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.     

Comparison of three enzyme immunoassays for detection of Clostridium difficile toxins A and B

AIM OF THE STUDY: Diagnosis of Clostridium difficile-associated diarrhea is classically based on detection of toxin A and/or toxins A+B by using several techniques. However, these techniques show important differences in terms of sensitivity and specificity. In this work, we compared three commercial immunoenzymatic tests for detecting C. difficile toxins. METHODS: We used three immunoenzymatic techniques: ELISA Premiertrade mark Toxins A and B (Meridian), ImmunoCard (IC) Toxins A and B (Meridian), and Triage (TRI) Antigen GDH and Toxin A (Biosite). Culture on the specific media C. difficile (bioMérieux) was performed in parallel. RESULTS: During two years, 898 stool specimens have been tested by using the three different techniques. According to the manufacturer's recommendations (sample positive if optical density [OD] value greater or equal to 0.15), 205 (22.8%) were positive for ELISA. Among them, 65 (31.7%) were negative for all other tests, and they have been considered as false-positive results. This discrepancy led us to choose other OD values (greater than 0.75: positive result; 0.15-0.75: limit result). For limit results, IC, TRI, antigen GDH, and culture methods were positive in 30, 2, 41, and 29% of cases, respectively, whereas for positive results (>0.75), they were positive in 82, 54, 84, and 76% of cases, respectively. CONCLUSION: ELISA and IC tests are the most powerful and are concordant together if interpretation is performed with OD values redefined by the microbiology laboratory. The choice of the technique to use depends on the number of samples to analyze, the rapidity of the result, and the cost.     

Biological activities of toxins A and B of Clostridium difficile.

Examination of the biological activities of the two known toxins of Clostridium difficile revealed that one of the toxins (toxin A) elicited a hemorrhagic fluid response in rabbit intestinal loops and a positive fluid response in infant mice. The other toxin (toxin B) did not produce a significant fluid response in either model, although the toxin was more lethal in infant mice. Both toxins elicited erythematous and hemorrhagic skin reactions and increased vascular permeability in rabbit skin.     

Purification and characterization of toxins A and B of Clostridium difficile.

Toxin preparations were obtained by growing Clostridium difficile VPI strain 10463 in 2-liter brain heart infusion dialysis flasks at 37 degrees C for 3 days. The initial step of the purification scheme involved ultrafiltration through an XM-100 membrane filter. Two toxic activities, designated toxins A and B, were separated by ion-exchange chromatography on DEAE-NaCl gradients. Toxin A was purified to homogeneity by an acetic acid precipitation at pH 5.5. Other separation techniques, including CM Sepharose CL-6B, (NH4)2SO4 and acetic acid precipitations, and hydrophobic interaction chromatography, were examined in attempts to further purify toxin B. Although these methods failed to increase the specific activity of toxin B, they provided additional evidence that the two toxins are distinct molecules. The toxins are acid and heat labile and are inactivated by trypsin and chymotrypsin, but not by amylase. The molecular weight of toxin A, as estimated by gel filtration and gradient polyacrylamide electrophoresis, ranged from 440,000 to 500,000. The estimated molecular weight of toxin B was 360,000 to 470,000.     

Evaluation of three commercial enzyme immunoassay kits for detecting faecal Clostridium difficile toxins.

The detection of faecal cytotoxicity using tissue culture was compared with three commercial Clostridium difficile enzyme immunoassay (EIA) kits; Premier C difficile toxin A (Meridian Diagnostic, Inc.); CD-TOX C difficile toxin A (Porton Cambridge); and Cytoclone A+B EIA (Cambridge Biotech Corporation). Of 160 faecal samples examined by all four methods, 52 (32.5%) were cytotoxic, 44 (27.5%) were positive by Premier, 48 (30%) by CD-TOX EIA, and 50 (31.3%) with Cytoclone. When compared with detection of cytotoxicity by tissue culture assay, the following performance indices were obtained: Premier, sensitivity 84.1%, specificity 99.1%, positive predictive value (PPV) 97.8%, negative predictive value (NPV) 93%; CD-TOX, sensitivity 92.3%, specificity 88.0%, PPV 78.7%, NPV 95.9%; Cytoclone, sensitivity 96.2%, specificity 93.5%, PPV 87.7%, NPV 98.1%. EIA results were available within three hours, whereas the results of the cytotoxin assay were available after 24-48 hours. All three kits provided satisfactory results and, although relatively expensive, all could be used in the laboratory effectively to screen for diarrhoeal disease associated with C difficile.     

Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies.

Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.     

Production and release of toxins A and B by Clostridium difficile

The production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated. Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells. Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay. Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle. The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed.     

Enzyme immunoassay for detection ofClostridium difficile toxins a and b in patients with antibiotic-associated diarrhoea and colitis

A sandwich enzyme immunoassay (ELISA) was developed to detectClostridium difficile toxins A and B in stools from patients with antibiotic associated diarrhoea and colitis. Immune serum to crudeClostridium 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 withClostridium bifermentans andClostridium 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 forClostridium difficile cytotoxin. With a predictive value of 96 % in clinical specimens, the ELISA for toxin A constitutes a sensitive and specific tool for diagnosis ofClostridium difficile associated diarrhoea and colitis.     

Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B

A mutant of Chinese hamster lung fibroblasts (Don cells), resistant against Clostridium difficile toxins A and B, was isolated after mutagenization with ethylmethanesulphonate and a two-step selection with toxin B. The mutant, termed CdtR-Q, was 10(4) times more resistant to toxin B than wild-type cells and cross-resistant to toxin A (10(3) times more resistant). The resistance was overcome by increasing the dose of toxin. The resistance has been stable after cultivation for 40 generations in the absence of toxin. The morphology of the mutant was more epithelial-like than that of the fibroblast parental cells. The plating efficiency was about half that of the wild-type, whereas the growth rate was the same. The mutant was significantly less sensitive than the wild-type to the microfilament-interacting cytochalasins B and D. It was as sensitive as the wild-type to endocytosed toxins (diphtheria, pertussis, ricin), to microtubule-interacting agents (colchicine, gossypol, nocodazole, taxol, vinblastine), and to membrane-damaging toxins with different mechanisms of action, with one exception; the mutant was more highly sensitive to the action of phospholipase C (with broad substrate-specificity) than the wild-type. The results suggest that the mutant has a normal endocytosis, and that the mutation does not affect the microtubuli. The results are consistent with a mutation affecting the microfilaments in the cytoskeleton.     

Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C. difficile.

Clostridium sordellii produces two toxins, designated HT (haemorrhagic toxin) and LT (lethal toxin), that are similar to toxins A and B of C. difficile. The physicochemical properties of toxins HT and A were remarkably similar. The specific biological activities of toxin HT were almost the same as those of toxin A, and their NH2-terminal sequences shared close homology. The properties of toxins LT and B were similar, as were their NH2-terminal sequences, but toxin B was much more cytotoxic than toxin LT. Immunodiffusion analysis with specific antibodies showed that although toxins B and LT shared major antigenic determinants, each had unique epitopes. The results suggest that toxins B and LT have diverged more than toxins A and HT. Immunoblotting with antibodies to the toxins of C. difficile showed that toxins HT and LT had common antigenic determinants.     

Clostridium difficile toxins A and B inhibit human immune response in vitro.

Two Clostridium difficile toxins isolated from strain VPI 10463 were tested for their effect on different human T-cell proliferation systems. In mitogen- and antigen-driven T-cell proliferation systems, toxins inhibited the proliferative response in a dose-dependent fashion. In interleukin-2-driven culture systems, no effect of toxins could be found on preactivated T cells. We suspected that monocytes were the influenced cells, since in antigen- and mitogen-driven systems monocytes were necessary for the proliferative response, whereas the interleukin-2-driven system was independent of monocytes. To prove this concept, purified monocytes were treated with toxins. The treatment was found to markedly reduce the capacity of monocytes to stimulate T-cell proliferation. No inhibition of the proliferative response was measured when, instead of monocytes, resting or preactivated T cells were treated with toxins. These experiments clearly show that C. difficile toxins interact with monocytes and not with T cells. The effect of toxins on cells of the immune system might be one factor in the development of pseudomembranous colitis.     

Evaluation of four immunoenzymatic tests for detecting Clostridium difficile toxins A and B

Four immunoenzymatic tests for detecting Clostridium difficile toxins A and B were studied: two rapid tests (Tox A/B QUIK CHEK-Techlab and NoviView Toxine-A-Hiss diagnostics) and two Elisa tests (C. difficile TOX A/B II -Techlab and Toxin A+B Elisa Test, Novitec-Hiss diagnostics). The results were compared to those obtained with ImmunoCard Tox A+B -ICTAB (Meridian), C. difficile Toxine A (Oxoid) for rapid test and Elisa Premier A+B Meridian for Elisa. A total of 41 stools and 16 isolates were studied with rapid tests. On stools, the sensitivity and specificity of QUIK CHEK test was 94.1% and 100% respectively compared to the test ICTAB. On the isolates, sensitivity and specificity was 100%. With the Noviview test, the sensitivity on stools and isolates was respectively 88.2 and 85.7% and the specificity was 100% compared to Oxoid. A total of 38 stools were studied with Elisa tests. With Techlab test compared to the test Premier, sensitivity and specificity was 100%. The Novitec test gave five false negative reactions with consequently a sensitivity of 70.6%.     

Clostridium difficile toxins A and B directly stimulate human mast cells

Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.     

Association between production of toxins A and B and types of Clostridium difficile.

One hundred and seventy two strains of Clostridium difficile isolated from 62 patients with antibiotic associated diarrhoea or pseudomembranous colitis were analysed for the production of toxins A and B and typed using 35S-methionine labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). There was a correlation between production of toxins A and B and the type of C difficile. One hundred and forty four of 172 strains were either high or low producers of both toxins. Toxins were not detected in 28 of 172 strains. Types A and Y were consistently non-toxin producers; types B and E were high toxin producers. Type X, the epidemic strain, showed variable toxin production. Symptoms of 49 patients with haematological malignant pathology who were part of a documented outbreak of antibiotic associated diarrhoea, were analysed in relation to toxin production and type of the clinical strains isolated. In general, there was a correlation between symptoms of antibiotic associated diarrhoea, the type of C difficile, and its potential for producing toxins.     

Comparison of three commercial methods for rapid detection of Clostridium difficile toxins A and B from fecal specimens

Three rapid enzyme immunoassays (X/pect Clostridium difficile Toxin A/B test, Wampole Tox A/B Quik Chek, and ImmunoCard Toxins A&B) were compared for the diagnosis of Clostridium difficile infection. Of the 367 stool specimens tested, 102 (27.8%) were positive for toxigenic C. difficile when a combination of direct cytotoxicity assay and cytotoxic culture was used as the gold standard. Sensitivity/specificity values were 49.0%/95.8%, 54.9%/95.5%, and 66.7%/95.1%, respectively. The median times to test five stool specimens were 28, 30, and 24 min, respectively. The ImmunoCard test was the quickest and most sensitive test of the three enzyme immunoassays evaluated.     

Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A.

The Premier Clostridium difficile toxin A enzyme immunoassay (PTA EIA) (Meridian Diagnostics, Inc., Cincinnati, Ohio) for rapid diagnosis of antibiotic-associated colitis (AAC) was evaluated in a multicenter study. Stool samples from 421 patients suspected of having AAC were tested for toxin A by the PTA EIA and for toxin B by three tissue culture assays (TCA) employing WI-38 cells (New England Deaconess Hospital) in conventional tubes or foreskin fibroblasts (Children's Hospital) or Vero cells (Beth Israel Hospital) in microwells. The tubes and plates were examined at 24 and 48 h for cytotoxicity. Clinical criteria, repeat testing at another site, and culture of frozen stool samples for C. difficile were used to evaluate discrepant results. Of 504 samples, 66 were positive and 409 were negative by both tests. Eight samples had indeterminate PTA EIA results and were excluded from this analysis. Of 21 discrepancies, 9 were PTA EIA positive and TCA negative and 12 were PTA EIA negative TCA positive. Following resolution of the discrepancies, 11 of 12 PTA EIA-negative-TCA-positive and 5 of 9 PTA EIA-positive-TCA-negative samples were considered true positive for AAC. The sensitivity and specificity were, respectively, 86.6 and 99.0% for the PTA EIA and 93.9 and 99.8% for TCA. The predictive values of positive and negative tests were, respectively, 94.7 and 97.4% for the PTA EIA and 98.7 and 98.8% for TCA. We conclude that the PTA EIA is a rapid, simple EIA technique whose accuracy in detecting enterotoxin A approaches that of reference TCA methods for detection of cytotoxin B.     

Enzyme immunoassay for detection of antibody to Epstein-Barr virus-specific early antigen.

Antibody to Epstein-Barr virus-induced early antigen could be measured in sera using enzyme-linked antibody. The sensitivity of this assay was comparable to that of the indirect immunofluorescent technique. Measurement of early antigen was accomplished on a producer cell line which was specifically treated to block late gene expression. It is now possible to measure Epstein-Barr virus-induced early antigen and viral capsid antigen antibodies by using the same cell line.     

Differential cytotoxic effects of toxins A and B isolated from Clostridium difficile

Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics. The cytotoxins were purified from dialyzed filtrates of C. difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration. The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique. Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B. Toxin B was resolved into one major and three minor cytotoxic proteins. One protein comigrating with toxin A had no cytotoxic activity. The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h. These effects correlated with morphological changes indicating cytotoxicity. At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident. The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay. Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete. In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min. The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.     

Positive regulation of Clostridium difficile toxins.

The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli. To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters. The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay. When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold. Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans. Our results suggest that TxeR is a positive regulator that activates expression of the C. difficile toxins.     

Clinical usefulness of components of the Triage immunoassay,enzyme immunoassay for toxins A and B,and cytotoxin B tissue culture assay for the diagnosis of Clostridium difficile diarrhea

We studied 557 nonduplicate fresh stool specimens from adult patients clinically suspected of having Clostridium difficile-associated diarrhea. All samples were tested in parallel with an in-house cytotoxin B tissue culture assay (CTA), the C DIFFICILE TOX A/B II test (TA/B; TechLab, Blacksburg, VA), and the Triage Micro C DIFFICILE Panel (Biosite Diagnostics, San Diego, CA). The Triage device detects toxin A (TA) and glutamate dehydrogenase (GDH) simultaneously. Of the specimens, 350 were negative and 95 were positive for all markers. Another 112 specimens yielded discrepant results. The CTA found 143 positive specimens. Results of the components of the Triage and TA/B were compared separately with those of CTA. GDH was the most sensitive but least specific marker, whereas TA and TA/B were less sensitive but highly specific. Because of these attributes and a quick turnaround time, GDH would be the best screening test for C difficile-associated diarrhea. CTA detected the highest number of cases of C difficile-associated diarrhea and would be most useful as a confirmatory test for GDH-positive and TA-negative specimens.