Genotypic and phenotypic characteristics of multidrug-resistant hepatitis B virus infection in Chineses hepatitis B patients

Objective To analyze the genotypic and phenotypic characteristics of multidrug-resistant hepatitis B virus (MDR HBV) infection in a large cohort of Chinese hepatitis B patients who had undergone long-term sequential nucleos(t)ide analogs (NAs) treatment. Methods NAs-resistance-associated mutation patterns and frequency in HBV reverse-transcriptase (RT) region were analyzed in 11 800 patients with chronic HBV infection. The MDR HBV strains from 46 patients were further identified by PCR direct sequencing and clonal sequencing (≥20 clones/sample). In vitrophenotypic resistance assay was used to investigate the inhibitory effect of combined use of nucleoside and nucleotide analogs on replication capacity of MDR HBV strains. Influence of clinical antiviral schedules on the development and control of MDR HBV infection was further analyzed. Results (1) HBV NAs-resistant mutations were detected in the 3658 (31.0%) of 11800 tested patients, lamivudine (LAM)-, adefovir (ADV)-, entecavir (ETV)-, and telbivudine (LdT)-resistant mutations were detected in 2592 (70.9%), 665 (18.2%), 293 (8.0%), and 62 (1.7%) patients, respectivel y. In addition, MDR mutations resistant to both nucleoside and nucleotide analogs were detected in 46 (1.3%) patients. (2) Clonal sequencing showed that 40 out of 46 MDR samples harbored mutations in the same HBV genome, and MDR mutations in the other 6 were in individual HBV genomes. Fifteen mutational patterns of MDR HBV strains were detected, including 10 both LAM-/LdT-and ADV-resistant mutations and 5 both ETV-and ADV-resistant mutations. (3) In vitrophenotypic analysis showed that combination use of ETV or LAM plus ADV efficiently inhibited the replication capacity of rtL180M+M204V+S202G+A181V MDR strain in HepG2 cells, whereas the synergistic inhibitory effect was not observed in the wild-type strain. (4) Clinical drug usage showed that MDR HBV infection mainly emerged in patients receiving long-term sequential NAs therapy. Among them, 22 with LAM switched to ADV and 10 with LAM switched to ADV, followed by switching to ETV, and they successively developed virological and/or biochemical breakthrough. Remedial therapy consisting of a combination of LAM and ADV or ETV and ADV could effectively inhibit HBV replication to undetectable level in most patients harboring MDR HBV strains. Conclusions The MDR HBV strains isolated from Chinese patients display diversity and complexity. Phenotypic analysis and clinical observation support combined use of nucleoside and nucleotide analogs for control of MDR HBV infection. The development of MDR HBV is closely associated with sequential suboptimal NAs treatment. It is necessary to intensively monitor viral response and resistant mutations during NAs therapy for planning an optimal therapeutic schedule as early as possible.