A point-of-care urine test can accurately determine if a person is adherent to tenofovir-based pre-exposure prophylaxis (PrEP), according to researchers at University of California – San Francisco, University of Washington – Seattle, Alere Rapid Diagnostics, Chiang Mai University, Sanpatong Hospital, Chiang Mai, Sanpasitthiprasong Hospital, Ubon Ratchathani and Harvard University presenting to the recent Conference on Retroviruses and Opportunistic Infections (CROI 2019) in Seattle.
The test checks for antibodies to tenofovir and was tested in a trial of 30 people in Thailand who had different levels of adherence to tenofovir/emtricitabine PrEP. A minimum cut-off value showing if a person had taken PrEP within the past 24 hours was determined. The accuracy of the urine test compared well to gold-standard assays.
A commercial point-of-care test has now been developed. The strip test provides results within five minutes and costs below $2. The investigators believe it could help motivate people to take their therapy and assist in the identification of people in need of adherence support.
However, some delegates expressed concern that the usefulness of the test in clinical practice could be undermined by the “white coat” effect: non-adherent individuals taking a dose shortly before a follow-up appointment when they knew their adherence would be tested.
With high adherence, PrEP can reduce the risk of infection with HIV by well over 90%. Data from clinical trials suggests that many people inaccurately assess or report their adherence to PrEP. In the VOICE study, for instance, 91% of people said they were adherent, but analysis of tenofovir levels in plasma showed that only 26% of individuals had optimum levels of adherence.
Tests for use in routine care to assess PrEP adherence are therefore needed. The current assays are costly and slow. A team of investigators therefore sought to develop a point-of-care urine test.
They successfully identified a tenofovir-specific antibody in animal tests. This was then validated in a proof-of-concept study. The investigators then designed the TARGET study to determine a level indicating PrEP dosing within the past 24 hours and also to compare the accuracy of urine analysis with current gold-standard assays.
The research was conducted in Thailand and involved 30 individuals. The people were divided into three groups, taking tenofovir/emtricitabine PrEP consistent with varying levels of adherence: high (daily PrEP); medium (four doses a week); low (two doses a week).
The participants provided a total of 637 urine samples over a six-week period after dosing and during washout.
A cut-off of 1500 ng/ml accurately identified 98% of people who took a PrEP dose within the previous 24 hours. The test had high sensitivity (94%) and specificity (99%), its accuracy comparing well to antibody assays (p < 0.0001).
Median levels were 12,000 ng/ml one day after dosing; 5000 ng/ml two days after dosing; 1500 ng/ml three days after dosing; and below the assay's lower limit of quantification four days after dosing.
However, the researchers acknowledged that the test is vulnerable to the “white-coat” effect, with individuals boosting their adherence when they knew a clinic appointment with urine analysis was imminent: a participant in the study admitted to this behaviour. Several delegates also stressed this potential limitation.
During discussion, the investigators noted that the study involved both men and women and the urine test worked well regardless of gender. However, further research is needed to specifically determine how well it performs in transgender women taking feminising hormones.
The current test provides a simple yes/no answer about adherence. A more expensive test capable of determining adherence level (high/medium/low) is in development.
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.
Monica Gandhi, Peter Bacchetti, Hideaki Okochi, Matthew A Spinelli, Jared Baeten, Warren Rodrigues, Guohong Wang, Michael Vincent, Rachel W Kubiak, Yardpiroon Tawon, Virat Klinbuayaem, Pra-ornsuda Sukrakanchana, Oraphan Siriprakaisil, Tim R Cressey, Paul K Drain