BACKGROUND: The two-step glutamate dehydrogenase antigencytotoxicity neutralization assay algorithm has been found to be reliable for the diagnosis of toxigenic Clostridium difficile. However, the high sensitivity of the screening method is compromised by the relative low sensitivity of the second step, the direct cytotoxin neutralization assay (DCNA) using a fecal filtrate. The objective of the present study was to compare the DCNA with an indirect cytotoxin neutralization assay (ICNA).METHODS: For ICNA, the cytotoxin B of C difficile was obtained from a broth culture of the stools and neutralized according to a standard cytotoxin assay using MRC-5 fibroblast cells.RESULTS: A total of 923 stool specimens from adults were tested during a three-month period from June to August 2008. The prevalence of toxigenic C difficile was 13.5%. The sensitivity of the two-step algorithm was 88%. With the ICNA, 12% toxigenic C difficile were detected that were missed by DCNA.CONCLUSIONS: The use of broth for the ICNA is convenient, and results in increased sensitivity of detection of toxigenic C difficile. It can be implemented in routine diagnosis.