Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence

Abstract

BackgroundPharmacologic adherence measures were critical to the interpretation of the tenofovir (TFV)-disoproxil-fumarate/emtricitabine (TDF/FTC) PrEP trials. These measures are being incorporated into PrEP demonstration projects, but currently-available metrics in plasma, cells, hair or urine involve expensive and time-intensive mass-spectrometry (MS)-based methods. No point-of-care method to assess PrEP adherence in real-time has yet been implemented. Antibody-based tests allow for low-cost, easy-to-perform, point-of-care drug detection. In this study, we developed an antibody-based TFV immunoassay and evaluated its test characteristics among individuals taking TDF/FTC.MethodsWe synthesized possible immunogens based on TFV's molecular structure, injected rabbits with the conjugated derivatives, and bled them monthly for subsequent ELISA-testing for TFV-specific antibodies. We purified an antibody with specific TFV binding and created dose–response curves for ELISA-quantification. We then quantified TFV in urine from human participants not taking TDF/FTC and from individuals taking daily TDF/FTC 300 mg/200 mg for 7 days with a 7-day washout period using ELISA with this TFV-specific antibody. ELISA results were compared with the gold-standard test for TFV detection/quantification using liquid-chromatography-tandem-MS (LC–MS/MS).FindingsNone of the urine samples from 115 participants not taking TDF/FTC showed ELISA- reactivity, indicating 100% specificity (95% CI 97–100%) of the immunoassay. Among participants taking TDF/FTC, 67 of 70 samples positive by LC–MS/MS were positive by the ELISA-immunoassay for an estimated diagnostic sensitivity of 96% (95% CI 88–99%). The precision of the assay was high (coefficient of variation < 15%). The rank correlation between ELISA and LC–MS/MS values in the 70 quantitative urine TFV levels positive by LC–MS/MS across a wide range of concentrations among participants on TDF/FTC was high (r = 0.96).InterpretationOur antibody-based immunoassay for measuring TFV in urine performed well compared to the gold-standard of LC–MS/MS among individuals taking TDF/FTC. A sensitive and specific immunoassay paves the way for real-time monitoring/feedback on recent adherence to TFV-based regimens, which should optimize interpretation and outcomes during PrEP and ART roll-out.FundingNIAID/NIH 2R01AI098472.

Antiretroviral Concentrations in Hair Strongly Predict Virologic Response in a Large Human Immunodeficiency Virus Treatment-naive Clinical Trial

“…However, when hair collection is incorporated at the start of a trial, with appropriate education of field staff and participants to "normalize" collection, rates of acceptability, both in the United States [11] and in Africa [3] and Asia [5], are much higher (>95%). Regarding cost of hair assays, our group and others are working on lower-cost methods to measure ARV concentrations in hair [12] and in developing urine-based methods to assess adherence in real time via a point-of-care assay [13]. Of note, the urine test will only assess short-term adherence, so hair levels combined with urine levels are needed to provide information on adherence patterns.…”

Comparing Self-Report Pre-Exposure Prophylaxis Adherence Questions to Pharmacologic Measures of Recent and Cumulative Pre-Exposure Prophylaxis Exposure

“…Research studies have employed biomarker quantification as an objective adherence measure. At present, these pharmacological assays are costly, require specialized laboratory equipment and personnel, and take time to yield results; however, there is research underway to develop point of care immunoassays in urine that could be used as clinical tools (Koenig et al, 2017;Gandhi et al, 2018). Until real-time drug concentration testing is available, biomarker quantification can be used to gauge the practicality and accuracy of alternative methods, including self-report measures.…”

Simplifying TREAtment and Monitoring for HIV (STREAM HIV): protocol for a randomised controlled trial of point-of-care urine tenofovir and viral load testing to improve HIV outcomes

“…[12][13][14][15][16][17][18][19][20][21] The recently developed and validated Abbott-manufactured POC test that objectively measures recent ART adherence via tenofovir (TFV) levels in urine shows promise as a rapid and inexpensive adherence monitoring tool to identify non-adherence when the establishment of optimal adherence patterns is critical, such before a client's first VL test. [22][23][24][25][26] Routine real-time ART adherence monitoring may also be beneficial in motivating subsequent good adherence among PLHIV and improving engagement in care. 27 28 However, little is known about the effects of implementing POC TFV adherence testing among PLHIV.…”