Comparison of vaginal microbiota sampling techniques: cytobrush versus swab

ABSTRACTBackground: Persistent infection by oncogenic HPV subtypes is causally associated with cervical cancer. While lifetime risk of acquiring an HPV infection is high, only a fraction of these persist and lead to pre-invasive and invasive cervical disease. The factors determining the outcome are poorly understood, although increasing evidence suggests that the vaginal microbiota (VM) may influence risk of persistent HPV infection and disease progression. Established biobanks of cytology samples, typically collected with a cytobrush, constitute a unique resource to study such associations. It is plausible that compared to rayon swabs; the most commonly used sampling devices, cytobrush sampling may disrupt biofilms leading to variation in VM composition.Results: Cervico-vaginal samples were collected with cytobrush and rayon swabs from 30 women with histology-proven high-grade squamous intra-epithelial lesions (HSIL) during the same visit. Quantitative polymerase chain reaction (qPCR) was used to compare the bacterial load for each sampling technique and was found to be higher in cytobrushes compared to swabs. VM composition was assessed using Illumina MiSeq sequencing of the V1-V3 regions of the 16S rRNA gene. Of a total of 70 taxa, 61 were identified in both swab and cytobrush samples, 8 exclusively in cytobrushes and 1 exclusively in swabs. Relative abundance of bacterial species was highly comparable between sampling devices (R2 = 0.993). However, in women with a Lactobacillus-depleted, high-diversity VM, significantly less correlation in relative species abundance was observed between sampling devices when compared to Lactobacillus species- dominant VM (p=0.0049).Conclusion: Cytobrush and swab sampling provide a comparable overview of the structure of the vaginal microbiota. In the case of a high-diversity vaginal microbiota, a swab did not detect the underlying high-diversity community structure. The ability to collect a higher bacterial load with the cytobrush may reduce the impact of potential contamination during processing low biomass samples. This study highlights the need to consider sampling devices as a potential confounders in high diversity samples, particularly when comparing across multiple studies and datasets. These findings open up the possibility of using biobanked samples, which can also be used for multiple other tests e.g. cytology, HPV DNA testing, thus reducing the need for excessive sampling of women recruited to research studies.