Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(11):e28184.
doi: 10.1371/journal.pone.0028184. Epub 2011 Nov 23.

Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings

Zhiyong Zhou  1 , Nick Wagar, Joshua R DeVos, Erin Rottinghaus, Karidia Diallo, Duc B Nguyen, Orji Bassey, Richard Ugbena, Nellie Wadonda-Kabondo, Michelle S McConnell, Isaac Zulu, Benson Chilima, John Nkengasong, Chunfu Yang
Affiliations

Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings

Zhiyong Zhou et al. PLoS One. 2011.

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 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 and nucleotide sequence concordance was 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 that 43.6% were 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 subtype F and unclassifiable, 0.4% each 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 saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Chunfu Yang, Zhiyong Zhou, Joshua R. DeVos and Nick Wager are the inventors in U.S. patent application no.: 61/504,522. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Difference of mixture chromatographs generated independently by 3 different operators using the optimized in-house assay from one PT sample.
Panel A shows 2 codons (37 and 41 of RT) with nucleotide base calling of AYR; Panel B shows the AWR at codon 41 (the second peaks at codon 37 were not detected in this replicate); Panel C shows ACR at codon 37 (minor T was not called by the ReCall at the cutoff of 15%) and AHR at codon 41 (almost equal height of second and third peak at the 2nd position).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Geretti AM. Epidemiology of antiretroviral drug resistance in drug-naive persons. Curr Opin Infect Dis. 2007;20:22–32. - PubMed
    1. Bennett DE. The requirement for surveillance of HIV drug resistance within antiretroviral rollout in the developing world. Curr Opin Infect Dis. 2006;19:607–614. - PubMed
    1. Bertagnolio S, Derdelinckx I, Parker M, Fitzgibbon J, Fleury H, et al. World Health Organization/HIVResNet Drug Resistance Laboratory Strategy. Antivir Ther. 2008;13(Suppl 2):49–57. - PubMed
    1. Jordan MR, Bennett DE, Bertagnolio S, Gilks CF, Sutherland D. World Health Organization surveys to monitor HIV drug resistance prevention and associated factors in sentinel antiretroviral treatment sites. Antivir Ther. 2008;13(Suppl 2):15–23. - PubMed
    1. Bennett DE, Camacho RJ, Otelea D, Kuritzkes DR, Fleury H, et al. World Health Organization surveys to monitor HIV drug resistance prevention and associated factors in sentinel antiretroviral treatment sites. PLoS One. 2008;13(Suppl 2):e4724. - PubMed

Publication types

MeSH terms

Associated data

-