Analysis for clinical detection and phenotypic features of HBV mutants resistant to both lamivudine and adefovir

CHEN Rong-juan; LIU Yan; LI Xiao-dong; DU Feng-xia; SI Lan-lan; LI Le; XU Zhi-hui; YAO Zeng-tao; DAI Jiu-zeng; XU Dong-ping; DUAN Chang-zhu

doi:10.3969/j.issn.1007-8134.2018.03.005

Abstract：Objective To analyze the clinical detection and phenotypic features of HBV mutants resistant to both lamivudine (LAM) and adefovir (ADV), and provide optimal schedules for clinical antiviral treatment. Methods LAM and ADV-resistance-associated mutation patterns and frequency, detection rate in HBV reverse-transcriptase region were analyzed in serum specimens sampled from 26 553 patients with chronic HBV infection. The LAM/ADV dual-drug resistant HBV mutants were further identified by PCR direct sequencing and clonal sequencing (≥20 clones/sample). Phenotypic resistance assay was used to investigate the inhibitory effect of potent nucleos(t)ide analogues (NAs) on the dual-drug resistant HBV mutants. Results LAM/ADV dual-drug resistant mutations were detected in 147 cases (0.6%) of 26 553 patients with chronic HBV infection and the major mutational patterns were rtL180M+A181V+M204V (65.3%) and rtA181V+M204I (10.2%). Most patients experienced LAM→ADV (40.8%) or LAM→ADV→ETV (38.8%) single sequential or combination therapy. The median term of antiviral treatment was 63 months. The dynamic clonal sequencing of serum specimens from 2 representative patients showed that LAM/ADV dual-drug resistant strains in viral quasispecies pool gradually disappeared as the maintenance time after the complete virological response prolonged during entecavir (ETV)+ADV combined rescue therapy. The dominant virus strains switched to single NAs-resistant strain or wild-type strain. In vitro phenotypic analysis showed that the dual-drug resistant strain had lower replication capacities than that of the wild-type strain in vitro. ETV+ADV, tenofovir (TDF), or TDF+ETV could effectively inhibit the replication of wild-type virus (inhibitory rate 98.5%-99.5%), while the inhibitory rates were 80.6%-92.5%, 86.1%-97.9% and 89.2%-97.5% on LAM/ADV dual-drug resistant strains, respectively. Conclusions Long-term LAM and ADV single sequential therapy can promote the occurrence of LAM/ADV dual-drug resistant HBV. The combination of ETV+ADV, TDF alone or TDF+ETV can effectively inhibit the LAM/ADV dual-drug resistant HBV replication, and the efficacy of the rescue therapy based on TDF is better.