应用高通量测序方法研究CRF01-AE亚型HIV-1毒株耐药突变的分子进化规律

Study on molecular evolutionary rules of drug resistance mutations in CRF01-AE subtype of HIV-1 strain by high-throughput sequencing

目的探讨CRF01-AE亚型人类免疫缺陷病毒1型(HIV-1)毒株在药物选择压力下耐药突变发生和演变的规律。方法选取5例一线方案治疗失败的CRF01-AE亚型HIV-1患者,收集其6~8年抗病毒治疗过程中的系列血样,应用高通量测序方法进行基因型耐药突变分析。结果①5例患者在启动治疗后早期即出现M184V突变以及非核苷类逆转录酶抑制剂(NNRTIs)类耐药突变,常见的有K101E、G190A和K103N,其中M184V+G190A+TAMs是常见突变组合,停药或更换二线药物,这些突变也始终存在。②胸腺嘧啶脱氧核苷类似物变异(TAMs)突变形成过程中,有3例患者表现为TAM2路径,1例患者为TAM1途径,还有1例患者前期只有TAM2路径的突变,后期累加上TAM1途径的突变。③有3例患者在治疗过程中分别形成TAMs、Q151M复合体和T69插入复合物等多重耐药突变,更换二线方案后仍然治疗失败。结论①CRF01-AE亚型耐药患者在用药早期即开始出现耐药突变,数种突变逐渐累加,最终导致临床耐药,应尽早进行耐药突变监测。②TAMs突变通过2种途径竞争性发展,CRF01-AE亚型更偏向于TAM2途径,但随着治疗时间延长,2种途径可以产生融合。③更换二线方案前应排查针对替诺福韦(TDF)的耐药突变,其可能会导致二线方案无效。

Objective To investigate the occurrence and development rules of drug-resistant mutations in CRF01-AE subtype of human immunodeficiency virus type 1(HIV-1)strain under the pressure of drug selection.Methods Totally 5 HIV-1 CRF01-AE subtype patients with failure of first-line antiretroviral therapy were selected,and blood samples were longitudinally collected during the course of 6 to 8 years of treatment.Genotypic analysis of drug resistance mutations was performed by high throughput sequencing.Results①All the patients had the M184V mutation and a certain type of non-nucleoside reverse transcriptase inhibitors(NNRTIs)high-frequency major drug resistance mutations at early stage after medication,common sites were K101E,G190A and K103N,and combination of M184V plus G190A plus TAMs was the common mutations.Mutations was still existed after stopping the corresponding drugs or switching to second-line drugs.②In the process of thymidine analogue mutations(TAMs)formation,3 patients had TAM2 pathway,1 patient had TAM1 pathway,and another patient had only TAM2 pathway mutations in the early stage and accumulated TAM1 pathway mutations in the later stage.③Three patients formed multi-drug resistance mutations including TAMs,Q151 complex and 69 insertion complexes,and they still failed in treatment after changing the second-line regimen.Conclusion①CRF01-AE subtype drug resistance patients begin to develop drug resistance mutations at the early stage of drug use,and several mutations gradually lead to clinical drug resistance,so drug resistance mutation monitoring should be carried out as soon as possible.②TAMs mutations are competitive in two pathways,CRF01-AE favors the TAM2 pathway.As the treatment time is prolonged,the two pathways can produce fusion.③It is recommended to carry out drug resistance testing before switching to the second-line protocol.If the resistance mutation against tenofovir(TDF)is found,the second-line regimen may be ineffective.