Design and evaluation of next-generation HIV genotyping for detection of resistance mutations to 28 antiretroviral drugs across five major classes including lenacapavir

The emergence and spread of HIV drug-resistant strains present a major barrier to effective lifelong Antiretroviral Therapy (ART). The anticipated rise in long-acting subcutaneous lenacapavir (LEN) use, along with the increased risk of transmitted resistance and Pre-Exposure Prophylaxis (PrEP)-associated resistance, underscores the urgent need for advanced genotyping methods to enhance clinical care and prevention strategies. We developed the Portable HIV Genotyping (PHG) platform which combines cost-effective next-generation sequencing with cloud computing to screen for resistance to 28 antiretroviral drugs across five major classes, including LEN. We analyzed three study cohorts and compared our drug resistance findings against standard care testing results and high-fidelity sequencing data obtained through unique molecular identifier (UMI) labeling. PHG identified two major LEN-resistance mutations in one participant, confirmed by an additional independent sequencing run. Across three study cohorts, PHG consistently detected the same drug resistance mutations as standard care genotyping and high-fidelity UMI-labeling in most tested specimens. PHG's 10% limit of detection minimized false positives and enabled identification of minority variants less than 20% frequency, pointing to underdiagnosis of drug resistance in clinical care. Furthermore, PHG identified linked cross-class resistance mutations, confirmed by UMI-labeling, including linked cross-resistance in a participant who reported use of long-acting cabotegravir (CAB) and rilpivirine (RPV). We also observed multi-year persistence of linked cross-class resistance mutations. PHG demonstrates significant improvements over standard care HIV genotyping, offering deeper insights into LEN-resistance, minority variants, and cross-class resistance using a low-cost high-throughput portable sequencing technology and publicly available cloud computing.