慢性乙型肝炎患者HBV耐药模式及rtA181变异准种分析

Analysis of hepatitis B virus resistant patterns and rtA181 mutation quasispecies

目的分析慢性乙型肝炎患者HBV逆转录酶区耐药变异情况及rtA181变异准种的分布。方法提取患者血清中HBVDNA,PCR扩增逆转录酶区域,产物测序后经软件比对分析耐药变异情况和基因型;对部分rtA181变异标本进行克隆后测序分析准种分布。结果 489例标本中,检出明确耐药变异265例,在B、C基因型中分布比例存在差异(56.6%vs43.0%,P=0.022)。拉米夫定相关耐药138例(52.1%),以M204I、M204I/V+L180M±L80I/V变异为主;阿德福韦相关耐药35例(13.2%),以N236T+A181T/V较为多见;拉米夫定+阿德福韦相关耐药70例(26.4%),几乎都和A181有关。rtA181准种分析发现1例位于同一病毒株的多耐药组合,且未发现单一A181T变异的病毒准种。结论 HBV耐药变异主要表现为M204和A181相关变异,耐药模式复杂;检测HBV逆转录酶区的变异有助于临床及时发现和确认耐药情况,指导临床合理进行抗病毒用药。

Objective To analyze the patterns of hepatitis B virus resistance mutation and quasispecies distribution of rtA181 mutation. Methods HBV reverse transcriptase gene was amplified after extracting HBV DNA from patients′ serum. The PCR products were directly sequenced and analyzed to determine the resistance mutation and genotypes. The quasispecies distribution of some rtA181 mutation specimens were clone-sequencing analyzed. Results Of the total 489 patients, resistance mutations were detected in 265 patients. The difference in the distribution between genotype B and C was significant （56.6% vs 43.0%, P=0.022）. Lamivudine related resistance was detected in 138 patients （52.1%）, M204I and M204I/V＋ L180M±L80I/V were more common in LAM-resistance mutations. N236T＋A181T/V mutation was frequently observed in ADV-resistant mutation. Lamivudine ＋ adefovir related resistance was detected in 70 patients （26.4%）, and was related with the A181T/V mutation. A multi-drug resistance form was detected in one strain of the virus in rtA181 quasispecies. Single A181T mutation was not detected in quasispecies. Conclusion M204 and A181 mutation were common in HBV resistance mutations. The patterns of resistance were complex. Detection of HBV reverse transcriptase may facilitate the discovery of drug resistance and is helpful to the application of clinical antiviral medication.