乙型肝炎病毒逆转录酶区预存耐药研究

Detection of pre-existing resistance of hepatitis B virus in the reverse transcriptase region

目的探讨未经核苷(酸)类似物治疗的乙型肝炎病毒(HBV)感染者HBV P基因逆转录(RT)区准种异质性及预存耐药情况,为临床抗病毒治疗药物选择提供理论依据。方法 2011年10月至2013年4月期间于中国医科大学附属盛京医院感染科住院的60例慢性HBV感染者为研究对象。检测HBV P RT区准种异质性及预存耐药情况;乙型肝炎病毒血清学标志物检测采用化学发光法;乙型肝炎病毒载量测定采用Tag Man实时荧光定量PCR法。HBV P RT准种异质性及预存耐药检测应用克隆测序法。结果 60例患者中32例(53.3%)检测出RT区基因变异,其中乙肝病毒携带者组、慢性乙型肝炎组和乙肝肝硬化组之间相比差异无统计学意义(P>0.05)。对HBV RT区的核苷酸序列分析共发现31种变异模式。拉米夫定预存耐药8例(1.33%)、恩替卡韦预存耐药5例(0.83%),阿德福韦酯预存耐药7例(1.16%)。乙肝肝硬化组准种复杂性(38.3%)明显高于慢性乙型肝炎(2.83%)和乙肝病毒携带者(1.33%)。e抗原(HBe Ag)阳性组RT区变异发生率为52.6%,阴性组为54.5%,两组差异无统计学意义。结论 HBV逆转录酶区核苷(酸)预存耐药天然存在,变异模式多种多样。HBV不同感染阶段RT区各耐药位点变异发生率差异无统计学意义,但乙肝肝硬化组准种复杂性明显高于慢性乙型肝炎和乙型肝炎病毒携带者。HBe Ag阳性和阴性HBV感染者RT区基因变异率差异无统计学意义。

Objectives To investigate the quasispecies heterogeneity and pre-existing resistance of hepatitis B virus （HBV） in the reverse transcriptase region in order to provide theoretical basis for clinical antiviral drug treatment. Methods Quasispecies heterogeneity and pre-existing resistance were detected in HBV P gene reverse transcriptase region of 60 patients with chronic HBV infection. HBV serological markers were detected with chemiluminescence method. HBV load was determined by TagMan real-time fluorescent quantitative polymerase chain reaction （PCR） method. Q.uasispecies heterogeneity and pre-existing resistance in HBV P gene reverse transcriptase region were detected with cloning and sequencing method. Results In 60 patients, 32 cases （53.3%） were found having mutations in RT gene. There was no significant difference among the groups of HBV carriers, chronic hepatitis B and cirrhosis. ＂Ihirty-one kinds of variation patterns were found in RT region through nucleotide sequence analysis. Pre-existing resistance of Lamivudine was found in 8 cases （1.33%）, Adefovir dipivoxil in 7 cases （1.16%） and Entecavir in 5 cases （0.83%）. The complexity of quasispecies in the group of cirrhosis （38.3%） was higher than that in the groups of chronic hepatitis B （2.83%） and HBV carriers （1.33%）. The incidence rates were 52.6% and 54.5% in HBeAg positive and negative groups respectively. There was no significant difference between the two groups. Conclusion Pre-existing resistance in HBV P gene reverse transcriptase region exist naturally. The modes of variation are various. The mutation rate is not significantly different between different stages of HBV infection, but the complexity of quasispecies in cirrhosis patients is higher than that in chronic hepatitis B patients and HBV carriers. The mutation rate is not significantly different between E antigen positive and negative groups.