title: Improvement of the oligonucleotide ligation assay for detection of the M184V drug-resistant mutation in patients infected with human immunodeficiency virus type 1 subtype CRF01_AE.
abstract: Methods based on genetic sequencing to monitor drug-resistance mutations in human immunodeficiency virus type 1 (HIV-1) require expensive instruments and are only capable of detecting mutant strains comprising >20% of virus populations. The National Institutes of Health's AIDS Research and Reference Reagent Program (NIH ARRRP) makes available a probe-based method, an oligonucleotide ligation assay (OLA-ARRRP), which is less expensive and more sensitive than sequencing to detect such mutations for HIV-1 subtype B. In this study, an OLA was designed to detect the Methionine to Valine mutation at codon 184 (M184V) of the reverse transcriptase (RT) gene in the circulating recombinant form AE strain of HIV-1 (HIV-1 CRF01_AE) common in Thailand, and was evaluated in Thai patients experiencing treatment failure. The subtype-specific OLA-CRF01_AE proved superior to OLA-ARRRP in detecting M184V, although this mutation existed in the genome of the multiple-drug-resistant virus at lower minimal detection levels of 3% prevalence of mutated virus, compared to 50% for OLA-ARRRP. On evaluation using clinical specimens, OLA-CRF01_AE showed excellent agreement with nucleotide sequencing (95.1% overall concordance, kappa>0.79), and the sensitivity was 100% for wild-type and 93.9% for mutant detection at codon 184. The OLA-CRF01_AE also detected M184V mutations in 2.4% (1/42) of specimens that were not detected by sequencing. The indeterminate detection by OLA-CRF01_AE was decreased, from 16.7% to 4.8%, in the samples containing mutant genotype when the strategy using unmodified- as a substitute of the modified-mutant detector probe was applied. Because of their low cost, sensitivity, and ease of use, the OLA-CRF01_AE is an attractive alternative to standard sequencing in resource-limited countries affected by this subtype of HIV-1.
authors.affiliation: Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Sirikwan
authors.initials: S
authors.lastname: Dokuta
authors.forename: Utaiwan
authors.initials: U
authors.lastname: Utaipat
authors.forename: Jutarat
authors.initials: J
authors.lastname: Praparattanapan
authors.forename: Jeitsada
authors.initials: J
authors.lastname: Keitkarn
authors.forename: Niwat
authors.initials: N
authors.lastname: Maneekarn
authors.forename: Thira
authors.initials: T
authors.lastname: Sirisanthana
authors.forename: Khuanchai
authors.initials: K
authors.lastname: Supparatpinyo
mesh_terms: D000328:Adult; D024882:Drug Resistance, Viral; D005260:Female; D015658:HIV Infections / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020125:Mutation, Missense; D015345:Oligonucleotide Probes / Q000235:genetics; D012680:Sensitivity and Specificity; D013785:Thailand
chemical_list: D015345:Oligonucleotide Probes; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase