infl uenza (H5N1) infection was unknown in areas where these outbreaks occurred. In 2005, we conducted a cross-sectional seroprevalence study of infl uenza virus (H5N1) antibodies among residents of 4 rural villages in Thailand where at least 1 human infl uenza (H5N1) case had occurred in 2004. Backyard poultry farming is common in these villages, but the villages have no live poultry markets.

Methods
The study was conducted during October 11-27, 2005, among residents of 4 rural villages in central and northern Thailand where infl uenza (H5N1) outbreaks in poultry and human infl uenza (H5N1) cases had occurred: village A in Prachin Buri Province (1 confi rmed case), village B in Kamphaeng Phet Province (1 probable case, 1 confi rmed case), village C in Sukhothai Province (1 confi rmed case), and village D in Phetchabun Province (1 confi rmed case) ( Figure). Illness onset in these case-patients occurred from August 31 through October 8, 2004. Residents of any of the villages for at least 2 weeks before and after illness onset of the respective case-patient in each village were eligible to participate in the study. Participants were enrolled by random selection from lists of village residents or by convenience sampling. Village residents were excluded if they had infl uenza (H5N1) diagnosed from August 17 through October 22, 2004, were <18 years of age and did not have parental consent, had an underlying coagulopathy, or were taking anticoagulant drugs within 2 weeks of enrollment. Written informed consent was obtained from all study participants or their proxies.
Using a standard questionnaire, trained interviewers collected demographic and exposure data through brief face-to-face interviews with study participants. Exposure was defi ned as either direct contact (touching) or close contact (within 1 m without direct contact) with chickens or other poultry or with a person with confi rmed infl uenza (H5N1) infection. A 5-mL blood specimen was collected Seroprevalence of Antibodies to Avian Infl uenza Virus A (H5N1) among Residents of Villages with Human Cases, Thailand, 2005 1 from participants >5 years of age, and a 3-mL specimen was collected from those <5 years of age. Serum samples were separated at a local hospital and transported on wet ice to a laboratory within 48 h after collection. Serologic testing by microneutralization (MN) assay was performed in an enhanced biosafety level-3 containment facility in accordance with a slightly modifi ed version of a protocol described previously (5-7). Infl uenza virus A/Thailand/1(KAN-1)/ 2004 (H5N1) was selected for the MN assay because of its antigenic similarity to infl uenza virus (H5N1) isolates from humans in Thailand (2). Immunofl uorescence with use of 293T cells transfected with hemagglutinin H5N1 recom-binant plasmid as the test antigen was used to confi rm MN assay results. In accordance with our modifi ed protocol, we considered an infl uenza virus (H5N1) neutralizing antibody titer >40 (equivalent to >80 in other protocols) to be a positive result (5)(6)(7).
Epi Info 2002 (Centers for Disease Control and Prevention, Atlanta, GA, USA) was used to enter and analyze study data. Mean, median, and proportion values were calculated for variables and compared by using bivariate analysis. The χ 2 test was used for most analyses, analysis of variance was used to compare means from the convenience sample with those from the random sample, and the Fisher exact test was used if expected cell values were <5. Differences between the 2 sample groups were considered signifi cant at p<0.05. The study was approved by the Ethical Review Committee for Research in Human Subjects, Thai Ministry of Public Health.

Results
The study population consisted of 901 participants: 228 from village A (28.1% of village residents), 203 from village B (28.4%), 209 from village C (30.5%), and 260 from village D (19.6%). Their median age was 40 years (range 2-101 years), and 42.4% were male. The 901 participants were enrolled in 2 ways: 131 (14.5%) by random selection (out of 838 randomly selected villagers: 15.6% participation), and 770 (85.5%) by convenience sampling. The 2 groups of study participants did not differ signifi cantly by demographic characteristics, history of illness, or exposure to poultry (Table 1). Most participants (68.1%) reported direct or close contact with backyard poultry, 25.7% reported direct or close contact with sick or dead chickens, and 7.1% reported close contact with a person with confi rmed infl uenza (H5N1) infection (Table 1). Of 110 participants who reported a history of acute respiratory symptoms, 74.5% reported direct or close contact with backyard poultry, 31.8% reported direct or close contact with sick or dead chickens, and 13.6% reported close contact with a person with confi rmed infl uenza (H5N1) infection (data not shown). All participants were seronegative for infl uenza virus (H5N1) neutralizing antibodies (Table 2).

Discussion
Participants in this study were from villages in central and northern Thailand where widespread, confi rmed outbreaks of infl uenza (H5N1) infection in poultry and at least 1 human infl uenza (H5N1) case had occurred during 2004. A substantial proportion of participants reported exposure to backyard poultry, including contact with sick or dead chickens, the primary risk factor for infl uenza (H5N1) infection (8,9). Nevertheless, we found no serologic evidence of mild or subclinical infl uenza (H5N1) infection, suggesting that clade 1 infl uenza virus A (H5N1) strains circulat-ing in Thailand among backyard poultry during 2004 did not transmit easily to our study population.
Our fi ndings differ from those from a study of poultry workers in Hong Kong, among whom the estimated seroprevalence of infl uenza virus (H5N1) neutralizing antibodies was 10% during the 1997 outbreak (10). The Hong Kong poultry workers, however, likely had much greater intensity of exposure to poultry infected with infl uenza virus (H5N1) than our study population had. Furthermore, the clade 1 infl uenza virus (H5N1) strains that infected poultry and humans in Thailand during 2004 were antigenically and genetically distinct from the clade 0 infl uenza virus (H5N1) strains that caused the 1997 outbreak in Hong Kong (11). Our fi nding of no serologic evidence of asymptomatic or mild infl uenza (H5N1) infection among Thai villagers is consistent with fi ndings from smaller infl uenza virus (H5N1) seroprevalence studies among workers in live poultry markets in the People's Republic of China (12), among villagers exposed to backyard poultry infected with clade 1 infl uenza virus (H5N1) in rural Cambodia (13), among poultry workers exposed to poultry infected with clade 2.2 infl uenza virus (H5N1) in northern Nigeria (14), and among poultry farmers exposed to poultry infected with clade 1 infl uenza virus (H5N1) in Thailand (7). Results of studies conducted since 2004 thus suggest that the risk for infl uenza (H5N1) infection is low among persons exposed to infected poultry; however, our fi nding of no serologic evidence of asymptomatic or mild infl uenza (H5N1) infection among Thai villagers suggests that the high case-fatality proportion in Thailand (17 deaths among 25 persons with confi rmed infection) may accurately refl ect the severity of the infection in Thailand.
Our study had 3 notable limitations. First, because study participants, most of whom were enrolled by convenience sampling, were generally older than the populations of the villages in which they resided (Thai Ministry of Public Health, unpub. data), our fi ndings may not be generalizable to these villages' populations. Second, because the study was conducted in 2005, some participants may not have accurately recalled relevant exposures or symp- Further data are needed on the natural history and kinetics of the immune response to infl uenza (H5N1) infection over time among severely ill persons who survived, as well as among those with clinically mild illness. Such prospective serial data may help researchers interpret the signifi cance of low levels of infl uenza virus (H5N1) neutralizing antibody titers, as well as the results of additional seroprevalence studies. In addition, because infl uenza virus (H5N1) strains continue to evolve, additional seroprevalence studies to estimate human risk for infection are needed worldwide among populations exposed to the virus, including poultry workers, rural residents, market workers, farm workers, healthcare workers, and family members of infected persons.  <5  5  10  20  40  80  >80  A  228  227  1  0  0  0  0  0  B  204  202  2  0  0  0  0  0  C  209  202  6  0  1*  0  0  0  D  260  257  2  1 †  0  0  0  0  Total  901  888  11  1 1 0 0 0 *Serum obtained from a 52-year-old woman (farmer) in village C without history of respiratory symptoms who reported contact with a sick/dead chicken and live poultry. †Serum obtained from an 18-year-old man in village D without history of respiratory symptoms who reported contact with a sick/dead chicken and live poultry.