Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France

Abstract

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.

FIG. 1.

Representation of the pathogenicity locus of C. difficile and the location of primers used for PCRs A1, A2, and A3 and PCRs with primers NK9-NK11 and NK104-NK105 (hatched areas represent repetitive domains of both toxin genes).

FIG. 2.

PCR amplification of C. difficile variant strains using tcdA primers NK9-NK11. Lanes: 1, strain 98-11522; 2, strain 98-15523; 3, strain 98-15845; 4, negative control; 5, ATCC 43596 (serotype C); 6, ATCC 43598 (serotype F); 7, strain 98-15632; 8, strain 98-16948; 9, strain 98-3050; 10, strain 98-4318; 11, strain 98-7426; 12, strain 60; λ, 100-bp ladder (Biolabs, Saint Quentin en Yvelines, France).