核苷和核苷酸类药物在慢性乙型肝炎病毒感染者中的应用及耐药突变分析

Application of Nucleoside and Nucleotide Analogs (NAs) and Analysis of Hepatitis B Virus (HBV) Gene Mutations Related to NAs in Patients with Chronic HBV Infection

关键词:  乙型肝炎病毒  核苷和核苷酸类药物  突变  基因测序

Key words:  hepatitis B virus  nucleoside and nucleotide analogs  mutation  gene sequencing

摘要:

目的 分析慢性乙型肝炎病毒(hepatitis B virus，HBV)感染者核苷和核苷酸类药物(nucleoside and nucleotide analogs，NAs)发生耐药后的突变模式，为规范抗病毒治疗和耐药管理提供参考价值。方法 选取2010年1月—2014年12月发生耐药突变的375例慢性HBV感染者的临床资料，采用实时荧光定量PCR方法对耐药患者血清HBV聚合酶基因逆转录酶区进行扩增，对PCR产物进行直接测序。结果 拉米夫定(lamivudine，LAM)耐药组和阿德福韦酯(adefovir dipivoxil，ADV)耐药组基因型耐药伴生物化学突破的构成比均高于恩替卡韦(entecavir，ETV)耐药组(χ²=12.111，P<0.001；χ²=7.992，P=0.005)。253例LAM耐药者中有16种突变类型，单位点突变134例(52.96%)，以rtM204I 最多见，多位点突变119例(47.04%)，以rtLl80M+M204V最多见；在rtM204突变模式中，rtM204I为单位点突变模式的主要位点(χ²=154.555，P<0.001)，rtM204V为多位点突变模式的主要位点(χ²=4.317，P=0.038)。88例ADV耐药者中有24种突变类型，单位点突变61例(69.32%)，以rtA181T最多见，多位点突变27例(30.68%)，以rtL180M+rtM204V+rtA181T最多见；无论是单位点突变模式还是多位点突变模式，相对于rtN236T，rtA181T均是主要突变位点(χ²=42.749，P<0.001；χ²=6.033，P=0.014)。34例ETV耐药者中有5种突变类型，均为多位点突变，以rtL180M+rtM204V+rtS202I/G最多见；相对于rtT184，rtS202为主要突变位点(χ²=5.882，P=0.015)。ADV耐药者突变模式的复杂性最高，其次为LAM耐药者，最低为ETV耐药者。结论 NAs的耐药突变复杂多样，尤其是ADV，应接受和执行优选和优化治疗策略，以实现预防耐药，减少或避免挽救治疗。

Abstract:

OBJECTIVE To analyze the characteristics of hepatitis B virus (HBV) gene mutations related to nucleoside and nucleotide analogs (NAs) in patients with chronic HBV infection and to provide some reference values on normative usage and management of NAs. METHODS The data of 375 chronic HBV infected patients with resistance mutations related to NAs from January 2010 to December 2014 were analyzed. The HBV reverse transcriptase region in polymerase gene were amplified by real-time fluorescence quantitative polymerase chain reaction (PCR), and the PCR products were analyzed by direct sequencing. RESULTS The constituent ratios of genotypic resistance accompanied biochemical breakthrough in lamivudine (LAM)-related resistance group and adefovir dipivoxil (ADV)-related resistance group were both higher than that in entecavir (ETV)-related resistance group (χ²=12.111, P<0.001; χ²=7.992, P=0.005). There were 16 types of mutations in 253 cases with LAM-related resistance, in which 134 cases (52.96%) had single-mutated sites and rtM204I was the most common mutation, 119 cases (47.04%) had multi-mutated sites and rtLl80M+M204V was the most common mutation. In rtM204 mutation patterns, rtM204I was the major locus of single-mutated?sites(χ²=154.555, P<0.001), and rtM204V was the major locus of multi-mutated sites (χ²=4.317, P=0.038). Twenty four types of mutations in 88 cases with ADV-related resistance, in which 61 cases (69.32%) had single-mutated sites and rtA181T was the most common mutation, 27 cases (30.68%) had multi-mutated sites and rtL180M+rtM204V+rtA181T was the most common mutation. Whether in single-mutated sites or in multi-mutated sites, rtA181T was both the major loci relative to rtN236T (χ²=42.749, P<0.001; χ²=6.033, P=0.014). Five types of mutations in 34 cases with ETV-related resistance, and all cases had multi-mutated?sites, rtL180M+rtM204V+rtS202I/G was the most common mutation. The rtS202 was the major locus relative to rtT184 (χ²=5.882, P=0.015). The mutated heterogeneities of HBV isolates were the highest in ADV-related resistance, the second in LAM-related resistance, and the lowest in ETV-related resistance. CONCLUSION The mutated sites related to NAs in HBV isolates are complex and diverse, especially in HBV isolates related to ADV resistance. We should identify?with and execute preferred selection of NAs and optimal treatment. Thus it will reach the aim for preventing resistance, reducing and avoiding salvage therapy.