6 research outputs found
Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence
STR-Typisierungen von DEPArrayTM-separierten Einzelzellen zur Aufsplittung von Blut-Blut-Mischungen in die jeweiligen Einzelbestandteile
Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis
Development of a Composite Measure of Product Adherence, Protocol Compliance, and Semen Exposure Using DNA and Protein Biomarkers for Topical HIV Prevention Studies
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.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.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.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