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Clostridium difficile Infections: Diagnosis, Treatment, and Prevention

Slide: 17 of 33

Assays Commonly Used in CDI Diagnostics

Cytotoxicity assay. The cultured cell cytotoxicity assay often has been used as a reference test for evaluating new diagnostic tests for toxigenic C. difficile. Briefly, a diluted and filtered aliquot of a stool sample is mixed with cultured test cells. The test cells are examined for toxin effects (cell rounding) that are not seen in comparator test cells where an excess amount of antitoxin is present. A cytotoxicity assay requires up to 48 hours for the toxin effects to appear. Cytotoxicity testing is not a perfectly accurate gold standard. Methodological differences in the time to process and dilution of stool samples, the age and type of cultured test cells being used for the test, the antitoxins, and the interpretation of results all can cause cytotoxicity assay results to vary.
Culturing C. difficile organisms. Culturing C. difficile by anaerobic incubation of fecal aliquots on selective cycloserine-cefoxitin, fructose agar, or other media can be more sensitive than the cytotoxicity assay for detecting the presence of C. difficile organisms. However, C. difficile culture techniques also are not standardized, are susceptible to methodological variation, and require expertise, equipment, and several days to complete. Cultured C. difficile organisms also have to test positive for disease-causing toxins. Therefore, culture of C. difficile from stool samples followed by a toxin-detection assay is considered the most sensitive method for detecting toxigenic C. difficile; however, the time it takes to complete these tests are not practical for clinical decisionmaking.
Immunoassays for toxins. A variety of faster (within a few hours), less costly commercial immunoassays for C. difficile toxins are available.  Initially, most immunoassays detected only toxin A. More recently it was discovered that a small but increasing number of clinically significant C. difficile strains produced only toxin B. When the performance of a diagnostic test depends on the level of toxins in test specimens and most organisms produce both toxins A and B, immunoassays that detect both toxins might be more sensitive if other critical factors such as dilution of the specimens are equal. Therefore, experts have recommended using immunoassays that can detect both toxins A and B. A highly sensitive and specific immunoassay for these toxins may be used as a second test after the stool culture. Data from the College of American Pathology proficiency testing program for C. difficile toxin detection indicated that 90 percent of labs used an immunoassay for toxins A and B in June 2009. The most commonly used tests were the Immunocard and Premier A & B test kits manufactured by Meridian, the TechLab Tox AB II and Toxin A/B QUIK CHEK kits, and the Remel ProSpecT and Xpect Toxin A/B tests.
Toxin gene detection tests. Three tests of stool specimens for the presence of genes involved in the production of C. difficile toxins have recently become commercially available. These tests use the polymerase chain reaction to amplify targeted gene fragments to detect the presence of a gene or genes involved in the production of toxins, not the actual toxins. The target of the assays can be the genes that produce toxin B and a gene C that negatively regulates the production of toxins A and B. A mutation in gene C has been detected in an increasingly common hypervirulent strain of C. difficile that produces large amounts of toxins A and B. One concern about using the tests based on amplification of toxin gene fragments is that very small, clinically unimportant genetic residue or specimen contamination may be detected. Clinically speaking these would be false positives that would reduce test specificity. Therefore, some experts have recommended using this type of test only when a patient has clinical signs and symptoms suggestive of CDI.