Laboratory methods

For drug level feedback, intracellular TFV-DP levels measured in DBS were performed at the University of Cape Town, which validated the DBS testing in collaboration with the University of Colorado, the developer of the TFV-DP DBS assay [18]. TFV-DP levels in DBS for endpoint analyses were performed at the University of Colorado. Intracellular TFV-DP in red blood cells has a 17-day half-life and 25-fold drug accumulation, which provides a cumulative measure of dosing and average adherence to PrEP in the prior 6 weeks [18]. Studies of directly observed PrEP dosing were used to define thresholds for DBS TFV-DP levels for categories of average weekly dosages taken of PrEP [17,19–21]. HIV status was determined by point-of-care rapid HIV tests at the site followed by an instrumented fourth generation antigen/antibody immunoassay (either GS IV Combo Ag/Ab EIA, Bio-Rad Laboratories, Hercules, California, USA or Architect HIV Ag/Ab Combo, Abbott Diagnostics, Wiesbaden, Germany) and RNA testing (Abbott RealTime HIV-1 Viral Load Assay, Abbott Molecular, Abbott Park, Illinois, USA) for acute infection for samples with positive rapid test or antigen/antibody immunoassays. HIV resistance testing on samples from seroconverters was performed using the ViroSeq HIV-1 Genotyping System v2.0 (Abbott Molecular, Des Plaines, Illinois, USA).

Creatinine clearance was estimated based on serum creatinine measurements using the Schwartz equation [19]. Neisseria gonorrhoeae and Chlamydia trachomatis were assessed by nucleic acid amplification (Cepheid GeneXpert, Sunnyvale, California, USA), and Trichomonas vaginalis by rapid test (OSOM Trichomonas Test, Sekisui Diagnostics, Burlington, Massachusetts, USA). Syphilis was assessed by rapid plasma reagin (Macro-Vue RPR Test, Becton Dickinson, Sparks, Maryland, USA) followed by a treponemal-specific confirmatory assay (Serodia-TPPA, Fujirebio, Tokyo, Japan or Randox-SYP-TPHA, Crumlin, County Antrim, United Kingdom).