Development and validation of the first point-of-care assay to objectively monitor adherence to HIV treatment and prevention in real-time in routine settings

Abstract

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.

Urine Tenofovir Levels Measured Using a Novel Immunoassay Predict Human Immunodeficiency Virus Protection

“…In summary, we show the high predictive utility of an adequate urine TFV 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 nonmedical personnel to help reach stigmatized or hidden populations.…”

“…During study follow-up, urine samples were archived at 3-, 12-, 24-, and 36-month visits. TFV concentrations were measured in all available archived urine samples using a quantitative enzyme-linked immunosorbent assay (ELISA; lower limit of quantification [LLOQ] = 1000 ng/mL) with this novel antibody [ 4 ]. TFV concentrations in date-matched plasma specimens were previously measured among the cohort using a validated LC-MS/MS assay (LLOQ = 0.31 ng/mL).…”

“…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.…”

Real-Time Monitoring and Point-of-Care Testing: A Review of the Current Landscape of PrEP Adherence Monitoring

“…51 Although a small study, these results suggest that patient adherence can be increased with increased data and targeted counselling. In addition, urine-based TFV monitoring has already been developed into a point-of-care test 52 , 53 that is currently awaiting FDA approval, where objective data on recent PrEP adherence can be reviewed in real-time with patients to guide further adherence support counseling. In contradistinction to measuring drug concentrations, the TARGET study measured an antibody-based urine TFV assay which was strongly correlated to the LC-MS/MS-based urine assay.…”

“… 47 , 55 Nevertheless, urine TFV testing is potentially more useful than plasma TFV testing in that the former can distinguish between PrEP dosing in the previous 1–2 days from dosing in the previous 2–7 days. 52 …”