An enzymatic assay with lateral flow readout for measurement of tenofovir diphosphate and long‐term HIV medication adherence

20 Although drug concentration feedback provides objective information that may help to improve HIV medication adherence and health outcomes, conventional testing with liquid chromatography tandem mass spectrometry (LC–MS/MS) is arduous and expensive. Urine tenofovir lateral flow assays (LFAs) for measuring adherence are rapid and inexpensive but cannot distinguish between recent (<1 week) and long-term (1–3 month) adherence. The REverse transcriptase ACTivity (REACT) assay rapidly measures tenofovir diphosphate (TFV-DP), a metabolite that indicates long-term oral PrEP/ART adherence, based on the drug's inhibition of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Recent clinical validation of REACT showed correlation with LC–MS/MS measurements and excellent accuracy at distinguishing TFV-DP concentrations that indicate sufficient adherence but required an external instrument for fluorescence readout. Here, we integrate REACT with CRISPR enzymes and reporters to provide an instrument-free LFA readout. We combined CRISPR RNA, Cas12a, DNA template, nucleotides, HIV-RT, biotin-FITC reporters and spiked TFV-DP concentrations corresponding to 2, 4 and 7 doses/week (350, 700 and 1250 fmol/3mm2 punch) into rehydrated dried blood spots from three donors not receiving any antiretroviral drugs. The test line of the LFA strip detects the presence of uncleaved CRISPR reporters, so we also included ‘no drug’ and ‘no enzyme’ conditions to determine the minimum and maximum signal achievable. We incubated REACT assays at 37°C for 45 min and visualized reaction products on commercially available LFA strips (Abingdon Health PCRD Flex) that were immersed in reaction tubes for 10 min. We scanned the LFAs to quantify the test line intensity, calculated signal-to-background ratios (SBR) and determined a positivity threshold at three standard deviations above the mean of the ‘no drug’ condition to allow quantitative analysis of our results. We optimized reagent concentrations (Cas enzyme, nucleotides and reaction time) to tune the LFA signal to be above the positive threshold. In the ‘no drug’ condition, LFA strips were unable to capture biotin-FITC reporters, resulting in faint test line intensities (SBR = 1.33 ± 0.22, N = 6) that set a positivity threshold at SBR = 1.99. Meanwhile, in the ‘no enzyme’ condition, LFA strips provided strong test line intensities well above the positivity threshold (SBR = 4.85 ± 0.27). LFA test line intensities correlated with TFV-DP concentrations (r = 0.995). At TFV-DP concentrations of 350, 700 and 1250 fmol/punch, the SBR values were 1.57 ± 0.10, 1.82 ± 0.13 and 2.60 ± 0.29, respectively. Additionally, the LFA signal intensity at 1250 fmol/punch (7 doses/week) was significantly different from the ‘no drug’ condition (p = 0.001), the 350 fmol/punch condition (p = 0.0007) and the 700 fmol/punch condition (p = 0.003). Taken together, our results constitute an important proof of concept of LFA readout of enzymatic assays for measuring TFV-DP concentrations and long-term HIV medication adherence that may be suitable for use in resource-constrained settings. Ongoing work is focused on tuning the LFA readout to TFV-DP thresholds relevant for different use cases among key populations (e.g., adherence monitoring or resistance screening) and validating our results with clinical samples.