慢性乙型肝炎患者在核苷(酸)类药物规范化治疗前耐药突变研究

Drug resistance screening in chronic hepatitis B patients prior to standardized therapy with nucleos(t)ide analogues

目的研究慢性乙型肝炎患者在核苷(酸)类药物(NAs)规范化治疗前耐药突变情况。方法在我国北方地区4家医院收集148例慢性乙型肝炎患者在NAs规范化治疗前的血清,提取血清中HBV DNA,用巢式聚合酶链反应-直接测序法获得HBV全长RT区序列,用生物信息学技术筛查该区内11个经典耐药突变位点(rt169、rt181、rt184、rt194、rt202、rt204、rt236、rt250、rt80、rt173和rt180)并鉴定患者HBV基因型。采用SPSS 13.0软件进行统计分析。计量资料采用x±s表示,计量资料的比较采用t检验,计数资料的比较采用χ2检验。P<0.05为差异有统计学意义。结果 148例慢性乙型肝炎患者中,有142例血清HBV RT区在11个经典耐药突变位点上均为野生型氨基酸;6例患者检出经典耐药突变,分别为rtM204I2例,rtA181V、rtA181T、rtL180M+rtM204I、rtL80V+rtL180M+rtM204I各1例,其在半年以前均有不规范NAs治疗史。148例患者基因型分布为:B基因型占4.1%(6/148),C基因型占95.3%(141/148),D基因型占0.6%(1/148)。结论依据PCR产物直接测序结果,本研究中慢性乙型肝炎患者抗病毒治疗基线时体内HBV以野生株为优势株,检出NAs耐药突变株的患者均有既往不规律抗病毒治疗史。基线耐药检测有助于NAs个体化治疗方案的优化。

Objective To study the drug resistant status of hepatitis B virus （HBV） in chronic hepatitis B （CHB） patients prior to standardized treatment with anti-HBV nucleos（t）ide analogues （NAs）. Methods Serum samples of 148 CHB patients were collected from 4 hospitals in Northern China before starting the standardized anti-HBV NAs therapy. HBV DNA was extracted and the full-length reverse transcriptase gene of HBV DNA was amplified and sequenced. Eleven classical NAs-resistance （NAr） mutation sites （rt169, rt181, rt184, rt194, rt202, rt204, rt236, rt250, rtS0, rt173 and rt180） were screened and HBV genotypes were identified. SPSS 13.0 software was used for data analysis. Results Wild type sequences of all 11 NAr mutation sites were found in 142 CHB patients. NAr mutations rtM204I were identified in 2 cases while rtA181V, rtA181T, rtL180M/rtM204I and rtL80V/rtL180M/rtM204I were detected in each of the remaining 4 CHB patients; all these 6 patients had received non-standardized treatment with NAs 6 months ago. Among the 148 HBV sequences, 4.1G （6/148） were genotype B, 95.3% （141/148） genotype C, and 0.6G （1/148） genotype D. Conclusions Wild type HBV strains in CHB patients were predominant prior to their standardized NAs anti-HBV therapy. The NAs resistance mutants were observed in patients previously received non-standardized antiviral therapy.