Hepatitis B in India: 3% HBsAg+
"Community-based epidemiology of hepatitis B virus infection in West Bengal, India: Prevalence of hepatitis B e antigen-negative infection and associated viral variants"
Journal of Gastroenterology and Hepatology
ABHIJIT CHOWDHURY*, AMAL SANTRA*, RUNU CHAKRAVORTY+, ARUP BANERJI+, SUPARNA PAL*, GOPAL K DHALI*, SIBNARAYAN DATTA+, SOMA BANERJI, BYOMKESH MANNA¤, SUSANTA ROY CHOWDHURY, SUJIT K BHATTACHARYA¤ and DEBENDRANATH GUHA MAZUMDER*
*Department of Gastroenterology, Institute of Post Graduate Medical Education and Research, +Indian Council of Medical Research Virus Unit, Indian Institute of Chemical Biology and ¤National Institute of Cholera and Enteric Diseases, Kolkata, India
"...... An effective childhood immunization program in most of the countries in East Asia that have previously been hyperendemic for HBV is fast reducing the HBV burden in these countries. As a result, the global epidemiology of HBV is in transition now. India, with a carrier rate of 3%, contributes nearly 10% of the HBV carriers in the world. Without any organized HBV prevention program, and with 25 million live births each year, nearly 1 million HBV infections are added to the HBV pool in India yearly, contributing to its rapid expansion. In this situation, HBV epidemiology in India is likely to be an important determinant of the global HBV burden in the future. Molecular epidemiological data regarding HBV infection in India provides information about the emerging worldwide epidemiology of HBV, which is likely to shift its focus to South Asia in general and India in particular in the years to come, in view of the growing HBV burden therein, in the absence of interventions......"
Background and Aims: There is a paucity of population-based epidemiological information regarding hepatitis B virus (HBV) infection in India. The present study was planned to outline the magnitude and pattern of HBV infection, hepatitis B e antigen (HBeAg)-negative infection and the associated viral mutants in India.
Methods: A community-based epidemiological study of HBV infection was carried out in West Bengal, India. Serological markers of infection and potential risk factors for HBV transmission were determined. Among the infected individuals, HBV-DNA, genotypes and mutations in the precore (PC) stop codon and basal core promoter (BCP) regions were determined by DNA sequencing and polymerase chain reaction (PCR) restriction fragment length polymorphism methods.
Results: Of the 7653 people included in the study, 227 (2.97%) tested positive for hepatitis B surface antigen (HBsAg), of whom 204 (90%) were HBeAg-negative and hepatitis B e antibody (anti-HBe)-positive, and 78% had normal alanine aminotransferase (ALT) levels. HBV-DNA could be detected by PCR in only 32% of people. G1896A PC stop codon mutants were present in 12% of people, BCP mutants in 18% and the remainder (70%) of the HBeAg-negative infections were associated with wild type sequences in these regions.
Conclusions: This first general population-based epidemiological study of HBV infection from India suggests that HBV acquisition starts in early childhood and peaks in adulthood. Most infections in the community are e-negative and inactive. The point prevalence of PC stop codon and BCP mutants is low in this primarily inactive and asymptomatic HBV-infected population sample in eastern India.
Study site, population description and community sampling
The study was carried out in a population sample from 19 preselected villages of Birbhum, which is one of the 18 districts (equivalent to counties) in the state of West Bengal in the eastern part of India. Birbhum district comprises 19 peripheral administrative units known as community development (CD) blocks. The method of administrative control, the population distribution of each CD block, and the sampling procedure adopted for this study have been described previously.20 Briefly, one village was randomly selected from each CD block. The selection of participants, and collection of demographic data and blood samples were carried out between September 2001 and March 2002. The study population was selected by a systematic 1:3 sampling procedure from the village population register. Of the 8785 subjects (4536 male and 4249 female) preselected in this way, 7653 people (87.11%; 4089 male and 3564 female) agreed to participate. Informed consent was obtained from the participants or, in the case of minors, their parents or guardians.
Each participating subject was administered a questionnaire related to demographic details. All study procedures were reviewed and approved by the ethical committee of the Institute of Post Graduate Medical Education and Research, Kolkata, India. At the end of the interview, the participants were asked to donate 5-6 mL of blood for assessment of HBV infection, and sera were separated and transported to the laboratory where they were stored at -70¡C for subsequent study. Subsequently, the 227 hepatitis B surface antigen (HBsAg)-positive subjects detected were recalled to the hepatitis clinic of the Institute of Post Graduate Medical Education and Research for clinical evaluation and collection of blood for HBV-DNA and serum alanine aminotransferase (ALT) level analysis. Sera were stored at -70¡C for HBV-DNA analysis and subsequent studies.
Serological HBV markers
Each sample was tested for the presence of HBsAg. All HBsAg-positive samples were re-assayed in duplicate to confirm the result. HBsAg-negative samples were tested for antibody to hepatitis B core antigen (anti-HBc). Each HBsAg-positive sample was tested for anti-HBc immunoglobulin M (IgM), HBeAg, hepatitis B e antibody (anti-HBe) and antibody to hepatitis C virus (HCV). Commercially available enzyme immunoassay kits (Organon Teknika, Boxtel, the Netherlands) were used for HBsAg, anti-HBc, anti-HBc IgM, HBeAg and anti-HBe assessment. For antibody to HCV, a third generation enzyme immunoassay kit (United Biomedical Co. Ltd, Beijing, China) was used.
Hepatitis B virus (HBV) infection is the most common cause of chronic liver disease in the Asia-Pacific region.1 Nearly 40 million people out of the global HBV infection pool of 350 million are from India, a country that is yet to adopt an organized HBV-prevention program.2-4 So far, studies on HBV epidemiology and estimates of burden of infection due to HBV in India have included patient populations as well as arbitrarily chosen population samples that have been small in size.4 Such studies may fail to reflect the demographic heterogeneity of the general population.
Apart from sero-epidemiology, an important facet of global HBV epidemiology is the increasing significance of hepatitis B e antigen (HBeAg)-negative infections and HBV mutants, particularly those associated with the precore (PC) stop codon and basal core promoter (BCP) regions of the HBV genome.5-9 The prevalence of this e-chronic hepatitis B (e-CHB) and its molecular basis varies geographically.10 In addition, genotypes of HBV have emerged as important determinants of outcome of infection, severity of disease and propensity for selection of mutants.11-14 The available information on HBeAg-negative infections and HBV genotypes has been derived mainly from studies among liver disease patients.10 The majority of Asian studies of e-CHB are from East Asia, where HBV is mostly acquired perinatally.10,15,16 In India, however, the available projections suggest a predominantly horizontal childhood transmission pattern as observed in Africa, and age at seroconversion and events thereafter (including the frequency of e-CHB and HBV mutants) that may be quite dissimilar to that seen in East Asia.17-19
We report here a population-based epidemiological study of HBV infection in a systematically sampled rural population from eastern India. The study specifically aimed to investigate: (i) age-related HBV infection and exposure rates; (ii) the predominant mode of HBV transmission in this population; and (iii) the prevalence of the PC stop codon (G1896A) and BCP mutation in HBeAg-negative infection in this community sample.
The demographic characteristics of the participants were comparable to the general population of Birbhum district as well as West Bengal (Table 1). The participation rate among female subjects (83.88%) was lower than that for male subjects (90.15%).
Seroprevalence by age and gender
A total of 227 individuals tested positive for HBsAg. None of the HBsAg-positive subjects were positive for anti-HBc IgM or HCV antibody. The prevalence of HBsAg positivity in this population was 2.97% (95% CI 2.59-3.35). The prevalence among male subjects (3.47%; 95% CI 2.9-4.04) was significantly higher (P < 0.01) than that among female subjects (2.38%; 95% CI 1.88-2.88). The infection rate was low in subjects younger than 5 years of age (1.94%), but progressively increased with age, reaching a peak in the fourth decade of life (3.56%) and declining thereafter (Fig. 1).
The prevalence of anti-HBc, a marker of HBV exposure, was 16.49% (95% CI 15.65-17.33) overall, but varied from 16.16% (95% CI 15.01-17.31) in male subjects to 16.87% (95% CI 15.63-18.11) in female subjects. The present study showed that exposure occurred at a very young age (<5 years) in 7.73% of participants, and gradually increased to a prevalence of 29.86% in the >=60 years age group.
Clinical and biochemical characterization of HBsAg-positive subjects
All of the 227 HBsAg-positive subjects were clinically asymptomatic. Most (177; 78%) had normal ALT levels (median value 28 IU/L) and only 50 (22%) had elevated ALT levels.
HBe and anti-HBe status of HBsAg-positive subjects
Among the HBsAg-positive individuals, 204 (89.86%) were positive for anti-HBe, whereas only 16 (7.05%) were positive for HBeAg. Seven samples were negative for both HBeAg and anti-HBe. Elevated serum ALT was observed in 15 (93.75%) HBeAg-positive subjects, whereas only 35 (17.16%) anti-HBe-positive subjects had raised serum ALT levels (Table 3).
HBV-DNA and its quantification
Serum HBV-DNA was detected by PCR in 73 (32.15%) samples out of the 227 samples tested. Fifteen out of the 16 HBeAg-positive subjects tested positive for HBV-DNA by PCR, whereas 58 (28.43%) anti-HBe positive subjects were positive for HBV-DNA. Serum HBV-DNA was detected by using the Digene Hybrid Capture assay in 20 (27.39%) out of the 73 samples that were HBV-DNA-positive according to PCR. Eleven (73.33%) HBe-positive individuals had detectable levels of HBV-DNA (median [range] 427.3321 x 106 copies/mL [6.0385-989.685 x 106]) compared with nine (15.52%) HBV-DNA-positive, anti-HBe-positive individuals (median [range] 47.515 x 106 copies/mL [0.1524-1491.406 x 106]).
Samples that tested positive to HBV-DNA according to PCR were genotyped, revealing a single genotype in 91.78% of isolates (Table 3). HBV genotype D (83.56%) was the predominant genotype in this population. HBV genotype C was detected in only seven subjects (9.59%), and five subjects (6.84%) had mixed infection with genotypes C and D.
BCP and PC mutations
The BCP sequence at nt 1762/1764 and PC sequence at nt 1896 remained wild type in the majority of cases (70%). In all of the HBeAg-positive individuals, the BCP sequence at nt 1762/1764 remained wild type and the PC sequence at nt 1896 remained wild type in 14 (93.33%) individuals. In contrast, nt A1762T1762 and/or nt G1764A1764 mutations in the basal core promoter region were detected in nine (15.52%) anti-HBe-positive individuals (Table 3). The nt G1896A1896 mutation was detected in five (8.62%) of the anti-HBe-positive subjects Two subjects (3.45%) had both PC and BCP mutations. In both these cases, the genotype was D. Of the six PC mutants, five belonged to genotype D and one to genotype C, whereas of the nine BCP mutants, seven belonged to genotype D and two to genotype C. Overall, of the 61 genotype D infections, 14 (22.95%) were associated with PC or BCP mutants.
Risk factors for HBV transmission
The frequency of various risk factors associated with HBV infection and the calculated OR, estimated by univariate analysis, are shown in Table 4. Multivariate analysis revealed that of these various factors, being older than 20 years of age (OR 1.5; 95% CI 1.3-1.7), poverty (OR 6; 95% CI 4.7-7.6), a low level of education (OR 7.8; 95% CI 5.1-12.0), use of reusable glass syringes (OR 2.8; 95% CI 2.3-3.4), being shaved by a community barber (OR 2.5; 95% CI 1.8-3.4), and being born at home (OR 3.5; 95% CI, 2.9-4.2) were associated with heightened transmission of HBV in this community.
The study we report here is the first community-based epidemiological study of HBV infection in India. A fairly large population sample was systematically obtained by using a multi-stage sampling methodology among the inhabitants of a wide but structurally defined geographic area that forms the basis of administrative territorial functioning in India. The validity and representative character of the sample were further upheld by the demographic similarity of the sample population with that of the local population as a whole. A low prevalence of PC and BCP mutants, despite a very high prevalence of HBeAg-negative infection, mostly inactive, characterize the asymptomatic HBV-infected cohort in the present study.
HBeAg-negative infections are assuming increasing significance in view of their high frequency globally, as well as because a subset of this serological state might connote active and ongoing viral replication, a condition called e-CHB.5,7,10 Most studies of e-CHB and its molecular characteristics have been carried out among liver disease patients, and thus might not reflect its significance in the vast majority of asymptomatic HBV-infected subjects in the general population. The majority (92%) of the HBV-infected individuals in the present population were HBeAg-negative in all the age groups studied, and most of them (78%) had normal ALT levels. This is consistent with the reported 70-95% frequency of HBeAg-negative infection in the asymptomatic population and blood donors in various studies all over the world, the majority of whom have normal amino transferases.5,15,24,25 Despite using a sensitive nested PCR reaction, the overall level of HBV-DNA positivity is low (32%) in the present study in comparison with the usual >50% positivity rate reported among HBV-infected subjects in diverse clinical states.10 This is intriguing, but is possibly due to the study design, which included inactive and asymptomatic subjects only, who were picked up by community sampling. In Italy, of a large follow-up cohort of 296 HBV-infected blood donors, only 30% were positive for HBV-DNA at baseline, and the majority had normal aminotransferase levels.26 Furthermore, even among e-CHB patients in a large study from Hong Kong, the frequency HBV-DNA positivity as determined by PCR was only 42% in the non-cirrhotic chronic hepatitis B patients with normal amino transferase levels.15 Most of the HBeAg-negative subjects in our study (83%) had normal transaminase levels, 73% had undetectable HBV-DNA by using a sensitive PCR method, and HBV-DNA could be detected by a non-amplification assay in only 4% of the entire HBeAg-negative cohort. Thus, only the 4% of HBV-infected subjects with elevated transaminases, along with those who had detectable HBV-DNA in a non-amplification assay conformed to the definition of e-CHB. We acknowledge that the present report is a single-point cross-sectional assessment. In the HBeAg-negative phase, the amount of HBV-DNA and serum transaminases often show fluctuations over time, particularly with the emergence of HBV mutants. This might result in an underestimation of HBeAg-negative hepatitis in the present report.
The overall prevalence of the G1896A PC mutant was relatively low, only 12%. Similarly, the prevalence of the TA changes (A1762T and G1764A) in the BCP region was 19%. Because of the presence of both the mutations in 3% of subjects, the overall frequency of the PC stop codon mutation and the basal core promoter mutation was 28%. The remainder of the HBeAg-negative infections were associated with wild type sequences in the nt 1896 and BCP region.
The low prevalence of the G1896A mutation in our study contrasts with its reportedly high prevalence (37-85%) among HBeAg-negative individuals with diverse outcomes of HBV infection.5,24,27,28 This is despite the fact that all the HBV sequences in our study had T at 1858, and most belonged to genotype D. There is a wide variation among studies regarding the prevalence of PC mutants in e-CHB. Its prevalence is highest in Mediterranean countries, where more than 85% of cases of e-CHB are associated with PC mutants.5,10 A similarly high prevalence has been reported in Korea (94%) and among blood donors in Taiwan (70%).16,24 A somewhat lower prevalence of PC mutants has been reported in a study of chronic hepatitis B patients from Hong Kong (37%) and also from Gambia (27%).15,29 A low prevalence of these PC mutants has also been reported from Western India, where 62% of e-negative chronic hepatitis B infection has wild type sequences in the PC region.30 HBV mutants exist as quasispecies, in a dynamic equilibrium (mix) with the wild type viral sequences, a process that is tightly regulated by host immune pressure, the replication competence of the mutant virus, and the evolutionary process of natural selection.31 It has been recently shown that the genetic diversity of HBV is highest during periods of high viral replication and a mounting immune response.32 Moreover, the mutation rate appears to be low, with little variability of sequences over time, in occult HBV infection, in which the host immune response is relatively quiescent.33 The majority of the HBeAg-negative subjects in the present study had very low levels of HBV-DNA, and biochemically inactive infection. It is possible that the relatively quiescent and inactive virological status and stable immunological status of HBV in the majority of the HBV-infected subjects we studied negatively influenced the emergence and evolution of PC mutants as the predominant species over the wild type sequences.
A low prevalence of BCP mutants was also noted in our study (19%). There had been a trend of higher prevalence of BCP mutants (66-90%) among patients with hepatocellular cancer and advanced liver disease as compared with asymptomatic subjects (11-47%).34 This is in accordance with the fact that the molecular virology of HBeAg-negative infections are heterogeneous, and are not invariably associated with the development of and predominance of viral variants.
We found that genotype D was the most frequent HBV genotype in this population (90%), either alone (83%) or in combination with genotype C (7%). Genotype D had previously been shown to be the predominant HBV genotype among chronic liver disease patients in northern and western India.30,35 The prevalence of genotype C had previously not been reported from India. However, it is the predominant genotype in neighboring Bangladesh.36 We note that the major population groups of Bangladesh are ethnically and linguistically closely related to the population we studied. It is known that the frequency distribution of HBV genotypes is geographically structured,16 and the geographic structuring of HBV genotypes is known to correlate with the anthropological history of human migration.37 More studies on HBV genotype distribution in India are required from this geographically and ethnically diverse country to test how well this distribution correlates with human migration in India.
The sero-epidemiology of HBV infection in this rural community provides information that might be useful in planning prevention strategies. The overall rate of HBsAg positivity in this study (2.97%) is similar (3-4%) to the average carrier rate reported from India.4,19 However, the peak age of acquisition of infection observed in our study is different from that reported by other studies. Thus, we found that the HBsAg positivity rate peaks in middle age, rather than in early childhood, as shown in other studies. This may be partly explained by the occupational and cultural practices of the predominantly poor rural population included in the present study. Middle-aged people engage in relatively more physical labor related to agriculture relative to the rest of the rural population. This frequently exposes them to minor cuts and bruises, which are mostly dealt with by untrained village paramedics. Moreover, the non-indicated use of parenteral injections is common in this rural community. This, along with non-sterile injection practices, would facilitate the transmission of HBV as well as HCV in this rural population.20,38 Intra-family transmission of HBV might also contribute to this rather delayed peak of infection observed in our study. Social factors, such as poverty and low levels of education, were positively correlated with heightened transmission, as was unsafe injection and obstetrics practices. These social factors are common to the developing world, particularly countries in the Asia-Pacific region. Health education is likely to play an important role in preventing HBV transmission in these countries.
The population-based data on the prevalence of HBV presented here is likely to improve our understanding of the evolution of these viral variants and their pathogenetic significance in disease states. Nearly three-quarters of the global HBV infection pool is in Asian countries. An effective childhood immunization program in most of the countries in East Asia that have previously been hyperendemic for HBV is fast reducing the HBV burden in these countries. As a result, the global epidemiology of HBV is in transition now.1,39 India, with a carrier rate of 3%, contributes nearly 10% of the HBV carriers in the world. Without any organized HBV prevention program, and with 25 million live births each year, nearly 1 million HBV infections are added to the HBV pool in India yearly, contributing to its rapid expansion.19 In this situation, HBV epidemiology in India is likely to be an important determinant of the global HBV burden in the future. Molecular epidemiological data regarding HBV infection in India provides information about the emerging worldwide epidemiology of HBV, which is likely to shift its focus to South Asia in general and India in particular in the years to come, in view of the growing HBV burden therein, in the absence of interventions.