Abstract
Regular HIV-1 viral load monitoring is the standard of care to assess antiretroviral therapy effectiveness in resource-rich settings. Persistently elevated viral loads indicate virologic failure (VF), which warrants HIV drug resistance testing (HIVDRT) to allow individualized regimen switches. However, in settings lacking access to HIVDRT, clinical decisions are often made based on symptoms, leading to unnecessary therapy switches and increased costs of care. This work presents a proof-of-concept assay to detect M184V, the most common drug resistance mutation after first-line antiretroviral therapy failure, in a paper format. The first step isothermally amplifies a section of HIV-1 reverse transcriptase containing M184V using a recombinase polymerase amplification (RPA) assay. Then, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow enzyme-linked immunosorbent assay (ELISA) differentiates between OLA-labeled products with or without M184V. Our method shows 100% specificity and 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory.
Highlights
The number of deaths each year due to HIV has been decreasing, from a high of 2.3 million in 2006 to the current 1 million in 2016 [1,2]
Because of the introduction of lateral flow antibody tests, patients on antiretroviral therapy (ART) are predicted to have near-normal life expectancy, and effective treatment of HIV to achieve viral load suppression is increasingly recognized as an important strategy for individual benefit and to prevent new infections at the population level [3,4]
We demonstrate proof-of-concept for a modular assay that includes recombinase polymerase amplification (RPA)-based isothermal amplification of the region of HIV-1 DNA that may contain four single nucleotide mutations associated with drug resistance, ligation of tagged oligonucleotide probes complementary to the amplified sequence (OLA), and sensitive and specific detection of the ligated products via a paper-based lateral flow enzyme-linked immunosorbent assay (ELISA) with visual readout
Summary
The number of deaths each year due to HIV has been decreasing, from a high of 2.3 million in 2006 to the current 1 million in 2016 [1,2]. Because of the introduction of lateral flow antibody tests, patients on antiretroviral therapy (ART) are predicted to have near-normal life expectancy, and effective treatment of HIV to achieve viral load suppression is increasingly recognized as an important strategy for individual benefit and to prevent new infections at the population level [3,4]. The World Health Organization (WHO) reports that the success of ART is threatened by the emerging problem of acquired and transmitted drug resistance [5]. In some areas, including East Africa, resistance rates to non-nucleoside reverse transcriptase inhibitors (NRTIs) are above 10% [5]. Between 10% and 30% of people receiving a first-line ART regimen will develop virological failure (VF) (viral load > 1000 copies/mL) at some point during their treatment [6,7,8].
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