title: Development of a composite measure of product adherence, protocol compliance, and semen exposure using DNA and protein biomarkers for topical HIV prevention studies.
abstract: BACKGROUND
  Poor and inconsistent use of study products has hindered clinical HIV prevention studies. It is important to be able to monitor product adherence and protocol compliance in order to determine microbicide efficacy and safety more accurately. Current methods for monitoring adherence are subjective, non-specific, or invasive. Herein, we present a composite, objective measure of product adherence and protocol compliance to assess vaginal insertion, semen exposure and drug expulsion utilizing DNA, protein, and drug isolated directly from returned, vaginally used gel applicators.
  
  
  METHODS
  DNA, vaginal cells, and residual tenofovir were isolated from vaginally inserted applicators. Vaginal and semen biomarkers were amplified using a multiplex PCR to determine vaginal insertion. Vaginal cells were fixed followed by cytokeratin 4 immunocytochemistry to confirm DNA assessment of vaginal insertion. Tenofovir was extracted and quantitated through LC-MS/MS.
  
  
  RESULTS
  DNA isolated from vaginally inserted applicators were positive for vaginal bacteria DNA and the control eukaryotic gene, amelogenin, while manually handled, "sham", applicators were negative for both. Semen exposure was independently determined by simultaneous amplification of one or both Y-chromosomal genes, SRY and TSPY4. Vaginal insertion determination by DNA analysis was further confirmed by positive cytokeratin 4 (CK4) immunocytochemistry of vaginal cells remaining on the gel applicators. On the contrary, sham applicators provided very few cells when swabbed, and they were all negative for CK4. CK4 was not found in epidermal cells from the hand. Drug expulsion was detected through quantitation of residual gel present on the surface of returned applicators. Sham applicators had no detectable tenofovir.
  
  
  CONCLUSIONS
  Utilizing a composite, triple marker based panel of DNA, protein, and drug present on the surface of returned vaginal gel applicators, it is possible to determine, objectively and non-invasively, product adherence, protocol compliance, and semen exposure in microbicide trials.
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.
authors.forename: Terry A
authors.initials: TA
authors.lastname: Jacot
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.
authors.forename: Ashley
authors.initials: A
authors.lastname: Nelson
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Thurman
authors.affiliation: University of North Carolina Eshelman School of Pharmacy and University of North Carolina Center for AIDS Research, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Angela D M
authors.initials: AD
authors.lastname: Kashuba
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.
authors.forename: David F
authors.initials: DF
authors.lastname: Archer
authors.affiliation: CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.
authors.forename: Gustavo F
authors.initials: GF
authors.lastname: Doncel
mesh_terms: D000225:Adenine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D000287:Administration, Topical; D000328:Adult; D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002482:Cellulose / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004781:Environmental Exposure / Q000032:analysis; D005240:Feasibility Studies; D005260:Female; D019983:Guideline Adherence / Q000706:statistics & numerical data*; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control* / Q000635:transmission; D006801:Humans; D053527:Keratin-4 / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D063065:Organophosphonates / Q000008:administration & dosage / Q000494:pharmacology; D017063:Outcome Assessment, Health Care / Q000379:methods*; D010349:Patient Compliance / Q000706:statistics & numerical data*; D012661:Semen / Q000821:virology*; D000068698:Tenofovir; D014621:Vagina / Q000187:drug effects / Q000821:virology; D055815:Young Adult
chemical_list: D019380:Anti-HIV Agents; D015415:Biomarkers; D053527:Keratin-4; D063065:Organophosphonates; D002482:Cellulose; C002283:hydroxyethylcellulose; D004247:DNA; D000068698:Tenofovir; D000225:Adenine