'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-acquir ed diarrhoea. Our objective was to prove that some stool samples with a dir ect negative cytotoxicity assay mag; indeed harbour toxigenic C. difficile and that this can be demonstrated by performing a 'second-look' cytotoxicit y 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 d iarrhoea (CDAD) were simultaneously plated on cycloserine cefoxitin fructos e agar. C. difficile strains isolated from samples with a negative direct c ell culture assay were re-tested for toxin production ('second-look' cell c ulture assay). Using both methods 6423 samples (78%) were negative. Of the remaining 1818 samples, 127 (7%) yielded C. difficile isolates which were c onfirmed as non-producers of toxin by both methods, 1437 (85%) were positiv e in direct cell culture assay; and 254 were positive only after the 'secon d-look' cell culture assay: Thus, our approach allowed us to detect an extr a 15% of toxin-producing strains that could have gone undetected otherwise. The combination of direct-cell culture assay culture for toxigenic C. diffi cile and 'second-look' cell culture assay enhances the potential for diagno sis of CDAD and enables us to be more efficient with our patient care resou rces. (C) 2001 The Hospital infection Society.