This large nationwide study of Chinese adults presents findings on both non-genetic and genetic risk factors associated with chronic HBV infection. While HBsAg prevalence was 3% in the overall cohort, this varied greatly by study site, with younger age, male sex, socioeconomic factors, alcohol intake and BMI strongly correlated with HBsAg positivity. We also replicated findings of existing GWAS for genetic variants previously associated with chronic HBV, and showed that several of these were additionally associated with risk of CLD. This is the largest population-based study in China to assess both non-genetic and genetic risk factors associated with chronic hepatitis B infection, highlighting the role that numerous risk factors may play in chronic hepatitis B infection.

Several large nationwide surveys in China have previously reported regional variation in HBsAg prevalence. Historically rates of chronic HBV have been higher in rural, western regions of China, but with widespread urbanization and mass migration of rural workers to large coastal cities and eastern provinces, these patterns have been shifting²⁶. A nationally representative serosurvey conducted in 2006²⁷ of ≈ 41,000 people aged 1–59 years recruited from 31 provinces measured HBsAg in serum blood samples using ELISA, reported higher HBsAg prevalence in western (8.3%) and rural (7.3%), compared to eastern (6.5%) and urban (6.8%) areas. However, there was large variation within these broad geographical regions—for example in Western China, HBsAg prevalence was 11.6%²⁸ and 3.9%²⁹ in Sichuan and Gansu province respectively. A more recent study in 2 million men aged 21–49 years in rural China enrolled in the National Free Preconception Health Examination project (NFPHEP) between 2010 and 2012 reported HBsAg prevalence of 7.7%, 5.5% and 6.5% in Eastern, Central and Western China respectively³⁰, while other large population based cross-sectional studies, mostly conducted in Eastern China, have reported higher HBsAg prevalence among areas of lower socioeconomic status³¹, coastal areas³² and areas containing a higher proportion of immigrants³³. In CKB, we also found large geographic variation in HBsAg prevalence, where study sites in southern and eastern China had higher HBsAg prevalence than western and north-eastern sites, and urban sites tended to have higher prevalence than rural sites. This regional variation in HBsAg prevalence highlights both the need to draw on populations from diverse areas of China, where the relative importance of correlates with chronic HBV may vary, and that pooled estimates across large regions may obscure important intra-region differences in HBsAg prevalence. The lower prevalence of HBsAg positivity in CKB compared to the 2006 National serosurvey²⁷ which reported an overall HBsAg prevalence of 7.2% (30–60 years: 8.6%) may reflect this regional variation in HBsAg prevalence, in addition to the CKB cohort including adults aged over 59 years (with lower HBsAg prevalence), and the HBsAg test used in CKB having lower sensitivity than ELISA³⁴.

The trend of HBsAg positivity in relation to age has shifted over recent decades; as the proportion of vaccinated younger adults increases, HBsAg prevalence peaks at older ages. For example, the 2006 National serosurvey²⁷ reported peak HBsAg prevalence in 20–29 years olds (10.5%), while another large cross-sectional study of ≈ 87,000 adults recruited in 2009–2010 in Eastern China reported HBsAg peaked in participants aged 35–40 years³¹ at 11.6%, and a third population based study in 2013 in Western China reported peak prevalence in 53–57 year olds at 10.5%³⁵. Prevalence tends to decrease with age beyond this peak, as more of the population undergoes HBsAg seroclearance, and a proportion of the infected individuals are diagnosed and treated, or die from liver related disease. The inverse association observed between older age and HBsAg prevalence is consistent with this, as participants in CKB are from the pre-vaccine era and thus largely unvaccinated.

The higher levels of HBsAg positivity among men compared with women has been described in past studies, including an absolute difference of 3% in the 2006 National serosurvey²⁷ (8.6% men; 5.7% women) and up to a twofold relative difference in odds of HBsAg positivity in other large population based studies^36,37,38. This is similar to our finding of a 1.4 fold greater risk in HBsAg positivity in men than in women. This sex disparity is hypothesized to be related to a differential HBV-related immune response where immune clearance of serum HBsAg is achieved in a higher proportion of women than men, in addition to women gaining better protection from HBV vaccination³⁹.

Past studies in China have also reported on the association between education level and HBsAg positivity^{27,36,40,41,42}, with most showing an inverse association, consistent with our findings. Findings for occupation have been mixed, although agricultural work has been associated with HBsAg positivity in several past studies^27,40,43, consistent with the higher prevalence of HBsAg positivity in agricultural workers in our study, which may reflect geographic variation and socioeconomic status. Two past studies in Henan³⁷ and Jilin⁴⁴ also reported no association between smoking and HBsAg positivity, while few studies have examined the association between self-rated health, alcohol intake or BMI and HBsAg positivity. Self-rated health is likely a marker of socioeconomic status, consistent with higher HBsAg prevalence among participants with lower levels of education described in past studies. Two existing studies reporting the association between HBsAg and BMI had conflicting findings—one population based study of ≈ 400,000 adults in Sichuan province found participants with BMI ≥ 25 kg/m² were significantly more likely to be HBsAg positive compared to normal weight (BMI 18.5–25 kg/m²) counterparts (OR 1.08, 95% CI 1.05–1.11)⁴⁴; while the other reported⁴⁵ in ≈ 3500 adults in Shanghai, an inverse association with odds of HBsAg positivity and BMI, where participants with BMI ≥ 28 kg/m² were 49% (95% CI 6–72%) less likely to be HBsAg positive than participants of normal weight. The association we observed between HBsAg positivity and BMI is consistent with this latter study, and may reflect socioeconomic status or reverse causation, whereby participants with chronic HBV may have lost weight in the course of their illness. Furthermore, a U-shaped association between BMI and cirrhosis in CKB has been previously described⁴⁶. Two past studies on Chinese adults in conducted in Sichuan and Guangdong province, reported lower risk of HBsAg positivity among occasional or low to moderate alcohol drinkers compared to never drinkers^35,47, while another study³² conducted in Zhejiang province found that any drinking was associated with a 30% (27–34%) higher risk of HBsAg positivity compared to no drinking. The apparent protective association between alcohol intake and HBsAg positivity in our study may reflect altered behaviour related to alcohol intake among known HBV positive people or people with CLD, for whom abstaining from alcohol may be recommended.

Since the first GWAS on chronic HBV was conducted in 2009, the number of SNPs significantly associated with chronic HBV has expanded from SNPs at HLA class II loci, to include those at HLA class I loci, non-classical HLA SNPs and non-HLA SNPs. Most previous GWAS were based on diagnosed clinical conditions such as CLD or liver cancer^{10,12,13,14,16,22}, meaning that participants with different HBV phenotypes such as less severe disease, or chronic HBV without progression to liver disease, may be under-represented. Furthermore, although most GWAS have been performed in participants of East-Asian ancestry, several used populations from particular geographic areas, and tended to be modest in sample size, ranging from between ≈ 4000¹⁶ to ≈ 15,000²³ people. Our study included > 65,000 participants and replicated the associations of 17 SNPs with HBsAg positivity. We did not replicate rs7000921 (INTS10) previously reported in a Chinese ancestry case–control study of ≈ 9500 people²⁴. However, the phenotype examined in that study was HBsAg positivity among anti-HBc positive individuals, which we had limited power to explore due to the small size of the sub-cohort with anti-HBc data.

Existing evidence suggests there is little overlap between SNPs associated with HBsAg positivity and those associated with progression to HBV-related liver disease, where a systematic review of published SNPs associated with different HBV phenotypes found that the overlap occurred between SNPs associated with HBV positivity and HBV vaccine response, rather than with disease progression¹¹. Most past GWAS on disease progression have reported HCC progression among HBsAg positive participants. These differences in population and phenotype reported in past GWAS may explain our finding of 14 of 18 SNPs being associated with CLD: we examined CLD more broadly among all participants regardless of HBsAg status, with limited power to investigate progression to CLD among HBsAg positive participants.

The strengths of this study include its large size from diverse geographic areas, both middle- and older-aged population and breadth of information that enabled investigation of a wide range of both conventional and genetic factors associated with HBsAg positivity. To date risk factors associated with chronic HBV have been focused on factors related to mother to child transmission and age of infection; while evidence around associations of socioeconomic, behavioural and medical factors with chronic HBV among adults is lacking. Given that the key burden of chronic HBV related disease occurs in middle-aged and older adults, and the low diagnosis rate of chronic HBV in China, our study findings help fill the evidence gap. However, our study also has several limitations. First, the RDT HBsAg test has lower sensitivity than laboratory-based tests such as ELISA used in most existing smaller studies³⁴, leading to a likely underestimation of HBsAg prevalence, which may be more pronounced in those with lower viral load, such as older participants. Second, due to lack of other hepatitis data (e.g. anti-HBc, e-antigen) in the whole cohort, we were only able to compare HBsAg positive to HBsAg negative individuals. We therefore were unable to detect individuals with occult infection, or investigate other phenotypes of interest such as HBsAg viral clearance. Although this approach is consistent with the approach taken by past GWAS^12,14,22, several others were able to investigate HBsAg clearance among a cohort of exposed participants^13,23,24. Third, we investigated SNPs identified in previous GWAS in different populations, mainly from the HLA region, but did not explore further the likely multiple independent effects from various SNPs in this part of the genome, which may vary among different populations. However, our work nonetheless adds to the evidence regarding the likely association between HLA variance and HBsAg positivity in Chinese populations. Four, we did not have information on other relevant risk factors, including drug use, number of sexual partners and vaccination status, in addition to information on viral subtype, which is an important source of disease heterogeneity. Finally, this is a cross-sectional study investigating associations between a range of non-genetic and genetic factors with prevalent chronic HBV, which does not capture risk of incident infection.

In summary, this study adds to the current knowledge of factors associated with HBV chronicity in adults, which may help to inform targeted HBsAg screening, enabling improved diagnosis and capturing of individuals on the HBV care continuum. Future research combining conventional and genetic risk factors, including viral genotypes, could further improve knowledge about the risk of HBV chronicity and disease progression.