Difficile Enterotoxin Research Articles

Comparison of Clostridium Difficile Associated Diarrhea Detection by Enzyme Linked Immunosorbent Assay (EIA) and Polymerase Chain Reaction (PCR) in Autologous Hematopoietic Stem Cell Transplant (AHSCT) Recipients

Abstract 4739 Background:Diarrhea is a frequent complication of autologous hematopoietic stem cell transplantation and is a common cause of morbidity. Clostridium difficile associated diarrhea (CDAD) is the most common cause of nosocomial infections. Detection of CDAD is widely performed using enzyme linked immunosorbent assay (EIA) to C. difficile toxins A and B because of its technical ease of use and rapid results. The sensitivity of EIA is 60%, but the sensitivity increases with repeated testing. Recently, detection of CDAD by Polymerase Chain Reaction (PCR) methodology has resulted in rapid detection of amplified C. difficile toxin (CDT) B chromosomal material and has been shown to be more sensitive than EIA in patients suspected of CDAD; however, no comparison of the two methodologies has been performed in recipients of an autologous hematopoietic AHSCT. Methods:This retrospective analysis of AHSCT recipients at our institution evaluates the incidence of CDAD when using either EIA or PCR. CDAD was assessed at the onset of diarrhea in both cohorts. The time period of interest was from 7 days prior to transplantation to 30 days posttransplanation. In the EIA cohort, three consecutive liquid stools were tested for the presence of CDT A and B by EIA. In the PCR cohort, only one liquid stool was sent for sampling. Our institution adopted PCR methodology on 1 February 2010. Demographic and clinical information were collected to assess CDAD risk. Successful treatment of CDAD was defined as resolution of diarrhea within seven days. Previously published data from our institution have shown the incidence of CDAD to be 15% when using EIA methodology. Nominal data were analyzed by Fisher's Exact Test and continuous data were analyzed by Student's T-Test. Results:A total of 159 patients were screened; and 16 patients were excluded because they did not have a CDAD test performed. Seventy-three were included in the EIA cohort and 69 were included in the PCR cohort. Clinical characteristics were similar between the groups. Known risk factors for CDAD including days in the hospital, days of neutropenia, and days of prophylactic and intravenous antibiotics used were similar between the cohorts. The incidence of a positive CDAD test was higher in the EIA cohort compared to the PCR cohort (18% vs. 7% [p = 0.049]). The duration of diarrhea was longer in the EIA cohort as compared to the PCR cohort (7.3 days vs. 5.5 days [0.016]). Of the three consecutive EIA tests sent, the first was positive only 30% (4/13) of the time. Metronidazole was the first-line agent used in all cases. Response to therapy was poor in both the EIA and PCR cohorts with rates of 30% and 20%, respectively. No fatalities occurred in either group as a result of CDAD. Conclusion:The incidence of diarrhea was significantly shorter in the PCR cohort as compared to the EIA cohort. The duration of diarrhea was significantly shorter in the PCR group as compared to the EIA cohort. It is possible that repeated testing for CDAD by EIA decreases the specificity of the test or that the EIA is detecting a non-toxigenic C. difficile enterotoxin. Because our patient population has additional causes of diarrhea including mucosal irritation caused by chemotherapy, additional studies are needed to discern the discrepancies between both methodologies.EIA Cohort (n=73)PCR Cohort (n=69)P valueAge (median, years)61600.163Race (n,%)36 (49)38 (55)0.521Caucasion19 (26)20 (29)African American17 (23)11 (16)Hispanic1 (1)0 (0)OtherMale (n,%)71 (97)64 (93)0.265Karnofsky Performance Status (median,%)90900.932Disease (n,%)56 (77)45 (65)0.625Myeloma15 (20)21 (30)Lymphoma2 (3)3 (5)OtherCDAD (n,%)13 (18)5 (7)0.049Diarrhea Duration (mean, days)7.35.50.016 (−3.32 to −0.16)CDAD treatment response (n,%)4 (30)1 (20)0.568Days of Neutropenia (mean, days)76.70.553 (−1.01 to 0.54)Days of oral antibiotics (mean, days)4.54.30.774 (−1.04 to 0.77)Days of IV antibiotics (mean, days)7.35.50.170 (−4.33 to 0.77) Disclosures:No relevant conflicts of interest to declare.

Clostridium difficile-associated diarrhoea, a frequent complication in patients with acute myeloid leukaemia

Diarrhoea occurs frequently in neutropenic patients with acute leukaemia receiving chemotherapy and may be caused by either infection- or drug-induced cytotoxicity. Since Clostridium difficile is the most common cause of nosocomial infectious diarrhoea in non-haematologic patients, we were interested in its incidence in patients with acute myeloid leukaemia (AML). In this retrospective study, we analysed 134 patients with AML receiving a total of 301 chemotherapy courses. Diarrhoea occurred during 33% of all courses in 58 patients. C. difficile-associated diarrhoea (CDAD) occurred in 18% of all patients and 9% of all treatment courses. Almost one third of diarrhoea episodes were caused by C. difficile. CDAD was associated with older age (58 vs. 50 years), number of antibiotics administered (2 vs. 1), duration of antibiotic therapy (7 vs. 4 days), ceftazidime as the antibiotic of choice (75% vs. 54%) and duration of neutropenia (12 vs. 7 days) prior to onset of diarrhoea. An increased risk for CDAD was seen for prolonged neutropenia. CDAD responded well to oral metronidazole and/or vancomycin and no patient died of this complication. In conclusion, CDAD is common in patients with AML receiving chemotherapy. C. difficile enterotoxin testing of stool specimens should be included in all symptomatic patients.

Covert Assessment of Concurrent and Construct Validity of a Chart to Characterize Fecal Output and Diarrhea in Patients Receiving Enteral Nutrition

An accurate and convenient method for characterizing fecal output and a consistent threshold for classifying diarrhea in patients receiving enteral nutrition are required. The aim of this study is to covertly assess the construct and concurrent validity of a chart for characterizing fecal output and classifying diarrhea in patients receiving enteral nutrition. The chart was used to monitor fecal output in patients receiving enteral nutrition for a total of 280 patient days. Nurses characterized 291 fecal samples, of which 84 underwent measurement of fecal water using lyophilization and 60 underwent Clostridium difficile enterotoxin analysis using enzyme-linked immunosorbent assay. Construct and concurrent validity was assessed covertly to measure the true performance of the chart in a real-life clinical and research context. Use of the chart demonstrated higher fecal frequency (P <or= .04), heavier stools (P <or= .167), more unformed stools (P <or= .001), higher daily fecal scores (P <or= .001), and higher incidence of diarrhea (P <or= .002) on days when patients had severe hypoalbuminemia, were receiving antibiotics, or had a recent positive C difficile assay, demonstrating construct validity. The water content of samples assigned to hard and formed (62.0%), soft and formed (72.1%), loose and unformed (79.3%), and liquid (87.9%) categories was significantly different (P < .001), demonstrating concurrent validity. Under covert assessment, the chart demonstrated construct validity for characterizing fecal output, daily fecal score, and diarrhea, together with concurrent validity for characterizing fecal consistency. Use of the chart in clinical practice and research will standardize the characterization of fecal output and classification of diarrhea in patients receiving enteral nutrition.

Klebsiella oxytoca: a rare cause of severe infectious colitis: first North American case report

Klebsiella oxytoca (K oxytoca) is a rare cause of infectious colitis. Only a handful of cases of K oxytoca colitis are described, all from France 1. Benoit R Dorval D Loulergue J Bacq Y Oliver J.M Audurier A et al. Post-antibiotic diarrheas: role of Klebsiella oxytoca. Gastroenterol Clin Biol. 1992; 16 ([French with English abstract]): 860-864 PubMed Google Scholar , 2. Bellaiche G Le Pennec M Choudat L Ley G Slama J.L Value of rectosigmoidoscopy with bacteriological culture of colonic biopsies in the diagnosis of post-antibiotic hemorrhagic colitis related to Klebsiella oxytoca. Gastroenterol Clin Biol. 1997; 21 ([French with English abstract]): 764-767 PubMed Google Scholar , 3. Beaugerie L Metz M Barbut F Bellaiche G Bouhnik Y Raskine L et al. Klebsiella oxytoca as an agent of antibiotic-associated hemorrhagic colitis. Clin Gastroenterol Hepatol. 2003; 1: 370-376 Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar , 4. Barbut F Beaugerie L Delas N Fossati-Marchal S Aygalenq P Petit J.C Comparative value of colonic biopsy and intraluminal fluid culture for diagnosis of bacterial acute colitis in immunocompetent patients. Clin Infect Dis. 1999; 29: 356-360 Crossref PubMed Scopus (39) Google Scholar , 5. Soussi F Tchirikhtchian K Ramaholimihaso F Yaziji N Coussinet S Diebold M.D et al. Diclofenac-induced colitis complicated by Klebsiella oxytoca infection. Gastroenterol Clin Biol. 2001; 25 ([French with English abstract]): 814-816 PubMed Google Scholar , 6. David X Pierrugues R Michel H Acute hemorrhagic colitis related to oral administration of cephalosporin. Gastroenterol Clin Biol. 1999; 15 ([French] [letter]): 659 Google Scholar , 7. Blanchi A Pariente A Acute hemorrhagic colitis after ingestion of amoxicillin. Gastroenterol Clin Biol. 1992; 16 ([French]): 1012-1014 PubMed Google Scholar , 8. Gineston J Watine J Bruna T Lamblin G Dubourdieu B Acute hemorrhagic colitis caused by pristinamycin: two cases with association of Klebsiella oxytoca and Clostridium difficile. Gastroenterol Clin Biol. 1993; 17 ([French]): 773-775 PubMed Google Scholar , 9. Grando V Bellaiche G Le Pennec M Slama J.L Ley G Nouts A et al. Ulcerative hemorrhagic proctitis caused by Klebsiella oxytoca after treatment with an amoxicillin-clavulanic acid combination. Gastroenterol Clin Biol. 1994; 18 ([French]): 536-537 PubMed Google Scholar , 10. Fort E Sevestre C Cahiez M Treppoz M Dorval E.D Acute hemorrhagic colitis after oral ingestion of synergistin [French]. Gastroenterol Clin Biol. 1993; 17: 231-232 Crossref PubMed Scopus (19) Google Scholar and Japan. 11. Koga H Aoyagi K Yoshimura R Kimura Y Iida M Fujishima M Can quinolones cause hemorrhagic colitis of late onset? Report of three cases. Dis Colon Rectum. 1999; 42: 1502-1504 Crossref PubMed Scopus (21) Google Scholar , 12. Sakurai Y Tsuchiya H Ikegami F Funatomi T Takasu S Uchikoshi T Acute right-sided hemorrhagic colitis associated with oral administration of ampicillin. Dig Dis Sci. 1979; 24: 910-915 Crossref PubMed Scopus (67) Google Scholar , 13. Chida T Nakaya R Tsuji N Shimizu N Masuda G Seo T et al. Intestinal microflora of patients with antibiotic-associated hemorrhagic colitis associated with Klebsiella oxytoca and Clostridium difficile enterotoxin. Kansenshogaku Zasshi. 1986; 60 ([Japanese]): 608-615 PubMed Google Scholar K oxytoca is a gram-negative, facultative aerobe/anaerobe and is found in the normal fecal flora from 30% to 40% of people. Over 100 strains have been identified, a few of which produce cytotoxins that can result in an infectious colitis. The clinical presentation of K oxytoca colitis is diarrhea with mucous or blood. Prior use of a penicillin-like antibiotic is characteristic and presumably results in selection of the cytopathic strains. Other medications, such as non-steroidal anti-inflammatory drugs (NSAID), also have been implicated. Leukocytosis is common at presentation. Patients range in age from 20 to 93 years. 1. Benoit R Dorval D Loulergue J Bacq Y Oliver J.M Audurier A et al. Post-antibiotic diarrheas: role of Klebsiella oxytoca. Gastroenterol Clin Biol. 1992; 16 ([French with English abstract]): 860-864 PubMed Google Scholar The colitis is non-pseudomembranous and can be segmental, i.e., left sided or right sided, or pancolonic. 1. Benoit R Dorval D Loulergue J Bacq Y Oliver J.M Audurier A et al. Post-antibiotic diarrheas: role of Klebsiella oxytoca. Gastroenterol Clin Biol. 1992; 16 ([French with English abstract]): 860-864 PubMed Google Scholar , 2. Bellaiche G Le Pennec M Choudat L Ley G Slama J.L Value of rectosigmoidoscopy with bacteriological culture of colonic biopsies in the diagnosis of post-antibiotic hemorrhagic colitis related to Klebsiella oxytoca. Gastroenterol Clin Biol. 1997; 21 ([French with English abstract]): 764-767 PubMed Google Scholar It is characteristically of mild to moderate severity and resolves spontaneously when treatment with the inciting antibiotic is stopped. Treatment with a fluoroquinolone may be needed in select cases. This report describes the first case in which K oxytoca colitis was severe and fulminant. As well, it is the first report of the recognition of this infectious colitis in North America.

Antibody responses to clostridial infection in humans.

Serum antibody responses to the major toxins produced by Clostridium difficile, Clostridium perfringens, Clostridium septicum, Clostridium tetani, and Clostridium botulinum have been documented following infection. Effective toxoid vaccines for tetanus and enteritis necroticans due to C. perfringens type C demonstrate the potential of antitoxin responses. Although individual serum and mucosal antibody responses to C. difficile enterotoxin (toxin A) vary, one-third of patients with C. difficile diarrhea develop neutralizing serum antibodies. IgA in the serum, not IgG, is typically responsible for this neutralization, suggesting a unique role for serum IgA in response to C. difficile infection, an infection that is usually limited to the intestinal mucosa. The relationship of naturally occurring antitoxin antibodies to the clinical course of C. difficile infection is controversial. However, patients with chronic relapsing C. difficile diarrhea and low levels of IgG to toxin A have shown clinical responses following intravenous therapy with immune globulin. Antibody responses to non-toxin C. difficile proteins also occur, but their significance is only partially known.

The effect of enteric bacterial toxins on the catecholamine levels of the rabbit

The rabbit catecholamine responses to bacterial toxins commonly found in Sudden Infant Death Syndrome (SIDS) victims were studied as part of a proposed animal model for SIDS. Six bacterial toxins commonly isolated from SIDS baby feces and a comparison endotoxin were injected intravenously (i.v.) and intraluminarily (i.l.) to determine their effects on catecholamine levels. I.v. injected toxins clearly altered catecholamine levels causing sharp rises in adrenaline and noradrenaline levels and at critical toxin concentrations sudden death ensued. Clostridium perfringens enterotoxin and alpha-toxin, Clostridium difficile enterotoxin (A) and cytotoxin (B), Escherichia coli STa toxin and staphylococcal enterotoxin B caused rises in catecholamine levels similar to that caused by E. coli endotoxin. Control rabbits showed very little or no obvious change in catecholamine levels. Clostridium difficile enterotoxin (A) and cytotoxin (B) injected i.v. exhibited synergy. Toxins injected into the duodenum, jejunum, ileum, cecum and large intestine caused behavioural changes ranging from reduced appetite and diarrhea to, in rare cases, death. Changes in the catecholamine levels of these animals however were not significantly different from those of the control animals. The results are discussed in relation to the possible effect of certain conditions (physiological, viral infections and environmental) which increase toxin permeability and allow absorption of these toxins, possibly resulting in sudden infant death.

Evaluation of an enzyme immunoassay kit for the detection of Clostridium difficile enterotoxin

The Premier Clostridium difficile toxin A enzyme immunoassay (EIA) kit was evaluated for the detection of C. difficile enterotoxin in fecal samples. A total of 314 samples was tested by culture, cytotoxin detection and EIA kit. Compared to a combined culture/cytotoxin result the Premier EIA kit had a sensitivity of 88.3%, a specificity of 100%, a predictive value positive of 100% and a predictive value negative of 87.4%. Test results were available within 3 hrs providing a rapid and reliable means of detecting C. difficile enterotoxin.

Detection of Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction.

A rapid assay was developed for detection of the Clostridium difficile enterotoxin gene in stool specimens by means of the polymerase chain reaction (PCR). The PCR primers amplified a 63-bp repetitive sequence of the enterotoxin gene, thereby generating a distinctive ladder pattern of DNA bands following electrophoresis. Crude DNA extracts from stools containing C. difficile produced one (63-bp) or more bands of the characteristic ladder. Of 172 stool specimens from 58 patients, 37 gave positive results by culture (15 specimens) or cytotoxin assay (36 specimens). When 36 available "positive" specimens were tested by the PCR assay, 34 (94%) gave positive results--24 by direct testing, and 10 after extraction of DNA by the Qiagen procedure. Insufficient material of the remaining two specimens was available for DNA extraction. Of 21 stools "negative" for C. difficile by culture or cytotoxin assay, one gave a positive result by PCR and seven produced atypical bands. The rapid PCR detection technique for C. difficile was more sensitive than standard culture, and of a sensitivity similar to cytotoxin testing. The method has the potential for adoption in routine laboratory practice.

Postoperative Clostridium Difficile Gastroenteritis

Clostridium difficile gastroenteritis can be the cause of an enigmatic postoperative syndrome of high temperature and marked leukocytosis, out of proportion to the initially mild constitutional symptoms. Patients may suffer delayed onset of diarrhea, which will test positive for the C. difficile enterotoxin by latex agglutination. We report 5 cases of C. difficile gastroenteritis that occurred within a 2-year period. We believe that the combination of preoperative bowel preparation, and intraoperative and postoperative systemic antibiotics is the primary operant factor. All patients responded rapidly when oral antibiotics specific for C. difficile were instituted. The sequelae of C. difficile colitis can include toxic megacolon with perforation and peritonitis, increasing the importance of early recognition and appropriate treatment.

An Unusual Case of Myxedema Megacolon With Features of Ischemic and Pseudomembranous Colitis

Myxedema megacolon is rare; usually, it manifests with abdominal distention, flatulence, and constipation. Herein we describe a 72-year-old man who had intermittent diarrhea, bloating, and abdominal pain for more than a year. Cultures of stool specimens for Clostridium difficile enterotoxin were variably positive and negative. Colonoscopic biopsy specimens were thought to be consistent with chronic ischemia. Thyroid function tests showed severe hypothyroidism; the patient's symptoms resolved with thyroid hormone replacement. We hypothesize that gross dilatation of the colon, attributed to myxedema, was followed by intestinal ischemia and complicated by recurrent episodes of pseudomembranous colitis. A review of the relevant literature is provided. This unusual manifestation of myxedema should be considered in the differential diagnosis when a patient has diarrhea, bloating, and abdominal pain.

Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.

Effect of Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells.

Electron microscopical immunocytochemistry and light microscopy were used to study the effect of Clostridium difficile enterotoxin A (EA) on cultured Chinese hamster ovary (CHO) cells. At 4 degrees C, immunocytochemically detected EA was randomly distributed along the plasma membrane; when cells were subsequently transferred to 37 degrees C, the EA moved into coated pits and coated vesicles within 2 min. Within 2 h of incubation at 37 degrees C with EA, the perinuclear cytoplasm of the CHO cells became highly refractile, and in 4 h, most of the cells were round; however, the majority of rounded cells excluded erythrosin B while remaining firmly attached to the culture dish plastic. When EA was removed from the cultured cells within 2 h, the cells returned to a normal morphology and formed a confluent monolayer. The nuclei of rounded cells were irregularly shaped; cytoplasmic intermediate filaments were collapsed toward the nucleus. Long bundles of parallel, 11-nm-diameter filaments appeared in the nuclei after 3 h of incubation with EA and disappeared by the fourth hour of incubation. Intoxicated cells did not undergo mitosis. Thus, EA was internalized, at least in part, by receptor-mediated endocytosis and subsequently affected the nuclei of CHO cells.

Monospecific antibodies for detection of Clostridium difficile enterotoxin.

Tests of polyclonal antisera to homogeneous enterotoxin preparations of Clostridium difficile by immunoblotting demonstrated that cross-reactions occurred with proteins from cell lysates of nearly all Clostridium species. There was a notable reduction in the amount of crossreaction when only culture supernatants were analysed. The enterotoxin could be easily identified by its unique high molecular weight of 230,000. The findings suggest that care must be taken when immunological tests other than immunoblotting are used for detection of Clostridium difficile enterotoxin.

Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc.

This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin. This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.

Cytotonic effect ofClostridium difficileenterotoxin on Chinese hamster ovary cells

Abstract The effect of purified enterotoxin produced by Clostridium difficile on Chinese hamster ovary (CHO) cells was examined. In a certain concentration range (0.9–3.6 μg/ml), purified toxin caused CHO elongation, namely a cytotonic effect, which is similar to a typical morphological change in CHO cells induced by cholera enterotoxin. At a higher concentration, purified enterotoxin had a cytotoxic effect on CHO cells which was neutralized by anti- C. difficile enterotoxin, but not by anti- C. difficile cytotoxin. Thus, enterotoxin had both cytotonic and cytotoxic effects on CHO cells. About 60 and 180 min were required for binding of enterotoxin to CHO cells, and its internalization, respectively, both times being much longer than those for C. difficile cytotoxin.

Cytotonic effect of Clostridium difficile enterotoxin on Chinese hamster ovary cells

The effect of purified enterotoxin produced by Clostridium difficile on Chinese hamster ovary (CHO) cells was examined. In a certain concentration range (0.9–3.6 μ g/ml), purified toxin caused CHO elongation, namely a cytotonic effect, which is similar to a typical morphological change in CHO cells induced by cholera enterotoxin. At a higher concentration, purified enterotoxin had a cytotoxic effect on CHO cells which was neutralized by anti-C. difficile enterotoxin, but not by anti-C. difficile cytotoxin. Thus, enterotoxin had both cytotonic and cytotoxic effects on CHO cells. About 60 and 180 min were required for binding of enterotoxin to CHO cells, and its internalization, respectively, both times being much longer than those for C. difficile cytotoxin.

薬剤関連出血性腸炎 (主として&lt;I&gt;Klebsiellla oxytoca&lt;/I&gt;分離例) について

Klebsiella oxytoca was detected from all of the bloddy stool specimens collected froni 11 patients with antibiotic-associated colitis in acute phase and the mean log10 number was 7.1 per gram of feces. The organisms were detected from 5 of 7 formed stool in convalescence with the mean log10 number of 5.5 per gram of feces.Clostridium difficile was not detected from any of the specimens, while C. difficile enterotoxin was detected from 2 of 10 bloody stool in acute phase. These two cases were diagnosed as antibioticassociated hemorrhagic colitis from clinical features and laboratory findings.The intestinal microflora of the acute phase showed a striking decrease in the total count and a simplification of bacterial groups as compared to that in the convalescence. In particular, the vaiable counts of the obligate anaerobes showed remarkable decrease so that some members were undetectable.

Detection of Clostridium difficile enterotoxin in neonates by latex agglutination.

Clostridium difficile enterotoxin (D-1) was detected in 13 symptomatic and nine asymptomatic neonates in the neonatal intensive care unit by latex agglutination test but was not found in 18 healthy neonates in two other newborn nurseries. Environmental contamination in the intensive care unit may have been the cause. An association between the presence of enterotoxin and clinical symptoms is discussed.