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Rapid detection of hypervirulent Clostridium difficile strains in a diagnostic microbiological laboratory

Abstract number: P1775

Antikainen J., Tarkka E., Pasanen T., Vaara M., Tissari P.

Objectives: The spread of hypervirulent Clostridium difficile strains, especially PCR ribotype 027, is a diagnostic challenge for microbiological laboratories. For rapid diagnostic purposes, we developed a simple one-tube multiplex PCR approach taking advantage of the virulence-associated markers of the hypervirulent C. difficile ribotype 027. The virulence profile of the C. difficile strains in Finnish patient samples has not been earlier determined; therefore we characterised the polymorphism in the tcdC-gene in toxin-positive C. difficile isolates.

Methods: A multiplex-PCR method for simultaneous amplification of C. difficile toxin genes (tcdA, tcdB), binary toxin genes (cdtA, cdtB) and regulator gene tcdC was developed. With this new method, we analysed consecutive C. difficile cultivation positive faecal samples during four weeks in November 2007. Genetic variation in tcdC gene from a representative set of the samples was analysed by sequencing.

Results: The developed multiplex-PCR method specifically identified the control strains used. From non-027 control strains toxin genes and tcdC gene were amplified. From 027 control strains, besides the toxin genes, also the binary toxin genes were amplified as well as the tcdC gene, where 18-bp deletion was clearly detectable. Of the clinical samples (n = 336), 73 (22%) were binary toxin positive and contained the 18-bp deletion in tcdC gene. TcdC gene sequencing revealed further mutations as described by Curry et al., 2007 (J Clin Microbiol 45:215–221). Two strains contained the 39-bp deletion in tcdC gene representing tcdC sequence type A as described by Spigaglia and Mastrantonio (J Clin Microbiol. 2002. 40:3470–3475). One strain with an 18-bp deletion, with a tcdC sequence corresponding to tcdC type B, and without binary toxin genes was detected. Two strains contained binary toxin genes and larger 54-bp deletion in tcdC, which has not previously been characterised to our knowledge.

Conclusion: With this method, we can easily analyse approx. 80 samples in less than six hours. The method proved specific and sensitive for identification of the C. difficile strains containing virulence genes associated with e.g. ribotype 027. In addition, we have initiated the development of a real-time PCR method and examination of the strains by rep-PCR. The development of rapid and reliable methods for diagnosis of these new hypervirulent strains may help to control epidemics caused by them.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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