单基因组测序分析病毒学失败HIV-1感染者耐药发生特征研究

Single genome sequencing analysis of dynamic characteristics of HIV-1 drug resistance in patients experiencing virologic failure

目的用单基因组测序技术分析HIV-1感染者体内基因型耐药动态变化,了解耐药相关突变发生特征。方法对4例抗逆转录病毒治疗失败病例及其基线冻存血浆样本进行标准基因型耐药检测和共享测序。采用倍比稀释法进行pol基因片段单基因组扩增和测序(SGS),分析准种变异和耐药动态变化特征。获得的基因序列用斯坦福大学HIV耐药数据库判断基因型耐药及耐药程度解析。结果NA2302-00病例存在传播性耐药突变(TDR,M184//G190A突变型),在治疗5个月后突变位点增多并导致治疗失败。3例未检测到TDR,但是CD4细胞计数均低于200个/μl,在治疗4~6个月后出现获得性耐药和治疗失败。所有病例均对所服用的拉米夫定和依非韦伦药物存在高度耐受,2例对替诺福韦高度耐受。从4个病例的治疗基线和失败两个采样时间点血浆样本中共获得291条pol基因SGS序列。2个无TDR的病例基线样本SGS序列存在劣势耐药相关突变(2.4%~2.9%),但这些突变在随访样本中未检测到。NA2302-00的27条SGS序列均检测到该TDR突变型。治疗失败样本SGS序列表现出复杂的准种变异,进化为不同耐药突变型。2个病例的治疗失败样本SGS序列突变型为1~2个,另2个病例分别有4和13个突变型,>20%优势毒株突变型各2个。4个病例在更换蛋白酶抑制剂为主的二线药物后,维持较好的病毒抑制效果。结论单基因组测序可以揭示感染者体内毒株准种变异和耐药发生特征。在药物选择压力下,感染者体内病毒发生适应性进化,产生复杂的耐药相关突变模式,尤其需要加强艾滋病期感染者治疗第一年的随访和监测。

Objective To investigate the intra-host dynamics of HIV-1 genotypic drug resistance using single genome sequencing(SGS)for better understanding the derivation of drug resistance mutations(DRMs).Methods Plasma specimens from four individuals experiencing virologic failure(VF)and the frozen plasma samples collected at their baseline of antiretroviral therapy(ART)were used to detect drug resistance by bulk PCR and consensus sequencing.Single genome amplification and sequencing were performed to harvest pol gene fragments with double dilutions of cDNA template.The quasispecies variation and dynamic characteristics of DRMs were analyzed.The obtained pol gene fragments were analyzed to identify transmitted and acquired drug resistance(TDR and ADR)using the Stanford University HIV drug resistance database.Results Case NA2302-00 carried TDR(M184//G190A)and experienced VF caused by severe DRMs after five months of ART.TDR was not detected in the other three individuals,but CD4 cell counts in them were all below 200 cells/μl.Moreover,ADR occurred and VF was caused in the three patients after 4-6 months of ART.All cases had a high tolerance to lamivudine and efavirenz and two to tenofovir.Totally,291 SGS sequences of pol gene were obtained from the samples collected at ART baseline and after VF.Minority DRMs(2.4%-2.9%)were identified in the baseline samples of two cases without TDR using SGS technique,but disappeared during follow-up.All 27 SGS sequences from case NA2302-00 carried homogeneous mutation pattern of TDR.SGS sequences obtained after VF indicated complicated quasispecie variation,which evolved into different mutation patterns.Two individuals carried 1-2 mutation patterns in the SGS sequences obtained after VF,while the other two cases respectively carried four and 13 mutation patterns with two majority patterns above 20%.All of the individuals showed well-maintained viral suppression after being switched to the second line regimen of lopinavir/ritonavir.Conclusions SGS was conducive to unveil the intra-host quasispecies variation and dynamic characteristics of mutations.Under the antimicrobial selection pressure,viruses in infected individuals would evolve adaptively,from which complex patterns of drug resistant mutations could derive.Follow-up and surveillance should be strengthened during the first year after ART,especially for patients at the stage of AIDS.