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PubMed1994

Immunoglobulin G directed against toxins A and B of Clostridium difficile in the general population and patients with antibiotic-associated diarrhea.

Serum immunoglobulin G (IgG) class antibodies directed against toxins A and B of Clostridium difficile were studied using an enzyme-linked immunosorbent assay and a serum-neutralizing assay based on the MRC-5 tissue cytotoxicity assay. Of 185 individuals, 46 sera (24%) in the general population demonstrated IgG antibody, 36 (19.4%) against toxin A and 15 (8.1%) against toxin B. Antibody titer in the general population did not correlate with serum-neutralizing activity. Antibody prevalence fell with age (P = 0.58) over 50 years. Six of ten patients with acute primary episodes of C. difficile-associated diarrhea demonstrated antibody in convalescent-phase sera, predominantly directed against toxin B. Only two (28%) of seven patients with a history of relapsing C. difficile disease had demonstrable antibody.

Adolescent↗

PubMed1996

Clinical prediction rules to optimize cytotoxin testing for Clostridium difficile in hospitalized patients with diarrhea.

BACKGROUND: Although routine testing of hospitalized patients with diarrhea for Clostridium difficile cytotoxin has been advocated as a high-yield procedure, the rationale for this practice has been questioned. To target a low-yield subgroup for whom routine testing could be deferred, we derived a clinical decision rule for predicting results of the C difficile cytotoxin assay in hospitalized adults with diarrhea. METHODS: We hypothesized a priori that two variables, antibiotic use (within 30 days prior to testing) and history of significant diarrhea (new onset of > 3 partially formed or watery stools per 24 hour period), would be highly predictive of cytotoxin results, and obtained these data on 480 consecutive patients who underwent diagnostic testing for C difficile at a university hospital and affiliated Veterans Affairs medical center. For more detailed modelling, we recorded symptoms, signs, comorbidity, and other potential causes of diarrhea for 68 test positive patients (cases) and 265 randomly selected test negative patients (controls) within the study cohort. RESULTS: The overall prevalence of positive cytotoxin assays was 14%. Prior antibiotic therapy (OR = 9.0, 95% CI 2.1-38.4), significant diarrhea (OR = 2.2, 95% CI 1.1-4.7), and abdominal pain (OR = 1.9, 95% CI 0.96-3.7) were independent predictors of cytotoxin assay results. The model discriminated patients with positive and negative assays with a receiver operating characteristic (ROC) area of 0.68; observed and predicted probabilities of a positive cytotoxin assay were well correlated over the entire range of observed probabilities (r2 = 0.86). A decision rule (defined as positive if prior antibiotic use and either significant diarrhea or abdominal pain are present) demonstrated sensitivity and specificity of 86 and 45%. When applied to the entire dataset (N = 480), a simplified a priori rule, defined as positive if both prior antibiotic use and history of significant diarrhea are present, demonstrated sensitivity, specificity, positive and negative predictive value of 80, 45, 18 and 94%, respectively (6% of those predicted to be cytotoxin-negative actually tested positive). Use of this rule would have averted 39% of cytotoxin assays in our study population. CONCLUSIONS: Patients without prior antibiotic use and either significant diarrhea or abdominal pain are unlikely to have positive C difficile cytotoxin assay results, and may not routinely require cytotoxin testing.

Abdominal Pain↗

PubMed2000

Comparison of the TOX A/B test to a cell culture cytotoxicity assay for the detection of Clostridium difficile in stools.

The TOX A/B Test (Techlab, Blacksburg, VA, USA) was compared to cell culture cytotoxicity assay on 1109 consecutive diarrheal stool samples collected from patients with the presumptive diagnosis of Clostridium difficile disease. The TOX A/B Test is an enzyme immunoassay in a microtiter format that detects both toxins A and B. The procedure used for this study takes approximately 1.5 h to perform. Cell culture cytotoxicity was performed by using a fibroblast cell line in a microtiter format read at 4 h, 24 h, and 48 h. One hundred ninety-four of the 1109 samples were positive by the "gold standard" cytotoxicity assay, whereas 189 were positive by EIA. There was a 98.5% agreement between the two assays. When compared to the cytotoxicity assay, the EIA had an initial sensitivity of 94.3% and a specificity of 99.3%. However, after resolution of six discrepants using another ELISA for toxin A detection the sensitivity, specificity, positive and negative predictive values for the TOX A/B test are as follows: 94.5%; 100%; 100%; 98.8%. The corresponding values for the cytotoxicity assay are: 97%; 100%; 100%; and 99.3%. This test seems to have excellent sensitivity and specificity as compared to an in-house cell culture cytotoxicity assay. It is sensitive enough to use as a stand-alone test for the detection of C. difficile toxin in laboratories that do not have cell culture cytotoxicity testing capability.

Cell Culture Techniques↗

PubMed2005

Crystal structure of receptor-binding C-terminal repeats from Clostridium difficile toxin A.

Clostridium difficile is a major nosocomial pathogen that produces two large protein toxins [toxin A (TcdA) and toxin B (TcdB)] capable of disrupting intestinal epithelial cells. Both belong to the family of large clostridial cytotoxins, which are characterized by the presence of a repetitive C-terminal repetitive domain (CRD). In TcdA, the CRD is composed of 39 repeats that are responsible for binding to cell surface carbohydrates. To understand the molecular structural basis of cell binding by the toxins from C. difficile, we have determined a 1.85-A resolution crystal structure of a 127-aa fragment from the C terminus of the toxin A CRD. This structure reveals a beta-solenoid fold containing five repeats, with each repeat consisting of a beta-hairpin followed by a loop of 7-10 residues in short repeats (SRs) or 18 residues in long repeats (LRs). Adjacent pairs of beta-hairpins are related to each other by either 90 degree or 120 degree screw-axis rotational relationships, depending on the nature of the amino acids at key positions in adjacent beta-hairpins. Models of the complete CRDs of toxins A and B suggest that each CRD contains straight stretches of beta-solenoid composed of three to five SRs that are punctuated by kinks introduced by the presence of a single LR. These structural features provide a framework for understanding how large clostridial cytotoxins bind to cell surfaces and suggest approaches for developing novel treatments for C. difficile-associated diseases by blocking the binding of toxins to cell surfaces.

Amino Acid Sequence↗

PubMed2002

Clostridium difficile in long-term-care facilities for the elderly.

Antimicrobial agents are among the most frequently prescribed medications in long-term-care facilities (LTCFs). Therefore, it is not surprising that Clostridium difficile colonization and C. difficile-associated diarrhea (CDAD) occur commonly in elderly LTCF residents. C. difficile has been identified as the most common cause of non-epidemic acute diarrheal illness in nursing homes, and outbreaks of CDAD in LTCFs have also been recognized. This position paper reviews the epidemiology and clinical features of CDAD in elderly residents of LTCFs and, using available evidence, provides recommendations for the management of C. difficile in this setting.

Aged↗

PubMed2005

Spatial and temporal analysis of Clostridium difficile infection in patients at a pediatric hospital in California.

OBJECTIVE: To examine the usefulness of temporal and spatial analysis in identifying nosocomial transmission of Clostridium difficile among pediatric patients hospitalized on four wards at The Children's Hospital of Central California from September 8, 1998, to January 16, 1999. DESIGN: Stool specimens obtained from the clinical microbiology laboratory during the study period were tested by culture and latex agglutination for C. difficile. Polymerase chain reaction was used to identify toxin genes. Isolates obtained were mapped to a grid for each ward and were analyzed using the Knox test. Results were compared with DNA fingerprints generated by arbitrarily primed polymerase chain reaction. RESULTS: Total occupancy of these 4 wards was 438 during the study period. Stool specimens were available for 256 (58%) of these patients, yielding 67 C. difficile isolates and generating 2,211 case pairs for analysis by the Knox test. After stratification by toxin status, 5 clustered pairs of toxigenic isolates were identified on 1 of the wards by this method. Fingerprint analysis identified 4 clusters with indistinguishable banding patterns on 2 of the 4 wards. Two of the identified clusters were toxigenic and 2 were nontoxigenic. None of these clusters corresponded to clusters identified by the Knox test. CONCLUSIONS: The Knox test is an ineffective method for identifying cases resulting from nosocomial transmission of C. difficile in a pediatric setting due to the persistence of C. difficile spores and the unique environment of a pediatric hospital. Molecular analysis remains the most effective method.

California↗

PubMedDate not supplied

Factors associated with failure of metronidazole in Clostridium difficile-associated disease.

GOAL: To identify patients likely to fail metronidazole as initial treatment of C. difficile infection. BACKGROUND: For moderate to severe Clostridium difficile-associated diarrhea, metronidazole is the drug of choice for treatment. Oral vancomycin is given to patients who fail metronidazole or have intolerable side effects. STUDY: Retrospective review identified all patients treated for C. difficile-associated diarrhea during hospitalization from January 2000 to September 2001. C difficile was documented by a positive toxin assay or pseudomembranes on colonoscopy. Metronidazole failure was defined as persistent symptoms of C. difficile-associated diarrhea after 5 days of uninterrupted therapy. Response was defined as improvement in symptoms at day 5 of therapy including reduction of diarrhea to <or=2 bowel movements per day. RESULTS: 119 C. difficile-associated diarrhea patients were identified, and 99 met inclusion criteria. There were 61 (62%) metronidazole responders and 38 (38%) treatment failures. Albumin <2.5g/l and intensive care unit stay at/prior to diagnosis were the only variables associated with treatment failure. The odds ratios for treatment failure were 11.7 (95% confidence interval: 4.0-31.6) and 4.1 (95% confidence interval: 1.3-12.2), respectively. When considering these 2 variables together (low albumin, intensive care unit care), the area under the receiver operating characteristic curve was 0.80 for predicting treatment failure. CONCLUSIONS: Albumin level <2.5g/l and intensive care unit stay were predictors of failure of metronidazole therapy for C. difficile-associated diarrhea. These patients may benefit from oral vancomycin therapy at outset.

Adult↗

PubMed1991

Clostridium difficile in inflammatory bowel disease relapse.

Stools of 65 patients with exacerbation of symptoms of inflammatory bowel disease were examined for the presence of enteric pathogens and Clostridium difficile. Ten (16%) had C. difficile toxin. Symptoms in all patients cleared after therapy, with improvement correlating with elimination of toxin from the stool.

Adolescent↗

PubMed2006

Pitfalls in the diagnosis of enteroviral infection in young children.

BACKGROUND: Three infants with enteroviral infections were admitted to a neonatal intensive care unit (NICU). Investigation into the possible nosocomial spread of enterovirus identified 31 other infected infants. Only 1 of the 31 was truly positive; 30 were false-positive results. METHODS: This was a retrospective observational study. Investigational laboratory studies were conducted to identify problems in diagnostic procedures. RESULTS: Stool cultures were used to identify asymptomatic infections. Investigation identified several problems in the procedures in identification of enterovirus: (1) stool specimens were not properly filtered, (2) insufficient number of tissue culture cell types were used, (3) nonspecific toxicity due to stool and the presence of Clostridia difficile toxin was responsible for cytopathic effects in tissue culture, (4) the failure to block nonspecific fluorescent staining led to misidentification of enteroviruses. CONCLUSIONS: The use of stool cultures to identify asymptomatic enteroviral infections in infants in an NICU can be fraught with problems because of high frequency of C. difficile toxin and the nonspecific toxic effects of stool on tissue culture cells. These problems can be rectified when proper procedures are followed.

Clostridioides difficile↗

PubMed2005

Detection of binary-toxin genes (cdtA and cdtB) among Clostridium difficile strains isolated from patients with C. difficile-associated diarrhoea (CDAD) in Poland.

Clostridium difficile A+ B+ and A- B+ strains isolated from stool samples of patients with C. difficile-associated diarrhoea (CDAD) were selected from the University Hospital Warsaw collection. The binary-toxin genes cdtA and cdtB were detected by PCR in five of the 41 A+ B+ strains tested, but in none of the 17 A- B+ strains tested, giving 8.6 % prevalence (5/58) of binary-toxin-positive strains. All of the strains that were positive for binary-toxin genes were grouped into toxinotype IV, suggesting that in this institution toxinotype IV might dominate among the population of C. difficile with binary-toxin genes.

Adult↗

PubMed2002

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins.

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.

Bacterial Adhesion↗

PubMed1998

Effects of toxin A from Clostridium difficile on mast cell activation and survival.

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-alpha). In contrast, 1 microg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 microg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-alpha upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

Animals↗

PubMed2002

Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea, a potentially serious condition frequently affecting elderly hospitalized patients. While tissue damage is primarily induced by two toxins, the mechanism of gut colonization, and particularly the role of bacterial adherence to the mucosa, remains to be clarified. Previous studies have shown binding of C. difficile whole cells to cultured cell lines and suggested the existence of multiple adhesins, only one of which has been molecularly characterized. In this paper, we have investigated tissue binding of C. difficile surface layer proteins (SLPs), which are the predominant outer surface components and are encoded by the slpA gene. The adherence of C. difficile to HEp-2 cells was studied by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis, which showed that antibodies to the high-molecular-weight (MW) SLP inhibited adherence. Immunohistochemical analysis of human gastrointestinal tissue sections revealed strong binding both to the surface epithelium lining the digestive cavities and to the subjacent lamina propria, while glands were negative. A similar pattern was observed in the mouse. By using purified recombinant SLPs, we show that binding is largely mediated by the high-MW SLP. By Western blotting analysis, we have identified two potential ligands of the C. difficile SLPs, one of which may be specific to the gut. By using purified extracellular matrix components immobilized on nitrocellulose, we also show SLP binding to collagen I, thrombospondin, and vitronectin, but not to collagen IV, fibronectin, or laminin. These results raise the possibility that the SLPs play a role both in the initial colonization of the gut by C. difficile and in the subsequent inflammatory reaction.

Adolescent↗

PubMed1991

Identification of toxigenic Clostridium difficile by the polymerase chain reaction.

Toxigenic strains of Clostridium difficile are causative agents of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis. The toxigenicity is routinely assayed by using highly sensitive cell cultures. We used a simple and rapid polymerase chain reaction (PCR) assay to differentiate toxigenic and nontoxigenic strains of C. difficile. Two sets of oligonucleotide primer pairs derived from nonrepeating sequences of the toxin A gene were used to amplify 546- and 252-bp DNA fragments. A primer pair derived from repeating sequences of the toxin A gene was used to amplify a 1,266-bp DNA product. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 35 cytotoxic strains of C. difficile tested generated the expected amplified DNA. In contrast, none of the 26 noncytotoxic strains tested gave positive results. Although the toxins of C. difficile have been demonstrated to cross-react serologically with the toxins of Clostridium sordellii, we did not detect any amplified DNA in two cytotoxic strains or seven noncytotoxic strains of C. sordellii. PCR was negative in all 30 strains of 20 other Clostridium species. Southern hybridization of HindIII-digested genomic DNA by use of subgenomic probes showed a single hybridization band in toxigenic strains but not in nontoxigenic strains. PCR appears to be a sensitive and specific assay for the rapid identification of toxigenic C. difficile. Nontoxigenic C. difficile appeared to lack the C. difficile toxin A gene.

Bacterial Proteins↗

PubMed1994

Investigation of an outbreak of Clostridium difficile infection in a general hospital by numerical analysis of protein patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

One hundred forty-five cultures of Clostridium difficile, including strains from an apparent nosocomial outbreak of infection, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. Each protein pattern was characterized by the presence of one to three dense bands which were highly reproducible. The first 100 strains (in chronological order) were used as the basis for a numerical analysis which divided the strains into 17 phenons (EP types 1 to 17). The protein patterns of the remaining 45 strains were identified to type by comparing their individual patterns against a data base made up of the protein patterns of the first 100 strains. EP type 1 was the most common, with 70 of 139 (50%) patient isolates having this pattern type, and it accounted for 26 of 35 strains (74%) from patients in a medical teaching ward from which the outbreak was believed to have originated. This type was also found as a high proportion of isolations in a number of other medical and oncology wards, but the majority of these isolates occurred subsequent to the isolations on the initial outbreak ward. This technique can therefore provide a method for tracing the possible spread of epidemic strains in hospitals and other institutions and may contribute to a better understanding of the epidemiology of C. difficile.

Adolescent↗

PubMed2000

Acute abdomen and Clostridium difficile colitis: still a lethal combination.

BACKGROUND: With the steadily prevalent appropriate and inappropriate use of antimicrobial agents, Clostridium difficile colitis has continued to be noticed as a common problem in hospitalized patients. The aim of this communication is to highlight a subset of C. difficile colitis patients who presented with an acute abdomen. METHODS: This is a retrospective study of 10 patients who underwent laparotomy for an 'acute abdomen' with an intraoperative or postoperative diagnosis of C. difficile colitis. RESULTS: All patients received antibiotics (mean 9.5 days) for other illnesses. The mean APACHE II score was 18.8 (range 8-25) and the mortality rate was 80%. Two patients had colostomies created. One patient underwent a subtotal colectomy, and another underwent a Hartmann procedure; the rest had a nontherapeutic procedure. CONCLUSION: We conclude that C. difficile colitis presenting as an 'acute abdomen' still represents a lethal entity. Patients who present with an 'acute abdomen', with a history of recent or current antibiotic intake, and without findings which mandate an exploration should have C. difficile colitis urgently excluded. Timely diagnosis of C. difficile colitis through bedside sigmoidoscopy or a CT scan could spare the critically ill patient an unneccessary and risky operation. Furthermore, if laparotomy is subsequently needed then having a preoperative diagnosis of C. difficile colitis will allow appropriate surgical therapy to be implemented.

Abdomen, Acute↗

PubMedDate not supplied

Saccharomyces cerevisiae fungemia in an elderly patient with Clostridium difficile colitis.

Saccharomyces boulardii is widely used as a probiotic compound and is generally thought to be safe. We report one case of fungemia caused by Saccharomyces cerevisiae occurring in an elderly patient treated orally with S. boulardii in association with vancomycin for Clostridium difficile colitis. We do not recommend administering this viable yeast particularly in debilited patient with active colitis.

Administration, Oral↗

PubMed2005

Steroid-refractory ulcerative colitis treated with corticosteroids, metronidazole and vancomycin: a case report.

BACKGROUND: Increasing evidence elucidating the pathogenic mechanisms of ulcerative colitis (UC) has accumulated and the disease is widely assumed to be the consequence of genetic susceptibility and an abnormal immune response to commensal bacteria. However evidence regarding an infectious etiology in UC remains elusive. CASE PRESENTATION: We report a provocative case of UC with profound rheumatologic involvement directly preceded by Clostridium difficile infection and accompanying fever, vomiting, bloody diarrhea, and arthritis. Colonic biopsy revealed a histopathology suggestive of UC. Antibiotic treatment eliminated detectable levels of enteric pathogens but did not abate symptoms. Resolution of symptoms was procurable with oral prednisone, but tapering of corticosteroids was only achievable in combination therapy with vancomycin and metronidazole. CONCLUSIONS: An infectious pathogen may have both precipitated and exacerbated autoimmune disease attributes in UC, symptoms of which could be resolved only with a combination of corticosteroids, vancomycin and metronidazole. This may warrant the need for more perceptive scrutiny of C. difficile and the like in patients with UC.

Adult↗