title: 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.
authors.affiliation: Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore, Singapore.
authors.forename: Chun Kiat
authors.initials: CK
authors.lastname: Lee
authors.forename: Hong Kai
authors.initials: HK
authors.lastname: Lee
authors.forename: Tze Ping
authors.initials: TP
authors.lastname: Loh
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Sethi
authors.forename: Evelyn Siew-Chuan
authors.initials: ES
authors.lastname: Koay
authors.forename: Julian Wei-Tze
authors.initials: JW
authors.lastname: Tang
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology* / Q000627:therapeutic use; D044466:Asian People; D024882:Drug Resistance, Viral / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques; D015658:HIV Infections / Q000175:diagnosis / Q000188:drug therapy / Q000821:virology; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D009154:Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D019380:Anti-HIV Agents; D054303:HIV Reverse Transcriptase; D016333:HIV Protease