Screening and characterization of vaginal fluid donations for vaginal microbiota transplantation

Abstract

Bacterial vaginosis (BV), the overgrowth of diverse anaerobic bacteria in the vagina, is the most common cause of vaginal symptoms worldwide. BV frequently recurs after antibiotic therapy, and the best probiotic treatments only result in transient changes from BV-associated states to "optimal" communities dominated by a single species of Lactobacillus. Therefore, additional treatment strategies are needed to durably alter vaginal microbiota composition for patients with BV. Vaginal microbiota transplantation (VMT), the transfer of vaginal fluid from a healthy person with an optimal vaginal microbiota to a recipient with BV, has been proposed as one such alternative. However, VMT carries potential risks, necessitating strict safety precautions. Here, we present an FDA-approved donor screening protocol and detailed methodology for donation collection, storage, screening, and analysis of VMT material. We find that Lactobacillus viability is maintained for over six months in donated material stored at - 80 °C without glycerol or other cryoprotectants. We further show that species-specific quantitative PCR for L. crispatus and L. iners can be used as a rapid initial screening strategy to identify potential donors with optimal vaginal microbiomes. Together, this work lays the foundation for designing safe, reproducible trials of VMT as a treatment for BV.

Figure 1

Microbial community profiling and absolute quantification of pilot donation fluid. (a) Lactobacillus colony forming unit (CFU) quantification for each donation and analysis aliquot. Total donation volume is listed below donation numbers. (b) Microbial species in donation (“D”) and analysis (“A”) aliquots of pilot donor (Donor 0) samples using bacterial 16S rRNA amplicon sequencing. D1 and D2 refer to two different donation aliquots from the same donation collection.

Figure 2

Donor screening and donation schedule. (a) 50 total women, including the pilot donor (light green), were pre-screened over the phone. Eight potential donors completed an in-person screen and testing, three of whom went on to become donors (dark green), four of whom were ineligible due to high Nugent scores (red), and one of whom was ineligible due to a recent blood donation and no subsequent follow-up (pink). Twenty-three potential donors (grey) were uninterested in following up or were disqualified for logistical reasons (e.g., travel), 18 completed pre-screening and asked for later follow up (yellow). (b) Donation screening and schedule with detailed testing for donors and donations before, during, and after donating.

Figure 3

Microbial community profiles of VMT donation samples. (a) Microbial community and metadata. Relative abundance of the bacterial community in the donation material was determined using 16S rRNA amplicon sequencing for each donation. Nugent score and white blood cell (WBC)/epithelial cell ratio are presented below each donation. (b) Absolute quantification of L. crispatus and L. iners in donation samples using species-specific qPCR. Detection limit of the assay is depicted by dashed lines. (c) Donation stability measured as Lactobacillus CFUs streaked and counted on MRS agar at long-term intervals post freezing.