1. A pragmatic approach to HIV-1 drug resistance determination in resource-limited settings by use of a novel genotyping assay targeting the reverse transcriptase-encoding region only.
- Author
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Aitken SC, Bronze M, Wallis CL, Stuyver L, Steegen K, Balinda S, Kityo C, Stevens W, Rinke de Wit TF, and Schuurman R
- Subjects
- Africa, Child, DNA Primers genetics, Developing Countries, Europe, Genotype, Humans, Microbial Sensitivity Tests methods, Plasma virology, RNA, Viral genetics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Drug Resistance, Viral, HIV Infections virology, HIV Reverse Transcriptase genetics, HIV-1 genetics, Molecular Diagnostic Techniques methods
- Abstract
In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples.
- Published
- 2013
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