Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, 8091 Zurich, Switzerland.
Institute of Medical Virology, University of Zurich, 8057 Zurich, Switzerland.
J Antimicrob Chemother. 2023 Sep 5;78(9):2323-2334. doi: 10.1093/jac/dkad240.
Genotypic resistance testing (GRT) is routinely performed upon diagnosis of HIV-1 infection or during virological failure using plasma viral RNA. An alternative source for GRT could be cellular HIV-1 DNA.
A substantial number of participants in the Swiss HIV Cohort Study (SHCS) never received GRT. We applied a method that enables access to the near full-length proviral HIV-1 genome without requiring detectable viraemia.
Nine hundred and sixty-two PBMC specimens were received. Our two-step nested PCR protocol was applied to generate two overlapping long-range amplicons of the HIV-1 genome, sequenced by next-generation sequencing (NGS) and analysed by MinVar, a pipeline to detect drug resistance mutations (DRMs).
Six hundred and eighty-one (70.8%) of the samples were successfully amplified, sequenced and analysed by MinVar. Only partial information of the pol gene was contained in 82/681 (12%), probably due to naturally occurring deletions in the proviral sequence. All common HIV-1 subtypes were successfully sequenced. We detected at least one major DRM at high frequency (≥15%) in 331/599 (55.3%) individuals. Excluding APOBEC-signature (G-to-A mutation) DRMs, 145/599 (24.2%) individuals carried at least one major DRM. RT-inhibitor DRMs were most prevalent. The experienced time on ART was significantly longer in DRM carriers (P = 0.001) independent of inclusion or exclusion of APOBEC-signature DRMs.
We successfully applied a reliable and efficient method to analyse near full-length HIV-1 proviral DNA and investigated DRMs in individuals with undetectable or low viraemia. Additionally, our data underscore the need for new computational tools to exclude APOBEC-related hypermutated NGS sequence reads for reporting DRMs.
基因耐药性测试（GRT）通常在 HIV-1 感染诊断或病毒学失败时进行，使用血浆病毒 RNA。GRT 的另一种替代来源可能是细胞 HIV-1 DNA。
瑞士 HIV 队列研究（SHCS）中的相当一部分参与者从未接受过 GRT。我们应用了一种方法，可以在不要求检测到可检测病毒血症的情况下，获得接近全长前病毒 HIV-1 基因组。
共收到 962 份 PBMC 标本。我们应用两步嵌套 PCR 方案生成 HIV-1 基因组的两个重叠长程扩增子，通过下一代测序（NGS）进行测序，并通过 MinVar 进行分析，这是一种检测耐药突变（DRMs）的管道。
681 个（70.8%）样本成功扩增、测序和分析。MinVar 中包含的 pol 基因的部分信息仅为 82/681（12%），可能是由于前病毒序列中自然发生的缺失。所有常见的 HIV-1 亚型都成功测序。我们在 331/599（55.3%）个体中至少检测到一种高频（≥15%）的主要 DRM。排除 APOBEC 签名（G 到 A 突变）DRMs，145/599（24.2%）个体携带至少一种主要 DRM。逆转录酶抑制剂 DRMs 最为普遍。DRM 携带者的 ART 经验时间明显更长（P=0.001），无论是否排除 APOBEC 签名 DRMs。
我们成功地应用了一种可靠高效的方法来分析接近全长的 HIV-1 前病毒 DNA，并研究了低病毒血症或无病毒血症个体中的 DRM。此外，我们的数据强调需要新的计算工具来排除 APOBEC 相关的高突变 NGS 序列读数，以报告 DRM。
