title: Development and validation of an internationally-standardized, high-resolution capillary gel-based electrophoresis PCR-ribotyping protocol for Clostridium difficile.
abstract: PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.
authors: Department of Microbiology, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom.
authors: Warren N
authors: WN
authors: Fawley
authors: Department of Medical Microbiology, Centre of Infectious Diseases, Leiden University Medical Centre, Leiden, Netherlands.
authors: C W
authors: CW
authors: Knetsch
authors: Centers for Disease Control and Prevention (CDC), Atlanta, United States of America.
authors: Duncan R
authors: DR
authors: MacCannell
authors: Department of Medical Microbiology, Centre of Infectious Diseases, Leiden University Medical Centre, Leiden, Netherlands.
authors: Celine
authors: C
authors: Harmanus
authors: Public Health Agency of Canada (PHAC), Winnipeg, Canada.
authors: Tim
authors: T
authors: Du
authors: Public Health Agency of Canada (PHAC), Winnipeg, Canada.
authors: Michael R
authors: MR
authors: Mulvey
authors: Centers for Disease Control and Prevention (CDC), Atlanta, United States of America.
authors: Ashley
authors: A
authors: Paulick
authors: Centers for Disease Control and Prevention (CDC), Atlanta, United States of America.
authors: Lydia
authors: L
authors: Anderson
authors: Department of Medical Microbiology, Centre of Infectious Diseases, Leiden University Medical Centre, Leiden, Netherlands.
authors: E J
authors: EJ
authors: Kuijper
authors: Department of Microbiology, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom; Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom.
authors: Mark H
authors: MH
authors: Wilcox
mesh_terms: D016360:Clostridioides difficile / Q000145:classification* / Q000235:genetics*; D021901:DNA, Intergenic; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012335:RNA, Ribosomal / Q000235:genetics; D015203:Reproducibility of Results; D021521:Ribotyping / Q000379:methods* / Q000592:standards
chemical_list: D021901:DNA, Intergenic; D012335:RNA, Ribosomal