Laboratory diagnosis ofClostridium difficile-associated diarrhoea

Abstract

This paper reviews the various laboratory procedures available for the isolation and identification of Clostridium difficile and the detection of toxins produced by this organism. Laboratories should be selective in determining which patients require investigation for Clostridium difficile-associated diarrhoea. Transport and storage of stool specimens at 4 degrees C is recommended when delays in processing may occur. Tissue culture techniques are still the best method for detection of cytotoxin and a variety of cell lines can be used. Other methods for detecting cytotoxin, and methods for detecting other toxins are not sufficiently developed yet to warrant introduction into diagnostic laboratories. Culture techniques remain the most sensitive for diagnosis, particularly since the development of a variety of enrichment techniques. Cycloserine cefoxitin fructose agar is still adequate, although reduced concentrations of antimicrobial agents are necessary, and improvements, such as the addition of sodium taurocholate, increase the recovery of spores. Enrichment cultures have markedly increased isolation rates for Clostridium difficile but the significance of these isolates needs to be carefully evaluated. Until simpler and more reliable tests are available in clinical laboratories for the detection of toxins, the isolation of Clostridium difficile from patients with diarrhoeal disease should be considered paramount.

Nationwide Surveillance Study of Clostridium difficile in Australian Neonatal Pigs Shows High Prevalence and Heterogeneity of PCR Ribotypes

“…The isolation of C. difficile was based on previously described methods (22), with some modifications. Feces were cultured both directly on C. difficile ChromID agar (CA; bioMérieux, Marcy l'Etoile, France) and in an enrichment broth containing gentamicin, cycloserine, and cefoxitin (GCC).…”

Toxigenic status of Clostridium difficile in a large Spanish teaching hospital

“…The presence of C. difficile toxin B was determined by demonstrating a specific cytopathic effect on MRC-5 cells, as described previously (Chang et al, 1979;Bowman & Riley, 1988;Bartlett, 1994), either directly from fecal samples or, if negative, from pure cultures of the micro-organism.…”

Comparison of alcohol shock enrichment and selective enrichment for the isolation ofClostridium difficile

“…One reason for this has been the use of inappropriate media at other stages of the isolation procedure; for example, many studies have used the original concentrations of cycloserine and cefoxitin in CCFA (500 mg/l and 16 mg/l respectively) although it has been clearly demonstrated that by using half these concentrations the isolation rate for C. difficile may be increased by 30-50% (Levett, 1984;Bowman, 1985). Other workers (Holst, Helin & Mardh, 1981) subcultured their enrichment broths on to non-selective blood agar, which would permit the growth of a number of inhibitory faecal bacteria 357 (Rolfe, Helebian & Finegold, 1981); unless alcohol treatment is used enrichment broths should be subcultured on to CCFA.…”

“…When a selective enrichment broth containing antibiotics is used it is possible to introduce too much inoculum; media containing cefoxitin are particularly susceptible to this 'inoculum effect'. Early studies by Bowman (1985) on a selective enrichment broth showed that an inoculum of one drop (0-02 ml) of fluid stool, or two drops of a 50% suspension, was sufficient; if a larger inoculum was used recovery of C. difficile was markedly reduced.…”