Hepatitis B virus genome mutations in precore and basal core promoter regions among HBeAg-negative chronic hepatitis B patients with high viral load in Indonesia

Hepatitis B e antigen (HBeAg) is widely used as a marker for active HBV replication and serves as a surrogate for HBV DNA > 200,000 IU/mL to determine eligibility for tenofovir disoproxil fumarate (TDF) prophylaxis to prevent vertical transmission, according to WHO guidelines. However, some HBeAg-negative patients still harbor high viral loads. Mutations in the precore (PC) and basal core promoter (BCP) regions may reduce or abolish HBeAg expression without necessarily suppressing viral replication. Next-generation sequencing (NGS)-based characterization of these mutations remains limited in Indonesia. This study aimed to analyze the mutation prevalence in the BCP and PC regions associated with HBeAg negativity in Indonesian patients. We conducted a cross-sectional study of 32 chronic HBV treatment-naïve, unvaccinated patients with HBV DNA > 200,000 IU/mL (16 HBeAg-negative, 16 HBeAg-positive) at Cipto Mangunkusumo General Hospital. BCP and PC mutations were analyzed using NGS, classifying mutations as major (mutation frequency index [MFI] ≥20%) or minor (MFI 1- < 20%). Associations were analyzed using the Chi-square or Fisher's exact test and p-values were adjusted using the Benjamini-Hochberg procedure. Among 29 major mutation sites, PC mutations A1846T/C and G1896A were more frequent in HBeAg-negative than HBeAg-positive patients (81.3% vs 6.3% and 75.0% vs 12.5%, respectively; all adjusted p = 0.019). Combined analysis showed higher mutation frequencies in HBeAg-negative patients (93.8%, 81.3%, and 62.5% for A1846T/C, G1896A, and G1899A, respectively; all adjusted p = 0.015). In conclusion, HBeAg-negative patients with high viral loads are strongly associated with PC mutations, particularly G1896A, A1846T/C, and G1899A. These exploratory findings provide regional NGS-based molecular evidence that established PC mutations may contribute to the coexistence of HBeAg negativity and continued high-level HBV replication in Indonesian patients. Larger studies incorporating broader virological and clinical comparison groups are required to determine the clinical significance of these findings.