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Introduction: Adolescent girls and young women (AGYW) account for two-thirds of new HIV infections in Africa. African AGYW have had high uptake of oral HIV pre-exposure prophylaxis (PrEP) but low adherence, which might be improved by point-of-care adherence monitoring with tailored counselling., Methods: From August 2022 to July 2023, we conducted a PrEP demonstration project with sexually active AGYW ages 16-30 years from 20 sites in South Africa, Eswatini, Kenya, Malawi, Uganda and Zambia. Participants were offered oral tenofovir-based PrEP at enrolment and followed up at 1, 3 and 6 months. PrEP adherence was assessed by a point-of-care qualitative lateral flow urine tenofovir (TFV) assay indicating PrEP use in the prior 4 days, which accompanied real-time adherence counselling that incorporated urine TFV results when testing was available (70.8% of month 1, 35.3% of month 3 and 83.9% of month 6 visits). We estimated overall adherence, correcting for missing test results, and analysed the association of having received urine TFV results at month 1 or 3 with subsequent urine TFV test positivity, using modified Poisson regression., Results: Of the 3087 AGYW enrolled, the median age was 24 years (interquartile range 21-27), 75.7% were from South Africa, 2878 (93.2%) initiated PrEP at enrolment and 107 (3.5%) after enrolment. Visit retention was 92.0-96.2% for months 1, 3 and 6, and 2518 (90.1%) exited the study with a PrEP refill. Adherence, based on the point-of-care urine tenofovir test positivity rate, was estimated as 72%, 71% and 65% at months 1, 3 and 6, respectively. Women who received one prior urine TFV test had a 42% higher likelihood of a subsequent positive urine TFV test (adjusted odds ratio, OR = 1.42, 95% confidence interval, CI 1.27-1.60), and those having received two prior tests had a 67% higher likelihood (adjusted OR = 1.67; 95% CI 1.41-1.98). Observed HIV incidence was 1.38/100 person-years (95% CI 0.97-2.08)., Conclusions: Oral PrEP uptake, recent adherence and persistence were high in a multisite cohort of young African women over 6 months of follow-up. The use of a novel point-of-care tenofovir assay with tailored real-time adherence counselling was associated with increased adherence to PrEP at subsequent visits, warranting further study., Clinical Trials Registration: clinicaltrials.gov NCT05746065., (© 2024 The Author(s). Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of International AIDS Society.)

Objectives: We evaluated a recently developed and validated point-of-care urine tenofovir (POC TFV) test to determine whether its use improves the accuracy of self-reported adherence to preexposure prophylaxis (PrEP) and sexual behavior., Design: We enrolled sexually active HIV-negative women ages 16 to 25 years in Kampala, Uganda., Methods: Women were followed quarterly for 24 months with HIV prevention counseling, PrEP dispensation, and adherence counseling. Midway through the study, the POC TFV test was introduced as part of routine study procedures. We examined changes in self-reported PrEP adherence, sexual behavior, and accuracy of self-reported PrEP adherence before and after the introduction of the POC TFV test., Results: A total of 146 women receiving PrEP refills had at least one visit with a POC TFV test administered before the study exit. At baseline, the median age was 19 years [interquartile range (IQR): 18-21] and the majority (76%) reported having condomless sex within the last 3 months. Participants more frequently self-reported low PrEP adherence [odds ratio (OR): 2.96, 95% confidence interval (CI): 1.89-4.67, P  = 0.001] and condomless sex (OR: 1.47, 95% CI: 1.04-2.06, P  = 0.03) during visits using the test compared with visits without the test. The accuracy of self-reported PrEP adherence (determined by concordance with TFV-diphosphate levels) was greater when the test was used (61 versus 24%, OR: 4.86, 95% CI: 2.85-8.30, P  < 0.001)., Conclusion: When the POC TFV test was used, we observed greater reporting of condomless sex, low PrEP adherence, and more accurate reports of PrEP adherence. The test could facilitate honest conversations between clients and providers and warrant further investigation., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Pre-exposure prophylaxis (PrEP) is highly effective at reducing HIV acquisition. We aimed to estimate usage of oral-PrEP, and factors associated with adherence among female sex workers (FSWs) in Nairobi, Kenya, using a novel point-of-care urine tenofovir lateral flow assay (LFA). The Maisha Fiti study randomly selected FSWs from Sex Worker Outreach Program clinics in Nairobi. Data were collected from 1003 FSWs from June-October 2019, including surveys on self-reported oral-PrEP adherence. Adherence was also measured using the LFA for HIV-negative FSWs currently taking oral-PrEP. Informed by a social-ecological theoretical framework, we used hierarchical multivariable logistic regression models to estimate associations between individual, interpersonal/community, and structural/institutional-level factors and either self-reported or LFA-assessed adherence. Overall, 746 HIV-negative FSWs aged 18-40 participated in the study, of whom 180 (24.1%) self-reported currently taking oral-PrEP. Of these, 56 (31.1%) were adherent to oral-PrEP as measured by LFA. In the multivariable analyses, associations with currently taking oral-PrEP included having completed secondary education, high alcohol/substance use, feeling empowered to use PrEP, current intimate partner, no recent intimate partner violence, having support from sex worker organisations, experiencing sex work-related stigma, and seeking healthcare services despite stigma. Associations with oral-PrEP LFA-measured adherence measured included having only primary education, experience of childhood emotional violence, belonging to a higher wealth tertile, and being nulliparous. Oral-PrEP adherence, measured by self-report or objectively, is low among FSWs in Nairobi. Programs to improve oral-PrEP usage among FSWs should work to mitigate social and structural barriers and involve collaboration between FSWs, healthcare providers and policymakers., (© 2024. Crown.)

Background: Young men who have sex with men and transgender women (YMSM/TGW) have disproportionately high HIV incidence and lower preexposure prophylaxis (PrEP) adherence. Point-of-care (POC) urine tenofovir (TFV) rapid assay (UTRA) testing permits real-time monitoring for nonadherence within clinical settings. We performed UTRA testing among PrEP users to examine the relationship between low PrEP adherence and future PrEP discontinuation, and the accuracy of POC testing compared to gold-standard liquid chromatography tandem mass spectrometry (LC/MS/MS)., Methods: YMSM/TGW participants ( n  = 100) were recruited during a daily PrEP visit. Logistic regression models analyzed the relationship between the primary predictor of urine POC assay results (cutoff 1,500 ng/ml) and the primary outcome of PrEP discontinuation, defined as no PrEP follow-up or prescription within 120 days., Results: Overall, 19% of participants had low urine TFV and 21% discontinued PrEP, while 11% of participants self-reported low PrEP adherence (<4 pills per week), which was only 43% sensitive/84% specific in predicting low TFV levels and was not associated with PrEP discontinuation. Low urine TFV level predicted PrEP discontinuation [adjusted odds ratio (AOR) 6.1; 95% confidence interval (CI): 1.4-11; P  = 0.005] and was 71% sensitive/90% specific for discontinuation after 120 days. Compared to LC/MS/MS, UTRA testing had a 98% positive and 100% negative predictive value., Conclusions: In a sample of YMSM/TGW on daily PrEP, POC UTRA testing predicted PrEP discontinuation more accurately than self-reported adherence, with high predictive values compared to LC/MS/MS. UTRA testing may be a clinical tool for directing preventive interventions towards those likelier to discontinue PrEP despite ongoing HIV vulnerability., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Adherence challenges with oral tenofovir-based pre-exposure prophylaxis (PrEP) are common. We developed a point-of-care assay to objectively assess tenofovir in urine and conducted a pilot trial examining the impact of counselling informed by use of this urine assay on long-term PrEP adherence., Methods: This randomised trial enrolled women not in serodiscordant partnerships 3 months after PrEP initiation at the Kenya Medical Research Institute to compare standard-of-care adherence counselling versus counselling informed by the urine assay (urine-test counselling group) every 3 months for 12 months. In the standard of care group, urine samples were stored and tested at study end without participant feedback. Here we report the adherence primary outcome of hair concentrations of tenofovir at 12 months as a long-term metric (undetectable levels defined long-term non-adherence), as well as urine concentrations of tenofovir at each visit as a short-term adherence metric and acceptability of the assay assessed by quantitative surveys. Data were analysed by randomisation group. This completed trial was registered with ClinicalTrials.gov (NCT03935464)., Findings: From March 17, 2021 to Jan 18, 2022 we enrolled 49 women in the urine-test counselling group and 51 in the standard of care group; retention was 86 (86%) of 100. Nine (21%) of 42 in the urine-test counselling group had hair samples at 12 months with tenofovir concentrations below the limit of quantification compared with 15 (37%) of 41 in the standard of care group. The relative odds of long-term non-adherence in the standard of care group compared with urine-test counselling were 3·53 (95% CI 1·03-12·03; p=0·044). Pre-intervention, urine tenofovir was detectable in 65% in the urine-test counselling group and 71% in the standard of care group (p=0·68). At 12 months, 31 (72%) of 43 in the intervention group had detectable urine tenofovir compared with 19 (45%) of 42 in the standard of care group (p=0·0015). 40 (93%) of 43 participants liked the test very much and only one disliked the test. One participant in the standard of care group was withdrawn at the 6-month visit due to HIV seroconversion., Interpretation: A low-cost urine tenofovir assay to inform PrEP counselling resulted in improvement in both short-term and long-term metrics of adherence. This urine tenofovir assay could help to improve long-term PrEP adherence., Funding: National Institute of Allergy and Infectious Diseases and National Institutes of Health., Competing Interests: Declaration of interests MG's Hair Analytical Laboratory co-owns the patent on the antibody directed against tenofovir with Abbott Laboratories. DVG has received consulting fees from Gilead Sciences. Beyond receiving National Institutes of Health grants which provide support to KN's institution, KN has received additional grant funding from the Bill & Melinda Gates Foundation, United States Agency for International Development, and Merck Pharmaceuticals. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Poor adherence to ART and pre-exposure prophylaxis (PrEP) can impact patient and public health. Point-of-care testing (POCT) may aid monitoring and adherence interventions., Objectives: We report the pharmacokinetics of tenofovir [dosed as tenofovir disoproxil (TDF) and tenofovir alafenamide (TAF)], emtricitabine (FTC), lamivudine (3TC) and dolutegravir (DTG) in plasma and urine following drug cessation to evaluate adherence targets in urine for POCT., Methods: Subjects were randomized (1:1) to receive DTG/FTC/TAF or DTG/3TC/TDF for 15 days. Plasma and spot urine were collected on Day 15 (0-336 h post final dose). Drug concentrations were quantified using LC-MS, and non-linear mixed-effects models applied to determine drug disposition between matrices and relationship with relevant plasma [dolutegravir protein-adjusted 90% inhibitory concentration (PA-IC90 = 64 ng/mL) and minimum effective concentration (MEC = 324 ng/mL)] and urinary thresholds [tenofovir disoproxil fumarate 1500 ng/mL]., Results: Of 30 individuals enrolled, 29 were included (72% female at birth, 90% Caucasian). Median (range) predicted time to plasma dolutegravir PA-IC90 and MEC were 83.5 (41.0-152) and 49.0 h (23.7-78.9), corresponding to geometric mean (90%) urine concentrations of 5.42 (4.37-6.46) and 27.4 ng/mL (22.1-32.7). Tenofovir in urine reached 1500 ng/mL by 101 h (58.6-205) with an equivalent plasma concentration of 6.20 ng/mL (4.21-8.18)., Conclusions: These data support use of a urinary tenofovir threshold of <1500 ng/mL (tenofovir disoproxil fumarate-based regimens) as a marker of three or more missed doses for a POCT platform. However, due to low dolutegravir concentrations in urine, POCT would be limited to a readout of recent dolutegravir intake (one missed dose)., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Introduction: Adherence counselling with point-of-care (POC) drug-level feedback using a novel tenofovir assay may support pre-exposure prophylaxis (PrEP) adherence; however, perceptions of urine testing and its impact on adherence are not well studied. We qualitatively examined how POC tenofovir testing was experienced by transgender women (TGW) in Uganda., Methods: Within a cluster randomized trial of peer-delivered HIV self-testing, self-sampling for sexually transmitted infections and PrEP among HIV-negative TGW showing overall low PrEP prevention-effective adherence (NCT04328025), we conducted a nested qualitative sub-study of the urine POC assay among a random sample of 30 TGW (August 2021-February 2022). TGW interviews explored: (1) experiences with POC urine tenofovir testing and (2) perceptions of PrEP adherence counselling with drug-level feedback. We used an inductive content analytic approach for analysis., Results: Median age was 21 years (interquartile range 20-24), and 70% engaged in sex work. Four content categories describe how TGW experienced POC urine tenofovir testing: (1) Urine tenofovir testing was initially met with scepticism: Testing urine to detect PrEP initially induced anxiety, with some perceptions of being intrusive and unwarranted. With counselling, however, participants found POC testing acceptable and beneficial. (2) Alignment of urine test results and adherence behaviours: Drug-level feedback aligned with what TGW knew about their adherence. Concurrence between pill taking and tenofovir detection in urine reinforced confidence in test accuracy. (3) Interpretation of urine tenofovir results: TGW familiar with the interpretation of oral-fluid HIV self-tests knew that two lines on the test device signified positivity (presence of HIV). However, two lines on the urine test strip indicated a positive result for non-adherence (absence of tenofovir), causing confusion. Research nurses explained the difference in test interpretation to participants' satisfaction. (4) White coat dosing: Some TGW deliberately chose not to attend scheduled clinic appointments to avoid detecting their PrEP non-adherence during urine testing. They restarted PrEP before returning to clinic, a behaviour called "white coat dosing.", Conclusions: Incorporating POC urine testing into routine PrEP adherence counselling was acceptable and potentially beneficial for TGW but required attention to context. Additional research is needed to identify effective strategies for optimizing adherence monitoring and counselling for this population., (© 2024 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of International AIDS Society.)

Background: Preexposure prophylaxis (PrEP) significantly reduces HIV infection risk but is dependent on adherence. Available approaches to measuring adherence have limitations related to accuracy, cost, practicality, and timeliness. This study compared the performance of two methods implementable in clinics and research studies [interview and urine point of care (POC) assay] to the gold-standard for measuring recent and longer term adherence in dried blood spots (DBS)., Methods: Participants were recruited from RADAR, a cohort study of young MSM, or via online advertisements. At 3 monthly visits, an interviewer administered 7-day timeline follow-back (TLFB) questionnaire, DBS samples were tested for tenofovir-diphosphate (TFV-DP) to estimate average dosing over the prior month and emtricitabine-triphosphate (FTC-TP) to assess recent dosing (past 2-3 days), and a urine POC TFV test to qualitatively assess recent adherence (past 4 days)., Results: Eighty-three PrEP users contributed 163 observations. At visit 1, self-reported adherence was 86% (4+ doses in last 7 days), versus urine TFV (74%), DBS FTC-TP (76%), and DBS TFV-DP (69%). The objective measures of short-term adherence performed similarly well in predicting longer term adherence. In multivariable logistic regression analyses, the urine assay was a significant predictor of DBS TFV-DP (adjusted OR = 19.4, P  < 0.0001); self-report did not add significantly., Conclusion: The urine POC TFV assay had excellent predictive values for adherence and self-report did not add significantly to prediction. The POC assay provides results in several minutes to enable same-visit counseling, requires no specialized training, and is projected to be low-cost., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: We sought to evaluate the utility of a point-of-care (POC) urine tenofovir (TFV) assay, developed to objectively assess adherence, to predict HIV drug resistance (HIVDR) in people failing first-line antiretroviral therapy (ART)., Design: We retrospectively analyzed TFV levels as a biomarker of adherence in urine specimens collected during a clinical trial that enrolled adults with virologic failure on first-line ART in Uganda and South Africa., Methods: Urine specimens were analyzed from participants on TFV-containing regimens who had a viral load >1000 copies/ml and paired genotypic resistance test (GRT) results. We assessed recent ART TFV adherence with a qualitative POC lateral flow urine assay with a cut-off value of 1500 ng/ml. We then calculated performance characteristics of the POC urine TFV assay to predict HIVDR, defined as intermediate or high-level resistance to any component of the current ART regimen., Results: Urine specimens with paired plasma GRT results were available from 283 participants. The most common ART regimen during study conduct was emtricitabine, tenofovir disoproxil fumarate, and efavirenz. The overall prevalence of HIVDR was 86% ( n = 243/283). Of those with TFV detected on the POC assay, 91% ( n  = 204/224) had HIVDR, vs. only 66% ( n  = 39/59) among those with no TFV detected ( P- value < 0.001). Positive and negative predictive values of the assay to predict HIVDR were 91% and 34%, respectively., Conclusions: In populations with a high prevalence of HIVDR, the POC urine TFV assay can provide a low-cost, rapid method to guide requirements for confirmatory resistance testing and inform the need for regimen change., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The detection of tenofovir (TFV) metabolites by point-of-care (POC) urine lateral flow immunoassays (LFIA) indicates adherence to tenofovir-containing HIV pre-exposure prophylaxis. However, the association between urine TFV metabolites as detected by LFIA and HIV viral load suppression in people receiving TFV-based antiretroviral therapy (ART) is unknown as is patient and clinician acceptability of POC urine LFIA testing in clinical practice in low- and middle-income country settings., Methods: We enrolled 409 people living with HIV from two HIV clinics in Lesotho and investigated the performance of POC urine LFIA TFV testing in predicting viral suppression. We interviewed 12 study participants and conducted a focus-group discussion with 5 clinicians to gather opinions on POC urine TFV testing., Results: Using a viral load threshold of 1000 copies/mL, 398 (98%) participants were virologically suppressed, and 8 were viremic. Tenofovir was detected in the urine of 405 (99%) participants. The sensitivity of the POC urine LFIA test in detecting TFV in participants with viral suppression was 99.3% (95% CI: 97.8-99.8); the specificity was 12.5% (95% CI: 0.3-52.6). The positive and negative predictive values were 98.3% and 25%, respectively. Point-of-care urine TFV testing was viewed favorably by both participants and clinicians. However, clinicians stated that the 2-3-days detection window of the assay studied limits adherence categorization., Conclusions: In our study cohort, urine POC TFV testing demonstrated high sensitivity in predicting viral suppression, but low specificity and negative predictive value. Urine POC TFV testing was highly acceptable to participants and clinicians; however, clinicians expressed concern about its clinical utility because of its limitations. While further research is needed to assess performance in less adherent populations, this test may support adherence counseling in some clinical settings.

Background: Tenofovir alafenamide (TAF) is increasingly used in HIV treatment, with or without agents that require pharmacologic boosters such as ritonavir/cobicistat. Boosters increase TAF levels, so the TAF dose is lowered in single-pill combinations. We hypothesized that individuals on dose-adjusted boosted TAF would have similar urine tenofovir (TFV) concentrations to those on unboosted TAF., Setting/methods: We collected urine samples from patients with HIV on TAF, with evidence of virologic suppression and high self-reported adherence at 2 San Francisco clinics from June 2019 to January 2020. We measured urine TFV levels by liquid chromatography/tandem mass spectrometry and used linear regression to compare natural log-transformed urine TFV levels for patients on boosted versus unboosted TAF., Results: Our analysis included 30 patients on unboosted TAF (25 mg daily TAF) and 15 on boosted TAF (12 on 10 mg daily TAF and 3 on 25 mg daily TAF). Patients on unboosted vs. boosted TAF had similar baseline age, weight, sex, and creatinine. In unadjusted univariate linear regression, there were no significant differences in urine TFV levels based on presence/absence of boosting after TAF dose reduction to 10 mg (geometric mean ratio 1.07; 95% confidence interval: 0.53 to 2.16). This finding was unchanged in adjusted analysis., Conclusions: No significant differences in urine TFV levels were seen for patients on unboosted vs. boosted dose-reduced TAF. These results have important implications for our forthcoming point-of-care urine immunoassay for TAF, implying that separate adherence cutoffs will not be necessary for patients on boosters and dose-reduced TAF. A single POC TAF immunoassay will, thus, support monitoring on most TAF-based antiretroviral therapy., Competing Interests: D.V.G. has accepted funds from Gilead Sciences. The remaining authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Approximately 32 million people have died of HIV infection since the beginning of the outbreak, and 38 million are currently infected. Among strategies adopted by the Joint United Nations Programme on HIV/AIDS to end the AIDS global epidemic, the treatment, diagnosis, and viral suppression of the infected subjects are considered crucial for HIV prevention and transmission. Although several antiretroviral (ARV) drugs are successfully used to manage HIV infection, their efficacy strictly relies on perfect adherence to the therapy, which is seldom achieved. Patient supervision, especially in HIV-endemic, low-resource settings, requires rapid, easy-to-use, and affordable analytical tools, such as the enzyme-linked immunosorbent assay (ELISA) and especially the lateral flow immunoassay (LFIA). In this work, high-affinity monoclonal antibodies were generated to develop ELISA and LFIA prototypes for monitoring tenofovir (TFV), an ARV drug present in several HIV treatments. TFV was functionalized by inserting a carboxylated C5-linker at the phosphonic group of the molecule, and the synthetic derivative was conjugated to proteins for mice immunization. Through a rigorous screening strategy of hybridoma supernatants, a panel of monoclonal antibodies strongly binding to TFV was obtained. Following antibody characterization for affinity and selectivity by competitive ELISA, a LFIA prototype was developed and tentatively applied to determine TFV in simulated urine. The point-of-care test showed ultra-high detectability (the visual limit of detection was 2.5 nM, 1.4 ng mL -1 ), excellent selectivity, and limited proneness to matrix interference, thus potentially making this rapid method a valuable tool for the on-site assessment of patient adherence to ARV therapy.

Background: Real-time, objective measures of adherence to antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) are needed to better assess adherence levels and to expedite clinical response for those with suboptimal adherence. Point-of-care tenofovir (POC-TFV) testing has been proposed as a solution to facilitate real-time antiretroviral adherence monitoring, but little is known about how health care providers, people living with HIV (PLWH) receiving ART, and people receiving PrEP will perceive POC-TFV testing., Methods: We conducted an exploratory qualitative study to assess perspectives on the utility and interest in POC-TFV testing from potential end users. We conducted three focus group discussions (FGDs) among 17 PLWH receiving ART and four individuals receiving PrEP, as well as eight in-depth interviews (IDIs) with health care providers in the Seattle area and presented participants with a hypothetical urine-based POC-TFV test. FGDs and IDIs were audio recorded, transcribed, coded, and analyzed to describe emerging themes., Results: Overall, study participants demonstrated divergent opinions about the POC-TFV test. Among study participants, PLWH were most ambivalent about POC-TFV testing, first demonstrating reluctance to TFV-level monitoring and shifting positions during the FGDs. However, all PLWH participants were receptive to POC-TFV testing if requested by their provider. PrEP participants were generally supportive of POC-TFV testing for routine adherence monitoring and emphasized potential value in self-administered testing. Providers' perceptions were equally divided - half suggested POC-TFV testing would be valuable, particularly for people receiving PrEP, while half indicated the test would have little benefit for most individuals receiving ART or PrEP in the U.S. All providers agreed that POC-TFV test results could be beneficial for assessing discrepancies in viral load results and self-reported adherence among PLWH. The study also revealed that a low-cost, non-urine-based POC-TFV test with a long-term limit of detection would be preferred over the hypothetical urine-based test., Conclusions: Our findings indicate POC-TFV testing may be beneficial for routine, clinic-based adherence monitoring, particularly for individuals receiving PrEP or for PLWH with persistent viremia or following recent ART initiation. These findings should also be used to formulate a target product profile for a POC-TFV test and to guide further developments in tools for objective antiretroviral adherence monitoring.

Objectives: The aim was to fully characterize the plasma and urine washout pharmacokinetics of tenofovir (TFV) in adults following 6 weeks of controlled levels of tenofovir disoproxil fumarate (TDF) adherence, in order to inform the utility of clinic-based adherence testing., Design: This was a three-arm, randomized, open-label study in adult volunteers. Participants were randomized to receive TDF 300 mg/emtricitabine (FTC) 200 mg as (1) 7 doses/week (perfect adherence), (2) 4 doses/week (moderate adherence), or (3) 2 doses/week (low adherence). Plasma and urine samples were collected regularly during the 6-week dosing phase and for 4 weeks following drug cessation., Results: Twenty-eight adults were included in this analysis. Median (range) age was 33 (20-49) years. No differences in TFV pharmacokinetic parameters during the washout were observed across the study arms. Small differences in TFV plasma concentrations occurred across arms between 4 and 10 h post-dose. The cumulative amount of TFV excreted in urine was not different at 24 h post-dose, but at 148 h it was 24.8 mg, 21.0 mg, and 17.2 mg for the perfect, moderate, and low adherence arms, respectively (p = 0.043)., Conclusions: Among adults with different TDF adherence patterns, relative differences in plasma concentrations and cumulative urine extraction of TFV were minor following cessation. TFV measurement in plasma or urine is more indicative of last drug ingestion, rather than prior dose patterns., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Therapeutic drug monitoring measures antiretroviral adherence more accurately than self-report but has not been available at the point-of-care (POC) until now. We compare a novel POC test for urine tenofovir to laboratory-based enzyme-linked immunosorbent assay (ELISA) testing in diverse patient populations urine pre-exposure prophylaxis (PrEP)., Setting: Urine samples were analyzed using ELISA and the POC lateral flow immunoassay (LFA) test from 2 cohorts of PrEP users taking tenofovir disoproxil fumarate/emtricitabine: the Partners PrEP Study, which recruited Kenyan and Ugandan heterosexual men and women, and the IBrEATHe Study, which recruited US transgender women and men using gender-affirming hormone therapy., Methods: We calculated the sensitivity, specificity, and accuracy of the POC test compared with ELISA at a cutoff of 1500 ng/mL., Results: Overall, 684 urine samples were tested from 324 participants in the 2 cohorts. In Partners PrEP, 454 samples from 278 participants (41% women) were tested with a median age of 33 years. In IBrEATHe, 231 samples from 46 individuals (50% transwomen) were tested with a median age of 31 years. Comparison of the LFA read-out to ELISA yielded 100% sensitivity [97.5% one-sided confidence interval (CI) = 99.3%], 98.3% specificity (95% CI = 95.2% to 99.7%), and 99.6% accuracy (95% CI = 98.7% to 99.9%)., Conclusion: The sensitivity, specificity, and accuracy of a novel POC test for urine tenofovir all exceeded 98% when compared with a laboratory-based ELISA method when tested in diverse patient populations. Given the LFA's high accuracy and expected low cost, this POC test is a promising tool to support antiretroviral adherence that could be widely scalable to real-world clinical settings.

Objective: HIV prevention and treatment studies demonstrate that pharmacologic adherence metrics are more accurate than self-report. Currently available metrics use liquid-chromatography/tandem-mass-spectrometry (LC-MS/MS), which is expensive and laboratory-based. We developed a specific and sensitive antibody against tenofovir, the backbone of treatment and prevention, but conversion to a lateral flow assay (LFA) - analogous to a urine pregnancy test - is required for point-of-care testing. We describe the development of the first LFA to measure antiretroviral adherence in real-time., Methods: Previous work in a directly observed therapy study of providing tenofovir disoproxil fumarate (TDF) to HIV-noninfected volunteers at various simulated adherence patterns defined the appropriate cut-off for the LFA (1500 ng tenofovir/ml urine). We developed the LFA using a sample pad for urine; a conjugate pad coated with TFV-specific antibodies conjugated to colloidal gold nanoparticles; a nitrocellulose membrane striped with tenofovir-antigen (test line) and a control line; with an absorbent pad to draw urine across the reaction membrane., Results: We tested 300 urine samples collected from the directly observed therapy study by this LFA and the gold-standard method of LC-MS/MS. The LFA demonstrated 97% specificity (95% CI 93-99%) and 99% sensitivity (94-100%) compared with LC-MS/MS. The LFA accurately classified 98% of patients who took a dose within 24 h as adherent., Conclusion: We describe the development and validation of the first point-of-care assay to measure short-term adherence to HIV prevention and treatment in routine settings. The assay is low-cost, easy-to-perform and measures the breakdown product (tenofovir) of both TDF and tenofovir alafenamide (TAF). This assay has the potential to improve HIV and PrEP outcomes worldwide by triggering differentiated service delivery with further study merited.

Tenofovir Disoproxil Fumarate (TDF) and tenofovir Alafenamide (TAF) are prodrugs of tenofovir and have excellent long-term efficacy and tolerability for the treatment of HIV. An objective marker of adherence to tenofovir-based therapy could be clinically useful in supporting adherence to TDF-based HIV pre-Exposure Prophylaxis (PrEP) in populations in whom, self-report has been shown to be unreliable, and could play a role in resource-limited settings to support HIV and hepatitis B treatment adherence. A semi-quantitative high-performance liquid chromatographymass spectrometry method for tenofovir quantification of urine samples was developed. This assay detects tenofovir concentration in log10 levels between 1 and 10,000 ng/mL, and was shown to distinguish between recent adherence and low/non-adherence to both TDF and TAF, with a concentration of >1000 ng/mL, highly predictive of medication ingestion in the last 24-48 hours. This assay was validated relative to other markers of adherence including dried blood spot and selfreport in a highly adherent population of PrEP patients, and tenofovir was shown to be stable at room temperature in urine for at least 14 days. The assay was successfully used in a clinical setting to maintain high PrEP adherence and retention in care of 50 young men who have sex with men (MSM) over 48 weeks, to assess PrEP adherence in youth with mental health conditions, and to monitor drug levels relative to plasma levels in a case study of chewed TDF/FTC (tenofovir/emtricitabine) for PrEP. Further studies are underway to implement the tenofovir urine assay to monitor adherence and pre-exposure prophylaxis, nationally and internationally., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background and Objectives: The elderly population receives the majority of prescription drugs but are usually excluded from Phase 1 clinical trials. Alternative approaches to estimate increases in toxicity risk or decreases in efficacy are therefore needed. This study predicted the pharmacokinetics (PK) of three renally excreted antiretroviral drugs in the elderly population and compared them with known exposures in renal impairment, to evaluate the need for dosing adjustments., Methods: The performance of the physiologically based pharmacokinetic (PBPK) models for tenofovir, lamivudine and emtricitabine were verified using clinical data in young and older subjects. Models were then used to predict PK profiles in a virtual population aged 20 to 49 years (young) and a geriatric population aged 65 to 74 years (elderly). Predicted exposure in the elderly was then compared with exposure reported for different degrees of renal impairment, where doses have been defined., Results: An increase in exposure (AUC) with advancing age was predicted for all drugs. The mean ratio of the increase in exposure were 1.40 for emtricitabine, 1.42 for lamivudine and 1.48 for tenofovir. The majority of virtual patients had exposures that did not require dosage adjustments. About 22% of patients on tenofovir showed exposures similar to that in moderate renal impairment, where dosage reduction may be required., Conclusion: Comparison of the exposure in the elderly with exposure observed in patients with different levels of renal impairment, indicated that a dosage adjustment may not be required in elderly patients on lamivudine, emtricitabine and the majority of the patients on tenofovir. Clinical trials to verify these predictions are essential.

Background: Urine provides a minimally invasive specimen that may allow for development of rapid tests to detect antiretroviral drugs and provide opportunities to improve individual adherence. This study sought to determine whether urine could provide a biomarker of adherence for currently approved pre-exposure prophylaxis and HIV treatment regimens., Methods: Urine and blood were collected from 34 HIV-negative men who have sex with men aged 18-49 years, enrolled in a clinical trial comparing 2 antiretroviral regimens. Specimens were collected 4 and 24 hours after a single oral dose of tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) (n = 10) or tenofovir alafenamide (TAF)/FTC/cobicistat (COBI)/elvitegravir (EVG) (n = 8), or after 4 and 10 days of daily oral TDF/FTC (n = 9) or TAF/FTC/COBI/EVG (n = 7). Tenofovir (TFV), FTC, and EVG were measured by high-performance liquid chromatography-mass spectrometry., Results: Median urine FTC concentrations at 4 and 24 hours were similar between men receiving TDF/FTC (4 hours 147 µg/mL; 24 hours 10 µg/mL) and men receiving TAF/FTC/COBI/EVG (4 hours 333 µg/mL, P = 0.173; 24 hours 13 µg/mL, P = 0.681). Median urine TFV concentrations were lower among men receiving TAF/FTC/COBI/EVG (4 hours 1.2 µg/mL; 24 hours 0.8 µg/mL) compared with men receiving TDF/FTC (4 hours 17 µg/mL, P < 0.001; 24 hours 7 µg/mL, P = 0.001). Urine TFV concentrations remained reduced among men receiving TAF/FTC/COBI/EVG compared with men receiving TDF/FTC after daily dosing. EVG was not consistently measurable in urine., Conclusions: High urine FTC and TFV concentrations could provide an indication of adherence to daily oral dosing with TDF or TAF-based regimens used for treatment and prevention.

Maximizing the impact of HIV pre-exposure prophylaxis (PrEP) requires optimizing access and adherence for those at risk of contracting HIV. This study examined challenges to the processes of accessing and adhering to PrEP encountered by participants from a large, U.S. urban clinical center and assessed the utility of objectively monitoring PrEP adherence via urine. Most participants (65%) reported starting PrEP within 1-3 months of hearing about it, although 35% of participants encountered a provider unwilling to prescribe PrEP. Self-reported adherence was high among this population, with remembering to take the medication reported as the major barrier to adherence (44%) rather than cost or stigma. Urine tenofovir (TFV) monitoring was highly acceptable to this population, and participants indicated greater willingness to undergo urine monitoring every 3 months compared to finger prick (dried blood spot), phlebotomy, or hair follicle testing. These findings highlight the importance of focusing efforts toward reducing obstacles to PrEP use and support the use of urine TFV adherence monitoring as a marker of PrEP adherence.

Background: Current pharmacologic adherence monitoring for antiretrovirals involves expensive, labor-intensive liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based methods. Antibody-based assays can monitor and support adherence in real time. We developed a tenofovir (TFV)-based immunoassay and further validated it in a directly observed therapy (DOT) study., Design: Pharmacologic DOT study of TFV disoproxil fumarate (TDF)/emtricitabine (FTC) administered to HIV-noninfected volunteers., Methods: The TARGET study provided directly observed TDF 300 mg/FTC 200 mg 7 (high adherence), 4 (moderate), and 2 doses/week (low) to 30 volunteers (10/group) in Thailand, collecting a total of 637 urine samples over 6 weeks of administration and during washout. ELISA measured urine TFV levels by the immunoassay and LC-MS/MS-based concentrations served as the gold standard. A mixed-effects regression model evaluated cutoffs for a point-of-care assay. Performance characteristics of the immunoassay were compared with LC-MS/MS at a chosen cutoff., Results: Median TFV levels were 12,000 ng/mL by the immunoassay 1 day after dosing; 5000 ng/mL 2 days after dosing; 1500 ng/mL 3 days after dosing; and below the lower limit of quantification thereafter (≥4 days). An immunoassay cutoff of 1500 ng/mL accurately classified 98% of patients who took a dose 24 hours ago as adherent. The specificity and sensitivity of the immunoassay compared with LC-MS/MS at the 1500 ng/mL cutoff were 99% and 94%; the correlation between TFV levels by the 2 assays was high (0.92, P < 0.00001)., Conclusions: We have developed a novel TFV immunoassay that is highly specific, sensitive, and correlates strongly with LC-MS/MS measurements in a large DOT study. Adherence benchmarks from this DOT study will guide the development of a low-cost rapid point-of-care test for pre-exposure prophylaxis and antiretroviral treatment adherence monitoring and interventions.

Objective: We examined the relationship between urine tenofovir (TFV) levels measured with a novel immunoassay, which permits point-of-care testing, with HIV seroconversion and objective adherence metrics in a large preexposure prophylaxis (PrEP) demonstration project., Design: Secondary analysis of stored specimens from an open-label PrEP cohort study., Methods: We examined the association between undetectable urine TFV levels and HIV seroconversion in iPrEx open-label extension using generalized estimating equations. We examined rank correlations between levels of TFV and emtricitabine in urine, dried blood spots (DBS), and hair and determined the sensitivity and specificity of undetectable urine TFV for predicting dosing cut-offs in DBS., Results: The median urinary TFV level was 15 000 ng/ml in those who remained HIV-negative (n = 105; interquartile range: 1000-45 000); 5500 in those who eventually seroconverted (n = 11; interquartile range: 1000-12 500); and all were undetectable at seroconversion (n = 9; P < 0.001). Decreasing strata of urine TFV levels were associated with future HIV seroconversion (P = 0.03). An undetectable urine TFV was 100% sensitive and 81% specific when compared with an undetectable DBS TFV-diphosphate level and 69% sensitive, but 94% specific when compared with low adherence by DBS (<2 doses/week)., Conclusion: Urine TFV detection by a novel antibody-based assay was associated with protection from HIV acquisition among individuals on PrEP. Urine TFV levels were correlated with hair and DBS levels and undetectable urine TFV was 100% sensitive in detecting nonadherence. By implementing the immunoassay into a point-of-care strip test, PrEP nonadherence could be detected in real-time, allowing rapid intervention.

Proper management of an HIV infection requires that a patient be at least 80-95% adherent to a prescribed drug regimen to avoid poor health outcomes and the development of drug-resistant HIV strains. Clinicians generally monitor adherence habits indirectly through patient self-reporting, pill counting, and electronic drug monitoring. While direct measurement of patient samples like urine for monitoring drug levels is possible, it requires specialized equipment and training that is not readily available in resource-limited settings where the need is greatest. In this work we report the development of an antibody that binds to tenofovir (TFV), a key small molecule drug for both the treatment and prevention of HIV, and a competitive lateral flow assay that uses that antibody to monitor urine samples for the presence of the drug. TFV was conjugated to an immunogenic protein and injected into rabbits to raise polyclonal antibodies sensitive to the drug. The antibodies were verified for TFV-sensitivity by immunoprecipitation and HPLC. A gold nanoparticle-based competitive assay was developed to detect the presence of TFV in urine samples with a sensitivity of 1 μg mL -1 . This TFV assay could be deployed as a point-of-care device for adherence monitoring in resource-limited settings as a low-cost, accurate, and speedy alternative to current methods to better inform changes in treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A combination of antiretroviral agents is frequently used in effective treatment of the human immunodeficiency virus infection. In this study, two different separation methods are presented for the simultaneous determination of emtricitabine, rilpivirine and tenofovir from raw materials and urine samples. Developed liquid chromatography and capillary electrophoresis methods were thoroughly optimized for high analytical performances. Optimization of multiple variables at the same time by performing a minimum number of experiments was achieved by the Box-Behnken design, which is an experimental design in response surface methodology, in capillary electrophoresis. The results of the experimental design ensure minimum analysis time with well-separated analytes. Separation conditions, such as different stationary phases, pH level, organic modifiers and temperatures in liquid chromatography method, were also optimized. In particular, among stationary phases, the core-shell column especially enhanced the effectiveness of separation in liquid chromatography. Both methods were fully validated and applied to real samples. The main advantage of the developed methods is the separation of the drug combination in a short time with high efficiency and without any time-consuming steps., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: Tenofovir disoproxil fumarate (TDF) is key component of pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) for HIV, but existing tools to monitor drug adherence are often inaccurate. Detection of tenofovir (TFV) in accessible biological samples, such as fingerprick blood, urine or oral fluid samples could be a novel objective measure of recent TDF adherence. To measure TFV concentrations associated with different levels of TDF adherence, we designed a randomized clinical trial to assess the blood, urine and oral fluid concentrations of TFV in adults with perfect, moderate and low drug adherence., Methods/design: A randomized, open-label, clinical pharmacokinetic study of tenofovir in healthy adult volunteers without HIV or Hepatitis B infection in Thailand. Consenting, eligible participants are randomized (1:1:1) among three groups to receive a controlled number of TDF (300 mg) doses in a combination pill with emtricitabine (FTC, 200 mg) for six weeks. Participants in Group 1 receive a single TDF/FTC tablet once daily (Perfect adherence); Group 2 receive a single TDF/FTC tablet 4 times/week (Moderate adherence); and Group 3 receive a single TDF/FTC tablet 2 times/week (Low adherence). Blood, plasma, urine and oral fluid samples are collected for drug measurement during three study phases: (i) initial 6-week treatment phase; (ii) intensive 24-h blood sampling phase after 6 weeks; (iii) 4-week washout phase. Thirty adults with evaluable pharmacokinetic samples (10 per group) will be enrolled [based on ensuring 25% precision in pharmacokinetic parameter estimates]. Pre-dose drug concentrations during the treatment phase will be descriptive and comparisons between groups performed using a Kruskal-Wallis test. A non-compartmental pharmacokinetic analysis will be performed on the intensive sampling data at Week 7 and the time course of TFV washout in the difference biological matrices will be reported based on the detected concentrations following drug cessation., Discussion: The results of this randomized trial will define the target concentration thresholds of TFV in blood, urine and oral fluid that can distinguish between different levels of TDF adherence. Such adherence 'benchmarks' can be applied to real-time drug testing and novel point-of-care tests to identify individuals with poor PrEP or ART adherence., Trial Registration: ClinicalTrials.gov Identifier NCT03012607 .

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 log 10 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., (© 2017 British HIV Association.)

The HIV virus and hepatitis B virus nucleotide reverse transcriptase inhibitor tenofovir has been associated with proximal tubular toxicity; the latter was found to be predicted by plasma concentrations and with single-nucleotide polymorphisms in transporters-encoding genes. A cross-sectional analysis in adult HIV-positive patients with estimated creatinine clearance >60 ml min -1 was performed. Twelve-hour plasma and urinary tenofovir concentrations and single-nucleotide polymorphisms in several transporter-encoding genes were analysed. In 289 patients 12-h tenofovir plasma, urinary and urinary to plasma ratios were 69 ng ml -1 (interquartile range 51.5-95), 24.3 mg ml -1 (14.3-37.7) and 384 (209-560). At multivariate analysis estimated creatinine clearance, protease inhibitors co-administration and SLC28A2 CT/TT genotypes were independently associated with plasma tenofovir exposure; ABCC10 GA/AA genotypes and protease inhibitor co-administration were independently associated with the urinary to plasma tenofovir ratio. Tenofovir clearance was associated with genetic polymorphisms in host genes and with co-administered drugs: if confirmed by ongoing studies these data may inform treatment tailoring and/or dose reductions.

Tenofovir disoproxil fumarate is a prodrug of tenofovir used in the treatment of HIV and HBV infections: it is the most used antiretroviral worldwide. Tenofovir is nucleotidic HIV reverse trascriptase inhibitor that showed excellent long-term efficacy and tolerability. However renal and bone complications (proximal tubulopathy, hypophosphatemia, decreased bone mineral density, and reduced creatinine clearance) limit its use. Tenofovir renal toxicity has been suggested as the consequence of drug entrapment in proximal tubular cells: measuring tenofovir urinary concentrations may be a proxy of this event and it may be used as predictor of tenofovir side effects. No method is currently available for quantifying tenofovir in this matrix: then, the aim of this work was to validate a new LC-MS method for the quantification of urinary tenofovir. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 5 μm T3, 4.6 mm × 150 mm, reversed phase analytical column. Detection of tenofovir and internal standard was achieved by electrospray ionization mass spectrometry in the positive ion mode. Calibration ranged from 391 to 100,000 ng/mL. The limit of quantification was 391 ng/mL and the limit of detection was 195 ng/mL. Mean recovery of tenofovir and internal standard were consistent and stable, while matrix effect resulted low and stable. The method was tested on 35 urine samples from HIV-positive patients treated with tenofovir-based HAARTs and did not show any significant interference with antiretrovirals or other concomitantly administered drugs. All the observed concentrations in real samples fitted the calibration range, confirming the capability of this method for the use in clinical routine. Whether confirmed in ad hoc studies this method may be used for quantifying tenofovir urinary concentrations and help managing HIV-positive patients treated with tenofovir., (Copyright © 2015 Elsevier B.V. All rights reserved.)