Biology subjects

Singh, M. A.

Publications and source records attributed to Singh, M. A..

3 recordsLinked to original sources

bioRxiv2025

REverse-transcriptase ACTivity with CRISPR (REACTR) Assay for Ganciclovir Triphosphate Monitoring

BackgroundCytomegalovirus (CMV) can cause severe disease and death in infants and immunocompromised people. Although effective at treating CMV, the first-line drug - ganciclovir (GCV) - has high rates of pharmacokinetic variability which leads to significant rates of underexposure or toxicity. Intracellular concentrations of ganciclovir triphosphate (GCV-TP) - GCVs active anabolite - are associated with dose-dependent neutropenia and could enable dose individualization to improve treatment efficacy and reduce adverse effects. However, GCV-TP is currently measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) which is impractical for routine use, especially in resource-limited settings, because of its high cost, labor-intensiveness, and need for specialized equipment. To address this gap, we adapted the REverse transcriptase ACTivity with CRISPR (REACTR) assay to measure GCV-TP. MethodsWe leveraged our earlier work using REACTR to measure reverse transcriptase (RT) inhibitors used to treat and prevent human immunodeficiency virus (HIV) because GCV-TP serendipitously also inhibits HIV RT. We designed custom DNA templates, primers, and CRISPR complexes to accurately measure GCV-TP spiked into buffer and blood. We evaluated the assays analytical performance with 40 dried blood spots from infants with congenital CMV. ResultsREACTR reproducibly measured clinically relevant GCV-TP concentrations using a simple workflow and equipment that are readily available in many clinical laboratories. REACTR measurements of clinical samples correlated with LC-MS/MS GCV-TP measurements (r = -0.7891; p<0.0001). ConclusionsThis study highlights the potential of REACTR as a rapid and accessible alternative to LC-MS/MS for therapeutic drug monitoring of GCV.

bioengineering↗

bioRxiv2024

Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout

Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTRs sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

bioengineering↗

bioRxiv2024

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 NNRTI 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 (Pearsons r=0.9807, P < 0.0001) and diluted plasma (Pearsons 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.

bioengineering↗