Detection of Clostridium-difficile Toxins in Stools - Comparison Between a New Enzyme-immunoassay for Toxin-a and Other Routine Tests

An enzyme-linked immunosorbent assay (ELISA) for detection of Clostridium difficile toxin A (enterotoxin) using a monoclonal antibody is described. No cross-reaction was observed with any of the Clostridium species tested except for toxigenic Clostridium difficile. One hundred and eight stool specimens from hospitalized patients harbouring C. difficile in their intestine and 43 samples negative for C. difficile isolation were studied to compare this test with a cytotoxicity assay, the isolation of toxigenic C. difficile and the commercial EIA Premier test (Meridian). As compared with the cytotoxicity assay and EIA, specificity was 91 and 100 %, while sensitivity 83 and 74 % respectively. This ELISA technique could be used as a routine test for toxin A detection in stools

Comparison of enterotoxin production, cytotoxin production, serogrouping, and antimicrobial susceptibilities of Clostridium difficile strains isolated from AIDS and human immunodeficiency virus-negative patients.

We analyzed and compared Clostridium difficile strains isolated from diarrheic stools of 49 human immunodeficiency virus (HIV)-negative and 50 AIDS patients. Our results suggest that distribution patterns of serogroups are different in these two populations. Serogroup C (which has been previously reported to be very resistant to antimicrobial agents) represents 66.0 and 18.4% of the isolates from AIDS and HIV-negative patients, respectively (P < 0.001); the selection of serogroup C could be explained by multiple antibiotic pressure to which AIDS patients have been subjected

Use of an enzyme-linked immunoassay for Clostridium difficile serogrouping.

An enzyme-linked immunoassay (ELISA) with 11 Clostridium difficile serogroup-specific antisera was applied for serogrouping of C. difficile colonies from 314 consecutive positive fecal samples. Two hundred forty-nine strains (79%) were correctly serogrouped, 57 (18%) belonged to serogroups other than the 11 which were evaluated and gave a negative reaction with all antisera, and 8 isolates (2.5%) did not react with their corresponding antisera. ELISA is a rapid and reliable method for serogrouping C. difficile and should allow for the automation of this procedure

Heterogeneity of Clostridium-difficile Isolates From Infants

In order to improve our understanding of the role of Clostridium difficile in infants we characterised the strains isolated from this population. The production of toxin A and toxin B was studied. The toxin A, playing a major role in the disease, was searched for in faecal samples. The serogroup of the isolates was determined because some serogroups have been shown to be more pathogenic than others. Over a 9-month period, 102 faecal samples from 102 hospitalised infants (0-12 months) were analysed and 26% of the children were colonised with C. difficile. Fifteen isolates secreted neither toxin A nor B (62.5%). Nine isolates were toxigenic and secreted both toxins (37.5%). Of the eight toxigenic strains tested, six were from serogroup H and two serogroup K. Of the 13 nontoxigenic strains tested, 8 belonged to serogroup D, 2 to serogroup X, and 1 each to serogroup A, serogroup B and serogroup C. Three infants out of 102 studied had toxin A in their faeces. In summary, the infants can be colonised by (1) nontoxigenic strains, most of them from nonpathogenic serogroup D, without toxin A in the faeces; (2) toxigenic strains of virulent serogroups H and K, with or without toxin A in the faeces. Although some infants had diarrhoea, none needed a specific treatment for C. difficile. No specific C. difficile pathology could be retained and different mechanisms are advanced to explain this absence of pathogenicity

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.

This study was undertaken to examine toxin production by Clostridium difficile 8864, a naturally occurring isolate that has been reported to produce toxin B in the absence of toxin A. To date, this is the only strain of C. difficile reported to produce only one of the toxins. The results of our initial studies with antibodies against toxins A and B confirmed these observations. Toxin B from strain 8864 and from VPI strain 10463, a strain that produces high levels of both toxin A and toxin B, was purified to homogeneity by sequential anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA22, and immunoadsorption chromatography, and their toxic activities were compared. Our results showed that toxin B from strain 8864 and toxin B from C. difficile VPI strain 10463 were comparable in their cytotoxic activities and that the 8864 toxin B was more lethal. In addition, we observed that toxin B from strain 8864 was weakly enterotoxic, which may explain the ability of this strain to cause intestinal disease in hamsters treated with antibiotics. Analysis with specific antibodies showed that the toxin B molecules from these strains were highly related but contained distinct epitopes. The results of hybridization studies with probes specific for the toxin B gene of VPI strain 10463 demonstrated differences between the toxin B genes of the two strains. In addition, probes specific for the toxin A gene of VPI strain 10463 showed that strain 8864 contains a region which shows identity with the 5' end of the toxin A gene but not the region of the gene which encodes a hydrophobic region and the repeating units

Serogroup-f Strains of Clostridium-difficile Produce Toxin-b But Not Toxin-a

Most toxigenic strains of Clostridium difficile produce two toxins: an enterotoxin (toxin A) and a cytotoxin (toxin B). Only one strain (strain 8864) has been reported to produce toxin B but no toxin A. Serogroup F strains (44) of C. difficile, often isolated from asymptomatic infants, have been examined for toxin production. These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in vitro when examined in three distinct immunoassays. Nevertheless, all the strains produced a cytotoxin. Immunological differences between the cytotoxin of the serogroup F strains and that produced by C. difficile strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C. difficile strain VPI 10463 or C. sordellii lethal toxin (LT). Polymerase chain reaction amplification with primers derived from C. difficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene. When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces. However, cytotoxin was detected. Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C. difficile strain VPI 10463. The serogroup F strains appeared to be homogeneous and distinct from other C. difficile strains with regard to toxin production