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.

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 300mg/FTC 200mg 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 wash-out. 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 cut-offs for a point-ofcare (POC) assay. Performance characteristics of the immunoassay were compared to LC-MS/MS at a chosen cut-off.Results: Median TFV levels were 12,000ng/mL by the immunoassay 1-day after dosing; 5000ng/mL 2-days after dosing; 1500ng/mL 3-days after dosing and below the lower-limit-of-

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Synthetic cannabinoids are often referred to as 'Spice' or K2 compounds. Detection of these compounds in oral fluid has, to date, been limited to chromatographic procedures such as liquid chromatography with tandem mass spectrometry detection. We report the first analytical immunoassay for the screening of some synthetic cannabinoids in oral fluid specimens collected with the Quantisal™ device. JWH-200 was chosen as the calibration standard, because parent compounds, not metabolites, are predominantly detected in oral fluid. The immunoassay is capable of detecting JWH-200, JWH-018, JWH-073, JWH-022, AM-2201, AM-2232 and AM-1220. The assay was validated according to accepted laboratory protocols and applied to 32 authentic oral fluid specimens previously analyzed using LC-MS-MS at an accredited laboratory. The assay is sensitive, with a cutoff concentration of 0.25 ng/mL, and has a wide working range from 0.1 to 5 ng/mL. Intra- and interday precision were determined to be <10%. The screening method was completely validated and characterized; critical aspects of the screening included the incorporation of a preincubation step that improves the sensitivity of the assay to allow relevant concentrations of synthetic compounds in oral fluid to be detected.

Objective: We examined the relationship between urine tenofovir (TFV) levels measured with a novel immunoassay, which permits point-of-care (POC) testing, with HIV seroconversion and objective adherence metrics in a large 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-OLE using generalized estimating equations. We examined rank correlations between levels of TFV and emtricitabine (FTC) 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; IQR:1,000-45,000); 5,500 in those who eventually seroconverted (n=11; IQR:1,000-12,500); 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 to an undetectable DBS TFV-DP level and 69% sensitive, but 94% specific, when compared to low adherence by DBS (<2 doses/week).Conclusions: 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