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Detection of Clostridium difficile and its toxin A (TcdA) in stool specimens from hospitalised patients.

The study has been carried out to determine the frequency of C. difficile recovery in stool cultures and the rate of C. difficile toxin A detection in faecal specimens of patients with nosocomial diarrhoea. Clinical specimens comprised 4414 stool samples collected from 1998 to 2002 from adult patients hospitalised in different wards of a university-affiliated hospital (1200 beds) and suspected of C. difficile-associated disease (CDAD). There have been 1308 (29.6%) specimens positive for C. difficile culture (15.1% in 1998, 29.5% in 1999, 33.8% in 2000, 31.2% in 2001 and 32.0% in 2002). The highest number of C. difficile strains was cultured from stool samples of patients hospitalised in the haematology/oncology ward (51.1% of all isolates), neurology (8.3%), nephrology (8.0%), gastrointestinal surgery (7.0%) and neurosurgery (6.2%) wards. The testing for C. difficile toxin A yielded 847 (19.2%) positive samples and 3567 (80.8%) toxin A-negative results. The percentage of C. difficile toxin A-positive samples was 29.4% in 1998, 17.5% in 1999, 23.2% in 2000, 17.1% in 2001 and 15.0% in 2002. In the analysed period we observed an increase in the number of stool specimens tested for C. difficile and an increase in the number of C. difficile culture-positive samples. A decrease in the number of C. difficile toxin A-positive samples was noted in the last 2 years of the study. This phenomenon may be due to an improved antibiotic policy of the hospital.

Anti-Bacterial Agents↗

Clostridium difficile in a department of surgery.

The colonization and transmission of Clostridium difficile strains was investigated at the Department of Surgery. Anaerobes were isolated from fecal samples of 27 patients among 308 investigated (mean age: 59.5 years). The majority of them (22 out of 27 patients) received anti-microbial agents prior to investigation for Clostridium difficile. Eighteen of them had undergone various surgical procedures including laparotomies. Only four Clostridium difficile positive patients had diarrhea, but pseudomembranes were not observed endoscopically. The highest colonization rate of C. difficile (20/27) was found to occur in autumn. All 54 samples from medical personnel were C. difficile free. C. difficile was isolated in 7% of samples taken from hospital environment. Toxigenicity of Clostridium difficile strains isolated from fecal samples taken from patients and from environment was 78% and 80%, respectively, as determined by cytotoxicity tissue assay, or 70% and 20%, respectively, in the latex agglutination test.

Adult↗

Diarrhea and Clostridium difficile-associated diarrhea on a surgical service.

OBJECTIVE: To identify the incidence, risk factors, and treatment of diarrhea and Clostridium difficile-associated diarrhea (CDAD) in surgery patients. DESIGN: Prospective and historical retrospective analysis. SETTING: Major urban tertiary care referral hospital. PATIENTS: Consecutive patients (N = 475) admitted to the vascular, trauma, and general surgical surgery services, prospectively evaluated during a 10-week period. A retrospective historical control of the same surgical services was used for comparison. INTERVENTION: None. MAIN OUTCOME MEASURES: Incidence of diarrhea and CDAD, use of bowel preparations, surgical procedure, use of C difficile toxin assay, white blood cell count, symptoms, treatment, and delay in hospital discharge. RESULTS: The incidence of diarrhea in surgery patients analyzed prospectively was 6.1%; the incidence of CDAD during the prospective and retrospective periods was 2%. Preoperative bowel preparations were associated with an increased risk of diarrhea (relative risk, 4.2; 95% confidence interval, 2.6-6.8; P < .001) and CDAD (relative risk, 3.2; 95% confidence interval, 1.5-7.2; P < .03). Leukocytosis (white blood cell count > 11 x 10(9)/L) was significantly higher in the CDAD group compared with the diarrhea group only on the day of diagnosis (P < .05). By subjective analysis, diarrhea was directly responsible for a delay in discharge in 7 of 29 patients for a mean (+/-SEM) of 4.0 +/- 1.0 days. CONCLUSIONS: Patients undergoing preoperative bowel preparations are at increased risk of experiencing diarrhea and CDAD. Among patients with diarrhea, an elevated white blood cell count may help identify those with C difficile. Early treatment of diarrhea with oral metronidazole while awaiting the results of the stool toxin assay is recommended for treating diarrhea in surgery patients. Prophylactic treatment of surgery patients undergoing bowel preparations should be considered.

Aged↗

Antecedent use of fluoroquinolones is associated with resistance to moxifloxacin in Clostridium difficile.

OBJECTIVE: Moxifloxacin is characterized by high activity against Gram-positive cocci and some Gram-positive and -negative anaerobes, including Clostridium difficile. This study investigates the role of prior quinolone use in relation to patterns of susceptibility of C. difficile to moxifloxacin. METHODS: Sixty-three clinical isolates of C. difficile were investigated for toxigenicity, susceptibility to moxifloxacin, and mutations in the DNA gyrase gene. The medical histories for 50 of these patients were available and used to identify previous fluoroquinolone use. RESULTS: Thirty-three (52.4%) strains showed resistance to moxifloxacin (MICs > or = 16 mg/L). All moxifloxacin-resistant strains harbored a mutation at amino acid codon Ser-83 of gyrA. Forty-five isolates (71.4%) were toxigenic; all moxifloxacin-resistant strains were in this group. Resistance to moxifloxacin was associated with prior use of fluoroquinolones (P-value 0.009, chi-square). CONCLUSIONS: Although the use of moxifloxacin to treat C. difficile-associated diarrhea is not likely to be common, these data show a relationship between antecedent fluoroquinolone use and resistance to moxifloxacin in C. difficile isolates, and raise questions regarding selection pressure for resistance placed on colonizing bacteria exposed to fluoroquinolones. Mutations in gyrA are involved in moxifloxacin resistance.

Anti-Infective Agents↗

Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals.

BACKGROUND: Large outbreaks of diarrhea caused by a newly recognized strain of Clostridium difficile occurred in four hospitals located in different parts of the United States between 1989 and 1992. Since frequent use of clindamycin was associated with the outbreak in one of the hospitals, we examined the resistance genes of the epidemic-strain isolates and studied the role of clindamycin use in these outbreaks. METHODS: Case-control studies were performed at three of the four hospitals to assess the relation of the use of clindamycin to C. difficile-associated diarrhea. All isolates of the epidemic strain and representative isolates of other strains identified during each outbreak were tested for susceptibility to clindamycin. Chromosomal DNA from these representative isolates was also analyzed by dot blot hybridization and amplification with the polymerase chain reaction (PCR) with the use of probes and primers from a previously described determinant of erythromycin resistance - the erythromycin ribosomal methylase B (ermB) gene - found in C. perfringens and C. difficile. RESULTS: In a stratified analysis of the case-control studies with pooling of the results according to the Mantel-Haenszel method, we found that the use of clindamycin was significantly increased among patients with diarrhea due to the epidemic strain of C. difficile, as compared with patients whose diarrhea was due to nonepidemic strains (pooled odds ratio, 4.35; 95 percent confidence interval, 2.02 to 9.38; P<0.001). Exposure to other types of antibiotics or hospitalization in a surgical ward was not significantly associated with the risk of C. difficile-associated diarrhea due to the epidemic strain. All epidemic-strain isolates were highly resistant to clindamycin (minimal inhibitory concentration, >256 microg per milliliter). DNA hybridization and PCR analysis showed that all these isolates had an ermB gene, which encodes a 23S ribosomal RNA methylase that mediates resistance to macrolide, lincosamide, and streptogramin antibiotics. Only 15 percent of the nonepidemic strains were resistant to clindamycin. CONCLUSIONS: A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.

Anti-Bacterial Agents↗

12th C. L. Oakley lecture. Pathogenesis of Clostridium difficile infection of the gut.

On the basis of the above findings it is possible to propose a sequence of events following exposure to C. difficile. Exposure of neonates to C. difficile leads to transient colonisation which is almost invariably asymptomatic; the reasons why colonisation is asymptomatic are not known. Exposure of antibiotic-treated adults to C. difficile does not invariably lead to colonisation; however, in those instances where colonisation occurs, it may be transient and asymptomatic or transient and symptomatic. The transient nature of the colonisation could be because the infecting strain is poorly virulent, or because the degree of compromise of the intestinal flora is insufficient to permit establishment and full expression of virulence. It is likely that it is easier to fully compromise the intestinal flora of the elderly so that they more readily become fully susceptible to colonisation by C. difficile. In a fully susceptible host and with a highly virulent strain, the following sequence of events could occur. The organism may associate with the intestinal mucosa possible via fimbriae, and form a microcolony of capsulate cells protected by a glycocalyx. The toxins, or other factors, produced may facilitate the interaction with mucosa and toxin A will result in increased vascular and mucosal permeability resulting in intra-luminal accumulation of serum-albumin-rich fluid. Although C. difficile does not appear to be capable of using serum albumin nutritionally, it may utilise other serum proteins, and the serum proteins in general may compete with host proteases and help prevent degradation of the toxins produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture.

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was conducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhesion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins. Indeed, adhesion to Caco-2 cell monolayers of C. difficile strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.

Antibodies, Bacterial↗

[Detection of Clostridium difficile toxin A from stool specimens by an enzyme immunoassay kit].

Toxin detection from stool specimens is prerequisite for Clostridium difficile-associated diarrhea and colitis. However, in Japan only one toxin detection kit is commercially available, which requires computerized VIDAS fluorescence reader. In this study we evaluated ImmunoCard Toxin A, which is an enzyme immunoassay with a format of individual cassette and needs no special equipment to perform, by comparing with the VIDAS CDA kit. Of 61 stool specimens 12 were positive and 39 were negative by both assays, 7 were VIDAS positive-ImmunoCard negative, 2 were VIDAS invalid-ImmunoCard negative, and 1 was invalid by both assays. Stool specimens which gave inconsistent results between two assays were subjected to cell culture assay to detect toxin B and culture for C. difficile followed by testing of toxin producibility of isolates. Of 7 VIDAS positive-ImmunoCard negative specimens 5 were negative for cell culture assay and toxigenic C. difficile culture and 2 were positive for cell culture assay. By omitting 3 VIDAS invalid specimens and counting 5 specimens, which were cell culture negative but VIDAS positive, as negative and 2, which were cell culture positive and VIDAS positive, as positive, 12 of the 14 VIDAS positive specimens were ImmunoCard positive (sensitivity, 85.7%) and all 44 VIDAS negative were ImmunoCard negative (specificity, 100%). These results indicate that although in comparison with VIDAS CDA, ImmunoCard Toxin A is slightly less sensitive, ImmunoCard is a rapid, simple, and reliable C. difficile toxin A detection kit, which has the advantage of requiring no special equipment to perform and no centrifuge step, as small as 25 muL of a specimen, and as short as approximately 15 min of time to complete the whole testing.

Bacterial Toxins↗

GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis.

GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with C. difficile, whereas cholestyramine protected only 10% of animals. Treatment of C. difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of C. difficile disease in humans.

Animals↗

Clostridium difficile brain empyema after prolonged intestinal carriage.

Clostridium difficile, the most common cause of antibiotic-associated diarrhea, is occasionally isolated from extraintestinal sites and is usually found as part of a polymicrobial flora. We report a case of brain empyema that occurred after the recurrent intestinal carriage of a nontoxigenic strain of C. difficile. Brain abscess cultures contained both toxigenic and nontoxigenic isolates. Pulsed-field gel electrophoresis showed that nontoxigenic isolates from the intestine and from the brain were identical.

Brain Diseases↗

Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification.

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus.

Bacterial Proteins↗

[Recurrent fatal pseudomembranous colitis].

BACKGROUND: Clostridium difficile pseudomembranous colitis may trace a fulminent course and require surgery. CASE REPORTS: Outcome was fatal despite subtotal colectomy in the reported case of recurrent Clostridium difficile pseudomembranous colitis. Infrequent localization, low serum albumin and an unfavorable clinical course were observed. DISCUSSION: Rapid surgical treatment with large resection is mandatory in such cases, particularly in patients of rectal and sigmoid remnant involvement.

Adult↗

Cytomegalovirus and Clostridium Difficile co-infection in severe ulcero-hemorrhagic colitis during induction chemotherapy for acute lymphoblastic leukemia.

Here we describe the first case of a biopsy-proven Cytomegalovirus ulcero-hemorrhagic colitis, associated with Clostridium Difficile co-infection, occurring during standard induction chemotherapy for common B cell acute lymphoblastic leukemia. We discuss the case and focalize clinical management and diagnostic issues arising from it.

Adult↗

Clostridium difficile typhlitis associated with cecal mucosal hyperplasia in Syrian hamsters.

A sudden increase in mortality occurred in a closed breeding colony of Syrian hamsters (Mesocricetus auratus). The colony consisted of approximately 40 hamsters, 8 of which were affected. Four adult males died suddenly. One pregnant female and one weanling died after having been observed as depressed for 1 day and 2 weeks respectively. One weanling and one adult male were euthanized. All affected hamsters had signs of diarrhea. At necropsy, hemorrhagic fluid-filled ceca were noted in five of eight animals. Clostridium difficile cytotoxin B was present in high titers [10(-3) to 10(-8)] in cecal contents of six of six animals tested, whereas C. difficile culture yielded positive results in only one of six animals. Histopathologically, findings consistent with Clostridium-induced typhlitis including necrosis, epithelial denudation, vascular congestion, and hemorrhage were present in six of six ceca evaluated. In addition, signs of a more chronic disease process included cecal mucosal hyperplasia in five of six hamsters. A silver stain of cecal hyperplastic mucosa for intracellular organisms including Campylobacter-like organisms was negative in all affected hamsters. Antibiotics had not been administrated to any hamster in this colony, nor had the affected animals been experimentally manipulated. Testing for antibiotic residues in the feed was negative, and C. difficile was not isolated from feed, water, or feces of unaffected hamsters. Thus C. difficile-induced typhlitis should be included in the differential diagnosis of deaths in hamsters which have no clinical histories of prior antibiotic administration or experimental manipulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Clostridium difficile and older adults: what primary care providers should know.

Clostridium difficile poses particular risk for older adults, who are subject to more serious symptoms than younger patients. Antibiotic exposure and other risk factors are associated with the pathogenesis of C. difficile-associated disease. Treatment goals include prescribing anti-C. difficile activity agents (when indicated), attending to volume status and prescribing oral rehydration therapy as needed, avoiding the use of antiperistaltic drugs, discontinuing any offending antibiotics, avoiding the indiscriminate use of broad-spectrum antibiotics, providing supportive therapy, following infection control protocols, and eliminating environmental contaminants.

Clinical Competence↗

Use of pulsed-field gel electrophoresis for investigation of an outbreak of Clostridium difficile infection among geriatric patients.

A six-month outbreak of Clostridium difficile infection among elderly residents of a middle-term-care facility was investigated. Pulsed-field gel electrophoresis was used to genotype 22 outbreak strains and 30 epidemiologically unrelated strains. A prospective case-control study was conducted to identify risk factors for epidemic Clostridium difficile-associated diarrhea. All epidemiologically unrelated Clostridium difficile strains of the same serogroup could be differentiated by their DNA patterns with two restriction enzymes (SmaI and KspI). Among clustered strains, two epidemic serogroups (C and K) were identified. Two different DNA patterns were identified among serogroup C strains and three among serogroup K strains. Multivariate analysis showed that the risk of Clostridium difficile infection increased with antimicrobial chemotherapy (beta-lactam agents and pristinamycin) and the presence of a feeding tube. This study confirms the high discriminative power of restriction fragment length polymorphism analysis by pulsed-field gel electrophoresis to describe Clostridium difficile epidemiology. The typing results confirm that infection was principally exogenous in this outbreak. Furthermore, they indicate the need to improve all measures limiting transmission of infection.

Aged↗

Splenic abscess caused by Clostridium difficile.

Splenic abscess is a previously unreported complication of Clostridium difficile colitis. A case of Clostridium difficile splenic abscess is reported that developed after the patient had been in intensive care for five weeks. A response was seen to radiologically guided drainage and antibiotic therapy with formal laparotomy, and surgical drainage not being required.

Abscess↗