Pharmacologic measures are widely used to assess adherence to tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine (FTC)-based PrEP and ART. Currently-available measures in plasma, dried blood spots, hair and urine involve liquid chromatography/tandem mass spectrometry (LC-MS/MS), which is expensive and labor intensive. Only a point-of-care (POC) test can monitor and support adherence in real-time and TFV-specific antibody-based assays allow for POC adherence monitoring. We developed an immunoassay to quantify TFV in urine and validated it against the gold standard of LC-MS/MS in a large directly-observed therapy (DOT) pharmacokinetics study.
The randomized TARGET study administered TDF 300mg/FTC 200mg directly-observed 7 (high adherence), 4 (moderate adherence) and 2 doses per week (low adherence) to 30 volunteers (10 per group) in Thailand, collecting urine samples over 6 weeks of administration and during wash-out. In total, 637 urine samples were collected (average 21 samples per participant). We measured urine TFV levels by the immunoassay using ELISA (lower limit of quantification LLOQ <1000 ng/ml) and by a validated LC-MS/MS-based method (LLOQ 500 ng/ml) and calculated the sensitivity and specificity of the novel assay compared to the gold standard. We calculated Spearman’s correlation between TFV levels via both assays.
Among all participants, median TFV urine levels were 12,000 ng/mL (IQR 7500-25,000) by the immunoassay 1 day after dosing; 5000 ng/mL (IQR 2500-8000) 2 days after dosing; 1500 ng/mL (IQR 500-2750) 3 days after dosing and below the immunoassay’s LLOQ thereafter (≥4 days). The specificity and sensitivity of the TFV immunoassay compared to the gold-standard of LC-MS/MS were 98.8% and 87.3% respectively. The correlation between TFV levels measured by the immunoassay and LC-MS/MS in all 637 urine samples from TARGET was 0.92 (p<0.00001) (Figure).
We have developed a novel TFV immunoassay that is highly specific (99%) and sensitive (87%), and correlates strongly with urine TFV concentrations measured by the gold standard of LC-MS/MS (rho=0.92) across a wide range of typical concentrations. TFV concentration cutoffs in urine for different degrees of adherence from this DOT study can now guide the development of an immunoassay into a POC rapid strip test. Real-time monitoring of TFV adherence using an easy-to-perform low-cost assay should allow for immediate intervention and optimization of outcomes for both HIV treatment and prevention.