BACKGROUND: Globally, the number of people living with HIV on antiretroviral treatment (ART) reached 28.7 million in 2021. To ensure the effectiveness of treatment, WHO issued guidelines and action plans for systematic surveillance of HIV drug resistance (DR), including monitoring HIVDR to dolutegravir. Currently, Sanger-based sequencing is the primary technology for HIVDR detection and surveillance. However, it has limitations, including low throughput, high cost, and less sensitivity in detecting variants below 20%. In this study, we assessed illumina-based next generation sequencing (NGS) technology on its accuracy, precision, reproducibility, and sensitivity compared to the Sanger sequencing method.
METHODS: PCR amplicons of HIV protease, reverse transcriptase (PRT), and integrase (INT) genes from 48 WHO-EQA and commercialized analytic samples representing 8 major subtypes and recombinants (>89% of HIV coverage) were generated using ThermoFisher HIVDR genotyping kit and sequenced with illumina Nextera-XT kit. NGS sequences were compared with Sanger sequences and analyzed statistically to assess accuracy, precision, reproducibility, and variants detection at 10%, 15%, and 20% thresholds.
RESULTS: Both PRT and INT NGS sequences from 48 amplicon samples exhibited overall >99.5% accuracy (CI 99.5-100%) compared to Sanger sequences. For detecting HIVDR mutations, NGS had 99.7% agreement in PRT and 100% in INT with Sanger sequencing. For precision, NGS produced an overall 100% (ranged 99.8-100%) similarity within 8 replicates from each 12 samples. With the same 8 replicates of 12 samples, NGS generated almost identical data in PRT (99.6%) and INT (99.9%) between 3 independent runs (p=1). In a 96-sample run, NGS generated an average of 30mb data and 33,157 reads coverage per sample, which is sufficient for variant calls. In this sample panel, NGS detected an average of 2.15 and 4.77 more variants in PRT, 1.85 and 4.35 more in INT at 15% and 10% threshold compared to the Sanger sequencing at 20% threshold, respectively.
CONCLUSIONS: We successfully validated illumina-based NGS for HIVDR genotyping with high accuracy and precision compared to Sanger sequencing. The validated NGS provides a higher throughput, potentially lower cost at scale, and sensitive sequencing method for detecting a full spectrum of HIVDR mutations, which can strengthen current HIVDR surveillance to guide effective ART regimens.

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