Design and Evaluation of Next-Generation HIV Genotyping for Detection of Resistance Mutations to 28 Antiretroviral Drugs Across 5 Major Classes Including Lenacapavir.

Abstract

The emergence and spread of human immunodeficiency virus (HIV) drug-resistant strains present a major barrier to effective lifelong antiretroviral therapy. The anticipated rise in long-acting subcutaneous lenacapavir (LEN) use, along with the increased risk of transmitted resistance and pre-exposure prophylaxis-associated resistance, underscore 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 5 major classes, including LEN. We analyzed 3 study cohorts and compared our drug-resistance findings with standard-care testing results and high-fidelity sequencing data obtained through unique molecular identifier (UMI) labeling. PHG identified 2 major LEN-resistance mutations in 1 participant, confirmed by an additional independent sequencing run. Across 3 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-positive results and enabled identification of minority variants with 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 and rilpivirine. We also observed multiyear 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.