Epidemiology, Diagnosis and Treatment of Clostridium Difficile Infection

Matteo Bassetti; Giovanni Villa; Davide Pecori; Alessandra Arzese; Mark Wilcox


Expert Rev Anti Infect Ther. 2012;10(12):1405-1423. 

In This Article

C. difficile Gold-standard Methods: Cytotoxicity Assays & Toxigenic Culture

Upon recognition of C. difficile as the major cause of AAD in the late 1970s[86] the main goal for microbiologists was initially to culture C. difficile. A specific selective medium was developed, allowing recovery of C. difficile in the presence of intestinal flora. Blood-enriched Cycloserine–Cefoxitin–Fructose agar and Taurocholate–Cycloserine–Cefoxitin–Fructose agar were developed and are still used in the laboratory in anaerobic conditions, in order to select C. difficile colonies from enteric microbiota.[87,88] However, since toxigenic and nontoxigenic strains are present both in carrier/asymptomatic subjects and during CDI, it was evident that the laboratory needed to assay directly for toxins. These can be tested by cytotoxicity assay, using a variety of cell lines (human foreskin cell monolayers, McCoy commercially available cell line, in-house cell lines that include Chinese hamster ovary K-1 cells and MRC-5 lung fibroblasts), with antitoxin neutralization. In this assay, fecal filtrates are tested for the ability to cause cell rounding: the specimen is considered positive if this cytotoxic effect can be neutralized by cell cytotoxicity neutralization assay (CCNA). C. difficile toxigenic culture or fecal toxin assay by CCNA are still regarded as the gold standard. The test detects picogram levels of C. difficile toxin (primarily toxin B), making it the most sensitive test available. Each of the adopted cell lines performs satisfactorily when conditions are carefully controlled and the technologist is experienced, but in the hands of an inexperienced technologist, in-house tissue culture assays may be inaccurate. Moreover, CCNA is also the least-controlled test, and nonspecific reactions are common in some laboratories. In experienced laboratories, C. difficile culture is believed to be more sensitive (but less specific) than cytotoxicity assays. It appears to be more reproducible and reliable than the CCNA test: C. difficile internal control reference strains are available, selective media are produced and controlled by several brands and anaerobic growth condition is easy to obtain today thanks to technological innovations. Moreover, by culturing stool for C. difficile, isolates are available for epidemiological studies and antibiotic-sensitivity testing, but not if CCNA is adopted.[85,89,90]

In conclusion, although CCNA is generally considered more sensitive than toxin detection by immunoassays, the turnaround time of up to 3 days, requirement of facilities for cell culture and technical expertise generally limit the application of this test to reference laboratories. C. difficile culture from stool appears easier to apply on a routine basis, although it still takes 48–72 h turnaround time, and it requires a further step in order to recognize the toxigenic potential of isolates (toxigenic culture). As a result, culture confirmation of C. difficile can take almost a week, whereas cytotoxicity assays take a minimum of 24 h for positives and 48 h for negatives. Both approaches are indeed labor intensive: all of these conditions could justify the adoption of toxigenic culture or CCNA as confirmatory tests, particularly for selected troublesome clinical cases or indeterminate results.