经治低水平病毒血症慢性乙型肝炎患者耐药突变的基因型特征

Characteristics of resistance-associated mutation in treatment-experienced chronic hepatitis B patients with low-level viremia

目的 分析经治低水平病毒血症(LLV)慢性乙型肝炎(CHB)患者反转录酶(RT)区耐药突变的基因型特征。方法 回顾性研究2007年9月-2019年8月在解放军总医院第五医学中心就诊的经治LLV CHB患者2096例,提取其血清乙型肝炎病毒(HBV)DNA,并采用巢式PCR扩增HBV RT片段,长度为1225 bp(nt54-nt1278),包含HBV RT基因(nt130-nt1161)及S基因(nt155-nt835)。对PCR产物进行双向测序,采用MEGA4软件对RT/S基因序列进行分子进化树分析,以肝炎病毒数据库(http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.Cgi)的标准序列为参照进行HBV基因分型。结果 2096例CHB患者中男1727例,女369例,年龄(45.6±12.0)岁,HBV DNA为(2.5±0.5)log10 IU/ml。拉米夫定耐药相关突变1216例,占58.0%,以M204I/V、L180M+M204V为主要形式;阿德福韦耐药相关突变444例,占21.2%,以A181V、N36T为主要形式;恩替卡韦耐药相关突变376例,占17.9%,以L180M+M204I/V+T184L/A/S/I、L180M+M204V+S202G为主要形式;多药耐药60例,占2.9%,以L180M+M204I/V+A181V、L180M+M204V+A181V+N236T+S202G为主要形式。结论 经治LLV的CHB患者耐药突变基因型以拉米夫定和阿德福韦耐药为主,且降低了恩替卡韦耐药的基因屏障,提高了恩替卡韦的耐药比例,抗病毒治疗应选择强效、低耐药率的一线药物。

Objective To analyze the genotypic characteristics of resistance-associated mutation in reverse-transcriptase(RT) domain of hepatitis B virus(HBV) in treatment-experienced chronic hepatitis B(CHB) patients with low-level viremia(LLV).Methods CHB patients with LLV, who admitted to the Fifth Medical Center of Chinese PLA General Hospital from September 2007 to August 2019, were retrospectively enrolled. Their serum HBV DNA was extracted, and a nested PCR was used to amplify an HBV RT fragment, 1225 bp in length(nt54-nt1278), containing the HBV RT gene(nt130-nt1161) and S gene(nt155-nt835).PCR products were bidirectionally sequenced, and a molecular evolutionary tree analysis of the RT/S gene sequence was performed with MEGA 4 software, with the Hepacivirus database(http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.Cgi) with the standard sequence as the reference for HBV genotyping. Results Among the 2096 patients, 82.4% were male, mean age was(45.6±12.0) years, HBV DNA was(2.5±0.5) log10 IU/ml. Lamivudine resistance related mutations accounted for 58.0%, with M204V and L180M+M204V as the main forms;Adefovir dipivoxil resistance associated mutations accounted for 21.2%, with A181V and N36T as the predominant forms;Entecavir resistance associated mutations accounted for 17.9%, with L180M+M204I/V+T184L/A/S/I and L180M+M204V+S202G being the predominant forms;Multidrug resistance accounted for 2.9%, with L180M+M204I/V+A181V and L180M+M204V+A181V+N236T+S202G being the predominant forms. Conclusion Lamivudine and Adefovir resistance are the main drug-resistant mutation genotypes in the treated CHB patients with LLV, which reduces the gene barrier of entecavir resistance and increases the proportion of entecavir resistance. Therefore, the first-line nucleos(t)ide analogues with strong effect and low resistance should be selected for antiviral treatment.