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ObjectivesWe evaluated how storing vaginal samples at room temperature in stabilising solutions versus immediate freezing affects 16S rRNA gene amplicon sequencing-based microbiota studies, aiming to simplify home and field collection. METHODS: Twenty participants self-collected six mid-vaginal swabs that were stored in two nucleic acid preservatives (three in modified Solution C2 (Qiagen) and three in Amies/RNALater (Sigma)) in January-February 2016. From each set, two were immediately frozen (-80°C) and one was shipped to the University of Idaho (Moscow, Idaho) with return shipping to the Institute for Genome Sciences (Baltimore, Maryland). Amplicon sequencing of the 16S rRNA gene was used to characterise the vaginal microbiota, VALENCIA was used to assign community state types (CSTs), and quantitative PCR (qPCR) of 16S rRNA genes was used to estimate bacterial abundance. Cohen's Kappa statistic was used to assess within-participant agreement. Bayesian difference of means models assessed within-participant comparisons between shipped and immediately frozen samples. RESULTS: There were 115 samples available for analysis. Average duration of transit for shipped samples was 8 days (SD: 1.60, range: 6-11). Within-participant comparisons of CSTs between shipped and immediately frozen samples revealed complete concordance (kappa: 1.0) for both preservative solutions. No significant differences comparing shipped and immediately frozen samples were found with taxon-level comparisons or bacterial abundances based on pan-bacterial qPCR. CONCLUSIONS: Short-term room temperature shipping of vaginal swabs placed in stabilising solutions did not affect vaginal microbiota composition. Home collection with mail-in of vaginal samples may be a reasonable approach for research and clinical purposes to assess the vaginal microbiota.
RESUMO
OBJECTIVES: Observational studies demonstrate an association between vaginal douching and bacterial vaginosis (BV) characterised by Gram stain. We sought to describe the effect of a douching cessation intervention on the composition and structure of the vaginal microbiota and molecular-BV, a state defined by low levels of Lactobacillus spp evaluated by molecular tools. METHODS: 33 women self-collected mid-vaginal swabs twice weekly (982 samples) during a douching observation phase (4 weeks) followed by a douching cessation phase (12 weeks) in a 2005 single crossover pilot study conducted in Baltimore, Maryland. Vaginal microbiota were characterised by 16S rRNA gene amplicon sequencing (V3-V4) and clustered into community state types (CSTs). Conditional logistic regression modelling allowed each participant to serve as their own control. Wilcoxon signed-rank tests were used to evaluate changes in microbiota between phases. Broad-range qPCR assays provided estimates of bacterial absolute abundance per swab in a subsample of seven participants before and after douching. A piecewise linear mixed effects model was used to assess rates of change in bacterial absolute abundance before and after douching. RESULTS: There was no statistically significant change in the odds of molecular-BV versus Lactobacillus-dominated CSTs comparing the douching cessation interval to douching observation (adjusted OR 1.77, 95% CI 0.89 to 3.55). Removal of L. iners-dominated CST III from the outcome did not affect the results. There were no significant changes in the relative abundance of four Lactobacillus spp and no meaningful changes in other taxa investigated. There was no significant change in bacterial absolute abundance between a participant's sample collected 3 days prior to and following douching (p=0.46). CONCLUSIONS: In this pilot study, douching cessation was not associated with major changes in vaginal microbiota. Douching cessation alone may not durably shift the vaginal microbiota and additional interventions may be needed to restore optimal vaginal microbiota among those who douche.