Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products

Background: Development of safe, effective products to prevent the sexual transmission of HIV remains a priority. Prior to clinical testing, the products must undergo strict safety evaluations to avoid mucosal drug toxicity, inflammation, and vaginal microbiome (VMB) shifts. Based on the Food and Drug Administration (FDA) guidance, we designed a study to measure the inflammatory markers and VMB changes after intravaginal treatment with products that have been associated with toxicity, with the objective to develop a Gram stain slide scoring system, similar to Nugent scoring, correlated with the proinflammatory cytokines in sheep. Methods: Non-pregnant Dorset ewes (n = 34) were randomized to receive 5 ml intravaginal 4% nonoxynol-9 (N9) contraceptive gel, positive control (0.2% benzalkonium chloride), placebo control [hydroxethyl cellulose (HEC)], or no application daily for 10 days, with 11-day post-treatment follow-up. The vaginal swabs were collected for the cytokines, VMB, and Gram-stained slides. An enzyme-linked immunosorbent assay (ELISA) analysis of cytokines interleukin (IL)-1β, IL-8, CXCL10, and tumor necrosis factor-α (TNF-α) was used to determine inflammatory state of the sample. Vaginal microbiome community types (CT) were utilized to create five equivalent slide subsets for iterative development of a Gram-stained slide scoring system with comparisons with inflammatory state based on the cytokine levels. Results: Digital images of the Gram-stained slides were scored based on Gram staining and morphology of bacteria, presence of sheep epithelial cells, and immune cells. The scoring system was modified in an iterative fashion with weighting based on cytokine categorization of inflamed samples, with three of four cytokine values above the mean indicating that the sample was inflamed. The parameters in the final version of the scoring system included mature epithelial cells, Gram-negative rods, and Gram-positive diplococci indicating normal and immune cells indicating inflammation. The area under the receiver operator characteristic curve (ROC AUC) was 0.725 (ROC AUCs range between 0.5 and 1.0) with a greater area indicating higher diagnostic ability of a test with a binary outcome: inflamed or normal. Conclusion: The scoring system, derived from the advanced VMB and cytokine analyses, provides a validated, practical method for quantification of Gram-stained slides that can be performed in most laboratories, increasing the potential for standardization. The training plan can assist laboratories to determine the safety of intravaginal products in their sheep studies or the methodological approach can be applied to other animal models where such data are also needed.