Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations

Abstract

Objective Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations—a metabolite that indicates long-term PrEP adherence. Setting The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle. Methods We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader—stronger fluorescence indicated 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). Results 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. Conclusions This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention

“…We previously demonstrated that we could achieve a CV of 13.5% when running RESTRICT with whole blood samples diluted in water. 30 , 31 Improvements in sample preparation—incorporating a heating step after blood dilution to denature blood proteins (e.g., hemoglobin) that nonspecifically inhibit reverse transcriptase 46 —have improved the accuracy even further (data not shown).…”

“…We did not observe any significant deviation from expected RESTRICT assay performance in our pilot evaluation study with 18 clinical samples. 31 This is likely because dNTP concentrations in red blood cells and plasma are too low to effectively compete with nucleotide analogs for incorporation into complementary DNA strands synthesized by HIV RT. Our inclusion of a 10‐fold blood dilution step—to reduce non‐specific enzyme inhibition by blood components—further reduces these already low dNTP concentrations.…”

“… 49 , 50 , 51 We have previously shown that blood dilution to lyse red blood cells is a simple and effective sample preparation strategy to release TFV‐DP from red blood cells and minimize non‐specific RT inhibition by blood matrix components in whole blood samples from PrEP clients. 31 RESTRICT requires <1 μl of whole blood per test. Each reaction requires addition of 5 μl of 10% whole blood (diluted in water) to 35 μl of reaction mix.…”

“…We use dilution in water as a simple and user‐friendly sample preparation mechanism to release intracellular TFV‐DP from RBCs and decrease non‐specific inhibition by blood matrix components. 31 In a pilot clinical evaluation, RESTRICT measured clinically relevant TFV‐DP concentrations in whole blood and identified participants with TFV‐DP concentrations corresponding to adequate adherence (≥700 fmol per 3 mm dried blood spot [DBS] punch) among PrEP clients. RESTRICT represents a new class of activity‐based diagnostic assays with potential for rapid, POC measurement of antiretroviral drugs used in HIV treatment and prevention.…”

HIV pre-exposure prophylaxis adherence test using reverse transcription isothermal amplification inhibition assay

“…Recently, our group reported on an enzymatic assay, termed REverSe TRanscrIptase Chain Termination (RESTRICT), that measures DNA duplex production by reverse transcriptase in the presence of NRTI drugs. 45,46 The assay accurately distinguished TFV-DP drug levels within the clinical range for adherence and has the potential to be a useful test to identify patients with poor adherence to ART and PrEP. 45 Real-time PCR has also been used to quantify inhibitory concentrations of NRTIs in drug development against HIV-1 RT.…”