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Urine Tenofovir Levels Measured by a
Novel Immunoassay Predict HIV Protection
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Here we demonstrate that urine TFV concentrations, measured by a novel immunoassay, predict protection from HIV acquisition among PrEP users in sub-Saharan Africa.
In summary, we show the high predictive utility of an adequate urine tenofovir level for HIV protection among men and women using PrEP in sub-Saharan Africa. The urine immunoassay has been developed into a lateral flow assay (LFA), which is low-cost, easy to perform, can be administered at the POC, and provides results within minutes.[4] The LFA is portable and requires no reagents so it may also be administered in the field by non-medical personnel to help reach stigmatized or hidden populations. The assay should be evaluated in a variety of populations for adherence monitoring and feedback.
Clinical Infectious Diseases
22 June 2020
Randy M. Stalter1,2, Jared M. Baeten1,2,3, Deborah Donnell1,4, Matthew A. Spinelli5, David V. Glidden5, Warren C. Rodrigues6, Guohong Wang6, Michael Vincent6, Nelly Mugo1,7, Andrew Mujugira1,8, Mark Marzinke9, Craig Hendrix9, Monica Gandhi5, for the Partners PrEP Study Team
1. Department of Global Health, University of Washington, Seattle, WA, USA
2. Department of Epidemiology, University of Washington, Seattle, WA, USA
3. Department of Medicine, University of Washington, Seattle, WA, USA
4. Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
5. Division of HIV, ID, and Global Medicine, University of California, San Francisco, San Francisco, CA, USA
6. Abbott Rapid Diagnostics, Pomona, CA
7. Center for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya
8. Infectious Diseases Institute, Makerere University, Kampala, Uganda
9. Division of Clinical Pharmacology, Department of Medicine, Johns Hopkins University, Baltimore, MD
Abstract
New tools are needed to support PrEP adherence for individuals at risk for HIV, including those that enable provision of real-time feedback. In a large, completed PrEP trial, adequate urine tenofovir levels measured by a novel immunoassay predicted HIV protection and showed good sensitivity and specificity for detectable plasma tenofovir.
Introduction
Use of oral pre-exposure prophylaxis (PrEP) is a highly efficacious strategy for preventing HIV when taken daily.[1] However, adherence to PrEP has proven challenging among many populations.[2] Due to the limited accuracy of self-reported adherence, objective biological adherence metrics, such as measurement of PrEP drug concentrations in plasma, dried blood spots (DBS) and hair, have become valuable tools for appraising recent or cumulative PrEP adherence.[3] However, these methods can be costly and often require shipment of samples to an external laboratory, skilled laboratory personnel and specialized equipment, making them impractical options for routine use, particularly in resource-limited settings.
Innovative approaches are needed to support PrEP adherence, including those that enable counseling at the point of care (POC). Recently, we reported on the development of a novel antibody with high selectivity for tenofovir (TFV),[4] with the resultant immunoassay demonstrating high sensitivity and specificity relative to the standard liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay. Here we further evaluate the immunoassay by comparing urine TFV measurements to TFV concentrations measured in plasma, which served as the gold standard metric for short-term adherence in most PrEP trials. Further, we assess the novel urine assay's ability to predict protection from HIV in a large completed PrEP trial.
Results
Of 4,432 individuals randomized to use of TDF or TDF/FTC in the Partners PrEP Study, 292 were included in the nested cohort. Among these participants, 39% were female and the median age was 33 (interquartile range [IQR]=28-39). Participants in the cohort contributed 722 paired urine and plasma samples. Of 52 individuals who seroconverted to HIV while using PrEP in the study, 22 had urine samples available from the visit where HIV was first detected and were included as cases. An additional 69 seroconverter samples collected prior to HIV infection were included as possible controls. Among cases, 55% were female and the median age was 33 (IQR=27-39).
The median duration from collection to assay of plasma and urine samples was 20 months and 103 months, respectively. In the cohort, the median TFV concentration was 37,500 ng/mL (IQR=500-90,000 ng/mL) in urine via ELISA and 65.4 ng/mL (IQR=1.6-103.0 ng/mL) in plasma via LC-MS/MS. Spearman's rank correlation coefficient () for the two measures was 0.46 (p<0.001). Of 558 plasma samples with detectable TFV (≥LLOQ of 0.31 ng/mL), 486 had a paired urine sample with detectable TFV (≥LLOQ of 1000 ng/mL) for a sensitivity of 87% (95% CI=84-90%). There were 164 plasma samples with undetectable TFV, of which 119 had a paired urine sample with undetectable TFV for a specificity of 73% (95% CI=65-79%). Of 468 individuals with plasma TFV >40 ng/mL, 420 had a paired urine sample with TFV ≥1500 ng/mL, for a sensitivity of 90% (95% CI=87-92%). Finally, 254 plasma samples had TFV levels ≤40 ng/mL, of which 146 had a paired urine sample with TFV <1500 ng/mL for a specificity of 57% (95% CI=51-64%).
In total, 770 control samples from 280 individuals were matched to the 22 case samples in our case-control study. Among participants in both active PrEP study arms, urine TFV ≥1500 ng/mL was associated with a 71% (95% CI=30-88%) reduction in HIV risk in the adjusted model (Table 1). By contrast, plasma TFV >40 ng/mL was associated with an 87% (95% CI=54-96%) reduction in HIV risk (Supplemental Table 1).
Discussion
The development of a low-cost POC assay to evaluate PrEP adherence would facilitate the implementation of real-time, drug-level feedback in current PrEP programs in resource-limited settings. Here we demonstrate that urine TFV concentrations, measured by a novel immunoassay, predict protection from HIV acquisition among PrEP users in sub-Saharan Africa. The concentrations measured by the urine assay also had good sensitivity and specificity for plasma levels, the gold-standard metric of adherence in placebo-controlled PrEP trials.
Previous studies have highlighted the advantages of using a urine-based assay to evaluate PrEP use. TARGET, a pharmacokinetic study that randomized Thai adults to directly-observed TDF in arms simulating low, medium and high adherence patterns, demonstrated that paired urine and plasma TFV concentrations measured by LC-MS/MS were highly correlated.[8] The study also showed that urine TFV concentrations can evaluate time since dosing, further demonstrated in other U.S.-based studies.[9,10] Data from the U.S. have also suggested that urine collection is highly acceptable, which may result in high uptake compared to other biological measures.[11] Additional evidence regarding the feasibility and acceptability of urine-based measures of PrEP use in other settings, including sub-Saharan Africa, is needed.
We previously demonstrated that urine levels via this immunoassay were correlated with other biomarkers, including TFV and FTC levels in hair and TFV-diphosphate and FTC-triphosphate concentrations in DBS, among men who have sex with men (MSM) and transwomen in the iPrEx open-label extension (OLE) study. Moreover, low urine concentrations among participants in iPrEx OLE were associated with subsequent HIV seroconversion.[12] The data presented here extend these prior results to heterosexual men and women on PrEP in sub-Saharan Africa.
There are several potential limitations to our results. First, we were unable to account for specific gravity of the urine samples or normalize to creatinine levels.[9] Second, longer storage of urine compared to plasma samples prior to analysis may have resulted in differential rates of sample degradation. Moderate correlation observed between urine and plasma TFV concentrations may be partially explained by these factors. Use of a specified TFV threshold for other analyses, however, may have mitigated the influence of measurement variability. Finally, most seroconverters did not have urine samples available from the visit of first HIV detection, limiting study power, and incomplete availability of control samples at urine archive months may have limited our control matching. However, our overall risk reduction estimates with plasma TFV >40 were nearly identical to those previously identified in this cohort,[7] suggesting bias may have been minimal.
In summary, we show the high predictive utility of an adequate urine tenofovir level for HIV protection among men and women using PrEP in sub-Saharan Africa. The urine immunoassay has been developed into a lateral flow assay (LFA), which is low-cost, easy to perform, can be administered at the POC, and provides results within minutes.[4] The LFA is portable and requires no reagents so it may also be administered in the field by non-medical personnel to help reach stigmatized or hidden populations. The assay should be evaluated in a variety of populations for adherence monitoring and feedback.
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