Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
Institute of Medical Virology, University of Zurich, Zurich, Switzerland.
J Antimicrob Chemother. 2023 Mar 2;78(3):656-664. doi: 10.1093/jac/dkac430.
Next-generation sequencing (NGS) is gradually replacing Sanger sequencing (SS) as the primary method for HIV genotypic resistance testing. However, there are limited systematic data on comparability of these methods in a clinical setting for the presence of low-abundance drug resistance mutations (DRMs) and their dependency on the variant-calling thresholds.
To compare the HIV-DRMs detected by SS and NGS, we included participants enrolled in the Swiss HIV Cohort Study (SHCS) with SS and NGS sequences available with sample collection dates ≤7 days apart. We tested for the presence of HIV-DRMs and compared the agreement between SS and NGS at different variant-calling thresholds.
We included 594 pairs of SS and NGS from 527 SHCS participants. Males accounted for 80.5% of the participants, 76.3% were ART naive at sample collection and 78.1% of the sequences were subtype B. Overall, we observed a good agreement (Cohen's kappa >0.80) for HIV-DRMs for variant-calling thresholds ≥5%. We observed an increase in low-abundance HIV-DRMs detected at lower thresholds [28/417 (6.7%) at 10%-25% to 293/812 (36.1%) at 1%-2% threshold]. However, such low-abundance HIV-DRMs were overrepresented in ART-naive participants and were in most cases not detected in previously sampled sequences suggesting high sequencing error for thresholds <3%.
We found high concordance between SS and NGS but also a substantial number of low-abundance HIV-DRMs detected only by NGS at lower variant-calling thresholds. Our findings suggest that a substantial fraction of the low-abundance HIV-DRMs detected at thresholds <3% may represent sequencing errors and hence should not be overinterpreted in clinical practice.
下一代测序（NGS）正逐渐取代 Sanger 测序（SS）成为 HIV 基因型耐药性检测的主要方法。然而，在临床环境中，关于这些方法的可比性的系统数据有限，特别是在低丰度耐药突变（DRMs）的存在及其对变异调用阈值的依赖性方面。
为了比较 SS 和 NGS 检测到的 HIV-DRMs，我们纳入了在瑞士艾滋病毒队列研究（SHCS）中接受 SS 和 NGS 序列检测的参与者，这些参与者的 SS 和 NGS 序列采集日期相差不超过 7 天。我们检测了 HIV-DRMs 的存在，并比较了不同变异调用阈值下 SS 和 NGS 之间的一致性。
我们纳入了来自 527 名 SHCS 参与者的 594 对 SS 和 NGS 序列。参与者中男性占 80.5%，在样本采集时 76.3%未接受 ART 治疗，78.1%的序列为亚型 B。总体而言，我们观察到在变异调用阈值≥5%时，HIV-DRMs 的一致性较好（Cohen's kappa >0.80）。我们观察到在较低的阈值下，低丰度 HIV-DRMs 的检测数量增加[28/417（6.7%）在 10%-25%到 293/812（36.1%）在 1%-2%阈值]。然而，在未接受 ART 治疗的参与者中，这种低丰度 HIV-DRMs 占比更高，且在大多数情况下，在之前的样本序列中未检测到，这表明阈值<3%时的测序错误很高。
我们发现 SS 和 NGS 之间具有高度一致性，但在较低的变异调用阈值下，NGS 还检测到大量低丰度 HIV-DRMs。我们的研究结果表明，在阈值<3%时检测到的大量低丰度 HIV-DRMs 可能代表测序错误，因此在临床实践中不应过度解释。
