A Validated In-House Assay for HIV Drug Resistance Mutation Surveillance from Dried Blood Spot Specimens

J Virol Methods. 2024 Apr 9:114939. doi: 10.1016/j.jviromet.2024.114939. Online ahead of print.ABSTRACTDespite increasing scale-up of antiretroviral therapy (ART) coverage, challenges related to adherence and HIV drug resistance (HIVDR) remain. The high cost of HIVDR surveillance is a persistent challenge with implementation in resource-constrained settings. Dried blood spot (DBS) specimens have been demonstrated to be a feasible alternative to plasma or serum for HIVDR genotyping and are more suitable for lower resource settings. There is a need for affordable HIVDR genotyping assays which can amplify HIV-1 sequences from DBS specimens, particularly those with low viral loads, at a low cost. Here, we present an in-house assay capable of reliably amplifying HIV-1 protease and partial reverse transcriptase genes from DBS specimens, which covers the complete World Health Organization 2009 list of drug resistance mutations under surveillance. DBS specimens were prepared using whole blood spiked with HIV-1 at concentrations of 10,000, 5,000, 1,000, and 500 copies/mL (n=30 for each concentration). Specimens were tested in triplicate. A two-step approach was used consisting of cDNA synthesis followed by nested PCR. The limit of detection of the assay was calculated to be approximately 5,000 (95% CI: 3,200-10,700) copies/mL for the protease gene and 3,600 (95% CI: 2,200-10,000) copies/mL for reverse transcriptase. The assay was observed to be most sensitive with higher viral load sp...
Source: Journal of Virological Methods - Category: Virology Authors: Source Type: research