Sarinoglu Rabia Can, Sili Uluhan, Hasdemir Ufuk, Aksu Burak, Soyletir Guner, Korten Volkan
Department of Medical Microbiology, Marmara University School of Medicine, Pendik Training and Research Hospital, Istanbul, Turkey.
Department of Infectious Diseases and Clinical Microbiology, Marmara University School of Medicine, Pendik Training and Research Hospital, Istanbul, Turkey.
Curr HIV Res. 2022;20(1):54-62. doi: 10.2174/1570162X19666211119111740.
The World Health Organization (WHO) recommends the surveillance of transmitted drug resistance mutations (TDRMs) to ensure the effectiveness and sustainability of HIV treatment programs.
Our aim was to determine the TDRMs and evaluate the distribution of HIV-1 subtypes using and compared next-generation sequencing (NGS) and Sanger-based sequencing (SBS) in a cohort of 44 antiretroviral treatment-naïve patients.
All samples that were referred to the microbiology laboratory for HIV drug resistance analysis between December 2016 and February 2018 were included in the study. After exclusions, 44 treatment-naive adult patients with a viral load of >1000 copies/mL were analyzed. DNA sequencing for reverse transcriptase and protease regions was performed using both DeepChek ABL single round kit and Sanger-based ViroSeq HIV-1 Genotyping System. The mutations and HIV-1 subtypes were analyzed using the Stanford HIVdb version 8.6.1 Genotypic Resistance software, and TDRMs were assessed using the WHO surveillance drug-resistance mutation database. HIV-1 subtypes were confirmed by constructing a maximum-likelihood phylogenetic tree using Los Alamos IQ-Tree software.
NGS identified nucleos(t)ide reverse transcriptase inhibitor (NRTI)-TDRMs in 9.1 % of the patients, non-nucleos(t)ide reverse transcriptase inhibitor (NNRTI)-TDRMs in 6.8 % of the patients, and protease inhibitor (PI)-TDRMs in 18.2 % of the patients at a detection threshold of ≥ 1 %. Using SBS, 2.3 % and 6.8 % of the patients were found to have NRTI- and NNRTI-TDRMs, respectively, but no major PI mutations were detected. M41L, L74I, K65R, M184V, and M184I related to NRTI, K103N to NNRTI, and N83D, M46I, I84V, V82A, L24I, L90M, I54V to the PI sites were identified using NGS. Most mutations were found in low-abundance (frequency range: 1.0 % - 4.7 %) HIV-1 variants, except M41L and K103N. The subtypes of the isolates were found as follows; 61.4 % subtype B, 18.2 % subtype B/CRF02_AG recombinant, 13.6 % subtype A, 4.5 % CRF43_ 02G, and 2.3 % CRF02_AG. All TDRMs, except K65R, were detected in HIV-1 subtype B isolates.
The high diversity of protease site TDRMs in the minority HIV-1 variants and prevalence of CRFs were remarkable in this study. All minority HIV-1 variants were missed by conventional sequencing. TDRM prevalence among minority variants appears to be decreasing over time at our center.
世界卫生组织（WHO）建议监测传播性耐药突变（TDRMs），以确保艾滋病毒治疗方案的有效性和可持续性。
我们的目的是在44例未接受过抗逆转录病毒治疗的患者队列中，使用下一代测序（NGS）和基于桑格测序（SBS）来确定TDRMs，并评估HIV-1亚型的分布情况，并进行比较。
2016年12月至2018年2月间送至微生物实验室进行艾滋病毒耐药性分析的所有样本均纳入本研究。排除后，对44例病毒载量>1000拷贝/mL的未接受过治疗的成年患者进行分析。使用DeepChek ABL单轮试剂盒和基于桑格测序的ViroSeq HIV-1基因分型系统对逆转录酶和蛋白酶区域进行DNA测序。使用斯坦福HIVdb 8.6.1版基因耐药软件分析突变和HIV-1亚型，并使用WHO监测耐药突变数据库评估TDRMs。使用洛斯阿拉莫斯IQ-Tree软件构建最大似然系统发育树来确认HIV-1亚型。
在检测阈值≥1%时，NGS在9.1%的患者中鉴定出核苷类逆转录酶抑制剂（NRTI）-TDRMs，在6.8%的患者中鉴定出非核苷类逆转录酶抑制剂（NNRTI）-TDRMs，在18.2%的患者中鉴定出蛋白酶抑制剂（PI）-TDRMs。使用SBS时，分别有2.3%和6.8%的患者被发现存在NRTI-和NNRTI-TDRMs，但未检测到主要的PI突变。使用NGS鉴定出与NRTI相关的M41L、L74I、K65R、M184V和M184I，与NNRTI相关的K103N，以及与PI位点相关的N83D、M46I、I84V、V82A、L24I、L90M、I54V。除M41L和K103N外，大多数突变存在于低丰度（频率范围：1.0% - 4.7%）的HIV-1变体中。分离株的亚型如下：61.4%为B亚型，18.2%为B/CRF02_AG重组亚型，13.6%为A亚型，4.5%为CRF43_02G，2.3%为CRF02_AG。除K65R外，所有TDRMs均在HIV-1 B亚型分离株中检测到。
本研究中少数HIV-1变体中蛋白酶位点TDRMs的高度多样性和CRFs的流行情况值得关注。传统测序遗漏了所有少数HIV-1变体。随着时间的推移，我们中心少数变体中TDRMs的流行率似乎在下降。
