Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings

PLOS ONE, Nov 2011

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.

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Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings

et al. (2011) 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(11): e28184. doi:10.1371/journal.pone.0028184 Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings Zhiyong Zhou 0 Nick Wagar 0 Joshua R. DeVos 0 Erin Rottinghaus 0 Karidia Diallo 0 Duc B. Nguyen 0 Orji Bassey 0 Richard Ugbena 0 Nellie Wadonda-Kabondo 0 Michelle S. McConnell 0 Isaac Zulu 0 Benson Chilima 0 John Nkengasong 0 Chunfu Yang 0 Cecilio Lopez-Galindez, Centro Nacional de Microbiologa - Instituto de Salud Carlos III, Spain 0 1 Division of Global HIV/AIDS, Center for Global Health, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, United States of America, 2 Department of Health and Human Services/US CDC , Hanoi, Vietnam, 3 CDC/GAP, Abuja , Nigeria , 4 Community Health Sciences Unit, Malawi Ministry of Health , Lilongwe , Malawi , 5 Thailand Ministry of Public Health/US CDC Collaboration, Nonthaburi, Thailand, 6 Global AIDS Program CDC-Zambia , Lusaka , Zambia 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, TRUGENEH and ViroSeqH, 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 TRUGENEH and ViroSeqH 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, TRUGENEH and ViroSeqH 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. - Funding: Dr. Erin Rottinghaus is a recipient of the 2009-2011 Emerging Infectious Disease (EID) Fellowship program sponsored by American Public Health Laboratory (APHL) and CDC. The authors thank Dr. Guoqing Zhang, a recipient of APHL/CDC 2011 International EID program for statistical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. Treatment of HIV-1 infection with highly active antiretroviral therapy (HAART) in the past decades has remarkably reduced HIV/ AIDS related mortality and morbidity. However, the emergence of drug resistance in persons on antiretroviral therapy (ART) and the transmission of drug-resistant HIV strains to newly infected persons are a major threat to the global success on HIV prevention and treatment effort [1,2,3]. Recent years, under multilateral supports for HIV treatment and prevention programs, especially the U.S. President Emergency Plan for AIDS Relief (PEPFAR) with the targets of treating two million HIV-infected people with ART, preventing five million new HIV infection and care for 10 million HIV-infected people and AIDS orphans, access to antiretroviral drugs (ARVs) has been scaled up rapidly in resource-limited countries where availability of laboratory monitoring is often limited or lacking [4,5]. This creates the potential for HIV drug resistance (HIVDR) emergence and transmission in these settings. Detection and monitoring of HIVDR by molecular g (...truncated)


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Zhiyong Zhou, 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. Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings, PLOS ONE, 2011, 11, DOI: 10.1371/journal.pone.0028184