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Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence

BACKGROUND: Pharmacologic adherence measures were critical to the interpretation of the tenofovir (TFV)-disoproxil-fumarate/emtricitabine (TDF/FTC) PrEP trials. These measures are being incorporated into PrEP demonstration projects, but currently-available metrics in plasma, cells, hair or urine inv...

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Autores principales: Gandhi, Monica, Bacchetti, Peter, Rodrigues, Warren C., Spinelli, Matthew, Koss, Catherine A., Drain, Paul K., Baeten, Jared M., Mugo, Nelly R., Ngure, Kenneth, Benet, Leslie Z., Okochi, Hideaki, Wang, Guohong, Vincent, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428441/
https://www.ncbi.nlm.nih.gov/pubmed/30906930
http://dx.doi.org/10.1016/j.eclinm.2018.08.004
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author Gandhi, Monica
Bacchetti, Peter
Rodrigues, Warren C.
Spinelli, Matthew
Koss, Catherine A.
Drain, Paul K.
Baeten, Jared M.
Mugo, Nelly R.
Ngure, Kenneth
Benet, Leslie Z.
Okochi, Hideaki
Wang, Guohong
Vincent, Michael
author_facet Gandhi, Monica
Bacchetti, Peter
Rodrigues, Warren C.
Spinelli, Matthew
Koss, Catherine A.
Drain, Paul K.
Baeten, Jared M.
Mugo, Nelly R.
Ngure, Kenneth
Benet, Leslie Z.
Okochi, Hideaki
Wang, Guohong
Vincent, Michael
author_sort Gandhi, Monica
collection PubMed
description BACKGROUND: Pharmacologic adherence measures were critical to the interpretation of the tenofovir (TFV)-disoproxil-fumarate/emtricitabine (TDF/FTC) PrEP trials. These measures are being incorporated into PrEP demonstration projects, but currently-available metrics in plasma, cells, hair or urine involve expensive and time-intensive mass-spectrometry (MS)-based methods. No point-of-care method to assess PrEP adherence in real-time has yet been implemented. Antibody-based tests allow for low-cost, easy-to-perform, point-of-care drug detection. In this study, we developed an antibody-based TFV immunoassay and evaluated its test characteristics among individuals taking TDF/FTC. METHODS: We synthesized possible immunogens based on TFV's molecular structure, injected rabbits with the conjugated derivatives, and bled them monthly for subsequent ELISA-testing for TFV-specific antibodies. We purified an antibody with specific TFV binding and created dose–response curves for ELISA-quantification. We then quantified TFV in urine from human participants not taking TDF/FTC and from individuals taking daily TDF/FTC 300 mg/200 mg for 7 days with a 7-day washout period using ELISA with this TFV-specific antibody. ELISA results were compared with the gold-standard test for TFV detection/quantification using liquid-chromatography-tandem-MS (LC–MS/MS). FINDINGS: None of the urine samples from 115 participants not taking TDF/FTC showed ELISA- reactivity, indicating 100% specificity (95% CI 97–100%) of the immunoassay. Among participants taking TDF/FTC, 67 of 70 samples positive by LC–MS/MS were positive by the ELISA-immunoassay for an estimated diagnostic sensitivity of 96% (95% CI 88–99%). The precision of the assay was high (coefficient of variation < 15%). The rank correlation between ELISA and LC–MS/MS values in the 70 quantitative urine TFV levels positive by LC–MS/MS across a wide range of concentrations among participants on TDF/FTC was high (r = 0.96). INTERPRETATION: Our antibody-based immunoassay for measuring TFV in urine performed well compared to the gold-standard of LC–MS/MS among individuals taking TDF/FTC. A sensitive and specific immunoassay paves the way for real-time monitoring/feedback on recent adherence to TFV-based regimens, which should optimize interpretation and outcomes during PrEP and ART roll-out. FUNDING: NIAID/NIH 2R01AI098472.
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spelling pubmed-64284412019-03-21 Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence Gandhi, Monica Bacchetti, Peter Rodrigues, Warren C. Spinelli, Matthew Koss, Catherine A. Drain, Paul K. Baeten, Jared M. Mugo, Nelly R. Ngure, Kenneth Benet, Leslie Z. Okochi, Hideaki Wang, Guohong Vincent, Michael EClinicalMedicine Research Paper BACKGROUND: Pharmacologic adherence measures were critical to the interpretation of the tenofovir (TFV)-disoproxil-fumarate/emtricitabine (TDF/FTC) PrEP trials. These measures are being incorporated into PrEP demonstration projects, but currently-available metrics in plasma, cells, hair or urine involve expensive and time-intensive mass-spectrometry (MS)-based methods. No point-of-care method to assess PrEP adherence in real-time has yet been implemented. Antibody-based tests allow for low-cost, easy-to-perform, point-of-care drug detection. In this study, we developed an antibody-based TFV immunoassay and evaluated its test characteristics among individuals taking TDF/FTC. METHODS: We synthesized possible immunogens based on TFV's molecular structure, injected rabbits with the conjugated derivatives, and bled them monthly for subsequent ELISA-testing for TFV-specific antibodies. We purified an antibody with specific TFV binding and created dose–response curves for ELISA-quantification. We then quantified TFV in urine from human participants not taking TDF/FTC and from individuals taking daily TDF/FTC 300 mg/200 mg for 7 days with a 7-day washout period using ELISA with this TFV-specific antibody. ELISA results were compared with the gold-standard test for TFV detection/quantification using liquid-chromatography-tandem-MS (LC–MS/MS). FINDINGS: None of the urine samples from 115 participants not taking TDF/FTC showed ELISA- reactivity, indicating 100% specificity (95% CI 97–100%) of the immunoassay. Among participants taking TDF/FTC, 67 of 70 samples positive by LC–MS/MS were positive by the ELISA-immunoassay for an estimated diagnostic sensitivity of 96% (95% CI 88–99%). The precision of the assay was high (coefficient of variation < 15%). The rank correlation between ELISA and LC–MS/MS values in the 70 quantitative urine TFV levels positive by LC–MS/MS across a wide range of concentrations among participants on TDF/FTC was high (r = 0.96). INTERPRETATION: Our antibody-based immunoassay for measuring TFV in urine performed well compared to the gold-standard of LC–MS/MS among individuals taking TDF/FTC. A sensitive and specific immunoassay paves the way for real-time monitoring/feedback on recent adherence to TFV-based regimens, which should optimize interpretation and outcomes during PrEP and ART roll-out. FUNDING: NIAID/NIH 2R01AI098472. Elsevier 2018-08-31 /pmc/articles/PMC6428441/ /pubmed/30906930 http://dx.doi.org/10.1016/j.eclinm.2018.08.004 Text en © 2018 Published by Elsevier Ltd. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Paper
Gandhi, Monica
Bacchetti, Peter
Rodrigues, Warren C.
Spinelli, Matthew
Koss, Catherine A.
Drain, Paul K.
Baeten, Jared M.
Mugo, Nelly R.
Ngure, Kenneth
Benet, Leslie Z.
Okochi, Hideaki
Wang, Guohong
Vincent, Michael
Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence
title Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence
title_full Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence
title_fullStr Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence
title_full_unstemmed Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence
title_short Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence
title_sort development and validation of an immunoassay for tenofovir in urine as a real-time metric of antiretroviral adherence
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428441/
https://www.ncbi.nlm.nih.gov/pubmed/30906930
http://dx.doi.org/10.1016/j.eclinm.2018.08.004
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