Dynamics of Genotypic Mutations of the Hepatitis B Virus Associated With Long-Term Entecavir Treatment Determined With Ultradeep Pyrosequencing: A Retrospective Observational Study.
Dynamics of Genotypic Mutations of the Hepatitis B Virus Associated With Long-Term Entecavir Treatment Determined With Ultradeep Pyrosequencing: A Retrospective Observational Study.
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超深焦磷酸测序回顾性观察研究确定与长期恩替卡韦治疗相关的乙型肝炎病毒基因型突变动态
DOI：
10.1097/md.0000000000002614
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发表时间：
2016-01
期刊：
影响因子：
1.6
通讯作者：
Xu XY
中科院分区：
文献类型：
--
作者：
Zhang XX;Li MR;Cao Y;Zhang RW;Zhang Y;Li F;Xi HL;Xu XY
本研究的目的是探讨接受长期恩替卡韦（ETV）治疗的部分病毒学应答者（PVR）逆转录酶区域内基因型突变的演变。共有32例患者被分类为完全病毒学应答者（CVR）（n = 12）或PVR（n = 20）。 5例部分应答者在长期治疗后出现B型肝炎病毒（HBV）DNA阳性，持续时间>3年。通过超深度焦磷酸测序（UDPS）分析了来自这32例患者的总计71份血清样本：32份样本来自基线时的所有患者，39份样本来自连续治疗间的PVR。每个样本生成大约84，708个序列。在基线时，两组之间的准种异质性没有显著差异。表明预先存在核苷（酸）类似物抗性（NAr）突变体的置换频率范围为0.10%至6.70%，组间也无统计学差异。然而，在13例患者中，与NAr突变体相关的取代与非NAr突变体相关的取代显著不同;其中6例患者为PVR，其他患者为CVR。5例患者在常规ETV单药治疗>3年后HBV DNA阳性，其中4例患者发生轻度NAr替代波动（<20%）。1例患者在携带单、双和三重（rtL 180 M、rtM 204 V、rtS 202 G）置换时发生病毒学突破。 除了常见的替换，未知的氨基酸替换，如rtL 145 M/S、rtF 151 Y/L、rtR 153 Q、rtI 224 V、rtN 248 H、rtS 223 A、rtS 256 C，需要进一步验证。 治疗前观察到NAr替换的频率为0.10%至6.7%。只要耐药突变的比例保持在相对较低的水平，长期ETV治疗通常会导致病毒学应答。在所有接受长期ETV治疗的PVR中检测到对ETV的基因型耐药。
The aim of the study is to explore the evolution of genotypic mutations within the reverse transcriptase region in partial virological responders (PVRs) receiving long-term entecavir (ETV) treatment. A total of 32 patients were classified as completely virological responders (CVRs) (n = 12) or PVRs (n = 20). Five partial responders were hepatitis B virus (HBV)-DNA positive after long-term therapy, which lasted for >3 years. A total of 71 serum samples from these 32 patients were assayed by ultra-deep pyrosequencing (UDPS): 32 samples were from all patients at baseline, and 39 were from PVRs with sequential inter-treatment. Approximately 84,708 sequences were generated per sample. At baseline, the quasispecies heterogeneity did not significantly differ between the 2 groups. The frequencies of substitutions indicating pre-existence of nucleos(t)ide analog resistant (NAr) mutants ranged from 0.10% to 6.70%, which did not statistically differ between groups either. However, the substitutions associated with the NAr mutants were significantly different from those associated with the non-NAr mutants in 13 patients; 6 of these patients were PVRs and the others were CVRs. Five patients were HBV DNA positive after regular ETV monotherapy for >3 years, and 4 of these patients underwent mild NAr substitution fluctuations (<20%). One patient developed virological breakthrough while bearing single, double, and triple (rtL180 M, rtM204 V, rtS202G) substitutions. In addition to the common substitutions, unknown amino acid substitutions, such as rtL145 M/S, rtF151Y/L, rtR153Q, rtI224 V, rtN248H, rtS223A, rtS256C, need to be further verified. NAr substitutions are observed at frequencies of 0.10% to 6.7% before therapy. Long-term ETV therapy generally results in virological responses, as long as the proportion of resistance mutations remains at a relatively low level. Genotypic resistance to ETV is detected in all PVRs receiving long-term ETV therapy.