Abstract
Background Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping, and can be used for long-term cytological biobanking. Herein, we investigate the feasibility of leveraging LBC specimens for use in CM surveys by amplicon sequencing. As methodological differences in DNA extraction protocols can potentially bias the composition of microbiota, we set out to determine the performance of four commonly used DNA extraction kits (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens.
Results LBC specimens from 20 patients (stored for 716 ± 105 days) with cervical intraepithelial neoplasia (CIN) 2/3 or suspected CIN2/3 were each aliquoted for the four kits. We observed that, regardless of the extraction protocol used, all kits provided equivalent accessibility to the cervical microbiome, with some minor differences. For example, the ZymoBIOMICS kit appeared to differentially increase access of several microbiota compared to the other kits. Potential kit contaminants were observed as well. Approximately 80% microbial genera were shared among all DNA extraction protocols. The variance of microbial composition per individual was larger than that of the DNA extraction protocol used. We also observed that HPV16 infection was significantly associated with community types that were not dominated by Lactobacillus iners.
Conclusions Collection of LBC specimens is routine for cervical cancer screening and HPV genotyping, and can be used for long-term cytological biobanking. We demonstrated that LBC samples, which had been under prolonged storage prior to DNA extraction, were able to provide a robust assessment of the CM and its relationship to HPV status, regardless of the extraction kit used. Being able to retroactively access the CM from biobanked LBC samples, will allow researchers to better interrogate historical interactions between the CM and its relationship to CIN and HPV. This alone has the potential to bring CM research one-step closer to the clinical practice.
Footnotes
Takeo Shibata: TShibata{at}uams.edu; Mayumi Nakagawa: MNakagawa{at}uams.edu; Hannah N. Coleman: ColemanHannahN{at}uams.edu; Sarah M. Owens: sarah.owens{at}anl.gov; William W. Greenfield: GreenfieldWilliamW{at}uams.edu; Toshiyuki Sasagawa: tsasa{at}kanazawa-med.ac.jp; Michael S. Robeson II: MRobeson{at}uams.edu.
Since our original upload to bioRxiv, we've received helpful comments to increase the clarity of our work. This resulted in the addition of just a few additional bits of text as well as some supplementary content. The overall content and perspectives of the manuscript remains unchanged. Many of the alterations and formatting had to do with meeting other journal requirements.