An in-house HIV genotyping assay for the detection of drug resistance mutations in Southeast Asian patients infected with HIV-1.

Genotyping for HIV drug resistance is costly and beyond the means for many Southeast Asian patients, who are self-funded. This prompted the development of a more cost-effective, in-house assay for an ethnically diverse, Southeast Asian population at the National University Hospital in Singapore, using Sanger-based sequencing. Plasma samples from 20 treatment-failure patients with a broad spectrum of HIV drug resistance mutations were used to validate this assay clinically. Blinded testing gave concordant results for 7/7 (100%) protease drug resistance-related mutations, including one major and six minor mutations, and 111/116 (95.7%) reverse-transcriptase (RT) drug resistance-related mutations, including 65 nucleoside RT inhibitors (NRTI) and 46 non-nucleoside RT inhibitors (NNRTI) mutations. There were five discordant results, involving three NRTI- and two NNRTI-resistance-associated mutations. Highly conserved primers designed to have a wide coverage of the HIV pol gene (covering the entire protease and 395 codons of the RT region) enabled efficient multi-ethnic population-based genotyping. Reagents for this in-house test cost around 60% less than those for commercially available assays (SGD150 vs. SGD350 per sample). In addition, this assay also identified mutations located within the C-terminal domain (codons 312-560) of RT that are beyond the reach of most published and commercial GRTs. Currently, most research on C-terminal drug-resistance-related mutations has been conducted on HIV subtype B infections. Therefore this assay enables further study of these C-terminal mutations in Southeast Asian populations, where there is a high prevalence of CRF01_AE and other non-subtype B HIV infections.