Microbiological quality control for intravaginal devices for the treatment of infectious diseases and bacterial...

Preclinical and clinical infectious disease research introducing intravaginal devices containing active agents and probiotic bacteria need to rely on materials that meet appropriate microbiologic standards. However, research methods for quality control of intravaginal devices are poorly described. Here, we develop a practical workflow that specifies media types, culture techniques, and modifications to commonly used assays to suit vaginal applications. A set of quality control assays were developed to test electrospun fibers intended for use in an experimental (mouse) model. The fibers carried probiotics, antibiotics, or no cargo (i.e., "blank"). To validate anti-bacterial action for metronidazole-containing fibers against a relevant target bacterium associated with bacterial vaginosis (BV), a modified Kirby Bauer disc diffusion assay confirmed a zone of Gardnerella clearance on NYCIII agar plates. For "blank" fibers containing no cargo, simple turbidity assays using TSB were effective in highlighting batches of electrospun fibers that were contaminated with culturable bacteria. CHROMagar™ Orientation agar was also a useful tool, particularly if fibers contained vaginal probiotic bacteria (Lactobacillus crispatus) as the intended cargo. For example, CHROMagar™ identified a probiotic strain other than L. crispatus SmR incorporated into fibers in one experiment (L. rhamnosus SmR). The limulus amebocyte lysate assay was also used to measure endotoxin levels in specimens from mice and humans, establishing what may be considered "normal" in these settings. Women with an abnormal BV-like microbiota (Nugent score > 4) had ∼250-fold higher levels of endotoxin compared to those without BV (median 134.2 vs. 0.528 EU/ml in vaginal swab elutions). Vaginal washes from uninfected female mice likewise had low concentrations of endotoxin (∼0.6900 EU/ml). Process improvements during the fabrication of electrospinning fibers identified contributing sources of endotoxin and resulted in a 10-fold improvement in median concentrations from 14.21 to 1.143 EU/device. Together, these studies demonstrate a feasible workflow that can quickly identify culturable contaminants or high levels of endotoxin in electrospun fibers intended for vaginal application.