Colorimetric detection of HIV drug metabolites in urine for adherence monitoring

Monitoring the adherence of patients taking highly-active anti-retroviral therapy (HAART) and Pre-Exposure Prophylaxis (PrEP) is a key step in treating an HIV infection. For most regimens, when patients are not at least 95% adherent to their drug schedule there is a loss of effectiveness in treatment resulting in increases of health care costs, increases of the rate of transmission, and reduction of positive patient outcomes. Currently, subjective methods such as pill counting, electronic drug monitoring, and patient self-reporting are the only ways clinicians can track adherence and intervene in cases of non-compliance. We address this issue by developing colorimetric solutions to directly assay for the presence of common HIV drug metabolites in urine. First we developed a dipstick based point-of-care azide-alkyne click chemistry assay with colorimetric readout that directly tests for the presence of azidothymidine (AZT). An alkyne-modified dextran was synthesized and characterized by NMR and then used to colorimetrically report the presence of AZT in urine samples. The assay is specific to azide-containing molecules that are not naturally present in the urine and is sensitive to physiologically relevant urine concentrations as low as 750μM. Second we adapted the Wheeler and Johnson test, originally discovered for the determination of uracil and cytosine, for colorimetric readout of lamivudine (3TC) and emtricitabine (FTC). The cytosine base of these molecules is reacted with bromine and precipitated by barium hydroxide to give a purple color to the solution. The protocol was adapted to be performed easily with urine samples at physiological concentrations. Finally, an antibody-based lateral flow device has been designed for the detection of tenofovir (TFV). Direct application of urine to the lateral flow device gives fast, clear, and reliable readouts of TFV detection. All of these colorimetric assays, which can be done in resource-limited settings with minimal equipment, are all strong alternatives over current direct measurement techniques that are expensive and require trained users such as HPLC and NMR. In addition the detection of AZT and TFV can be deployed at the point-of-care for direct monitoring where the need is greatest