Frequencies and dynamics of HIV variants characterized by NGS exhibiting ART-associated DRM

Frequency and dynamics of drug resistance mutations (DRM) in treatment naïve HIV-infected men-who-have-sex-with-men (MSM) were studied longitudinally. Data were collected from 10 (ten) HIV-infected individuals (P) studied longitudinally from early/acute-to-chronic HIV infection stages involving up to 5 years of follow-up. HIV-polymerase gene was amplified by PCR and further used for HIV subtype characterization and genotypic antiretroviral therapy (ART) resistance testing. HIV-genotypic ART resistance testing was performed using next-generation sequencing (NGS) by MiSeq (Illumina) on clinical samples. Consecutive samplings from each individual were numbered (p1.1; p1.2; p1.3, etc.). The spreadsheet shows major drug-associated resistance mutations (DRM) defined according to Stanford University database (https://hivdb.stanford.edu/hivseq/by-mutations/). The analyzed region spanned up to 34 DRM positions. The frequencies of DRM were determined considering as valid those positions having ≥ 1000 reads of depth (red values are those that were discarded). Taking into consideration the frequency for each codon, the aminoacid were classified as "major" (highest frequency), and "second" or "tertiary" according to its frequency. These codon sites were classified into 5 categories according to the nature of the detected substitution, as follows: (i) “Wild type” (WT): the most abundant triplet was the only one with more than 0.01 of abundance and the encoded aminoacid was associated with susceptibility to treatment (non-colored rows); (ii) “Major Drug Resistance Mutation” (Major DRM): either the dominant or the secondary variant encoded a treatment resistance aminoacid (violet rows); (iii) “Stop”: the secondary variant encoded a stop codon or non-sense mutation (dark gray rows); (iv) “Synonymous mutation Wild Type” (smWT): both the dominant and the secondary variant encoded the same susceptibility associated aminoacid (blue rows); and (v) “Unknown”: either the dominant or the secondary variant encoded an aminoacid with an unknown impact on the resistance profile (yellow rows). Among HIV-1 therapy-naïve MSM longitudinally studied, the frequency of DRM was low and mostly circumscribed to minority variants detected at acute/early stages of infection. At intra-patient, their frequency decreases as the infection progresses to chronicity. This information should be useful when considering the still controversial HIV baseline genotypic antiretroviral resistance testing implementation before starting HIV treatment.本研究对初治HIV感染男男性行为者（men-who-have-sex-with-men, MSM）体内的药物耐药突变（drug resistance mutation, DRM）发生频率与动态变化进行了纵向队列研究。研究共纳入10名HIV感染者（编号为P），对其从HIV感染早期/急性期至慢性期开展长达5年的纵向随访。通过聚合酶链式反应（PCR）扩增HIV聚合酶基因，后续用于HIV亚型鉴定以及基因型抗反转录病毒治疗（antiretroviral therapy, ART）耐药性检测。采用Illumina公司MiSeq测序平台的下一代测序（next-generation sequencing, NGS）技术对临床样本进行HIV基因型ART耐药性检测。每名受试者的连续采样样本均进行编号（如p1.1、p1.2、p1.3等）。本数据集表格展示了根据斯坦福大学数据库（https://hivdb.stanford.edu/hivseq/by-mutations/）定义的主要药物相关耐药突变（DRM）。本次分析覆盖最多34个DRM位点。DRM的频率统计以测序深度≥1000的位点为有效位点（红色数值为被剔除的位点）。结合每个密码子的突变频率，氨基酸变异可按频率分为"主要"（频率最高）、"次要"或"三级"三类。这些密码子位点可按检测到的碱基替换类型分为5类，具体如下：(i) "野生型（Wild type, WT）"：丰度最高的三联密码子为唯一丰度＞0.01的密码子，且其编码的氨基酸与药物敏感性相关（无填充色行）；(ii) "主要药物耐药突变（Major DRM）"：显性或次要变异体编码耐药性相关氨基酸（紫色填充行）；(iii) "无义突变（Stop）"：次要变异体编码终止密码子或无义突变（深灰色填充行）；(iv) "同义突变野生型（Synonymous mutation Wild Type, smWT）"：显性与次要变异体均编码与药物敏感性相关的同种氨基酸（蓝色填充行）；(v) "未知影响"：显性或次要变异体编码的氨基酸对耐药性表型的影响尚不明确（黄色填充行）。在本研究纳入的纵向随访HIV-1初治MSM人群中，DRM的发生频率较低，且大多局限于感染急性期/早期检出的低丰度变异株。在个体内部，DRM的频率随感染进展至慢性期而逐渐降低。本研究结果可为临床启动HIV治疗前仍存在争议的HIV基线基因型ART耐药检测实施提供参考依据。