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.

Fig 1. Process for multi-centre consensus method validation.

STAGE 1: 70 well characterised ribotypes disseminated from Netherlands to each laboratory for ribotyping (data (i) held locally for future comparsion/ribotype assignment, (ii) sent to UK laboratory); STAGE 2: 50 anonymised isolates disseminated from Netherlands to each laboratory for ribotype identification (assignments sent to UK laboratory for analysis); STAGE 3: 60 anonymised data files disseminated from UK to each laboratory for ribotype identification (assignments sent to UK for analysis).

Fig 2. PCR-ribotypes with very similar profiles: (a) ribotypes 027 and 081 and (b) ribotypes 015 and 046.

PCR-ribotypes 027 and 081 differ from one another by only a ~20bp difference at position d. Similarly PCR-ribotypes 015 and 046 differ by only a ~20bp difference at position b. Discriminating features between these very similar profiles are indicated (arrows) and associated fragment sizes are highlighted in bold. Relative fragment size was the only parameter used to discriminate between ribotype profiles. Relative peak heights (relative fluorescent units, y-axis) within profiles lacked reproducibility for some ribotypes and therefore this parameter was not used.