Differentiating virulent 027 and non-027 Clostridium difficile strains by molecular methods

Abstract

Objective: Hypervirulent Clostridium difficile clade has been shown to include several lineages of ribotype 027 and also other ribotypes. We present data on additional non-027 strains, identified as presumptive 027 by two commercial molecular C. difficile assays. Methods: The tested clinical isolates were selected from the national reference laboratory collection on the basis of toxin gene profile similarities with ribotype 027 and tested with Xpert^®C. difficile/Epi and Amplidiag™ C. difficile+027 assay. Result: Xpert misclassified five ribotypes (016, 019, 080, 176 and variant of type 046) as presumptive 027 and Amplidiag two ribotypes (016, 176). The misclassified strains were rare, covering 1.6% of reference laboratory strain collection. Conclusion: Our findings confirm the concept that there are closely related outliers to hypervirulent 027 clones that can be misclassified as 027, and that these comprise numerous ribotypes, including previously reported four ribotypes (198, 176, 244, 019), and additional three (016, v046, 080) identified in the present study.

Keywords:

• Clostridium difficile
• hypervirulence
• identification
• PCR
• ribotype
• toxin

Acknowledgements

The Cepheid GeneXpert instrument was kindly provided by Immuno Diagnostics, Finland and Amplidiag C. difficile+027 testing was done in blinded fashion by Mobidiag, Finland.

Financial & competing interests disclosure

S Laakso and J Kirveskari are employees of Mobidiag Ltd., Finland. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues

• Closely related outliers to hypervirulent 027 clones can be misclassified as 027, and these comprise numerous ribotypes, (198, 176, 244, 019, 016, 080, variant 046).

• Many commercial molecular tests can be used without molecular facilities and expertise, though expertise should be available for consultation.

• New C. difficile strains with high-level transmission capacity, antibiotic resistance and extended virulence properties are bound to evolve.

• Individual Paloc variants are rare, but they should not be overlooked since their relative pathogenicity is likely to be high.

• Closely related strains should be distinguished when tracking transmission, but for assessing hypervirulence, all clinically and epidemiologically important strains should be pointed out.

• Proportion of all non-027 strains that present 027-like genetic virulence characteristics (A, B and binary toxins, and a deletion in TcdC) was as high as 14% in the reference laboratory collection.