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

Abstract

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.

Epidemiology of GB virus type C among patients infected with HIV in Singapore

“…Plasma samples were isolated from blood as described previously [Lee et al, ]. All specimens had HIV‐1 viral load tested by the COBAS AmpliPrep/COBAS TaqMan HIV‐1 Test, version 2.0 (Roche Diagnostics, Mannheim, Germany) prior to this study.…”

“…Sequencing of the purified product was carried out on the Applied Biosystems 16‐capillary ABI Prism3130xl genetic analyzer, according to instructions from the manufacturer. Gel‐purification, sequencing reaction, and post‐reaction clean up protocols were performed as described previously [Lee et al, ]. The purified product was sequenced bi‐directionally, using the forward and reverse sequencing primers, along with the internal sequencing primers (Table ).…”

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

“…The new method was originally designed to work with six sequencing primer to sequence all 99 codons of the protease gene and the first 300 codons of the reverse transcriptase gene 20,32 . Similar methods also use six to eight primers 33,34 . Some recently published methods have used less than six primers, although sometimes sequencing the protease and RT genes seprately 35,36 .…”

“…Several low cost in-house methods have been described in efforts to try to make HIV drug resistance genotyping more affordable 33,34,36 . There is no doubt of the need to integrate drug resistance testing into the continuum of care for individuals on antiretroviral therapy in resource-limited settings.…”

“…However, if the method would be used in other parts of the world where CRF02_AG is prevalent, there is a need for the modification of the primers since the method failed to amplify one of the panel samples that was shown to have CRF02_AG. Alternatively, a degenerate set of primers sensitive to all group M viruses 33,36 could be used in regions where the subtype distribution is more heterogeneous 38 .…”