Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C. difficile, cytotoxin assay, culture, and latex agglutination.

A three-center study was undertaken to compare several test methods for the detection of Clostridium difficile, associated toxin, or related markers by using 927 stool specimens. Methods included direct assay of cytotoxin in stool by tissue culture, C. difficile bacterial culture followed by cytotoxin assay, bacterial culture alone, latex agglutination assay, and the ImmunoCard C. difficile test (Meridian Diagnostics, Inc.). The sensitivities, as determined against direct cytotoxin assay results, of the ImmunoCard C. difficile and latex agglutination assays were 84 and 67%, respectively (92 and 77%, respectively, when adjusted for bacterial culture outcomes). Evaluation for C. difficile-associated disease (CDAD) among 864 patients was based on clinical criteria for antibiotic-associated diarrhea combined with laboratory evidence of toxin or toxin-producing C. difficile in stool specimens. The sensitivity of each test method for screening of CDAD was as follows: bacterial culture, 95%; culture with cytotoxin assay of isolates, 90%; ImmunoCard C. difficile test, 83%; cytotoxin assay 82%; and latex agglutination assay, 67% (P < or = 0.05 versus all other methods). The standard deviations of the test sensitivity statistics between study sites were ranked as follows: cytotoxin assay (+/- 3.1%) < ImmunoCard C. difficile test (+/- 5.7%) < latex agglutination assay (+/- 12.3%) < culture (+/- 24.7%) < culture with cytotoxin assay (+/- 28.0%). The data support the use of the ImmunoCard C. difficile test as an adjunct for the diagnosis of CDAD.     

Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease.

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.     

Commercial latex agglutination test for detection of Clostridium difficile-associated diarrhea.

A commercially available latex agglutination test for Clostridium difficile was compared with a cell culture cytotoxin assay and bacteriological culture for the laboratory diagnosis of C. difficile-associated diarrhea and colitis (CAD). Stool specimens from 626 patients were tested by the three methods, and specimens from 118 patients (19%) were positive by at least one of the methods. The results of the three tests agreed in 88% of the specimens tested, overall, but they agreed in only 34% of the 118 positive specimens. Ninety-three patients were evaluated to assess the significance of positive and negative results for each assay. Of 40 patients found to have CAD, 70% were positive by the cytotoxin assay, 78% were positive by the latex agglutination test, and 90% were culture positive. Of 53 patients who did not have CAD, 2% were positive by the cytotoxin assay, 8% were positive by the latex test, and 4% were culture positive. The detection of CAD was improved by using the tests in combination, and 97% of specimens positive by two or three methods were from patients who had CAD. Testing of multiple specimens from individual patients also increased the sensitivity of detection of CAD. The results suggest that the latex agglutination test may be useful for rapid diagnosis of CAD, especially in laboratories that lack cell culture facilities. However, the accuracy of CAD detection is improved when the latex test is used in combination with culture or the cytotoxin assay.     

Latex agglutination test for detection of Clostridium difficile toxin in stool samples

A total of 163 stool specimens were tested for detection of Clostridium difficile and its toxin by cytotoxicity assay with tissue culture, latex agglutination test, and isolation of the organism. From 33 specimens which were positive for toxin by cytotoxicity, 30 were positive by the latex agglutination test; the organism was isolated from 21. The total number of samples which were positive with the latex agglutination test was 44. The predictive value of a positive latex agglutination result relative to the cytotoxicity test was 68%, and the predictive value of a negative result was 97.5%. The specificity and sensitivity of the latex agglutination test relative to the cytotoxicity assay and the low cost and simple facilities required indicate that the latex agglutination test is a useful procedure for screening for C. difficile toxins, provided that positive latex results are confirmed by cytotoxicity assay.     

An evaluation of a rapid latex test for the diagnosis of Clostridium difficile-associated diarrhea

We present here the results of an evaluation of a rapid latex test for detection of Cl. stridium difficile-associated in comparison with our standard cytotoxin assay and culture for C. difficile. Some 515 diarrheal stools were examined. C. difficile was cultured from 70 specimens (13.5%); 53 specimens (10.2%) were positive with the latex test, and 50 (9.6%) by cytotoxin assay. The latex test did not differ significantly from the cytotoxin assay in sensitivity or specificity compared to culture results. There was also no significant difference in the specificity of the latex test compared to cytotoxin assay in patients in whom the diagnosis of C. difficile-associated diarrhea was negative. Positive and negative predictive values of the latex test for C. difficile-associated diarrhea were similar to those of cytotoxin assay. The latex test thus appears to be a rapid and practical test for the laboratory diagnosis of C. difficile-associated diarrhea. To optimize specificity and sensitivity its use should be restricted to patients where the diagnosis is strongly suspected and a rapid answer is required. As it does not distinguish between toxigenic virulent C. difficile strains and non-toxigenic avirulent strains, it would seem prudent to confirm positive results subsequently by demonstrating in-vivo or in-vitro cytotoxin production.     

Detection of Clostridium difficile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens. Evaluation of a latex agglutination test

A new latex test, Culturette Brand Rapid Latex Test for detection of Clostridium difficile toxin A, was tested on 408 stool samples. In 247 frozen tissue culture supernate specimens previously obtained from patients with C. difficile-associated diarrhea (CAD), the latex test (enterotoxin) was positive in 182 (74%) as compared with 194 (79%) for the repeat tissue culture (P greater than 0.1) cytotoxin (toxin B) test. Testing of 161 fresh stool samples found the latex test superior to tissue culture (P less than 0.05) in cases of CAD (90% positivity vs. 70%), with the two tests being equal in both non-CAD diarrheal and non-diarrheal control groups. In vitro evaluation of 61 C. difficile isolates found all (100%) to be producers of enterotoxin A, while only 53 (87%) produced toxin B. The latex test for C. difficile toxin detection is a rapid, simple test for use in the diagnosis in CAD.     

Comparison of the VIDAS Clostridium difficile toxin A immunoassay with C. difficile culture and cytotoxin and latex tests.

The VIDAS Clostridium difficile toxin A immunoassay (CDA) is a new, automated, enzyme-linked fluorescent-antibody assay for detection of C. difficile toxin A antigen in stool specimens. Simultaneous, parallel testing was performed by using the VIDAS CDA, the Culturette brand CDT latex test for C. difficile antigens, and conventional laboratory cell culture tests for C. difficile, cytotoxicity and C. difficile culture. One hundred ninety-four consecutive fresh soft or liquid stool samples submitted for C. difficile testing between July and September 1990 were evaluated. Of the 194 samples tested, 19 (10%) were from 16 patients who met our case definition for C. difficile-associated disease. The in vitro tests were evaluated in relation to two forms of a clinical case definition. In one form, a positive culture for toxin-producing C. difficile or a positive cytotoxin result obtained directly from the stool specimen was required as laboratory evidence of C. difficile. In the other, a positive result of any of the four laboratory tests was accepted for the laboratory portion of the case definition. No significant difference between the sensitivity of the VIDAS CDA and that of the Culturette brand CDT latex test was found (48 to 58% sensitivity for the CDT latex test and 52 to 63% sensitivity for the VIDAS CDA compared with 93 to 100% sensitivity for culture and 70 to 100% sensitivity for cytotoxin testing). The performance of the VIDAS CDA, however, was hampered by a high percentage of tests (19%) which gave an uninterpretable result.     

Evaluation of the latex agglutination test for detection of Clostridium difficile.

We compared two Clostridium difficile latex agglutination tests, Meritec from Meridian Diagnostic (Cincinnati, Ohio) and CDT from Becton-Dickinson (Cockeysville, Md), on 289 specimens submitted for tissue culture cytotoxicity using MRC-5 cells. When compared with CDT, the Meritec latex agglutination test had a sensitivity of 90% (26/29), a specificity of 97% (251/260), and a correlation of 96%. Meritec was compared with tissue culture cytotoxicity on 357 specimens. Meritec had a sensitivity of 77% (30/39), a specificity of 93% (298/318), and a correlation of 92%. Clinical review of 10 Meritec +/- tissue culture cytotoxicity minus patients revealed one likely, two probable, and seven doubtful cases of C difficile disease. In contrast, review of 10 Meritec +/- tissue culture cytotoxicity plus patients showed seven likely and three probable cases of C difficile disease. The Meritec is comparable with the CDT latex agglutination test, but is not nearly as sensitive as either tissue culture assay or culture for detection of C difficile disease. A positive latex agglutination test should be confirmed by a tissue culture cytotoxicity assay.     

Evaluation of a latex agglutination test for Clostridium difficile in two nursing home outbreaks.

The Culturette Brand Clostridium difficile test (CDT; Marion Laboratories, Inc., Kansas City, Mo.) is a latex agglutination test for C. difficile. The recent controversy involving the identity of antigens detected by CDT has made decisions on its use difficult. We compared the test results with those of selective culture and stool cytotoxin assays in investigations of two nursing home outbreaks of C. difficile-associated disease in order to formulate usage recommendations. Selective culture for C. difficile identified 27 (19%) of 142 subjects as carriers. CDT and the stool cytotoxin assay identified only 52 and 48% of these carriers, respectively. Compared with the stool cytotoxin assay, CDT had a high sensitivity (92%) and specificity (89%) for the detection of C. difficile disease, but the positive predictive value of the test was only 17% when the prevalence of disease was 2%. We conclude that the CDT should not be used to identify carriers but that it is a sufficiently sensitive and specific screening test for diagnosing C. difficile disease. However, since the positive predictive value of the CDT is low when the prevalence of disease is low, positive test results should be confirmed by the stool cytotoxin assay.     

Analysis of latex agglutination test for Clostridium difficile toxin A (D-1) and differentiation between C difficile toxins A and B and latex reactive protein.

Virulent toxigenic and avirulent non-toxigenic strains of Clostridium difficile gave a positive result in the latex agglutination test (LAT) for C difficile toxin A (D-1). Similar concentrations of latex agglutinating antigen were produced by these strains in vivo. Positive reactions were also given by C sporogenes, proteolytic C botulinum Types A, B, and A/F, and Bacteroides assaccharolyticus. The latex agglutinating antigen was denatured by boiling for 10 minutes, but not by heating at 56 degrees C for 30 minutes. The reaction was abolished by incubation of test material with crude C difficile antitoxin but not with other clostridial antitoxins or specific antitoxin to C difficile toxin A. The latex agglutinating antigen present in C difficile eluted between 0.39% and 0.47% M sodium chloride, and that produced by the other clostridia, between 0.35% and 0.43% M sodium chloride by fast protein liquid chromatography. The latex agglutinating antigen of C difficile was neither cytotoxic nor mouse lethal and was distinct from toxin A and toxin B. In the analysis of faecal specimens from patients with diarrhoea the latex agglutination test correlated better with the presence of C difficile than with toxin B and detected both toxigenic and non-toxigenic strains. The latex agglutination test should only be used in the laboratory as an alternative to culture for C difficile and not as a method for the detection of C difficile toxins.     

Comparison of a dot immunobinding assay, latex agglutination, and cytotoxin assay for laboratory diagnosis of Clostridium difficile-associated diarrhea.

C. diff-CUBE, a dot immunobinding assay (DIA) (Difco Laboratories, Ann Arbor, Mich.) for detection of Clostridium difficile toxin A in stool specimens, was compared with latex agglutination (LA) (Marion Laboratories, Kansas City, Mo.) and cytotoxin assay (CTA) for the laboratory diagnosis of C. difficile-associated diarrhea. A total of 200 stool specimens collected from 169 patients with suspected C. difficile diarrhea were tested. Of the 198 specimens evaluated by all three methods, 36 (18%) from 36 patients were positive by one or more of the tests. Twenty-five, 26, and 23 specimens were positive by CTA, DIA, and LA, respectively; 14 were positive by all three methods. Eight specimens yielded nonspecific LA test results; all eight were negative by CTA, and one was positive by DIA. DIA results agreed with CTA results in 183 (92%) cases and with LA results in 175 (88%) cases. CTA and LA results agreed in 179 (90%) cases. Freezing of the specimen did not appear to adversely affect either the DIA or LA test. These preliminary results suggest that C. diff-CUBE may be useful as a rapid screen for the diagnosis of C. difficile-associated diarrhea. However, for optimum laboratory diagnosis, further testing of all stools that are negative by DIA is warranted.     

Evaluation of methods for detection of toxins in specimens of feces submitted for diagnosis of Clostridium difficile-associated diarrhea

Clostridium difficile is the principal pathogen associated with hospital-acquired acute diarrheal disease. We have evaluated the performances of six approaches for diagnosis of C. difficile-associated diarrhea (CDAD). Consecutive stool specimens (n = 200) from 133 patients were examined by cytotoxin assay, by culture of C. difficile on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test). A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens). The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture. The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.     

Comparison of two commercially available enzyme immunoassays for detection of Clostridium difficile in stool specimens.

Clostridium difficile is the cause of most cases of pseudomembranous colitis, the most severe form of antibiotic-associated diarrhea. Rapid diagnosis guides both the treatment and the control of nosocomial spread of infection. Two enzyme immunoassay (EIA) kits developed for the rapid detection of C. difficile toxin A in fecal specimens, Premier (Meridian Diagnostics, Cincinnati, Ohio) and Tox-A test (TechLab, Virginia Polytechnic Institute Research Park, Blacksburg), were evaluated by using 410 fecal specimens. Seventy-six specimens were positive for C. difficile toxin B by the cytotoxin assay (prevalence rate, 19%). The Meridian EIA was positive for 71 of the 76 samples, yielding a sensitivity of 93%. The TechLab EIA detected 75 of the 76 positive samples, yielding a sensitivity of 99%. The Meridian and TechLab EIAs had specificities of 100 and 93%, respectively. These data indicate that both EIAs are suitable alternatives to the cytotoxin assay in routine diagnostic laboratories. However, confirmation of TechLab EIA-positive test results by the cytotoxin assay remains necessary.     

Stool caproic acid for screening of Clostridium difficile

Clostridium difficile is the prime etiologic agent in the production of pseudomembranous colitis by its powerful cytotoxin. The most common test for the toxin is a tissue culture method with neutralization of cytopathic effect by a C. difficile antiserum. This method is expensive and requires a minimum of 72 hours before results can be obtained. Attempts to create a rapid method, counterimmunoelectrophoresis, enzyme-linked immunosorbent, latex agglutination, and fluorescent antibody test are fraught with many problems. This report describes a rapid method for the identification of C. difficile, using gas-liquid chromatography (GLC) for the demonstration of caproic acid, a product of the organisms fatty acid metabolism.     

Rapid diagnosis of Clostridium difficile-associated diarrhoea using a latex agglutination test

A rapid latex agglutination test, Culturette Brand CDT from Marion Laboratories, was evaluated and compared to a tissue culture assay (TCA) and isolation of Clostridium difficile in 380 faecal specimens from 226 patients with clinically suspected Clostridium difficile-associated diarrhoea. The sensitivity and specificity of the latex test compared with the TCA were 83% and 80% respectively, and the positive and negative predictive values were 55% and 94% respectively. In patients with repeated sampling the sensitivity increased to 95%. The latex test may be useful as a screening test for negative specimens in laboratories where TCA is not available, but positive specimens have to be confirmed by TCA.     

Evaluation of four methods for rapid detection of adenovirus

Four methods for rapid detection of adenovirus were evaluated by testing retrospectively 28 frozen clinical specimens from which an adenovirus strain had been isolated. After thawing all specimens were retested for the presence of adenovirus by conventional culture on KB cells and found to be positive. The four tests used for rapid detection of adenovirus were a 48-hour culture technique, and an immunoassay, a latex agglutination test and an immunofluorescence assay for direct detection of viral antigen using commercially available reagents. Of the 28 specimens all were positive in the 48-hour culture, 25 (89 %) positive in the immunoassay and 10 (36 %) positive in the latex agglutination test. Six of eight nasopharyngeal aspirate specimens were positive in the immunofluorescence assay. Twenty-five clinical specimens negative for adenovirus on conventional culture were also negative in the 48-hour culture technique. Overall, the rapid (48-hour) culture technique was 100 % sensitive and 100 % specific compared to conventional culture. The direct detection of viral antigen by immunoassay was less sensitive, however results were available within a few hours. Prospective comparative studies are warranted to determine whether these rapid techniques could replace conventional culture in the routine diagnosis of adenovirus infection.     

Evaluation of the immunoenzyme test (Elisa) in detecting Clostiridium difficile toxin A in fecal samples]

Currently, the method of choice in diagnosis of Clostridium difficile-associated intestinal diseases is the detection of toxin B in fecal specimens. This method is long (72 h) and can be realized in laboratories which have tissue culture facilities. Commercial agglutination test have been evaluated but they lack in specificity. An immunoenzymatic test has been recently commercialized for detection of toxin A. We have compared the results of this assay on 275 fecal specimens from patients suspected of having Clostridium difficile-associated intestinal diseases with the results obtained with the cytotoxicity test and the culture. Of the 275 fecal specimens, 58 were positive in cytotoxicity and 53 in Elisa. The overall sensitivity and specificity of the Elisa compared with cytotoxicity were 89.5% and 99.0% respectively. The immunoenzymatic test detecting Clostridium difficile toxin A is an easy test to perform in 2 h 15; it displays a good correlation with detection of toxin B and can be very useful in daily laboratory diagnosis.     

Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens.

Rapid (2.5- to 3.5-h) enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxins have been developed. We report the results of simultaneous testing of 700 fresh stool specimens by the tissue culture cytotoxin assay and four EIAs (Bartels Prima System C. difficile Toxin A EIA, Cambridge Biotech Cytoclone A+B EIA, Meridian Diagnostics Premier C. difficile Toxin A EIA, and TechLab C. difficile Tox-A Test EIA). In cases of disagreement, culturing for toxigenic C. difficile was performed. A total of 61 (8.7%) specimens from 46 patients were positive for C. difficile toxin. The sensitivity of the cytotoxin assay was 87%, and that of culture was 93%. In comparison with the cytotoxin assay results, the sensitivity and specificity of the EIAs were as follows: Bartels, 87 and 96%; Cambridge, 89 and 99%; Meridian, 87 and 98%; and TechLab, 87 and 95%, respectively. In comparison with the cytotoxin assay plus toxigenic culture results, the sensitivity and specificity of the EIAs were as follows: Bartels, 84 and 97%; Cambridge, 85 and 99%; Meridian, 79 and 98%; and TechLab, 80 and 96%, respectively. The EIAs varied in positive predictive values (PPVs). A high PPV was seen with the Cambridge EIA (96%); lower PPVs were seen with the TechLab (64%), Bartels (72%), and Meridian (80%) EIAs because of high false-positive rates. The negative predictive values (98 to 99%) were excellent with all EIAs. Results were indeterminant with 0.3% of the samples by the Meridian EIA and 3% by all the other EIAs. Although the EIAs were less sensitive than the cytotoxin assay, they provide same-day results and may be useful in laboratories without tissue culture facilities.     

Commercial latex test for Clostridium difficile toxin A does not detect toxin A.

The rapid latex test recently marketed by Marion Scientific (Div. Marion Laboratories, Inc., Kansas City, Mo.) for the detection of Clostridium difficile toxin A does not react with the toxin, based on the following findings: culture filtrates from nontoxigenic strains of C. difficile gave positive reactions in the test, culture filtrate in which toxin A had been removed gave positive reactions, purified toxin A did not react in the test, and the latex reagent bound an antigen which is distinct from toxin A and which is produced in various amounts by both toxigenic and nontoxigenic strains of C. difficile.     

Laboratory diagnosis of Clostridium difficile disease

The laboratory diagnosis of Clostridium difficile -associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.