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Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes

2012; Elsevier BV; Volume: 54; Issue: 1 Linguagem: Inglês

10.1016/j.jcv.2012.01.010

1873-5967

Susan C. Aitken, Aletta Kliphuis, Carole L. Wallis, Mei Ling J. N. Chu, Quirine Fillekes, Roos E. Barth, Wendy Stevens, Tobias F. Rinke de Wit, Rob Schuurman,

HIV/AIDS Research and Interventions

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. 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. 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). 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. 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).