REverse transcriptase ACTivity (REACT) assay for point-of-care measurement of established and emerging antiretrovirals for HIV treatment and prevention.

Maintaining adequate levels of antiretroviral (ARV) medications is crucial for the efficacy of HIV treatment and prevention regimens. Monitoring ARV levels can predict or prevent adverse health outcomes like treatment failure or drug resistance. However, conventional ARV measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is slow, expensive, and centralized delaying clinical and behavioral interventions. We previously developed a rapid enzymatic assay for measuring nucleotide reverse transcriptase inhibitors (NRTIs) - the backbone of HIV treatment and prevention regimens - based on the drugs' termination of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here, we expand our work to include non-nucleoside reverse transcriptase inhibitors (NNRTIs) - an ARV class used in established and emerging HIV treatment and prevention regimens. We demonstrate that the REverse Transcriptase ACTivity (REACT) assay can detect NNRTIs including medications used in oral and long-acting/extended-release HIV treatment and prevention. We demonstrate that REACT can measure NNRTIs spiked in either buffer or diluted plasma and that fluorescence can be measured on both a traditional plate reader and an inexpensive portable reader that can be deployed in point-of-care (POC) settings. REACT measured clinically relevant concentrations of five NNRTIs spiked in aqueous buffer. REACT measurements showed excellent agreement between the plate reader and the portable reader, with a high correlation in both aqueous buffer (Pearson's r = 0.9807, P < 0.0001) and diluted plasma (Pearson's r = 0.9681, P < 0.0001). REACT has the potential to provide rapid measurement of NNRTIs in POC settings and may help to improve HIV treatment and prevention outcomes.
Competing Interests: Declarations. Conflict of interest: A.O. is an inventor on a patient filed (PCT/US2020/037609) based on related work on enzymatic assays for measuring reverse transcriptase inhibitors. Provisional patent applications have been filed on several components of the Harmony reader. N.P. and B.L. are inventors on two patent applications: PCT/US2021/041035 (pending international patent application) and 63/165,029 (pending U.S. provisional patent). N.P. and B.L. hold equity in a startup company that has licensed related technology and supports ongoing work in the B.L. laboratory at the University of Washington. B.L. serves as a scientific advisor for the company. The company played no role in the funding, study design, data analyses, or reporting of results for this study. C.B., M.A.S., J.T., C.C., S.G., and M.M.C. have no conflicts of interest to declare.
(© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)