Vashisht Ashutosh, Mondal Ashis K, Vashisht Vishakha, Ananth Sudha, Alptekin Ahmet, Jones Kimya, Farmaha Jaspreet K, Kolhe Ravindra
Georgia Esoteric and Molecular Biology Laboratory, Department of Pathology, Augusta University, Augusta, GA 30912, USA.
Reagent Sciences Department, Research and Development, Illumina, San Diego, CA 92122, USA.
Diagnostics (Basel). 2024 Aug 14;14(16):1766. doi: 10.3390/diagnostics14161766.
Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy.
This study aimed to validate the Vela Diagnostics NGS platform for HIV drug resistance mutation analysis, rigorously assessed with clinical samples and CAP proficiency testing controls previously analyzed by Sanger sequencing.
The experimental approach involved the following: RNA extraction from clinical specimens, reverse transcription polymerase chain reaction (RT-PCR) utilizing the Sentosa SX 101 platform, library preparation with the Sentosa SQ HIV Genotyping Assay, template preparation, sequencing using the Sentosa SQ301 instrument, and subsequent data analysis employing the Sentosa SQ Suite and SQ Reporter software. Drug resistance profiles were interpreted using the Stanford HIV Drug Resistance Database (HIVdb) with the HXB2 reference sequence.
The Vela NGS system successfully identified a comprehensive array of drug resistance mutations across the tested samples: 28 nucleoside reverse transcriptase inhibitors (NRTI), 25 non-nucleoside reverse transcriptase inhibitors (NNRTI), 25 protease inhibitors (PI), and 10 integrase gene-specific variants. Dilution experiments further validated the system's sensitivity, detecting drug resistance mutations even at viral loads lower than the recommended threshold (1000 copies/mL) set by Vela Diagnostics.
This study underscores the validation and clinical applicability of the Vela NGS system, and its implementation may offer clinicians enhanced precision in therapeutic decision-making for individuals living with HIV.
耐多药HIV毒株对治疗效果构成挑战并增加死亡率。下一代测序（NGS）技术能快速检测变异，有助于实现个性化抗逆转录病毒治疗。
本研究旨在验证Vela诊断公司的NGS平台用于HIV耐药突变分析的效果，通过临床样本以及先前经桑格测序分析的CAP能力验证检测对照进行严格评估。
实验方法包括以下步骤：从临床标本中提取RNA，使用Sentosa SX 101平台进行逆转录聚合酶链反应（RT-PCR），使用Sentosa SQ HIV基因分型检测进行文库制备，模板制备，使用Sentosa SQ301仪器进行测序，以及随后使用Sentosa SQ Suite和SQ Reporter软件进行数据分析。使用带有HXB2参考序列的斯坦福HIV耐药数据库（HIVdb）解读耐药谱。
Vela NGS系统成功在测试样本中鉴定出一系列全面的耐药突变：28种核苷类逆转录酶抑制剂（NRTI）、25种非核苷类逆转录酶抑制剂（NNRTI）、25种蛋白酶抑制剂（PI）和10种整合酶基因特异性变异。稀释实验进一步验证了该系统的灵敏度，即使在病毒载量低于Vela诊断公司推荐阈值（1000拷贝/毫升）时也能检测到耐药突变。
本研究强调了Vela NGS系统的验证及其临床适用性，其应用可为临床医生为HIV感染者制定治疗决策提供更高的精准度。
