Validation of a dye stain assay for vaginally inserted hydroxyethylcellulose-filled microbicide applicators.

The reliability and validity of self-reports of vaginal microbicide use are questionable given the explicit understanding that participants are expected to comply with study protocols. Our objective was to optimize the use of Population Council's previously validated dye stain assay (DSA) and related procedures, and to establish predictive values for the DSAs ability to identify vaginally inserted single-use, low-density polyethylene microbicide applicators filled with hydroxyethylcellulose gel. Applicators, inserted by 252 female sex workers enrolled in a microbicide feasibility study in Southern India, served as positive controls for optimization and validation experiments. Before validation, optimal dye concentration and staining time were ascertained. Three validation experiments were conducted to determine sensitivity, specificity, and negative and positive predictive values. The dye concentration of 0.05% (wt/vol) FD&C Blue No. 1 Granular Food Dye (Prime Ingredients, Inc, Saddlebrook, NJ) and staining time of 5 seconds were determined to be optimal and were used for the 3 validation experiments. There were a total of 1848 possible applicator readings across validation experiments; 1703 (92.2%) applicator readings were correct. On average, the DSA performed with 90.6% sensitivity, 93.9% specificity, and had a negative predictive value of 93.8% and a positive predictive value of 91.0%. No statistically significant differences between experiments were noted. The DSA was optimized and successfully validated for use with single use, low-density polyethylene applicators filled with hydroxyethylcellulose gel. We recommend including the DSA in future microbicide trials involving vaginal gels so as to identify participants who have low adherence to dosing regimens. In doing so, we can develop strategies to improve adherence as well as investigate the association between product use and efficacy.