Urine assay for tenofovir to monitor adherence in real time to tenofovir disoproxil fumarate/emtricitabine as pre-exposure prophylaxis

Abstract

Objectives: Tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) is approved for pre-exposure prophylaxis (PrEP) against HIV infection. Adherence is critical for the success of PrEP, but current adherence measurements are inadequate for real-time adherence monitoring. We developed and validated a urine assay to measure tenofovir (TFV) to objectively monitor adherence to PrEP.

Methods: We developed a urine assay using high-performance liquid chromatography coupled to tandem mass spectrometry with high sensitivity/specificity for TFV that allowed us to determine TFV concentrations in log₁₀ categories between 0 and 10 000 ng/mL. We validated the assay in three cohorts: (1) HIV-positive subjects with undetectable viral loads on a TDF/FTC-based regimen, (2) healthy HIV-negative subjects who received a single dose of TDF/FTC, and (3) HIV-negative subjects receiving daily TDF/FTC as PrEP for 24 weeks.

Results: The urine assay detected TFV with greater sensitivity than plasma-based measures and with a window of measurements within 7 days of the last TDF/FTC dose. Based on the urine log-linear clearance after the last dose and its concordance with all detectable plasma levels, a urine TFV concentration > 1000 ng/mL was identified as highly predictive of the presence of TFV in plasma at > 10 ng/mL. The urine assay was able to distinguish high and low adherence patterns within the last 48 h (> 1000 ng/mL versus 10-1000 ng/mL), as well as nonadherence (< 10 ng/mL) extended over at least 1 week prior to measurement.

Conclusions: We provide proof of concept that a semiquantitative urine assay measuring levels of TFV could be further developed into a point-of-care test and be a useful tool to monitor adherence to PrEP.

Keywords: HIV prevention; Truvada; adherence; pre-exposure prophylaxis; urine tenofovir assay.

Figure 1

Tenofovir (TFV) calibration curve in human urine, which was found to be highly accurate over a concentration range of 10 to 1000 ng/mL, with an accuracy range of 93.6% to 100% (r² = 0.9962). A known concentration of TFV was injected into a human urine sample (x axis) and measured using LC-MS/MS (y axis). The measurement urine TFV was found to be linear over the range of 10 – 1000 ng/mL with the limit of detection (LOD) of 5 ng/mL. [TFV] = tenofovir concentration.

Figure 2

Qualitative assessment, at a single time point, of the relationship of urine tenofovir (TFV) to plasma TFV in 10 HIV-positive subjects with undetectable HIV viral loads on a Tenofovir/Emtricitabine (TDF/FTC)-based regimen. All subjects with TFV in plasma also had detectable TFV in urine. Subject 4 had an undetectable viral load yet had no detectable TFV in blood or urine, and was later found to have stopped taking his antiretroviral therapy shortly after his viral load had been collected yet within 4 weeks of study sampling. Concentrations in plasma (P) and urine (U) are shown for each subject.

Figure 3

Measurement of tenofovir (TFV) clearance in plasma and urine over 7 days in 10 HIV-negative subjects who received a single dose of Tenofovir/Emtricitabine (TDF/FTC). After taking one dose of TDF/FTC, tenofovir (TFV) was measurable in plasma up to 4 days, and in urine for at least 7 days. TFV concentration decays in a log-linear fashion. When TFV was detected in plasma (out to 3 days post-dose), urine TFV concentration was > 1000 ng/mL in almost all subjects.