Pre-treatment HIV-drug-resistance (PDR, HIVDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is increasing globally. NNRTIs continue to be used as 1st-line antiretroviral therapy (ART) in some communities due to the cost of dolutegravir-based ART or dolutegravir-associated adverse events. A simplified version of the oligonucleotide ligation assay (OLA), “OLA-Simple”, is a low-cost, near point-of-care assay that provides ready-to-use lyophilized reagents and reports HIVDR mutations as colored lines on lateral flow strips. Our objective was to design and validate OLA-Simple for a Mexican cohort.
OLA-Simple probes to detect K65R, K103N/S, Y181C, M184 V, and G190A were optimized for HIV Mexican sequences. Sixty clinical plasma specimens were analyzed by OLA-Simple by technicians blinded to Illumina-MiSeq sequences, and HIVDR results were compared.
Plasma RNA was tested using OLA-Simple kits. OLA-Simple lateral flow strips were read by in-house software and were classified as mutant or wild-type at each codon. The comparison of results by OLA-Simple and Miseq was used to generate receiver operating characteristic (ROC) curves.
OLA-Simple PCR amplified 59/60 specimens and successfully genotyped 287/295 codons, with 8/295 (2.7%) indeterminate results. Compared to MiSeq, OLA-Simple gave 5/295 (1.7%) false-positive and 4/295 (1.4%) false-negative results. Excluding indeterminate results, OLA-Simple classified mutant with an accuracy of 97.4% and 98.8% when using thresholds at 10% and 25% mutant within an individual's HIV quasispecies, respectively.
Compared to MiSeq, OLA-Simple detected HIVDR with high sensitivity and accuracy. OLA-Simple could expand access to affordable and rapid HIVDR testing to guide appropriate ART choices in populations using NNRTI-based ART.