Ion Torrent Genexus as a Fast and Reliable Solution for HIV-1 Drug Resistance Testing: Comparison with the GeneStudio S5 Workflow

Next-generation sequencing (NGS) has improved HIV-1 genotypic resistance testing (GRT) by enabling the detection of minority drug-resistance variants, although interpretation of low-frequency mutations remains challenging because of sequencing artifacts. This study compared the analytical performance and workflow efficiency of two Ion Torrent platforms, GeneStudio S5 (S5) and Genexus (GX), for routine HIV-1 GRT. A total of 134 plasma samples from people with HIV were prospectively collected, and 100 samples successfully sequenced on both platforms were included in the comparative analysis. Overall concordance for resistance-associated mutations was 88.0%, with agreement rates of 97.0% for protease, 90.0% for reverse transcriptase, and 100% for integrase. Both platforms generated clinically interpretable resistance profiles; however, 13 discordant mutations were identified. Application of a standardized confirmation algorithm, integrating Stanford HIVdb analysis with manual read-level inspection in Geneious software (version 2025.2.2), reclassified several discordant mutations as low-confidence or non-confirmed variants. Operationally, GX provided a fully automated workflow with approximately 24 h turnaround time and minimal hands-on processing, whereas S5 required approximately 72 h and substantially greater operator involvement. These findings support both platforms for routine HIV-1 GRT while emphasizing the importance of standardized bioinformatic review for reliable variant interpretation.