Clostridium difficile (C.diff.) Testing Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol



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-center studies and inter-laboratory variability has not been formally measured or assessed.

Here, we achieved in a multi-center 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 center 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 centers spanning Europe and North America (98.8% after analyzing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centers and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.


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