超深度焦磷酸测序技术用于HIV-1RT基因区原发性耐药突变的研究

贺健梅; 邹潇白; 陈曦; 郑军

Abstract

贺健梅,邹潇白,陈曦,郑军.超深度焦磷酸测序技术用于HIV-1RT基因区原发性耐药突变的研究[J].Chinese journal of Epidemiology,2014,35(10):1142-1145

超深度焦磷酸测序技术用于HIV-1RT基因区原发性耐药突变的研究

The use of ultra deep sequencing technique in the screening program on HIV-1 drug resistance mutation among ART-naïve patients in Hunan province

Received:April 30, 2014

DOI：10.3760/cma.j.issn.0254-6450.2014.10.015

KeyWord: HIV-1 原发性耐药超深度焦磷酸测序 Sanger法测序核苷类药物非核苷类药物

English Key Word: HIV-1 Drug resistance Ultra deep sequencing Sanger sequencing Nucleotide reverse-transcriptase inhibitor Nonnucleoside reverse transcriptase inhibitor

FundProject:国家科技重大专项（2012ZX10001001）

Author Name	Affiliation	E-mail
He Jianmei	Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China
Zou Xiaobai	Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China
Chen Xi	Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China	chenxi161@sohu.com
Zheng Jun	Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China

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Abstract:

目的利用超深度焦磷酸测序技术（UDS）研究湖南省HIV感染者中原发性耐药流行趋势。方法 90 份未接受抗病毒治疗的HIV感染者同时采用UDS测序和Sanger 测序进行HIV基因型耐药检测，测序结果采用斯坦福大学HIV 耐药突变数据库进行分析比对，分析湖南省HIV-1 反转录（RT）基因区原发性耐药突变情况。结果 UDS 检测成功获取90 份测序结果，84.4%为AE亚型（76/90），发现38 例（42.2%，38/90）针对RT基因区的耐药突变位点，其中14 例样本仅发生核苷类反转录酶（NRTI）耐药突变（15.6%），15 例仅发生非核苷类反转录酶（NNRTI）耐药突变（16.7%），9 例样本同时发生了NRTI 和NNRTI 类耐药突变。在16 个位置上共发现54 次耐药突变。17 例（18.9%，17/90）样本有针对RT基因区的中度及以上的耐药突变。传统Sanger 法仅检出7 例；其中针对NRTI 类药物耐药的6 例；针对NNRTI 类药物耐药的5 例。结论 Sanger 法测序能够鉴定出显著的耐药性突变（≥20%），但不能及时发现尚未达到显著突变量的突变位点或稀有型抗药型突变（<20%）。而UDS则能更灵敏检测到低频突变毒株，这些耐药突变同样可在药物选择压力下快速的增长引起病毒学治疗失败，导致临床治疗失败。因此选择更高灵敏度和精确度的技术检测低水平耐药突变位点，对于HIV抗病毒临床治疗有着十分重大的意义。

English Abstract:

Objective To determine the prevalence rates of nucleotide reverse-transcriptase inhibitor（NRTI）and nonnucleoside reverse transcriptase inhibitor（NNRTI）TDRs among HIV-1 ART-naïve patients in Hunan province using the ultra deep sequencing（UDS）technique. Methods ART-naïve subjects diagnosed in Hunan between 2010 and 2011 were evaluated by both UDS technique and Sanger sequencing techniques,to the 1% variant level. Mutations were scored using the Stanford HIVdb algorithm to infer the status on drug resistance. Results UDS method was performed on 90 ART-naïve subjects that seeking service of care,in Hunan. In total,42.2%（38/90）of the subjects showed major NRTI or nonnucleoside reverse transcriptase inhibitor NNRTI TDRs by UDS technique,at a HIV variant frequency level of ≥1%,15.6%（14/90）showed NRTI TDR,16.7% （15/90） showed a major NNRTI TDR and 10%（9/90）were both resistant to NRTI and NNRTI when variants were analyzed by Stanford HIVdb. Conclusion ART-naïve subjects from Hunan province, which had been predominately infected by subtype AE,would frequently possess HIV variants with NRTI/NNRTI TDRs that would affect the use of first line ART in the region,identified by the UDS technique. Further studies were needed to describe the prevalence of TDRs and to gather related information.

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