Abstract
Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.
Highlights
Since human immunodeficiency virus (HIV) was recognized in the 1980s [1], 75 million people have become infected, of which about half have died of AIDS [2]
Leftover blood specimens from HIV-infected Kenyans collected as part of an observational study of transmitted drug resistance conducted in Kenya in 2010 were tested and analyzed for a retrospective study approved by Human Subjects Protection Committees at Seattle Children’s Hospital and Kenyatta National Hospital
Clinical specimens (N = 26) from Kenyan’s infected with HIV-1 subtypes A, C, D, AE and G, and plasmid standards at 0, 5 and 50% MUT were tested for drug resistance mutations at four HIV codons (K103N, Y181C, M184V and G190A, N total = 104) by both paper CDD and plate CDD oligonucleotide ligation assay (OLA) (Figs 4 and 5)
Summary
Since human immunodeficiency virus (HIV) was recognized in the 1980s [1], 75 million people have become infected, of which about half have died of AIDS [2]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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