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目的检测未接受过核苷(酸)类似物(NA)治疗的急性乙型肝炎患者感染的HBV是否存在耐药变异。方法收集96例急性乙型肝炎患者住院早期血清,提取HBV DNA,采用巢式PCR方法扩增HBV反转录酶(RT)全基因,对PCR产物进行DNA双向测序,对RT/S基因序列进行分子进化树分析反基因分型,对rt80、rt173、rt180、rt181、rt184、rt202、rt204、rt236和rt250等位点上的耐药相关变异进行分析,并用克隆测序法进行验证,每个样本测定10～20个克隆。结果用直接测序法检出NA耐药变异8例(8.3%),C型6例,B型2例。其中6例为拉米夫定(LAM)耐药变异,包括4例rtM204I、1例rtL80I+rtM204I和1例rtL180M+rtM204I;另2例检出与阿德福韦酯(ADV)耐药相关的rtA181V变异。变异株多数与野生型病毒株共存。克隆测序法的结果与直接测序法的结果大体相符,部分样本中有2种或多种耐药变异株共存,其中1例患者的样本中除LAM变异株外,还检出了ADV和恩替卡韦(ETV)耐药变异株,变异形式分别为rtA181T+rtN236T和rtL180M+rtS202G+rtM204V。结论未接受过NA治疗的急性乙型肝炎患者可以感染NA耐药株病毒,NA耐药病毒可以在人群中传播引起急性乙型肝炎,变异病毒的传播致病不只局限于LAM耐药株。
Objective To detect the presence of resistance mutations in HBV infected with acute hepatitis B patients who did not receive NAA. Methods Ninety-six patients with acute hepatitis B were enrolled in the hospital early stage serum. HBV DNA was extracted. The complete reverse transcriptase (RT) gene was amplified by nested PCR. The PCR products were double-stranded DNA sequenced. The RT / S gene sequences Molecular phylogenetic tree analysis of anti-genotyping, resistant mutations at rt80, rt173, rt180, rt181, rt184, rt202, rt204, rt236 and rt250 isolates were analyzed and cloned sequencing method to verify the determination of each sample 10 to 20 clones. Results Eighty-one (8.3%) NA-resistant mutations were detected by direct sequencing, six in type C and two in type B. Six of them were resistant to lamivudine (LAM), including four rtM204I, one rtL80I + rtM204I and one rtL180M + rtM204I. The other two cases were associated with resistance to adefovir dipivoxil (ADV) rtA181V mutation. Mutants most coexist with wild-type strains. Cloning and sequencing results and direct sequencing results generally in line with some of the samples in two or more resistant strains coexist, including 1 patient samples in addition to LAM variants, but also detected ADV and entecavir ( ETV) resistant mutants, the variant forms were rtA181T + rtN236T and rtL180M + rtS202G + rtM204V, respectively. CONCLUSION: NA-resistant virus can be infected in patients with acute hepatitis B who have not been treated with NA. NA-resistant virus can spread in people causing acute hepatitis B. Pathogenicity of the variant virus is not limited to LAM-resistant strains.