Department of Biological Sciences, University of Cyprus, 1 University Avenue, Aglantzia 2109, Nicosia, Cyprus.
Department of Biological Sciences, University of Cyprus, 1 University Avenue, Aglantzia 2109, Nicosia, Cyprus.
Infect Genet Evol. 2020 Jul;81:104243. doi: 10.1016/j.meegid.2020.104243. Epub 2020 Feb 13.
Comprehensive PCR assays for the genotypic drug resistance analysis of all HIV-1 antiretroviral agents (reverse transcriptase, protease and integrase inhibitors) are increasingly in demand due to introduction of integrase inhibitors in the first line regimens and the increasing presence of non-B HIV-1 clades around the world. This study focused on the development and evaluation of a new PCR-based assay for the amplification and sequencing of the entire HIV-1 pol region of major circulating group M HIV-1 strains in Europe for genotypic drug resistance analysis. The comprehensive touchdown PCR assay developed in this study utilized HIV-1 RNA extracted from the plasma of blood samples of consenting HIV-1 infected patients in Cyprus, collected from 2017 to 2019. The HIV-1 pol region was amplified by touchdown PCR for both the primary RT-PCR and the secondary PCR steps. Successful PCR amplicons were determined by population DNA sequencing, using the Sanger method and the genotypic drug resistance analysis was performed with the Stanford University HIV Drug Resistance Database Program. The newly developed assay successfully amplified the entire HIV-1 pol region (2844 nucleotides long) of 141 out of 144 samples of group M HIV-1 subtypes and recombinant strains of the Cyprus HIV-1 Transmission Cohort Study (CHICS) isolated from 2017 to 2019 and genotypic analyses were conducted for all currently available HIV-1 reverse transcriptase, protease and integrase inhibitors. The drug resistance, epidemiological and demographic data of these study subjects will be expanded upon in the CHICS (L.G. Kostrikis et al., manuscript in preparation for publication). The newly developed HIV-1 genotypic drug resistance assay would benefit clinical settings, and research focusing on the world-wide spread of HIV-1 drug-resistant strains, especially in geographic regions characterized by polyphyletic HIV-1 infections.
由于整合酶抑制剂被纳入一线治疗方案，以及世界各地非 B 型 HIV-1 亚型的出现越来越多，对所有 HIV-1 抗逆转录病毒药物（逆转录酶、蛋白酶和整合酶抑制剂）基因型耐药分析的综合 PCR 检测的需求日益增加。本研究专注于开发和评估一种新的基于 PCR 的检测方法，用于扩增和测序欧洲主要流行的 M 型 HIV-1 株的整个 HIV-1 pol 区，用于基因型耐药分析。本研究中开发的综合降落 PCR 检测法利用了 2017 年至 2019 年期间从塞浦路斯同意的 HIV-1 感染患者的血浆中提取的 HIV-1 RNA。该检测法在主 RT-PCR 和次 PCR 步骤中均利用降落 PCR 扩增 HIV-1 pol 区。通过群体 DNA 测序成功确定了 PCR 扩增子，使用 Sanger 法进行，并使用斯坦福大学 HIV 耐药性数据库程序进行基因型耐药分析。新开发的检测法成功地扩增了 2017 年至 2019 年期间从塞浦路斯 HIV 传播队列研究（CHICS）分离的 144 份 M 型 HIV-1 亚型和重组株中的 141 份样本的整个 HIV-1 pol 区（2844 个核苷酸长），并对所有目前可用的 HIV-1 逆转录酶、蛋白酶和整合酶抑制剂进行了基因型分析。这些研究对象的耐药性、流行病学和人口统计学数据将在 CHICS 中进一步扩展（L.G. Kostrikis 等人，即将发表的手稿）。新开发的 HIV-1 基因型耐药检测法将有益于临床环境，以及关注 HIV-1 耐药株在全球传播的研究，特别是在具有多源性 HIV-1 感染的地理区域。
