Department of Medical Microbiology, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX Utrecht, Netherlands; Department of Molecular Medicine and Hematology, University of the Witwatersrand, 7 York Road, 2193 Johannesburg, South Africa; PharmAccess International, Pietersbergweg 17, 1105BM Amsterdam, Netherlands; Department of Clinical Pharmacy, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525GA Nijmegen, Netherlands; Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525GA Nijmegen, Netherlands; Department of Internal Medicine and Infectious Diseases, University Medical Centre, Heidelberglaan 100, 3584CX Utrecht, Netherlands; National Health Laboratory Services, 1 Modderfontein Road, Johannesburg, South Africa; Global Health Department, Amsterdam Institute for Global Health and Development (AIGHD), Academic Medical Center, Meibergdreef 9, 1105AZ Amsterdam, Netherlands
Aitken, S.C., Department of Medical Microbiology, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX Utrecht, Netherlands, Department of Molecular Medicine and Hematology, University of the Witwatersrand, 7 York Road, 2193 Johannesburg, South Africa; Kliphuis, A., Department of Medical Microbiology, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX Utrecht, Netherlands, PharmAccess International, Pietersbergweg 17, 1105BM Amsterdam, Netherlands; Wallis, C.L., Department of Molecular Medicine and Hematology, University of the Witwatersrand, 7 York Road, 2193 Johannesburg, South Africa; Chu, M.L., Department of Medical Microbiology, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX Utrecht, Netherlands; Fillekes, Q., Department of Clinical Pharmacy, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525GA Nijmegen, Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525GA Nijmegen, Netherlands; Barth, R., Department of Internal Medicine and Infectious Diseases, University Medical Centre, Heidelberglaan 100, 3584CX Utrecht, Netherlands; Stevens, W., Department of Molecular Medicine and Hematology, University of the Witwatersrand, 7 York Road, 2193 Johannesburg, South Africa, National Health Laboratory Services, 1 Modderfontein Road, Johannesburg, South Africa; Rinke de Wit, T.F., PharmAccess International, Pietersbergweg 17, 1105BM Amsterdam, Netherlands, Global Health Department, Amsterdam Institute for Global Health and Development (AIGHD), Academic Medical Center, Meibergdreef 9, 1105AZ Amsterdam, Netherlands; Schuurman, R., Department of Medical Microbiology, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX Utrecht, Netherlands
Background: High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes. Objectives: Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping. Study design: A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs). Results: All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n= 52; 21.7%), B (n= 12; 5.0%), C (n= 127; 52.9%), D (n= 25; 10.4%), CRF01_AE (n= 10; 4.2%), and CRF02_AG (n= 10; 4.2%), and unassigned variants (n= 10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful. Conclusions: A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS). © 2012 Elsevier B.V.