Title: Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.
Authors: Zhou Z, Wagar N, Devos JR, Rottinghaus E, Diallo K, Nguyen DB, Bassey O, Ugbena R, Wadonda-Kabondo N, Mcconnell MS, Zulu I, Chilima B, Nkengasong J, Yang C .
Journal: PLoS One,6:e28184 (2011)
Abstract
Abstract
Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low-cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of the pol gene.
The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6%, and 99.1%, respectively.
The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P < 0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped, with a nucleotide sequence concordance of 98.8%.
Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped.
Phylogenetic analysis of the 236 sequences revealed the following distribution:
- 43.6% CRF01_AE
- 25.9% Subtype C
- 13.1% CRF02_AG
- 5.1% Subtype G
- 4.2% Subtype B
- 2.5% Subtype A
- 2.1% each of Subtype F and Unclassifiable
- 0.4% each of CRF06_CPX, CRF07_BC, and CRF09_CPX
Conclusions
The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE®, and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost savings make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.
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Citation: Zhou Z, Wagar N, Devos JR, Rottinghaus E, Diallo K, Nguyen DB, Bassey O, Ugbena R, Wadonda-Kabondo N, Mcconnell MS, Zulu I, Chilima B, Nkengasong J, Yang C . Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings. PLoS One,6:e28184 (2011).