At least one-quarter of the ≥20 million people receiving antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention have drug concentrations outside the therapeutic range and are at risk of treatment failure or adverse reactions. Regular antiretroviral drug measurement could help people living with HIV who receive antiretroviral therapy (ART) to suppress viral replication and also help people receiving pre-exposure prophylaxis (PrEP) to prevent HIV infection. However, the gold standard for ARV measurement is liquid chromatography tandem mass spectrometry (LC-MS/MS) which is slow, centralized, and expensive. Developing and implementing a rapid point-of-care test could help to monitor and improve medication dosing and ART/PrEP outcomes.

In this talk, I will describe the REverSe TRanscrIptase Chain Termination (RESTRICT) assay for rapid measurement of nucleotide analog drugs – used in over 90% of ART and all approved PrEP regimens – based on their inhibition of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Guided by a probabilistic model, RESTRICT uses readily available nucleic acid synthesis reagents and user-friendly sample preparation techniques to detect clinically relevant antiretroviral drug concentrations in <1 hour. Using DNA templates designed to account for the chemical structure of nucleotide analogs, we selectively measure clinically relevant concentrations of the drugs tenofovir diphosphate (TFV-DP), emtricitabine triphosphate (FTC-TP), and azidothymidine triphosphate (AZT-TP) with agreement between experiment and theory.

We completed a pilot evaluation using 18 clinical samples from clients at the Madison HIV Clinic in Seattle. Blood samples are diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader—stronger fluorescence indicating higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC–MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week). Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC–MS/MS. RESTRICT fluorescence correlated with LC–MS/MS measurements (r = − 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity.

RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring and precision dosing that could be extended to other diseases that are treated with nucleotide analogs or enzyme inhibitors.