PLoS ONE (Jan 2015)

Development and validation of an internationally-standardized, high-resolution capillary gel-based electrophoresis PCR-ribotyping protocol for Clostridium difficile.

  • Warren N Fawley,
  • C W Knetsch,
  • Duncan R MacCannell,
  • Celine Harmanus,
  • Tim Du,
  • Michael R Mulvey,
  • Ashley Paulick,
  • Lydia Anderson,
  • E J Kuijper,
  • Mark H Wilcox

DOI
https://doi.org/10.1371/journal.pone.0118150
Journal volume & issue
Vol. 10, no. 2
p. e0118150

Abstract

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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.