Emory Vaccine Center, Emory University, Atlanta, Georgia, USA.
Institute of Biomedical Investigations in Retrovirus and AIDS (INBIRS), School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.
AIDS. 2020 Dec 1;34(15):2201-2210. doi: 10.1097/QAD.0000000000002697.
Short-read next-generation sequencing (NGS) has been implemented to study the resistance profile of HIV as it provides a higher sensitivity than Sanger sequencing. However, short-reads only generates a consensus view of the viral population rather than a reconstruction of the viral haplotypes. In this study, we evaluated the resistance profile of HIV quasispecies in patients undergoing treatment failure using SMRT sequencing.
Whole-pol RT-PCR was performed on viral RNA extracted from plasma samples of 38 HIV-positive individuals undergoing treatment failure, and sequenced in the RSII instrument. Error correction and viral haplotype phasing was performed with the Multilayer Directed Phasing and Sequencing (MDPSeq) algorithm. Presence of resistance mutations reported by the IAS-USA in 2017 was assessed using an in-house script.
The SMRT sequencing-based test detected 131/134 resistance mutations previously detected using a Sanger sequencing-based test. However, the SMRT test also identified seven additional mutations present at an estimated frequency lower than 30%. The intra-host phylogenetic analysis showed that seven samples harbored at least one resistance variant at 20--80% frequency. The haplotype-resolved sequencing revealed viral diversification and selection of new resistance during suboptimal treatment, an overall trend toward selection and accumulation of new resistance mutations, as well as the co-existence of resistant and susceptible variants.
Our results validate the SMRT sequencing-based test for detection of HIV drug resistance. In addition, this method unraveled the complex dynamic of HIV quasispecies during treatment failure, which might have several implications on clinical management.
短读下一代测序（NGS）已被用于研究 HIV 的耐药谱，因为它比 Sanger 测序具有更高的灵敏度。然而，短读只生成病毒群体的共识视图，而不是病毒单倍型的重建。在这项研究中，我们使用 SMRT 测序评估了治疗失败患者中 HIV 准种的耐药谱。
对 38 名治疗失败的 HIV 阳性个体血浆样本中的病毒 RNA 进行全-pol RT-PCR，在 RSII 仪器上进行测序。使用多层定向相位和测序（MDPSeq）算法进行错误校正和病毒单倍型相位。使用内部脚本评估 2017 年 IAS-USA 报告的耐药突变。
基于 SMRT 测序的检测方法检测到了 131/134 个先前使用基于 Sanger 测序的检测方法检测到的耐药突变。然而，SMRT 检测方法还鉴定了另外 7 个估计频率低于 30%的突变。宿主内系统发育分析表明，7 个样本至少携带一种耐药变异，频率在 20%到 80%之间。单倍型解析测序揭示了在治疗效果不佳时病毒的多样化和新耐药的选择，总体趋势是选择和积累新的耐药突变，以及耐药和敏感变异的共存。
我们的结果验证了基于 SMRT 测序的检测方法用于检测 HIV 耐药性。此外，这种方法揭示了治疗失败期间 HIV 准种的复杂动态，这可能对临床管理产生多种影响。
