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J Clin Microbiol. 2004 Oct; 42(10): 4863–4865.
PMCID: PMC522321

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

We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile isolates were tested for the presence of the toxin genes by PCR. If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive. Eighty-seven of 93 and 79 of 93 C. difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively. No test was able to detect toxin in all samples with true-positive results. Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results. Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C. difficile-associated diarrhea, respectively, if used alone.

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 −70°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 497 (18.7%) of the specimens included in the study. Of these 93 isolates, 20 (21.5%) strains were negative for the toxin A and B genes and were thus nontoxigenic. Triage panels could not be evaluated in 15 (3%) cases because of blackening of the panels. Only one of these samples was truly positive for toxin; the remaining samples were negative for all other markers. By our definition, 52 of the 497 (10.5%) samples were truly positive for toxin. Of these, TL-GDH was positive with all and TR-GDH was positive with 50 samples. Results of TL-GDH and TR-GDH for the detection of C. difficile in specimens and results of C-Tox, Tox A/B, and TR-Tox-A for the detection of true toxin-positive samples are shown in Tables Tables11 and and2,2, respectively. Though none of the assays could detect all samples with a true-positive toxin test, in combination with C-Tox, Tox-A/B and TL-GDH were positive in 52 instances and TR-Tox-A and TR-GDH were positive in 50 instances. All specimens with a false-positive result by TL-GDH or TR-GDH were negative by all other tests. False-positive toxin tests were seen in association with other negative markers or nontoxigenic C. difficile.

TABLE 1.
Detection of GDH by TL-GDH and TR-GDH in specimens positive for C. difficile by culture
TABLE 2.
Detection of CDAD by C-Tox, Tox-A/B, and TR-Tox-A

In hospital populations, colonized patients outnumber patients with CDAD by severalfold (7), and CDAD develops in less than 10% of colonized patients (17). Still, C. difficile is the leading cause of diarrhea in hospitalized patients (10, 12). Furthermore, complications of this infection are associated with substantial morbidity and mortality, and they impose a significant financial burden on health care (17, 18).

Rapid diagnosis and treatment of CDAD is important to check the progression of the disease to pseudomembranous colitis and also to control the spread of the organism to other patients. C. difficile toxin is detected in only 15 to 25% of patients with less-severe antibiotic-associated diarrhea, and in the majority of these patients, the etiology remains unidentified (5). Since there exists no sufficiently discriminatory or specific screening or diagnostic test, the Society for Healthcare Epidemiology of America recommends that, in order to achieve maximal diagnostic sensitivity and specificity, cultures, as well as a cytotoxin assay, should be performed on stool specimens submitted for determination of CDAD (12). This approach is problematic, as both these tests are associated with slow turnaround times and require a minimum of 2 days to yield results.

Detection of toxin B by tissue culture is the most sensitive test for the diagnosis of CDAD, and because of its high sensitivity (94 to 100%) and specificity (99%), this assay is considered the “gold standard” (8, 9, 15). However, the test is costly, is technically demanding, and has slow turnaround-times, so most laboratories use commercial immunoassays to detect toxin A or toxins A and B (4). Immunoassays are less sensitive than cytotoxin assays (15). An advantage of these tests is their rapid turnaround time; results are available within hours. A large number of commercial enzyme-linked immunoassays that employ either monoclonal or polyclonal antibodies to detect C. difficile toxins are available. In a recent review by Brazier, sensitivities and specificities of commercial kits were found to range from 65 to 95 and 75 to 100%, respectively (6). Kits designed to detect toxins A and B generally showed greater sensitivity than those designed to detect toxin A alone (21). More recently, an association between toxin A-negative but toxin B-positive strains and CDAD has been shown (1). In our study, the sensitivities of C-Tox, Tox-A/B, and TR-Tox-A for the detection of CDAD were 96.1, 84.6, and 69.2%, respectively. The specificity of all methods was high (98.2 to 100%). These results are similar to those published previously (6). Unfortunately, no single method was able to detect all samples with a true-positive toxin test result.

GDH tests, like stool cultures for C. difficile, are unable to differentiate between toxigenic and nontoxigenic strains, and they cannot establish a diagnosis of CDAD, because colonization with C. difficile in hospitalized patients is frequent and CDAD is a toxin-mediated disease. A number of previous studies have shown that testing for GDH is a useful screening test (3, 14, 16, 20), and the present study confirms this observation.

This study verifies the findings of previous studies that the best laboratory approach for the diagnosis of CDAD is to test stool specimens for GDH and, if positive, to test the specimens with a toxin detection enzyme immunoassay (3, 16). The GDH-positive but toxin-negative specimens should be further tested with a tissue culture cytotoxin assay. Based on the results of this study, this approach would reduce the number of specimens required for testing for toxin detection considerably.

Acknowledgments

We thank L. Zheng from TechLab Inc. for providing the TL-GDH and Tox-A/B kits.

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