Medical Faculty Johannes Kepler University, Center for Medical Research, Krankenhausstraße 5, Linz, Austria.
Institute of Laboratory Medicine, Kepler Universitätsklinikum, Med Campus III, Krankenhausstraße 9, Linz, Austria.
Curr HIV Res. 2022;20(3):213-221. doi: 10.2174/1570162X20666220324111418.
Typically, genotypic resistance testing is recommended at the start of antiretroviral therapy and is even mandatory in cases of virologic failure. The material of choice is plasma viral RNA. However, in patients with low viremia (viral load < 500 copies/ml), resistance testing by population-based sequencing is very difficult.
Therefore, we aimed to investigate whether next generation sequencing (NGS) from proviral DNA and RNA could be an alternative.
EDTA blood samples (n = 36) from routine clinical viral load testing were used for the study. Viral loads ranged from 96 to 390,000 copies/mL, with 100% of samples having low viremia. Distribution of subtypes; A (n = 2), B (n = 16), C (n = 4), D (n = 2), G (1), CRF02 AG (n = 5), CRF01 AE (n = 5), undefined/mixed (n = 4). The extracted consensus sequences were uploaded to the Stanford HIV Drug Resistance Data Base and Geno2pheno for online analysis of drug resistance mutations and resistance factors.
A total of 2476 variants or drug resistance mutations (DRMs) were detected with Sanger sequencing, compared with 2892 variants with NGS. An average of 822/1008 variants were identified in plasma viral RNA by Sanger or NGS sequencing, 834/956 in cellular viral RNA, and 820/928 in cellular viral DNA.
Both methods are well suited for the detection of HIV substitutions or drug resistance mutations. Our results suggest that cellular RNA or cellular viral DNA is an informative alternative to plasma viral RNA for variant detection in patients with low viremia, as shown by the high correlation of variants in the different viral pools. We show that by using UDS, a plus of two DRMs per patient becomes visible, which can make a big difference in the assessment of the expected resistance behavior of the virus.
通常情况下，在开始抗逆转录病毒治疗时建议进行基因耐药性检测，在病毒学失败的情况下甚至要求进行该检测。首选的检测材料是血浆病毒 RNA。然而，在病毒载量低（病毒载量<500 拷贝/ml）的患者中，基于群体的测序进行耐药性检测非常困难。
因此，我们旨在研究从前病毒 DNA 和 RNA 进行下一代测序（NGS）是否可以作为替代方法。
使用来自常规临床病毒载量检测的 EDTA 血液样本（n=36）进行研究。病毒载量范围为 96 至 390,000 拷贝/ml，所有样本均有 100%的低病毒载量。亚型分布：A（n=2）、B（n=16）、C（n=4）、D（n=2）、G（1）、CRF02 AG（n=5）、CRF01 AE（n=5）、未定义/混合（n=4）。提取的共识序列被上传至斯坦福 HIV 耐药性数据库和 Geno2pheno，以在线分析耐药突变和耐药因素。
与 NGS 相比，通过 Sanger 测序共检测到 2476 个变异或耐药突变（DRMs），而通过 NGS 则检测到 2892 个变异。通过 Sanger 或 NGS 测序在血浆病毒 RNA 中平均鉴定出 822/1008 个变异，在细胞病毒 RNA 中平均鉴定出 834/956 个变异，在细胞病毒 DNA 中平均鉴定出 820/928 个变异。
两种方法都非常适合检测 HIV 替换或耐药突变。我们的结果表明，在病毒载量低的患者中，细胞 RNA 或细胞病毒 DNA 是检测变异的一种有意义的替代方法，不同病毒池中的变异具有高度相关性。我们证明，通过使用 UDS，每个患者可额外检测到两个耐药突变，这在评估病毒的预期耐药行为方面可能会产生很大影响。
