Dynamics of Genotypic Mutations of the Hepatitis B Virus Associated With Long-Term Entecavir Treatment Determined With Ultradeep Pyrosequencing: A Retrospective Observational Study

The aim of the study is to explore the evolution of genotypic mutations within the reverse transcriptase region in partial virological responders (PVRs) receiving long-term entecavir (ETV) treatment. A total of 32 patients were classified as completely virological responders (CVRs) (n = 12) or PVRs (n = 20). Five partial responders were hepatitis B virus (HBV)-DNA positive after long-term therapy, which lasted for >3 years. A total of 71 serum samples from these 32 patients were assayed by ultra-deep pyrosequencing (UDPS): 32 samples were from all patients at baseline, and 39 were from PVRs with sequential inter-treatment. Approximately 84,708 sequences were generated per sample. At baseline, the quasispecies heterogeneity did not significantly differ between the 2 groups. The frequencies of substitutions indicating pre-existence of nucleos(t)ide analog resistant (NAr) mutants ranged from 0.10% to 6.70%, which did not statistically differ between groups either. However, the substitutions associated with the NAr mutants were significantly different from those associated with the non-NAr mutants in 13 patients; 6 of these patients were PVRs and the others were CVRs. Five patients were HBV DNA positive after regular ETV monotherapy for >3 years, and 4 of these patients underwent mild NAr substitution fluctuations (<20%). One patient developed virological breakthrough while bearing single, double, and triple (rtL180 M, rtM204 V, rtS202G) substitutions. In addition to the common substitutions, unknown amino acid substitutions, such as rtL145 M/S, rtF151Y/L, rtR153Q, rtI224 V, rtN248H, rtS223A, rtS256C, need to be further verified. NAr substitutions are observed at frequencies of 0.10% to 6.7% before therapy. Long-term ETV therapy generally results in virological responses, as long as the proportion of resistance mutations remains at a relatively low level. Genotypic resistance to ETV is detected in all PVRs receiving long-term ETV therapy.