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. 2024 Aug 14;14(16):1766.
doi: 10.3390/diagnostics14161766.

Enhancing Precision in HIV Treatment: Validation of a Robust Next-Generation Sequencing System for Drug Resistance Mutation Analysis

Ashutosh Vashisht  1 , Ashis K Mondal  1 , Vishakha Vashisht  1 , Sudha Ananth  1   2 , Ahmet Alptekin  1 , Kimya Jones  1 , Jaspreet K Farmaha  1 , Ravindra Kolhe  1
Affiliations

Enhancing Precision in HIV Treatment: Validation of a Robust Next-Generation Sequencing System for Drug Resistance Mutation Analysis

Ashutosh Vashisht et al. Diagnostics (Basel). .

Abstract

Background: Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy.

Aim: 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.

Method: 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.

Results: 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.

Scope: 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.

Keywords: HIV; antiretroviral therapy; clinical validation; drug resistance; next-generation sequencing.

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Conflict of interest statement

Sudha Ananth was employed by the company Illumina. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the steps involved in the workflow of Vela NGS sample preparation, sequencing, and reporting.
Figure 2
Figure 2
Frequency of the occurrence of DRMs in the (a) NRTI, (b) NNRTI, (c) PI, and (d) IN regions among the 40 samples.
Figure 3
Figure 3
A Bland–Altman plot was used to assess the agreement (how closely the measurements obtained from the two different methods or systems align with each other) between the Vela NGS and Abbott ViroSeq systems by plotting the differences between them against their average. Individual data points, depicted as black dots, represent differences in values obtained from both techniques for identical mutations. The central thick line represents the zero bias, while the dotted lines above and below denote the resulting bias (depicting that one method consistently measures higher or lower values compared to the other method). The clustering of these dots around the zero-difference line indicates an overall statistically significant agreement between the systems. The upper (UL) and lower limits (LL) of agreement are shown by a colored area representing the range within which 95% of the differences between measurements from the two systems fall.
See this image and copyright information in PMC

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