An enzymatic assay for rapid and minimally instrumented ganciclovir triphosphate monitoring in infants with congenital cytomegalovirus

7 One in 200 infants is born with cytomegalovirus (CMV), and 25% experience lifelong consequences such as hearing loss or developmental delay. Intravenous ganciclovir (GCV) and its oral prodrug valganciclovir are the first-line treatments for congenital CMV (cCMV) and have been found to improve hearing and developmental outcomes. However, GCV drug levels vary widely among infants, making optimal dosing difficult without therapeutic drug monitoring (TDM). With standard dosing, underexposure and inadequate treatment occur in 30% of infants, while overexposure and significant toxicity occur in 25% of infants. Intracellular levels of the active form of the drug, GCV triphosphate (GCV-TP), are associated with dose-dependent neutropenia toxicity. However, TDM of GCV-TP is currently performed using liquid chromatography mass spectrometry (LC–MS), which is costly, labor-intensive and requires specialized equipment, making it impractical for routine clinical use. To address this gap, we have developed an enzymatic assay for GCV-TP measurement that does not rely on complex instruments or highly trained users. This test is adapted from the rapid, user-friendly and minimally instrumented REverse transcriptase ACTivity crispR (REACTR) assay that we recently developed for measuring human immunodeficiency virus (HIV) medications. REACTR measures intracellular levels of nucleotide analog drugs based on inhibition of DNA synthesis by HIV reverse transcriptase (RT) enzyme. GCV-TP inhibits HIV RT, allowing us to adapt REACTR to measure GCV-TP. REACTR assays were performed by combining CRISPR-Cas12a, CRISPR RNA, cytidine-rich DNA template, DNA primer, fluorescent DNA reporter, HIV RT and GCV-TP concentrations spiked into aqueous buffer or dried blood spots (DBS). Spiked GCV-TP concentrations (1.45e-9 M to 4.63e-8 M) corresponded to the clinical range (100–3200 fmol/6 mm punch) of GCV-TP in DBS of infants with cCMV in a recently completed clinical trial (NCT03107871). GCV-TP-spiked DBS samples were processed with a simple workflow that included dilution to a final volume of 25% in water, protease digestion with thermostable proteinase K at 23°C for 15 min and heat denaturation at 95°C for 10 min. The assay was incubated at 37°C in a microplate reader for 60 min, and fluorescent measurements were taken every minute. We normalized results using 'no drug' and 'no enzyme' controls to indicate maximum and minimum signal, respectively, and fit dose response curves to sigmoidal four-parameter logistic regression curves in GraphPad Prism. Statistically significant differentiation between spiked concentrations was confirmed using repeated measures one-way ANOVA. REACTR selectively measured clinically relevant GCV-TP concentrations in both aqueous buffer and dried blood spots within 30 min of incubation. The 50% inhibitory concentration (IC50) of GCV-TP in REACTR was 2.01e-8 M, well within the target clinical range. In aqueous buffer samples, REACTR was able to distinguish between five clinically relevant concentrations of GCV-TP corresponding to 100, 400, 800, 1600 and 3200fmol/6 mm punch (p = 0.001). This study demonstrates the potential of the REACTR assay as a rapid, accessible and cost-effective alternative to LC–MS for GCV-TP monitoring in infants with cCMV, paving the way for personalized antiviral dosing and improved clinical outcomes.