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

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

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries

HIV drug resistance mutation Profiles of DBS specimens collected on W-903, A-226, and M-TFN filter papers.

<p>HIV-1 drug resistance genotyping analyses of the <i>pol</i> region were performed for all the DBS specimens with a viral load ≥1,000 copies/mL and with all three types of the filter papers using a broadly sensitive genotyping assay (N = 26). Drug resistance mutations against nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were identified using the HIVdb program, and HIV-1 drug resistance profiles were determined by the HIValg program at the Stanford HIV Drug Resistance Database website. Discordant mutations that were identified in only one type of filter paper are shown in boldface type. Specimens that had a difference in drug susceptibility ratings with one of the filter paper types are indicated by asterisk (*). Eight specimens with no mutations detected in any of the filter paper types were excluded from the table.</p><p>HIV drug resistance mutation Profiles of DBS specimens collected on W-903, A-226, and M-TFN filter papers.</p

Genotyping efficiency and nucleotide sequence identities of 26 DBS specimens with a VL ≥1000 copies/mL and collected on W-903, A-226, and M-TFN filter papers.

<p>*:Fisher's exact test; ^#SD: Standard deviation.</p><p>Genotyping efficiency and nucleotide sequence identities of 26 DBS specimens with a VL ≥1000 copies/mL and collected on W-903, A-226, and M-TFN filter papers.</p