Reverse Transcriptase Activity with CRISPR (REACTR) Assay for Rapid and User-Friendly Therapeutic Drug Monitoring in...

Cytomegalovirus (CMV) can cause severe disease and death in infants and immunocompromised people. The first-line drug ganciclovir has high pharmacokinetic variability which leads to significant rates of underexposure or toxicity. Intracellular ganciclovir triphosphate (GCV-TP)─ganciclovir's active anabolite─is associated with concentration-dependent neutropenia, and monitoring could enable dose individualization to improve treatment efficacy. 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, due to its high cost, labor-intensiveness, and reliance on specialized equipment. To address this gap, we adapted the REverse transcriptase ACTivity with CRISPR (REACTR) assay to measure GCV-TP. We leveraged previous work using REACTR to measure reverse transcriptase (RT) inhibitors used to treat and prevent human immunodeficiency virus (HIV) because GCV-TP serendipitously inhibits HIV RT. We designed custom DNA templates, primers, and CRISPR complexes to accurately measure GCV-TP spiked into buffer and blood. REACTR reproducibly measured clinically relevant GCV-TP concentrations using a simple workflow and equipment that are readily available in many clinical laboratories. We evaluated the assay's performance with 40 dried blood spots from infants with congenital CMV and REACTR measurements of clinical samples correlated with LC-MS/MS GCV-TP measurements (Spearman ρ = -0.811; p < 0.0001). REACTR correctly identified samples above and below simulated 50% and 75% adherence thresholds with areas under receiver operating characteristic curves (AUCs) of 0.910 and 0.957, respectively. This study highlights the potential of REACTR as a rapid and accessible alternative to LC-MS/MS for therapeutic drug monitoring of GCV-TP.