Performance of the TechLab C. DIFF CHEK-60 Enzyme Immunoassay (EIA) in Combination with the C. difficile Tox A/B II EIA Kit, the Triage C. difficile Panel Immunoassay, and a Cytotoxin Assay for Diagnosis of Clostridium difficile -Associated Diarrhea

Abstract

Clostridium difficile-associated diarrhea (CDAD) is the most commonly identified cause of nosocomial diarrhea (10,12,17). The pathogenicity of C. difficile is due to the production of two exotoxins: toxin A, an enterotoxin, and toxin B. Both toxins A and B contribute to human disease (19).TechLab Inc. (Blacksburg, Va.) has recently marketed an enzyme immunoassay, C. DIFF CHEK-60, which detects glutamate dehydrogenase (GDH), a common C. difficile antigen (the assay is hereinafter referred to as TL-GDH), and also markets an enzyme immunoassay, C. difficile Tox A/B II (Tox-A/B), for the detection of toxins A and B. The aim of this study was to evaluate and compare these two assays with another immunoassay, the Triage C. difficile Panel assay (Biosite Diagnostics, San Diego, Calif.), which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); an in-house cytotoxin assay (C-Tox); and stool cultures for C. difficile.All nonformed stool specimens from inpatients suspected of having CDAD were included in the study. Except for stool cultures, all tests were performed within 48 h of the arrival of specimens in the laboratory. Specimens were kept at 4°C if not processed immediately. A portion of stool was stored at Ϫ70°C for subsequent culture. Specimens of insufficient quantity were excluded from all tests.The results of TL-GDH and Tox-A/B were read by using a dual-wavelength spectrometer (450 and 620 nm). TR-GDH, TR-Tox-A, TL-GDH, and Tox-A/B were carried out according to the manufacturer's instruction. Details of the Triage panels and Tox-A/B and C-Tox have been described previously (16).After thawing, fecal samples were planted onto prereduced cycloserine-cefoxitin-fructose agar (cefoxitin, 8 mg/liter; cycloserine, 250 mg/liter; fructose agar; Oxoid, Ottawa, Canada) and incubated for 96 h at 35°C in an anaerobic glove box. Suspected colonies were identified based on their growth on selective media, their colonial morphology, and a positive reaction by the MicroScreen C. difficile latex slide agglutination test (Microgen Bioproducts Ltd., Surrey, United Kingdom) (2, 11).All C. difficile isolates were tested by PCR for the presence of toxin A and B genes by use of primers NK9 and NK11, derived from the repeating portion of toxin A, and NK104 and NK105, derived from the nonrepeating portion of toxin B. These primers have been described by Kato and coworkers (13). The composition of our master mix and the amplification conditions were the same as used by those investigators.For the purpose of this study, a true positive sample was defined as a sample that was positive for toxin A and/or B and from which a toxigenic C. difficile strain (positive by PCR for a toxin gene) was isolated. If a positive toxin detection test result was seen for a sample which was negative for C. difficile by culture or yielded a nontoxigenic strain, the reaction was considered to be falsely positive. Sensitivity, specificity, and positive and negative predictive values for each test were calculated on this basis.C. difficile was isolated from 93 of 49...

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection

“…The Tox A/B II assay demonstrated poor sensitivity (52.4%), near the lower end of the ranges reported by others compared to TC (66 to 88.6%) (6,8,13,20,27) or CCNA (38 to 90.7%) (8,13,20,32). The high specificity of the Tox A/B II assay of 97.5% is congruent with the work of others (95.7 to 100%) (6,8,13,20,27,32).…”

“…They can detect glutamate dehydrogenase (GDH) (so-called common antigen) and/or major toxins A and B and are inexpensive, rapid, and easy to perform. A drawback of EIA toxin tests is a lack of sensitivity (8,9,13,15,16,20,22,27,30,32). Conversely, EIA GDH tests have good sensitivity but lack specificity, as they cannot distinguish toxigenic from nontoxigenic C. difficile (8, 13, 15, 23-25, 30, 32).…”

Yield of Stool Culture with Isolate Toxin Testing versus a Two-Step Algorithm Including Stool Toxin Testing for Detection of Toxigenic Clostridium difficile

“…This highlights the high negative predictive value of the GDH screen relative to culture, which has also been evaluated relative to CCNA (31,34,37). GDH screening is less specific, which may reflect antigenic homology among clostridia, as was suggested by a false-positive rapid stool toxin test result for a patient with Clostridium sordellii bacteremia (13).…”

Evaluation of Repeat Clostridium difficile Enzyme Immunoassay Testing

“…In 2004, Snell et al (22) demonstrated that using a two-step method in the diagnosis of C. difficile-associated diarrhea offered a marked increase in sensitivity compared to that of EIA alone. When using glutamate dehydrogenase (GDH) EIA followed by a toxin detection immunoassay for positive cases, the sensitivity was over 96% and reached 100% when a tissue culture cytotoxin assay was used for GDH-positive, toxin-negative cases (22).…”