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目的研究拉米夫定(LAM)治疗后出现HBV病毒学突破患者HBVRT区变异位点和变异类型。方法研究对象选自2004年4月至2007年3月在苏州大学附属第一医院门诊或住院治疗的慢性乙型肝炎患者,用聚合酶链式反应(PCR)方法扩增LAM治疗后出现HBV病毒学突破患者的血清HBVRT区基因,对PCR产物直接测序,用Chromas2.0软件分析HBVRT区基因的核苷酸和氨基酸差异、变异类型。结果109例患者在拉米夫定耐药后出现病毒学突破,其中94例出现拉米夫定耐药相关性变异,包括YMDD变异93例,单独rtA181T变异1例。13例(11.93%)患者经测序分析未发现YMDD变异,但用限制性内切酶片段长度多态性(RFLP)方法检测,均发现有YMDD变异。测序结果发生变异位点和出现频率:rtM204V/I93例(85.3%)、rtL180M51例(46.9%)、rtV173L/M7例(6.4%)、rtV207M/L/I4例(3.7%)、rtA181T4例(3.7%)、rtT184I/S/M2例(1.8%)、rtM250L2例(1.8%)。变异类型:rtM204V/I、rtA181T、rtM204V/I+rtL180M、rtM204V/I+rtL180M+rtV173M等。结论拉米夫定耐药主要变异类型为rtM204V/I变异,常伴随rtL180M和rtV173L/M变异;少数拉米夫定耐药患者在阿德福韦和恩替卡韦治疗前即已产生阿德福韦和恩替卡韦耐药相关性变异。
Objective To investigate the HBV mutation site and mutation types in patients with HBV virological breakthrough after lamivudine (LAM) treatment. Methods The subjects were selected from outpatients or hospitalized patients with chronic hepatitis B from April 2004 to March 2007 in the First Affiliated Hospital of Soochow University. HBV was amplified by polymerase chain reaction (PCR) after treatment with LAM To study the serum HBVRT gene of patients and to directly sequence the PCR products. Chromos 2.0 software was used to analyze the nucleotide and amino acid differences and mutation types of HBVRT gene. Results A total of 109 patients developed virological breakthrough after lamivudine resistance, of which 94 cases showed lamivudine resistance-related mutation, including 93 cases of YMDD mutation and 1 case of rtA181T mutation alone. No mutation of YMDD was found in 13 patients (11.93%) by sequencing analysis. However, YMDD mutation was detected by restriction fragment length polymorphism (RFLP). The mutation site and frequency of sequencing were as follows: rtM204V / I 93 cases (85.3%), rtL180M 51 cases (46.9%), rtV173L / M7 cases (6.4%), rtV207M / %), RtT184I / S / M2 cases (1.8%), rtM250L2 cases (1.8%). Mutation types: rtM204V / I, rtA181T, rtM204V / I + rtL180M, rtM204V / I + rtL180M + rtV173M and the like. Conclusions The main variation type of lamivudine resistance is rtM204V / I variation, often accompanied by rtL180M and rtV173L / M variation. A small number of lamivudine-resistant patients have adefovir and adefovir before treatment with adefovir and entecavir Entecavir resistance-related variation.