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

Abstract

Human immunodeficiency virus type 2 (HIV-2) is naturally resistant to several antiretroviral drugs, including all of the non-nucleoside reverse transcriptase inhibitors and the entry inhibitor T-20, and may have reduced susceptibility to some protease inhibitors. These resistance properties make treatment of HIV-2 patients difficult, with very limited treatment options. Therefore, early detection of resistance mutations is important for understanding treatment failures and guiding subsequent therapy decisions. With the Global Fund Initiative, a substantial number of HIV-2 patients in West Africa will receive antiretroviral therapy. Therefore, development of cheaper and more sustainable resistance assays, such as the oligonucleotide ligation assay (OLA), is a priority. In this study, we designed oligonucleotide probes to detect the Q151M mutation, associated with phenotypic resistance to zidovudine, didanosine, zalcitabine, and stavudine, and the M184V mutation, associated with phenotypic resistance to lamivudine and emtricitabine, in HIV-2. The assay was successfully developed and evaluated with 122 samples from The Gambia, Guinea Bissau, The Netherlands, and Sweden. The overall sensitivity of the assay was 98.8%, with 99.2% for Q151M and 98.4% for M184V. OLA results were compared with sequencing to give high concordances of 98.4% (Q151M) and 97.5% (M184V). OLA demonstrated a higher sensitivity for detection of minor variants as a mixture of wild-type and mutant viruses in cases when sequencing detected only the major population. In conclusion, we have developed a simple, easy-to-use, and economical assay for genotyping of drug resistance in HIV-2 that is more sustainable for use in resource-poor settings than is consensus sequencing.

Virological Response to Highly Active Antiretroviral Therapy in Patients Infected with Human Immunodeficiency Virus Type 2 (HIV-2) and in Patients Dually Infected with HIV-1 and HIV-2 in The Gambia and Emergence of Drug-Resistant Variants

“…HIV-2 RNA was extracted from 140 l of EDTA-plasma using the QIAamp viral RNA kit (Qiagen, Venlo, The Netherlands) and was eluted into 50 l nuclease-free water. The entire HIV-2 protease and RT were amplified using a single-tube reverse transcription-PCR method (Titan one-tube RT-PCR; Roche Applied Science, Lewes, United Kingdom) starting from 3 l of RNA, followed by a nested PCR with Expand High-Fidelity DNA polymerase and 0.5 to 1 l of the first-round product as previously described (24). Purified PCR products were directly sequenced on both strands and were analyzed with DNAStar software (Lasergene; DNAStar, Madison, WI) (24).…”

“…These mutations are associated with 1,000-fold phenotypic resistance to 3TC (24,27). The Q151M mutation, frequently found in HIV-2 patients failing an NRTI-containing regimen (42), was absent in these patients.…”

Human Immunodeficiency Virus Type 1 Resistance or Cross-Resistance to Nonnucleoside Reverse Transcriptase Inhibitors Currently under Development as Microbicides

“…Primer SJH12a (5Ј-CCTAATGCATACTGTGAGTCTG-3Ј) was used for the reverse transcription of vRNA and in combination with SJH11A (5Ј-AAAAGGGCTGTTGGA AATGTGG-3Ј) for the amplification of a 2,042-bp Gag-Pol fragment (nucleotide positions 2,018 to 4,060 according the HxB2 reference sequence). Nested PCR was performed on 1 l of the first-round product, using primers SJH13a (5Ј-GAGAGACAGGCTAATTTTTTAGGG-3Ј) and SJH14 (5Ј-CCTATTAGC TGCCCCATCTACATA-3Ј), as previously described (32,33 Drug susceptibility of NNRTI-resistant viruses using the TZM-bl assay. The antiviral activities of four candidate microbicide RTIs (i.e., TMC120, MIV-160, UC781, and TFV) and four therapeutic RTIs (i.e., DLV, NVP, EFV, and AZT) was measured by preincubating 10,000 TZM-bl cells (at 10 5 cells/ml in culture medium supplemented with 30 g/ml DEAE-dextran) in a 96-well plate for 30 min at 37°C in 5% CO 2 in the presence or absence of serial dilutions of the respective compound (Fig.…”

Transmitted HIV Resistance to First-Line Antiretroviral Therapy in Lima, Peru

“…22,27 More sensitive assays, such as the oligonucleotide ligation assay (OLA), can detect low concentrations of drug-resistant virus to a substantially greater degree compared to consensus sequencing. [28][29][30][31][32][33][34][35][36] In this study we evaluated the prevalence of TDR using a sensitive OLA assay in a population of ARV-naive Peruvian patients. Four drug-resistant codons were selected to screen for transmitted drug resistance based on the drug regimen being used as first-line ART in Lima, Peru, and the prevalence of transmitted resistance mutations observed in ARV-naive populations living in communities that primarily use nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART.…”