Departmental Faculty, UniCamillus, Saint Camillus International University of Health Sciences, 00131 Rome, Italy.
Department of Experimental Medicine, University of Rome "Tor Vergata", 00133 Rome, Italy.
Viruses. 2024 Sep 6;16(9):1422. doi: 10.3390/v16091422.
Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting.
Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm.
Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-B subtypes. Mutations at an NGS frequency >10% showed fair concordance among different interpretation tools.
This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.
下一代测序（NGS）正逐渐取代 Sanger 测序，用于 HIV 基因型耐药性检测（GRT）。本研究评估了不同 NGS-GRT 解释工具在实际环境中的一致性。
使用 AD4SEQ HIV-1 Solution v2 商业试剂盒，11 家意大利实验室从病毒 RNA 中生成常规 NGS-GRT 数据。NGS 结果由试剂盒提供的 SmartVir 系统和两个在线工具（HyDRA Web 和斯坦福 HIVdb）进行解释。当每个 PR/RT/IN 耐药相关位置的覆盖率>100 个读（100×）时，NGS-GRT 被认为是有效的，这些位置列在 HIVdb 9.5.1 算法中。
在 629 份 NGS-GRT 中，根据 SmartVir、HyDRA Web 和 HIVdb，分别有 75.2%、74.2%和 70.9%是有效的。考虑到至少两种解释工具，463 份（73.6%）NGS-GRT 对耐药分析具有有效的覆盖率。有效样本的比例受到病毒载量<10,000-1000 拷贝/mL 和非 B 亚型的影响。在 NGS 频率>10%的突变在不同的解释工具之间具有良好的一致性。
这项关于 NGS 耐药检测的意大利研究表明，病毒载量水平和 HIV 亚型会影响 NGS-GRT 的覆盖率。在当前 NGS-GRT 的常规方法中，只有频率>10%的突变似乎可以在不同的解释工具中可靠地检测到。
