A validated in-house assay for HIV drug resistance mutation surveillance from dried blood spot specimens

Bronwyn Neufeld; Chantal Munyuza; Alexandria Reimer; Rupert Capiña; Emma R Lee; Marissa Becker; Paul Sandstrom; Hezhao Ji; François Cholette

doi:10.1016/j.jviromet.2024.114939

A validated in-house assay for HIV drug resistance mutation surveillance from dried blood spot specimens

J Virol Methods. 2024 Apr 10:327:114939. doi: 10.1016/j.jviromet.2024.114939. Online ahead of print.

Authors

Bronwyn Neufeld¹, Chantal Munyuza², Alexandria Reimer², Rupert Capiña², Emma R Lee², Marissa Becker³, Paul Sandstrom², Hezhao Ji⁴, François Cholette⁴

Affiliations

¹ National Sexually Transmitted and Blood-Borne Infections Laboratory, J.C. Wilt Infectious Diseases Research Centre at the National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada. Electronic address: Bronwyn.Neufeld1@alumni.lshtm.ac.uk.
² National Sexually Transmitted and Blood-Borne Infections Laboratory, J.C. Wilt Infectious Diseases Research Centre at the National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada.
³ Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Winnipeg, Canada; Department of Community Health Sciences, University of Manitoba, Winnipeg, Canada.
⁴ National Sexually Transmitted and Blood-Borne Infections Laboratory, J.C. Wilt Infectious Diseases Research Centre at the National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada; Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Winnipeg, Canada.

PMID: 38604585
DOI: 10.1016/j.jviromet.2024.114939

Abstract

Despite 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, 5000, 1000, 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 5000 (95% CI: 3200-10,700) copies/mL for the protease gene and 3600 (95% CI: 2200-10,000) copies/mL for reverse transcriptase. The assay was observed to be most sensitive with higher viral load specimens (97.8% [95% CI: 92.2-99.7]) for both protease and reverse transcriptase at 10,000 copies/mL with performance decreasing with the use of specimens with lower viral loads (46.7% [36.1-57.5] and 60.0% [49.1-70.2] at 500 copies/mL for protease and reverse transcriptase, respectively). Ultimately, this assay presents a promising opportunity for use in resource-constrained settings. Future work should involve validation under field conditions including sub-optimal storage conditions and preparation of DBS with fingerprick blood in order to accurately reflect real-world collection scenarios.

Keywords: Dried blood spots; Drug resistance; HIV; Surveillance.