拉米夫定和阿德福韦酯双重耐药HBV株临床检出特点与表型特性分析  

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

作　　者：陈容娟 刘妍[2] 李晓东[2] 杜凤霞 思兰兰 李乐[2] 许智慧[2] 姚增涛[2] 戴久增[2] 徐东平[2] 段昌柱[1] 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(College of Basic Medicine,Chongqing Medical University,400016,China)

机构地区：[1]重庆医科大学基础医学院,400016 [2]解放军第三〇二医院临床研究管理中心/全军传染病研究所,北京100039 [3]解放军第三〇二医院药学部,北京100039

出　　处：《传染病信息》2018年第3期215-219,共5页Infectious Disease Information

基　　金：国家自然科学基金项目(81772178;81371852;81721002);国家"十三五"传染病重大专项子课题(2017ZX10302201-001)

摘　　要：目的分析拉米夫定(lamivudine,LAM)和阿德福韦酯(adefovir,ADV)双重耐药HBV株临床检出特点和表型特性,为临床优化抗病毒方案提供参考。方法回顾性分析26 553例慢性HBV感染者血清样本中HBV反转录酶区与LAM和ADV耐药相关的突变类型及频率,采用PCR产物直接测序法和克隆测序法(≥20个克隆/样本)对双重耐药HBV株进行鉴定,采用表型耐药方法分析强效核苷(酸)类似物[nucleos(t)ide analogues,NAs]对双重耐药HBV的抑制效果。结果 26 553例慢性HBV感染者的血清样本中,LAM/ADV双重耐药HBV突变株的检出率为0.6%(147/26 553),主要突变类型为rt L180M+A181V+M204V(65.3%)和rt A181V+M204I(10.2%)。多数患者经历了LAM→ADV(40.8%)或LAM→ADV→ETV(38.8%)单药序贯/联合治疗,平均抗病毒时间为63个月。其中2例代表性LAM和ADV双重耐药患者的动态血清样本克隆结果显示,在恩替卡韦(entecavir,ETV)+ADV联合挽救治疗期间,随着获得完全病毒学应答后维持时间的延长,病毒准种池中LAM/ADV双重耐药株逐渐消失,病毒株以单一NAs耐药株或野生株为主。体外表型结果显示,各种双重耐药HBV突变株的体外复制力均低于野生株,ETV+ADV联合、替诺福韦酯(tenofovir,TDF)单独或TDF+ETV联合可有效抑制野生株复制(抑制率为98.5%~99.5%),而对各种LAM/ADV双重耐药突变株的抑制率分别为80.6%~92.5%、86.1%~97.9%和89.2%~97.5%。结论长期LAM和ADV单一序贯治疗可促进LAM/ADV双重耐药HBV发生,ETV+ADV联合、TDF单独或TDF+ETV联合均可有效抑制LAM/ADV双重耐药HBV复制,而基于TDF的挽救治疗效果更佳。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

关 键 词：乙型肝炎病毒 双重耐药突变 挽救治疗 抑制作用 

分 类 号：R512.6[医药卫生—内科学]