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Previous Methodologies: Culture and Cell Cytotoxicity Neutralization Assay (CCNA)

Bacterial culture, utilizing selective/differential media, is an effective method for recovering Clostridium difficile. Its drawbacks are the length of time required (up to four days), as well as the inability to distinguish toxigenic strains from non-toxigenic strains. Positive cultures require follow-up testing for the ability to produce toxin.

Cell Cytotoxicity Neutralization Assay (CCNA)
This assay detects the presence of C. difficile toxin in fecal samples. A filtrate of stool sample is prepared and inoculated onto sensitive tissue culture cells. Typically human fibroblast cells are utilized; if toxin is present in the filtrate, it causes the fibroblasts to round up in a characteristic cytopathic effect.

To verify that the cytopathic effect is caused by C. difficile toxin (and not some other toxic component or viral agent), the filtrate is also inoculated in parallel onto a second set of tissue culture cells, to which C. difficile specific anti-toxin has been added. Absence of cytopathic effect in the second set of cell cultures provides evidence that the cellular changes in the first set were caused by C. difficile toxin.

Although CCNA is considered a gold standard for the detection of C. difficile toxin, it is labor intensive, requires the use of cell cultures, and requires at least 48 hours of incubation.