Optimal sampling and analysis methods for clinical diagnostics of vaginal microbiome

Area of Science:

• Microbiology
• Genetics
• Bioinformatics

Background:

• Vaginal microbiome analysis using next-generation sequencing is sensitive to pre-analytical steps.
• Standardized sample collection methods are lacking, impacting data reliability.
• Comparing different swab types is crucial for consistent results.

Purpose of the Study:

• To compare Puritan DNA/RNA swab (PS) and Copan FLOQ swab (CS) for vaginal microbiome profiling.
• To assess the impact of room temperature storage (PS) versus immediate freezing (CS) on results.
• To evaluate consistency and reliability of microbiome profiles obtained from different collection methods.

Main Methods:

• Collected paired vaginal samples from 26 women using PS (room temp) and CS (frozen).
• Amplified 16S rRNA gene's V4 region via PCR and sequenced using Illumina MiSeq.
• Analyzed data for quality control, operational taxonomic units, alpha/beta diversity, richness, and evenness.

Main Results:

• Nineteen sample pairs yielded usable DNA and microbial profiles.
• Alpha bacterial diversity was not affected by the collection method.
• No significant differences in beta diversity metrics were observed between PS and CS methods.
• Lactobacillus-dominated profiles were found in 43% of women, irrespective of the swab type.
• Key vaginal microbiome phyla (Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria) were detected with varying abundances.

Conclusions:

• Both PS and CS swabs allow for consistent vaginal microbiota sampling.
• The PS swab is suitable for on-site clinical collection due to no requirement for immediate freezing.
• Dual sampling with consistent results is feasible, potentially improving diagnostic yield.