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Journal of Clinical Microbiology, July 2009, p. 2142-2148, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.02498-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens{triangledown}

Danielle Wroblewski,1 George E. Hannett,1 Dianna J. Bopp,1 Ghinwa K. Dumyati,2 Tanya A. Halse,1 Nellie B. Dumas,1 and Kimberlee A. Musser1*

Wadsworth Center, New York State Department of Health, Albany, New York,1 University of Rochester Medical Center, Rochester, New York2

Received 29 December 2008/ Returned for modification 15 February 2009/ Accepted 20 April 2009

Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.

* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, P.O. Box 22002, Albany, NY 12201-2002. Phone: (518) 474-4177. Fax: (518) 486-7971. E-mail: musser{at}wadsworth.org

{triangledown} Published ahead of print on 29 April 2009.

Journal of Clinical Microbiology, July 2009, p. 2142-2148, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.02498-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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