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Subject: Clostridium Difficile Detection
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Clostridium difficile is a major cause of antibiotic-associated diarrhea and pseudomembranous colitis, and it represents a significant and serious agent of nosocomial infection. C. difficile infection (CDI) may be mild and self-limited after discontinuation of antibiotics, but a significant number of patients have persistent and/or severe diarrheal illness that may progress to pseudomembranous colitis or toxic megacolon. C. difficile is an anaerobic, spore-forming, gram-positive bacillus. It forms several toxins (toxins A and B) that have been used as the basis for detection. C. difficile detection is recommended for patients in whom diarrheal illness develops after antibiotic therapy or during hospitalization, especially when colitis (increased fecal leukocytes) is a prominent feature.
Cytotoxicity: In this method, a suspension of stool, passed through a membrane filter to remove bacteria, is inoculated on cultured eukaryotic cells (e.g., WI-38). The cell monolayer is examined for 48 hours for evidence of typical “actinomorphic” cytotoxicity (disruption of the normal cellular morphology in the monolayer). In order to exclude nonspecific cytotoxicity that may be seen with stool filtrates, neutralization of the cytotoxic effect, using anti–C. difficile antiserum, must be demonstrated.
Toxigenic culture: C. difficile may be isolated from stool using a spore selection technique (by heat shock or alcohol pretreatment of a stool suspension prior to medium inoculation) and selective media. Because not all C. difficile isolates produce the toxins associated with disease, culture supernatants must be tested for toxin production to make a diagnosis of C. difficile–associated disease.
Toxin detection using immunodiagnostic procedures: A number of commercially available latex agglutination or EIA kits have been developed for the detection of C. difficile toxin A and/or toxin B in stool samples. These assays provide a more rapid turnaround time, but lower sensitivity and specificity, compared to culture or cytotoxicity assays.
C. difficile glutamate dehydrogenase antigen detection: Detection of C. difficile glutamate dehydrogenase antigen may be used to screen stool for the presence of C. difficile. Because glutamate dehydrogenase is not specific for toxigenic C. difficile, positive specimens must be tested for toxin to confirm the diagnosis of C. difficile–associated disease.
Molecular diagnostic methods: Molecular diagnostic methods for detection of the C. difficile are commercially and provide the most sensitive assay for CDI. The test is very specific when used on patients with typical clinical signs and symptoms of C. difficile disease.
Molecular diagnostic, immunodiagnostic assays, and the glutamate dehydrogenase assays: 24 hours
Cytotoxicity assays: 24–72 hours
Culture: 96 hours
Liquid stool specimens collected in clean containers with tight-fitting lids should be transported to the laboratory at room temperature within 2 hours. If transport will be prolonged, the specimen should be held at refrigerator temperature. Do not freeze.
Expected results: Negative
The available assays vary somewhat in sensitivity and specificity for diagnosis of C. difficile disease. The choice of diagnostic methods must take cost, assay performance, turnaround time, and other factors into consideration. Positive C. difficile test results must be interpreted with caution in infants; toxin may be detected in the stool of healthy infants without signs of diarrheal illness or colitis.