"Second-look" cytotoxicity: an evaluation of culture plus cytotoxin assay of Clostridium difficile isolates in the laboratory diagnosis of CDAD

Clostridium difficile is one of the most frequent causes of hospital-acquired diarrhoea. Our objective was to prove that some stool samples with a direct negative cytotoxicity assay may indeed harbour toxigenic C. difficile and that this can be demonstrated by performing a "second-look" cytotoxicity assay using the isolated C. difficile strains. Over an eight-year period (1992-1999), the 8241 stool samples submitted for direct cell culture from patients with suspected C. difficile-associated diarrhoea (CDAD) were simultaneously plated on cycloserine cefoxitin fructose agar. C. difficile strains isolated from samples with a negative direct cell culture assay were re-tested for toxin production "second-look" cell culture assay). Using both methods 6423 samples (78%) were negative. Of the remaining 1818 samples, 127 (7%) yielded C. difficile isolates which were confirmed as non-producers of toxin by both methods, 1437 (85%) were positive in direct cell culture assay, and 254 were positive only after the "second-look" cell culture assay. Thus, our approach allowed us to detect an extra 15% of toxin-producing strains that could have gone undetected otherwise.The combination of direct-cell culture assay, culture for toxigenic C. difficile and "second-look" cell culture assay enhances the potential for diagnosis of CDAD and enables us to be more efficient with our patient care resources.