Culture
Using selective/differential media, bacterial culture is an effective method for recovering Clostridioides difficile. Its drawbacks are the length of time required (up to four days) and the inability to distinguish toxigenic strains from non-toxigenic strains. Positive cultures require follow-up testing for the ability to produce toxins.
Cell Cytotoxicity Neutralization Assay (CCNA)
This assay detects the presence of C. difficile toxin in fecal samples. A stool sample filtrate is prepared and inoculated onto sensitive tissue culture cells. Typically, human fibroblast cells are utilized; if a 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 a C. difficile-specific anti-toxin has been added. The absence of a 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 the gold standard for detecting C. difficile toxin, it is labor intensive, requires cell cultures, and requires at least 48 hours of incubation.
