Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol for Clostridium difficile

Details

• Alternative Title:
  PLoS One
• Personal Author:
  Fawley, Warren N. ; Knetsch, C. W. ; MacCannell, Duncan R. ; Harmanus, Celine ; Du, Tim ; Mulvey, Michael R. ; Paulick, Ashley ; Anderson, Lydia ; Kuijper, E. J. ; Wilcox, Mark H.
• Description:
  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.
• Subjects:
  Clostridioides Difficile DNA, Intergenic Electrophoresis, Capillary Humans Polymerase Chain Reaction Reproducibility Of Results Ribotyping RNA, Ribosomal
• Source:
  PLoS One. 10(2).
• Document Type:
  Journal Article
• Volume:
  10
• Issue:
  2
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:179a9344fd95331de7b88cfc32cb969530987f7ccd764f67a90eb53500a1fc67
• Download URL:
  https://stacks.cdc.gov/view/cdc/28061/cdc_28061_DS1.pdf
• File Type:
  [PDF - 608.90 KB ]