Optimal sampling and analysis methods for clinical diagnostics of vaginal microbiome

Abstract

Next-generation sequencing-based microbiological analysis is a complex way to profile vaginal microbiome samples since each step affects the results gained. Methodologies for sample collection lack golden standards. We compared Puritan DNA/RNA swab (PS) and Copan FLOQ swab (CS) and provided consistent and reliable microbiome profiles analyzed by 16S rRNA gene sequencing. We collected two consecutive vaginal samples utilizing PS with room temperature storing and CS with instant freezing from 26 women. Variable region 4 of bacterial 16S rRNA gene was amplified with single PCR by custom-designed dual-indexed primers and sequenced with Illumina MiSeq system. Read quality control, operational taxonomic unit tables, and alpha and beta diversities analysis were performed, and community richness, diversity, and evenness were evaluated and compared between the two samplings and tests. Nineteen sample pairs produced detectable, intact DNA during the extraction protocol and/or further microbial profiles. Alpha bacterial diversity indices were independent on the collection protocol. No significant statistical differences were found in the measured beta diversity metrics between the collection methods. Of the women, 43% had Lactobacillus-dominated vaginal microbiome profile despite of collection method. Previously reported important vaginal microbiome phyla Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria were present in the sample set although their relative abundances varied among individuals. PS and CS enable constant vaginal microbiota sampling. The PS method with no need for instant freezing is suitable for on-site collections at clinics. Furthermore, it seems to be possible to take two samples instead of one with constant microbiological results.

Interactions between microbiota and cervical epithelial, immune, and mucus barrier

“…Second, sampling site and methodology may also have influence on microbiome sequencing as well as cytokines detection. As discussed before, there are difference in the microbiome composition of vagina wall, posterior fornix and endocervix, as well as sampling device (Kim et al, 2009;Virtanen et al, 2017;Kero et al, 2023). Cytokine and protein concentration were also different collected by endocervical swabs, lavage samples, and vaginal swabs (Dezzutti et al, 2011).…”

Validation of vaginal microbiome proxies for in vitro experiments that biomimic Lactobacillus‐dominant vaginal cultures

“…Given the importance and delicate balance of the vaginal microbiome and the genital tract mucosa, it is critical to develop advanced in vitro methods of incorporating these components into traditional cell culture systems. However, there are several limitations hindering the scientific community from incorporating vaginal microbiomes directly into in vitro models including: clinical challenges and limitations in collecting and preserving microbiome samples from diverse population groups, 18,19 in vitro changes in culture-multi-species microbiome colonies, 20 lack of glycogen/mucin backbone molecules to support microbial colonization, 21 and recreating cell-microbe interactions without inducing pathologic overgrowth. 20 Therefore, to provide an in vitro proxy for the proper cell-multi-species microbe interactions found in the vaginal tract, metabolites were assessed as a surrogate to determine their sufficiency in modeling the in vivo environment.…”