Genotypic distribution & clinical profile of chronic hepatitis B cases: insights from a tertiary care hospital in North India

Materials & Methods

This observational study was conducted in the department of Microbiology, ESIC Medical college & Hospital, Faridabad, Haryana, India, a tertiary care hospital, after the approval for the study was obtained from the Institutional Review Board and Ethics Committee. The study participants were patients attending the Gastroenterology outpatient facility from October 2019 to October 2022. The study population consisted of individuals enrolled in the Employees’ State Insurance Corporation (ESIC) scheme, which provides health and social security benefits to workers in the organised sector, primarily covering low-income workers and their families.

The criteria for patient selection were the presence of HBsAg in the serum for more than six months and no history of ongoing treatment. Exclusion criteria were incomplete demographic details, patients on treatment and HBsAg positivity for less than six months (Figure). Screening for hepatitis B surface antigen (HBsAg) was performed using a one-step HBsAg test strip (Meriscreen HBsAg), in accordance with the manufacturer’s instructions. Levels of key hepatic enzymes, including aspartate aminotransferase (AST), ALT, and alkaline phosphatase, were recorded. Serological markers such as HBeAg, and anti-HBe antigen were determined using commercially available VIDAS^® Hepatitis panel (Biomérieux, Marcyl’ Etoile, France) and the record was maintained. The participants’ details included age, sex, occupation, a family history of Hepatitis B positivity, alcohol consumption, intravenous drug use, tattooing, surgical history, and blood transfusion. All this information was collected through a structured proforma adapted from National Action Plan - Combating Viral Hepatitis in India (2019)¹³.

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Figure.

Algorithm used for testing samples in the present study.

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Clinical categorisation of CHB patients was done according to European Association for the Study of the Liver (EASL) criteria, and APRI was utilised as a prognostic criterion⁵. APRI was calculated by using the formula: [AST (upper limit of normal) × 100]/platelet (10⁹/l)⁶. For high-risk patients, imaging (elastography) was done (data not included). HBV viral load quantification was done for all the patients at least once using TRUPCR® HBV. HBV genotyping was done in 183 samples using the Sacace HBV Genotype A, B, C, D Real-TM Quant Detection kit.Twenty samples were amplified, and HBV DNA polymerase genes (S/POL) were sequenced as described previously to determine the concordance of genotyping¹⁴. HBV DNA sequences were aligned with reference sequences using the CLUSTAL method in Molecular Evolutionary Genetics Analysis (MEGA) version 10.The alignment was carried out to guarantee sequence quality and to identify variable and conserved segments, which facilitated further analysis. Sequences were manually edited with the BioEdit Sequence Editor (version 7.2.5) and then subjected to blast analysis to compare with sequence databases to calculate the similarity percentage. The sequences were submitted to GenBank and following accession numbers were generated (OP556578-OP556596).

Results

In the study period, 183 patients with CHB were enrolled in the study. Of these, 102 (55.7%) were males and 81 (44.3%) were females (Table I). The higher prevalence of HBeAg positivity was seen in the older age group (46-90 yr), suggesting persistent viral replication in a subset of patients. In younger patients (0-45 yr), a lower prevalence of HBeAg positivity was observed, indicating a possible transition towards immune control, as shown in table I.

Previous history of surgical intervention (77/183; 42.1%) was the most common risk factor associated with the disease, followed by tattooing and body piercing (39; 21.3%), blood transfusion (14; 7.7%), dialysis (33; 18%), mother-to-child transmission (4; 2.2%), IV drug use (10; 5.5%), and dental procedures (3; 1.6%). Three patients (1.6%) reported other risk factors. A total of 108 patients (59%) had a positive family history of HBV, 64 patients (35%) had a negative family history, and for 11 patients (6%) such information was unknown (Table I).

Eighty-two patients (44.8%) were HBeAg seropositive and 101 (55.2%) were HBeAg seronegative. ALT and HBV DNA levels were assessed for predicting liver inflammation and treatment decisions. Liver enzymes (ALT) were elevated in 112 patients (61.2%) and normal in 71 patients (38.8%). Among those with elevated ALT, 40 were HBeAg seropositive (40/112; 36%), while among those with normal ALT, 42 were HBeAg seropositive (42/71; 59% Table I). HBeAg seropositivity was significantly correlated (P = 0.00313) with elevated ALT levels, as shown in table I.

Among those with detectable viral load, 19 patients (10.4%) had a viral load <10 IU/ml, 31 patients (16.9%) had viral load ranging from 10-2,000 IU/ml, 57 patients (31.1%) had viral load ranging from 2,001-20,000 IU/ml, 44 patients (24%) had viral load ranging from 20,001-2,000,000 IU/ml, and 32 patients (17.5%) had viral load >2,000,000 IU/ml (Table I).

A significant association (P< 0.00001) was found between HBeAg status and HBV viral load (Table I). HBeAg seropositive status was present in all individuals with a viral load greater than 2,000,000 IU/ml, whereas a considerable number of HBeAg seronegative patients had detectable HBV DNA levels in the lower viral load ranges (<2,000,000 IU/ml). In patients with a detectable viral load of 10-2,000 IU/ml, it was observed that 11.4 per cent were HBeAg seropositive and 88.6 per cent were HBeAg negative. However, different observations were seen in patients with a viral load of 2,001-20,000 IU/ml, where 31.6 per cent were HBeAg seropositive and 68.4 per cent were HBeAg seronegative. Out of the 44 patients with a viral load of 20,001-2,000,000 IU/ml, 63.6 per cent were HBeAg seropositive and 36.4 per cent were HBeAg seronegative. These findings suggest a strong relationship between higher viral load and HBeAg positivity, with a significant proportion of patients in the lower viral load categories being HBeAg negative.

All CHB patients were classified into different phases using HBeAg and ALT levels. The immunotolerant phase comprised 6.5 per cent of cases, while immune-active cases accounted for 38.2 per cent. Inactive carrier patients represented 6.01 per cent of the cohort (Table II). Most patients, approximately 49.2 per cent, were in the HBeAg seronegative/immune escape phase (Table II).

HBV DNA was genotyped in 183 HBsAg-positive samples, and the results were concordant with the DNA sequencing. The most prevalent genotype was D 170 /183 (92.9%), followed by genotype A 11/183 (6.0%) and C 2/183 (1.1%). The cross-sectional analysis of genotypic association with respect to gender, HBeAg Status, phases of chronic HBV infection and viral load was not found to be statistically significant. Genotypic association with the probable transmission source was found to be statistically significant (P<0.0001; Table III). Genotype D was significantly associated with active and progressive phases of chronic HBV (both HBeAg-positive and -negative chronic hepatitis), supporting its link to more severe disease (P=0.04; Table III). Genotype A was identified in earlier or less active phases (like immunotolerant or inactive carrier), possibly indicating slower disease progression. However, the number of patients in this category was small (Table III).

Discussion

The current study provides insights into the demography, genetic diversity and clinical characteristics of CHB patients from North India. We did not identify any case of co-infection with hepatitis C, similar to a study conducted in the Himalayan region¹⁵. In the Indian context, hepatitis B infection is primarily acquired through horizontal transmission¹⁶, leading in many cases to CHB. Our study showed a statistically significant association between healthcare-related or invasive procedures (surgery, dialysis, blood transfusion, and tattooing) and genotype D infection, suggesting that genotype D may be more commonly transmitted through parenteral routes. In contrast, genotype A may be acquired through a different transmission pattern, such as vertical or familial spread. European surveillance data have indicated that the healthcare-associated transmission of hepatitis B and C continues to occur in hospital settings. However, the extent of the situation in Asia remains unclear due to under-reporting¹⁷. The mother-to-child transmission (MTCT) and intrafamilial route were more commonly seen in studies from Brazil and China where HBeAg negative clinical forms were seen at 94 and 81.5 per cent, respectively¹⁸. A recent study on transmission dynamics of Hepatitis B infection among Indian families showed that close contact with a carrier, sharing of bed/bedding or personal hygiene items, and eating in shared utensils were associated with the transmission of HBV¹⁹. Another study from Odisha identified tattooing and barbering as risk factors in low-socioeconomic populations and in areas with high HBV endemicity²⁰.

In our study, a high percentage of CHB were HBeAg seronegative (84.7%), probably due to pre-core and core promoter mutations as seen in the endemic areas. The frequency of virus expressing HBeAg might vary according to its genotype, which may explain the differences in the MTCT rates¹⁸. An earlier study showed a strong association of 1896 pre-core stop codon mutations with the protein of HBeAg more often in genotype D²⁰. Consistent with this, our study found that genotype D was the most prevalent, followed by A and C. The predominance of HBV/D in India has been shown by various studies from North as well as South India^21,22. HBV Genotype C has also been reported in Eastern and North-Eastern India²³. Globally, genotype A is the most prevalent genotype in African countries where MTCT is the commonest mode of transmission. Genotypes B and C are commonly found in Asia, while genotypes and D are common in Europe. In the Americas, genotype F is the most common¹⁸.

The knowledge of HBV genotype is crucial for understanding the infection’s clinical behaviour, transmission, treatment responses, and long-term outcomes. Our study did not find any association between the two most frequent sub-genotypes (A and D) with the viral load, demographic characteristics (age, sex), or HBeAg status (Table III). The findings from our study are consistent with previous studies suggesting that nosocomial transmission and non-sterile practices could be a major source of infection, especially for genotype D¹⁸.

Viral loads were lower in HBeAg-negative patients compared to those who were HBeAg-positive. Additionally, patients with abnormal ALT levels had a higher viral load level among the HBeAg-negative group (Table I). Similarly, findings were reported earlier by Nita et al²⁴. HBeAg positivity and higher levels of ALT and HBV DNA have been reported to be associated with a higher cumulative incidence of HCC²⁵. Among HBeAg-negative patients with low viral loads, HCC risk is determined by levels of HBsAg and ALT and age, but not HBV DNA²⁵. Genotype A, found in 11 patients, was associated with a relatively less severe progression, as it was linked to the immune-tolerant phase. This finding could be important when considering the prognostic implications of genotype A in HBV infection, suggesting that it may have a less aggressive course compared to genotype D.

The findings of our study should be interpreted in light of the following limitations. First, the study did not include longitudinal follow up of patients, particularly those who received treatment during the study period. Furthermore, although we identified several potential risk factors for HBV transmission (such as surgical interventions and dental procedures), there is a possibility of underreporting or misclassifying these factors, which could limit the generalisability of these findings.

In conclusion, the study highlights genotypic diversity and its association with disease progression and transmission patterns of HBV in North India in a population from a low socio-economic setting. Genotype D was most prevalent, associated with healthcare-associated transmission and more severe disease phases, while genotype A was seen in the immune-tolerant phase. The study emphasises the role of infection control measures in healthcare settings and the need for family-centred strategies in reducing HBV transmission, especially in vulnerable populations.