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Journal of Clinical Microbiology, June 2002, p. 2079-2083, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2079-2083.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Prevalence and Genetic Characterization of Toxin A Variant Strains of Clostridium difficile among Adults and Children with Diarrhea in France

Frédéric Barbut,* Valérie Lalande, Béatrice Burghoffer, Huong Vu Thien, Emmanuel Grimprel, and Jean-Claude Petit

Research Group on Clostridium difficile, Centre Hospitalo-Universitaire Saint-Antoine, Assistance Publique-Hôpitaux de Paris, Université Pierre et Marie Curie, Paris, France

Received 3 December 2001/ Returned for modification 2 March 2002/ Accepted 27 March 2002

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of Clostridium difficile have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (tcdA) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with C. difficile-associated diarrhea in France. The repeating segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol. 36:2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire tcdA gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11, C. difficile variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other tcdA variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of C. difficile-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion of C. difficile-associated diarrhea but a negative immunoassay for toxin A.

* Corresponding author. Mailing address: Unité d'Hygiène et de Lutte contre les Infections Nosocomiales (UHLIN), Hôpital Saint-Antoine, 184, rue du faubourg Saint-Antoine, 75571 Paris cedex 12, France. Phone: 33 1 49 28 30 08. Fax: 33 1 49 28 30 09. E-mail: frederic.barbut{at}sat.ap-hop-paris.fr.

Journal of Clinical Microbiology, June 2002, p. 2079-2083, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2079-2083.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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