Towards Point-of-Care Measurement of Medication Levels To Improve Human Immunodeficiency Virus (HIV) Health Outcomes
Ayokunle Olanrewaju a, Benjamin Sullivan a, Alicia Gim a, Derin Sevenler b, Andrew Bender a, Paul Drain a, Jonathan Posner a
a University of Washington, Seattle, USA
b Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Boston
Proceedings of Emerging Investigators in Microfluidics Conference (EIMC)
Online, Spain, 2021 July 20th - 21st
Organizers: Adrian Nightingale, Darius Rackus and Claire Stanley
Invited Speaker, Ayokunle Olanrewaju, presentation 036
Publication date: 5th July 2021

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.

We are grateful for funding from the NIH (R01AI136648, R21AI127200, R01EB022630), the University of Washington CoMotion Innovation Gap Fund, and the Mistletoe Research Foundation. Part of this work was conducted using equipment in the Biochemical Diagnostics Foundry for Translational Research supported by the M.J Murdock Charitable Trust. Research reported in this publication was supported by the University of Washington/Fred Hutch Center for AIDS Research, an NIH-funded program under award number AI027757 which is supported by the following NIH Institutes and Centers: NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NIA, NIGMS, NIDDK. We thank Lindsay Legg, MarkJason Cabudol, Kate Thanel, Abby Howell, Charlene Hayes, and Nina Kim at the CFAR for their help with participant recruitment and sample collection. We also thank Colleen Kimsey and Harald Haugen at the International Clinical Research Center at the University of Washington for their support with DBS sample transportation, storage, and shipping; and Yardpiroon Tawon at the PHPT-AMS laboratory at Chiang Mai University for DBS drug level measurements. We are also grateful for helpful conversations and support from Andrew Bender, Marta Fernandez-Suarez, Jay Rutherford, Rebecca Sandlin, Derin Sevenler, Mehmet Toner, and Jane Zhang.

© Fundació Scito
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