Evaluation of a new commercialClostridium difficile toxin A enzyme immunoassay using diarrhoeal stools

Delmée, Michel; Mackey, Tami-Lea A.; Hamitou, A

doi:10.1007/bf02098089

Cited by 30 publications

(13 citation statements)

References 12 publications

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“…Current methods for diagnosing C. difficile infection are based on detection of the organism or the toxin genes and proteins or on the effect of the cytotoxin on tissue culture cells (2,(10)(11)(12). The only method that can provide information about the activities of the toxins is the cell cytotoxicity assay.…”

Section: Discussionmentioning

confidence: 99%

Harnessing the Glucosyltransferase Activities of Clostridium difficile for Functional Studies of Toxins A and B

Darkoh¹,

Kaplan²,

DuPont

2011

J Clin Microbiol

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The incidence of Clostridium difficile infection (CDI) has been increasing within the last decade. Pathogenic strains of C. difficile produce toxin A and/or toxin B, which are important virulence factors in the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect either the bacterium or the toxins. We have developed an assay (Cdifftox activity assay) to detect C. difficile toxin A and B activities that is quantitative and cost-efficient and utilizes a substrate that is stereochemically similar to the native substrate of the toxins (UDP-glucose). To characterize toxin activity, toxins A and B were purified from culture supernatants by ammonium sulfate precipitation and chromatography through DEAE-Sepharose and gel filtration columns. The activities of the final fractions were quantitated using the Cdifftox activity assay and compared to the results of a toxin A-and B-specific enzyme-linked immunosorbent assay (ELISA). The affinity for the substrate was >4-fold higher for toxin B than for toxin A. Moreover, the rate of cleavage of the substrate was 4.3-fold higher for toxin B than for toxin A. The optimum temperature for both toxins ranged from 35 to 40°C at pH 8. Culture supernatants from clinical isolates obtained from the stools of patients suspected to be suffering from CDI were tested using the Cdifftox activity assay, and the results were compared to those of ELISA and PCR amplification of the toxin genes. Our results demonstrate that this new assay is comparable to the current commercial ELISA for detecting the toxins in the samples tested and has the added advantage of quantitating toxin activity.

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Section: Discussionmentioning

confidence: 99%

Harnessing the Glucosyltransferase Activities of Clostridium difficile for Functional Studies of Toxins A and B

Darkoh¹,

Kaplan²,

DuPont

2011

J Clin Microbiol

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“…Numerous studies have investigated techniques for the rapid detection of C. difficile toxins (40,46,62,69,70,77,91,103,142). An enzyme-linked immunosorbent assay for the presence of toxins A and B was used to examine more than 90 isolates of C. difficile for toxin production.…”

Section: Enzyme-linked Assay Systemsmentioning

confidence: 99%

Clostridium difficile: clinical disease and diagnosis

1993

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Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions.

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“…Laboratory diagnosis relies on culture of the microorganism and/or detection of the toxins in stool specimens [8]. Many immunoenzymatic assays that detect either toxin A or toxins A+B have become commercially available in the 5 last years [9–18]. They represent good alternatives to cytotoxicity assays for laboratories without cell‐culture facilities.…”

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confidence: 99%

Rapid detection of Clostridium difficile toxin A in stool specimens

Barbut

Macé

Lalande

et al. 1997

Clinical Microbiology and Infection

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OBJECTIVE: To evaluate a rapid (15-min) enzyme immunoassay in the format of an individual cassette (ImmunoCard toxin A, Meridian, BMD, Marne-la-Vallée, France) for the detection of Clostridium difficile toxin A in stool specimens. METHODS: We compared this new test with the cytotoxicity assay using MRC-5 cells, the ToxA test (TechLab, BioWhittaker, Fontenay-sous-bois, France) and toxigenic culture for the diagnosis of C. difficile-associated diseases (CDAD). A total of 236 stool specimens collected from 220 patients was simultaneously tested with the four methods. Discordant results were resolved by reviewing patients' clinical records. RESULTS: The prevalence of CDAD was 13.9%. Test sensitivities and specificities were 100% and 99% respectively for the cytotoxicity assay, 87.5% and 100% for ImmunoCard toxin A, 77.4% and 100% for the ToxA test and 100% and 98% for toxigenic culture. CONCLUSIONS: The ImmunoCard Toxin A is a very rapid, individual and easy-to-perform test for the diagnosis of CDAD. It provides same-day results and may be useful for both guiding appropriate treatment and controlling nosocomial spread of C. difficile.

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Evaluation of a new commercialClostridium difficile toxin A enzyme immunoassay using diarrhoeal stools

Cited by 30 publications

References 12 publications

Harnessing the Glucosyltransferase Activities of Clostridium difficile for Functional Studies of Toxins A and B

Harnessing the Glucosyltransferase Activities of Clostridium difficile for Functional Studies of Toxins A and B

Clostridium difficile: clinical disease and diagnosis

Rapid detection of Clostridium difficile toxin A in stool specimens

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