Isolation of C. difficile from stool samples is not diagnostic of CDAD but is important for the epidemiological typing of strains. Isolation is best performed after alcohol shock treatment. Stool samples are treated with an equal volume of methylated spirit or absolute alcohol, homogenised using a vortex mixer and left at room temperature for 1 hour, then inoculated on to Clostridium difficile selective agars e.g. CDSA with 7% horse blood; Cycloserine, Cefoxitin, egg yolk agar (CCEY), and incubated anaerobically for 40 - 48hrs.

Following primary selective culture, rapid latex agglutination tests allow identification of presumptive colonies of C. difficile within minutes. The latex particles are coated with antibodies specific for C. difficile somatic antigen. These screening tests can be used to reduce the number of samples that need to be progressed onto a toxin assay. If required confirmation may be done by biochemical identification tests.

For diagnosis of CDAD it is necessary to demonstrate the presence of C. difficile toxins. Toxin B may be detected by the production of a cytopathic effect (CPE) of faecal extracts on Vero cell cultures that is neutralised by C. sordellii antitoxin. It takes between 24 and 48 hours for a final result to be obtained. A CPE that is not neutralised by C. sordellii antitoxin may indicate that another pathogen is present in the faeces e.g. C. perfringens or Vero-toxic E. coli. Many labs use locally developed assays but commercial cytotoxity kits are available..

Direct testing of stool sample extracts for toxin presence using a variety of immunoassay techniques has largely replaced the culture method. The toxin is unstable at room temperature so the sample needs to be processed quickly or held refrigerated. Both toxins of C. difficile may be detected by these tests and there are a range of commercially available kits in different formats. As strains which only produce either toxin A or B have been reported as pathogenic, methods which detect both are preferable. For the high volume laboratory, microtitre plate or automated methods are available.

Assays can also be presented in a lateral flow format as single tests which give faster results, normally within a few minutes and with less operator skill requirements. Some assays are designed to detect C. difficile common antigen (glutamate dehydrogenase or GDH) produced by both toxigenic and non-toxigenic strains, and can also have a toxin test included in the panel so both toxin and organism may be revealed.

Studies using molecular detection of toxin A and B genes have been performed but they remain technically more demanding and hence more expensive and do not confirm expression of toxin. They do however allow identification of hypervirulent strains of C. difficile e.g. PCR ribotype 027 [or NAP1 in North Americas].