Detection Of Clostridium Difficile Toxin Research Articles

Evaluation of two rapid assays for detection of Clostridium difficile toxin A in stool specimens.

Rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment. We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B. A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays. With the "gold standard" of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods. Total agreement for all assays was 90.5% (592 of 654). The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA. While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).

The Frequency of Detection ofClostridium Difficile Toxins A and B in Symptomatic Pediatric Patients.

The Frequency of Detection ofClostridium Difficile Toxins A and B in Symptomatic Pediatric Patients.

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A.

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false...

Rapid detection of Clostridium difficile toxin A in stool specimens

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.

Evaluation of two commercial microtiter cytotoxin assays for the detection of Clostridium difficile toxin B in stool specimens

Two commercial microtiter cytotoxin assays using a fibroblast cell line (Bartels, Baxter Diagnostics, Inc., Deerfield, IL) and an epithelial cell line (Cytotoxi Test, Advanced Clinical Diagnostics, Toledo, OH) were evaluated for their ability to detect Clostridium difficile toxin B in stool specimens. After 48 hours, the assays had comparable sensitivity (90 versus 92%) and specificity (99 versus 98%). Although not statistically significant, the Bartels assay detected more toxin-positive specimens at 24 hours (84 versus 72%, P = 0.089, Fisher's Exact Test) and the Cytotoxi assay had fewer nonspecific reactions requiring repeat testing (2.2 versus 1.1%, P = 0.186, Fisher's Exact Test). Both cytotoxin assays had comparable analytic performance.

Specific detection of Clostridium difficile toxin A gene sequences in clinical isolates

The polymerase chain reaction (PCR) was used to specifically detect toxin A gene sequences of Clostridium difficile in DNA isolated from human faeces. A set of oligonucleotide primers derived from the non-repetitive of the toxin A gene was developed to amplify a 634-bp DNA fragment. All 28 cytotoxic strains of C. difficile, previously characterized by a toxin B-PCR assay, were positive for the presence of toxin A gene sequences. No amplification products were obtained from DNAs extracted from non-toxigenic strains, strains of C. sordellii, or C. bifermentans. In addition, amplification of DNA extracted from C. difficile 8864, a strain which does not produce toxin A, resulted in multiple bands which probed negative for toxin A gene sequences. DNAs extracted from nine stool specimens which were positive for toxin B the cytotoxicity assay and by the toxin B-PCR assay were also positive in this assay. Toxin A gene sequences were detected in DNAs obtained from 4/11 stool specimens which were negative by the toxin B cytotoxicity assay. These four specimens were from patients who had a history of relapses due to C. diffcile-associated colitis, and whose stools had previously been found to be positive by the toxin B-PCR test despite no detectable toxin B in the specimens. These data indicate a comparable degree of clinical sensitivity between these two toxin-gene PCR-based assays. This rapid, sensitive and specific assay may be useful not only in the diagnosis of C. difficile infections, but also molecular studies of the toxin A gene in C. difficile strains.

Evaluation of an oligonucleotide probe and an immunological test for direct detection of toxigenic Clostridium difficile in stool samples.

A 33 basepair oligonucleotide probe, designed from the sequence of the Clostridium difficile toxin B gene, was evaluated for its ability to detect toxigenic Clostridium difficile directly in stool samples, without culture or DNA isolation. Two different labelling techniques were investigated: radiolabelling and digoxigenin-labelling. One hundred ninety-six stools were tested, with a good correlation (96%) obtained between the oligonucleotide probe and the gold standard, the cytotoxicity tissue culture assay. The sensitivity and specificity were 83% and 100%, respectively. In parallel, a new commercially available enzyme immunoassay for the detection of Clostridium difficile toxin A in stool specimens was investigated. In 162 samples tested, a sensitivity of 80% and a specificity of 98% were obtained.

Sulphasalazine-induced forms of colitis

Administration of oral or rectal sulphasalazine may lead to a number of potentially serious and life-threatening adverse reactions, including specific gastrointestinal complications. Mimicry and/or exacerbation of either ulcerative colitis or Crohn’s colitis may occur and has been described in several previous case reports. In addition, challenge studies after oral and rectal sulphasalazine have demonstrated unequivocal worsening of rectal biopsies. More recently, sulphasalazine treatment resulted in the development of pseudomembranous colitis associated with detection ofClostridium difficile toxin in a patient with ankylosing spondylitis and no evident pre-existing intestinal disease. This observation is particularly interesting because of the potential effects of the medication in altering the enteric microflora.

Evaluation of a commercial enzyme immunoassay kit for the detection of Clostridium difficile toxin A.

A new enzyme immunoassay (EIA) kit developed for the rapid detection of Clostridium difficile toxin A in faecal specimens, Premier (Meridian Diagnostics), was evaluated using 101 faecal specimens. Sixty-nine specimens were positive for Clostridium difficile by isolation of the organism and by cytotoxicity in tissue culture. The EIA for toxin A was positive in 49 of these 69 cases. No specimen that was negative for cytotoxicity was positive by EIA. Eight of the 32 specimens negative by both EIA and cytotoxicity assay yielded Clostridium difficile by culture. In five of these cases the cytotoxigenic status of the isolate was determined, and four were positive. There was no direct relationship between cytotoxin titre and EIA reading.

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.

Enzyme immunoassay (ELISA) for detection of Clostridium difficile toxin B in specimens of faeces

Antisera against Clostridium difficile toxin B were prepared in sheep and rabbit and were used in indirect and sandwich enzyme-linked immunosorbent assays (ELISA) for the detection of toxin B. Polyvinyl chloride and polystyrene microtitration plates were tested as solid phases for the assay. Both assays had a lower limit of detection for toxin B of 1 ng/ml. They were used to detect the presence of toxin B in 210 human faecal specimens and also in the culture supernatant fluids of C. difficile strains isolated from the faecal samples. There was a close correlation between the results of sandwich ELISA and those of cytotoxicity tests and isolation of C. difficile. Our sandwich ELISA method seems to be useful as a presumptive test for detection of C. difficile toxin B.

Clinical Comparison of Latex Agglutination and Cytotoxin Assay for Detection of Clostridium difficile Toxin in Feces

To determine if a latex agglutination (LA) test (Marion Scientific, Kansas City, MO) could be used to diagnose antibiotic-associated colitis, 206 stool specimens submitted for detection of Clostridium difficile toxin were tested by both LA and cytotoxin assay (CTA). Five specimens, all negative by CTA, gave nonspecific results by LA; these specimens were excluded from analysis. Of the remaining 201 specimens, 33 (16%) were positive and 161 (80%) were negative by both test methods, three were CTA positive and LA negative, and four were negative by CTA but LA positive. The sensitivity, specificity,and positive and negative predictive values of LA were 89%, 98%, 92%, and 98%, respectively. There was no significant difference between LA and CTA results. When materials and technologist's time were considered together, the cost of LA and CTA were comparable. Although we found that LA offered a rapid alternative to CTA, because the kit has recently been shown not to be specific for toxin A, we do not believe that LA should replace CTA. LA, however, could be used as a screening procedure, confirming stools yielding nonspecific and positive LA results with CTA.

Detection of Clostridium difficile toxin in various tissue culture monolayers.

Thirty stool filtrates known to contain Clostridium difficile toxin based on previous testing on McCoy cells were tested for toxicity on primary African green monkey kidney (AGMK), McCoy, MRC-5, primary rhesus monkey kidney (RMK), and Vero cells. All 30 filtrates showed cytotoxic effect at greater than or equal to 1:100 dilution on McCoy and Vero cells. A total of 22 filtrates were positive on MRC-5 monolayers, while only 16 and 10 filtrates showed positive cytotoxic effect on AGMK and RMK cells, respectively. Another 630 stool specimens were tested on McCoy and Vero cells only. Of these stool filtrates, 70 were positive and 560 were negative with both cell lines, which thus gave 100% agreement. Vero cells can be used interchangeably with McCoy cells for the detection of C. difficile toxin in stool filtrates.

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.

Evaluation of a commercial cytotoxicity assay for detection of Clostridium difficile toxin.

A comparative study was performed to determine the accuracy of Clostridium difficile toxin detection. A commercial cytotoxicity assay (Bartels Immunodiagnostic Supply, Bellevue, Wash.) was compared with conventional microcytotoxicity assays, using Vero and MRC-5 cells. The Bartels system was found to be essentially equivalent to conventional cytotoxicity assays currently being performed for routine C. difficile toxin detection.

Detection of Clostridium difficile toxin A by immunoblotting

A clinical isolate of Clostridium difficile has been tested for its toxin production. Both toxins, toxin A and toxin B, could be detected by tissue culture and in animal models as well. Antibodies against a crude toxin A preparation have been prepared. These antibodies are able to neutralize the toxin both in the mouse lethality test and tissue culture test systems. The specificity of this antiserum has been analysed by electroimmunoprecipitation methods. Using immunoblotting, it could be demonstrated that the antigenicity of toxin A after SDS polyacrylamide gel electrophoresis under denaturing and reducing conditions was still preserved. The molecular weight of toxin A has been estimated to be 250000. Immunoblotting offers a simple and reliable procedure for toxin A detection from culture supernatants of C. difficile.

Enzyme immunoassay for detection of Clostridium difficile toxins A and B in patients with antibiotic-associated diarrhoea and colitis.

A sandwich enzyme immunoassay (ELISA) was developed to detect Clostridium difficile toxins A and B in stools from patients with antibiotic associated diarrhoea and colitis. Immune serum to crude Clostridium difficile toxin and non-immune serum were coated onto polystyrene microtiter plates to act as capture antibodies; toxins A and B in human stools were detected by antibodies from rabbits immunized with purified toxins A and B. The ELISA for toxin B showed cross-reactions with Clostridium bifermentans and Clostridium sordellii and lacked diagnostic sensitivity in clinical samples. The ELISA for toxin A showed no cross-reactions with other clostridiae investigated and was positive in 33% (62/189) of patients with antibiotic associated diarrhoea. This compared with 38% (71/189) positive in the tissue culture assay for Clostridium difficile cytotoxin. With a predictive value of 96% in clinical specimens, the ELISA for toxin A constitutes a sensitive and specific tool for diagnosis of Clostridium difficile associated diarrhoea and colitis.

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.

The use of CIE for the detection of Clostridium difficile toxin in stool filtrates: laboratory and clinical correlation.

Two CIE procedures (CIE-1, CIE-2) for the detection of Clostridium difficile in diarrheal stools were evaluated by comparison to cytotoxin assay and culture results and by comparison to a clinical likelihood of C. difficile-induced diarrhea. Using a combination of toxin assay and culture results for reference, the CIE-1 and CIE-2 procedures had sensitivities of 33% and 47%, specificities of 89% and 91%, and positive predictive values of 42% and 54%, respectively. Using clinical likelihood for reference, the best results were obtained by the CIE-2 method, which yielded a positive predictive value of 77%. Neither CIE procedure provided an acceptable sensitivity for the detection of C. difficile in stools.