Oligonucleotide Ligation Assay for Detection of Mutations Associated with Reverse Transcriptase and Protease Inhibitor Resistance in Non-B Subtypes and Recombinant Forms of Human Immunodeficiency Virus Type 1

Abstract

The oligonucleotide ligation assay is a genotypic assay for the detection of resistance-associated mutations to reverse transcriptase and protease inhibitors in human immunodeficiency virus type 1 subtype B. This assay has been modified and developed for non-B subtypes and recombinant strains and has been evaluated with sequencing, resulting in a more sensitive assay than sequencing for non-B subtypes.Methods to identify resistance mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene are needed since the use of protease (PR) and/or reverse transcriptase (RT) inhibitors may increase the prevalence of resistance mutations (9, 11). DNA sequencing is the most commonly used method to evaluate the presence of drug resistance mutations, but its high cost and equipment requirements make it unsuitable when economic resources are limited (1,8).The oligonucleotide ligation assay (OLA) is a genotypic assay which has been used to identify point mutations in DNA for a variety of diseases (3, 5) and to detect drug resistanceassociated mutations in HIV-1 subtype B (1,7,8,15). This is the most prevalent subtype in developed countries, although non-B subtypes and recombinant forms (RFs) are dramatically increasing in developing countries, where antiretroviral therapy is starting to be accessible (13), as well as in several areas of the developed world (9, 13).In this work, we describe the development of OLA for the detection of point mutations associated with high-level resistance to PR and RT inhibitors in HIV-1 non-B subtypes and RFs, and we evaluate OLA and sequencing methods for resistance-associated mutation detection in non-B and subtype B strains.RNAs extracted from plasmas of 66 HIV-1 specimens (22 non-B subtypes and 44 subtype B strains) were selected. The distribution of the 22 non-B subtypes, according to their pol genes, was as follows: (i) three subtype G strains and one each of subtypes A 1 , C, and F 1 ; (ii) 10 circulating recombinant forms (CRFs) (nine CRF02_AG and one CRF14_BG strain); and (iii) two GKU strains and one each of subtypes AG, UA 2 , and UAJ (U, unknown fragment). Five non-B strains included in this study were characterized in our laboratory by full-length genome sequencing (4).To carry out RT-PCR, 5 to 15 l of RNA extracted from plasma was reverse transcribed following purification and sequencing with an automated fluorescence sequencer (Applied Biosystems). Reaction conditions, oligonucleotides, and the thermocycling profile were described previously (14).The OLA is based on the covalent joining of two adjacent oligonucleotides by a DNA ligase when they are hybridized to a cDNA target. A set of three oligonucleotides was designed for the detection of the following mutations: K103N, Q151M, Y181C, M184V, and T215Y in RT and D30N, V82A, and L90M in PR. Table 1 shows the drugs to which these mutations appear to confer resistance, the amino acid substitutions detected, and the oligonucleotide sequences. The 5Ј end of the common oligonucleotide is phosphorylated and designed to anneal t...

Optimization of the Oligonucleotide Ligation Assay, a Rapid and Inexpensive Test for Detection of HIV-1 Drug Resistance Mutations, for Non-North American Variants

“…Rates of indeterminate results were similar to our present study when testing subtype B and non-B viruses from Spain 15. Testing of RT codons 65, 103, 151, 181, 184, and 215 in HIV-1 subtype C South African specimens16 had an overall low reactivity rate due to high genetic variation in the region of the OLA probes.…”

Evaluation of two human immunodeficiency virus-1 genotyping systems: ViroSeq™ 2.0 and an in-house method

“…In addition to this, comparison between the two assays using a wider panel of the different HIV-1 subtypes and CRFs would be useful. Additional issues include the development of new and emerging assays for minority variants (Church et al, 2008; Johnson et al, 2007; Villahermosa et al, 2001; Beck et al, 2002; Vega et al, 2005) and assays for genotyping in integrase and envelope, particularly as new drugs against these targets become available (Grant and Zolopa, 2008; Paar et al, 2008; He et al, 2008). In-house approaches to implementing molecular diagnostics can build capacity and provide training in resource-limited settings at a cost substantially lower than commercial assays ($ 100 vs. $ 230).…”

Development and Evaluation of an Oligonucleotide Ligation Assay for Detection of Drug Resistance-Associated Mutations in the Human Immunodeficiency Virus Type 2 pol Gene

“…Like all ligation assays, the potential disadvantage of OLA is that the presence of other mutations around the ligation site may result in failure of the assay (indeterminate result). The occurrence of indeterminate results with OLA is related to the high level of genetic variability in HIV, such that the HIV-1 OLA has been modified for some non-B subtypes (33). For HIV-2, only two subtypes, A and B, are of epidemiological importance.…”