Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes
Journal of Clinical Virology
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
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.
amino acid; efavirenz; Human immunodeficiency virus proteinase; lamivudine; lopinavir plus ritonavir; nevirapine; RNA; RNA directed DNA polymerase; stavudine; zidovudine; adult; article; child; controlled study; enzyme assay; female; genotype; highly active antiretroviral therapy; human; Human immunodeficiency virus 1; Human immunodeficiency virus 1 infection; Human immunodeficiency virus infected patient; major clinical study; male; nonhuman; priority journal; reverse transcription polymerase chain reaction; single drug dose; validation process; virus detection; virus load; Adolescent; Adult; Africa; Child; Child, Preschool; DNA Primers; Drug Resistance, Viral; Female; HIV Infections; HIV Protease; HIV Reverse Transcriptase; HIV-1; Humans; Male; Microbial Sensitivity Tests; Polymerase Chain Reaction; Sensitivity and Specificity; Young Adult