高通量下一代测序技术分析 HIV 耐药性和病毒载量的性能。
Performance of a high-throughput next-generation sequencing method for analysis of HIV drug resistance and viral load.
机构信息
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Big Data Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
出版信息
J Antimicrob Chemother. 2020 Dec 1;75(12):3510-3516. doi: 10.1093/jac/dkaa352.
OBJECTIVES
To evaluate the performance of a high-throughput research assay for HIV drug resistance testing based on whole genome next-generation sequencing (NGS) that also quantifies HIV viral load.
METHODS
Plasma samples (n = 145) were obtained from HIV-positive MSM (HPTN 078). Samples were analysed using clinical assays (the ViroSeq HIV-1 Genotyping System and the Abbott RealTime HIV-1 Viral Load assay) and a research assay based on whole-genome NGS (veSEQ-HIV).
RESULTS
HIV protease and reverse transcriptase sequences (n = 142) and integrase sequences (n = 138) were obtained using ViroSeq. Sequences from all three regions were obtained for 100 (70.4%) of the 142 samples using veSEQ-HIV; results were obtained more frequently for samples with higher viral loads (93.5% for 93 samples with >5000 copies/mL; 50.0% for 26 samples with 1000-5000 copies/mL; 0% for 23 samples with <1000 copies/mL). For samples with results from both methods, drug resistance mutations (DRMs) were detected in 33 samples using ViroSeq and 42 samples using veSEQ-HIV (detection threshold: 5.0%). Overall, 146 major DRMs were detected; 107 were detected by both methods, 37 were detected by veSEQ-HIV only (frequency range: 5.0%-30.6%) and two were detected by ViroSeq only. HIV viral loads estimated by veSEQ-HIV strongly correlated with results from the Abbott RealTime Viral Load assay (R2 = 0.85; n = 142).
CONCLUSIONS
The NGS-based veSEQ-HIV method provided results for most samples with higher viral loads, was accurate for detecting major DRMs, and detected mutations at lower levels compared with a method based on population sequencing. The veSEQ-HIV method also provided HIV viral load data.
目的
评估一种基于高通量全基因组下一代测序（NGS）的 HIV 耐药性检测研究检测方法的性能，该方法还可定量 HIV 病毒载量。
方法
从 HIV 阳性男男性行为者（HPTN 078）中获得血浆样本（n=145）。使用临床检测方法（ViroSeq HIV-1 基因分型系统和 Abbott RealTime HIV-1 病毒载量检测）和基于全基因组 NGS 的研究检测方法（veSEQ-HIV）对样本进行分析。
结果
使用 ViroSeq 获得了 HIV 蛋白酶和逆转录酶序列（n=142）和整合酶序列（n=138）。使用 veSEQ-HIV 获得了 142 个样本中 100 个（70.4%）的所有三个区域的序列；对于病毒载量较高的样本，结果获得的频率更高（93.5%的 93 个样本病毒载量>5000 拷贝/ml；50.0%的 26 个样本病毒载量为 1000-5000 拷贝/ml；23 个样本病毒载量<1000 拷贝/ml，检测结果均为 0%）。对于两种方法均有结果的样本，使用 ViroSeq 检测到 33 个样本存在耐药相关突变（DRMs），使用 veSEQ-HIV 检测到 42 个样本存在耐药相关突变（检测阈值：5.0%）。总体上，检测到 146 种主要 DRMs；107 种由两种方法检测到，37 种由 veSEQ-HIV 检测到（频率范围：5.0%-30.6%），2 种由 ViroSeq 检测到。veSEQ-HIV 检测到的 HIV 病毒载量与 Abbott RealTime 病毒载量检测结果高度相关（R2=0.85；n=142）。
结论
基于 NGS 的 veSEQ-HIV 方法为大多数病毒载量较高的样本提供了结果，对于检测主要 DRMs 是准确的，与基于群体测序的方法相比，能够检测到更低水平的突变。veSEQ-HIV 方法还提供了 HIV 病毒载量数据。
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