Clostridium difficile in haematological malignancy.

Twenty patients with haematological malignancies who developed Clostridium difficile bowel infection or colonisation are described. All isolates of C difficile were toxigenic in vitro and faecal cytotoxin (toxin B) was detected in 20/26 episodes. Ten of 20 episodes with detectable faecal cytotoxin were associated with typical antibiotic associated diarrhoea. In the other 10 episodes (nine patients), there was a severe unusual illness which was associated with detection of C difficile. The unusual features of the illness were pronounced jaundice (total bilirubin greater than or equal to 44 mumol/l), abdominal pain and distension, and initial constipation followed either by diarrhoea or by large bowel stasis. Four of these patients died within seven days. Bacteraemia was often a presenting feature in neutropenic patients subsequently shown to have C difficile. This was not the case in non-neutropenic patients. Bacteraemia was commonly polymicrobial and in two cases C difficile was isolated from blood culture. The clinical implications of recognition of this atypical C difficile associated syndrome are discussed.     

Clostridium difficile cytotoxin B in adults with diarrhea: a comparison of patients treated or not treated with antibiotics prior to infection

Objective   To study the detection rate of Clostridium difficile cytotoxin B in stool specimens from adults with diarrhea as related to previous antimicrobial treatment.
Methods   Stool specimens from 802 adult patients with diarrhea and 203 healthy controls were tested for C. difficile cytotoxin B using a cell cytotoxicity assay. Antibiotic susceptibility testing of C. difficile was performed with the E test.
Results   Of 173 patients treated with antimicrobial medication within 5 weeks of onset of diarrhea, 60 (35%) were positive for C. difficile cytotoxin B (group A) compared to only 41 (7%) of 629 untreated patients (group B) and two of the 203 (1%) healthy controls. Compared to patients in group A, patients in group B possessed characteristics not usually connected with C. difficile disease. They were generally younger (median age 40 years vs. 73 years), had been hospitalized less frequently (10% vs. 67%), had more often travelled abroad within the previous 2 weeks (46% vs. 1%), and more often had multiple enteropathogens (41% vs. 3%). Minimal inhibitory concentrations for vancomycin, metronidazole and fucidic acid to C. difficile isolates ranged from 0.5 to 4 mg/L, from 0.125 to 256 mg/L and 0.25 to 4 mg/L, respectively.
Conclusions   The detection rate of C. difficile cytotoxin B in patients with diarrhea, not associated with antibiotic treatment, is comparable to that in healthy control subjects. It probably merely reflects a carrier state without clinical significance.     

Evaluation of the Clearview Clostridium difficile Toxin A Test and various selective culture media in comparison with the cytotoxin assay for the diagnosis of Clostridium difficile-associated diarrhoea

AIM: Clostridium difficile is the major pathogen associated with nosocomial diarrhoea. We evaluated the performances of a commercially available toxin A enzyme immunoassay (EIA; Clearview C. difficile Toxin A Test), culture and tissue culture cytotoxin assay in the diagnosis of C. difficile-associated diarrhoea. METHODS: Comparative test performance was determined from data obtained from 166 faecal samples. The initial analysis compared the performance of toxin A EIA and culture with that of cytotoxin assay, this being defined as a 'laboratory gold standard'. A second analysis compared the individual performance of the toxin A EIA, culture and cytotoxin assay using a combined clinical and laboratory diagnostic assessment as a 'clinical gold standard'. In a parallel, study three selective culture media were compared. RESULTS: From the initial analysis, the sensitivity and specificity of the methods were, respectively, 84.6 and 65.4% for the toxin A EIA, and 38.5 and 93.5% for culture. From the second analysis, the sensitivity and specificity of the methods were, respectively, 100 and 67.5% for the toxin A EIA, 63.6 and 96.7% for culture and 72.7 and 98.0% for cytotoxin assay. Media containing d-cycloserine 250mg/L and cefoxitin 8mg/L performed best, growing 88.2% of the isolates. CONCLUSION: The toxin A EIA we evaluated had poor specificity in the diagnosis of C. difficile-associated diarrhoea. We conclude that in our laboratory the combination of culture and cytotoxin assay is a preferred approach to the diagnosis of C. difficile-associated diarrhoea.     

New selective medium for isolating Clostridium difficile from faeces.

AIMS: To compare CCFA (cycloserine, cefoxitin fructose agar) with a new selective medium CDMN (containing cysteine hydrochloride, norfloxacin, and moxalactam) for the isolation of Clostridium difficile after direct faecal culture. METHODS: The minimum inhibitory concentration (MIC) of norfloxacin was determined for 64 strains of C difficile, 17 strains of other Clostridium sp, and 66 various isolates of faecal origin, together with MIC determinations of moxalactam against the 81 strains of Clostridium sp and 15 isolates of Bacteroides sp. Using C difficile agar base with 0.5 g/l of cysteine hydrochloride, norfloxacin and moxalactam were incorporated into the medium and compared with CCFA for the isolation of C difficile after direct faecal culture. RESULTS: Norfloxacin (12 mg/l) inhibited the growth of enterobacteriaceae and faecal streptococci; moxalactam (32 mg/l) inhibited the growth of most strains of Bacteroides sp tested, together with Clostridium sp other than C difficile. Using the antibiotics in combination (CDMN), the growth and colonial morphology of 64 strains of C difficile were unaffected. When CDMN medium was compared with CCFA for the isolation of C difficile from 832 faeces from inpatients with diarrhoea, the CDMN agar isolated 20% more strains and reduced the number of contaminating colonies by 30%. CONCLUSIONS: CDMN both improves the isolation rate of C difficile from faecal specimens and reduces the growth of other organisms compared with CCFA.     

Prevalence of Clostridium difficile and its cytotoxin in infants in Mexico

The incidence of Clostridium difficile and its cytotoxic activity were determined in the feces of 122 children under 1 year of age. Samples were obtained from children receiving antibiotics and with (52 cases) or without (26 cases) diarrhea, from children with diarrhea who did not receive antibiotics (22 cases), and from healthy children (22 cases). Isolation of C. difficile in feces from children in all groups was similar (mean 23.4%) except for the group with non-antibiotic-associated diarrhea (4.5%). In both groups of children receiving antibiotics, with or without diarrhea, the cytotoxin was detected in 7.6% of the cases. In the group with non-antibiotic-associated diarrhea, none of the samples was positive for cytotoxicity. In healthy children, cytotoxin was positive in 4.5% of the cases.     

Colonization of the large bowel by Clostridium difficile in healthy infants: quantitative study.

Colonization of the large bowel of healthy infants by Clostridium difficile was studied. Feces were collected from five breast-fed aand five formula-fed infants throughout the first year of life, and levels of C. difficile were quantitated. Three breast-fed and five formula-fed infants were colonized for periods of between 8 and 42 weeks, and another infant harbored the organism only during week 1. Colonization of breast-fed infants commenced before or during weaning, with levels reaching 10(3) to 10(5) organisms per g of wet feces. Colonization of formula-fed infants commenced before solid foods were given, with levels of 10(3) to 10(7) organisms per g of wet feces. Isolates from eight of the babies were shown to produce cytotoxin in vitro. Single fecal specimens from 60 more children aged up to 4 years were also examined, and it was found that the carriage rate of C. difficile fell sharply after 1 year of age, although in the second year it was still higher than in adults. These findings are discussed in relation to the microbial ecology of the large bowel and the paradox that levels of C. difficile in the large bowel of healthy infants are similar to those causing pseudomembranous colitis in patients.     

Colonisation and transmission of Clostridium difficile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis

Healthy adults who had not been exposed to antimicrobial agents for the preceding 4 weeks were examined for intestinal carriage of Clostridium difficile. The 1234 individuals examined were composed of seven groups: three classes of university students, hospital workers at two hospitals, employees of a company and self-defence force personnel at a local station. Overall, 94 (7.6%) individuals were positive for C. difficile by faecal culture but carriage rates among the study groups ranged from 4.2% to 15.3%. Typing by PCR ribotyping and pulsed-field gel electrophoresis demonstrated clusters of carriers colonised by a single type in each of three groups, indicating that cross-transmission of C. difficile can occur in community settings. Follow-up culture was performed on 38 C. difficile-positive individuals and C. difficile was isolated again from 12 (32%) of them 5-7 months after the initial culture; six (50%) of these 12 individuals had a new strain on repeat culture. Two or more family members were C. difficile-positive in five of 22 families examined. C. difficile with an identical type was isolated from persons within a family in only one family. These results suggest that intestinal carriage by healthy adults may play a role as a reservoir for community-acquired C. difficile-associated diarrhoea, but that cross-transmission of C. difficile does not occur frequently among family members at home.     

Clostridium difficile and its cytotoxin in diarrhoeic stools of hospitalized patients. Toxigenic potential of the isolates

Cytotoxin assay and culture for Clostridium difficile were performed on 303 diarrhoeic stools from 261 hospitalized patients. Specimens from 42 patients were positive by at least one of the methods, and 40 of them had an antibiotic-associated diarrhoea. The cytotoxin assay was positive in 5 of 7 patients with pseudomembranous colitis. Thirteen had an appropriate response to specific therapy and the remainder have resolved of diarrhoea without C. difficile directed chemotherapy. These findings show the lack of reliability of the cytotoxin assay for the diagnosis of C. difficile antibiotic-associated diarrhoea. The 6 strains isolated from patients with pseudomembranous colitis were examined for enterotoxin by the rabbit ileal loop test: 4 produced both toxins, 2 only enterotoxin. Both toxins could therefore not be essential for the clinical expression of the disease.     

Is Campylobacter involved in antibiotic associated diarrhoea?

Campylobacter jejuni is an important cause of acute bacterial diarrhoea. In developing countries like India, children gain immunity early during infancy. However, the incidence is higher in non-immune hosts. Antibiotic use destabilizes the gut flora and can inhibit the local immune responses, thereby compromising resistance to a variety of infections. It is not yet known whether antibiotic intake can also precipitate C. jejuni enteritis as the infectious dose is low and attack rates are high. We made a preliminary study to determine the prevalence of C. jejuni in hospitalized patients receiving antibiotics for various ailments. One hundred and thirty eight stool samples submitted for Clostridium difficile toxin assay were additionally cultured for C. jejuni in blood-free campylobacter selectivity agar. All suspected colonies were subjected to Gram staining, oxidase, catalase and nalidixic acid sensitivity tests. Confirmation of C. jejuni was done by the hippurate hydrolysis test. Of the 138 faecal samples investigated, 14 (10.1%) grew C. jejuni and 11 of them belonged to adults. Two of these 14 samples were also positive for C. difficile toxin. Though not as yet reported, C. jejuni may also be involved in antibiotic associated diarrhoea due to lowered immunity in the host. It may cause enteritis either by itself or in synergy with C. difficile infection.     

Asymptomatic carriage of Clostridium difficile in patients with cystic fibrosis.

Faecal samples from 37 patients with cystic fibrosis and 40 control patients at the Brompton Hospital and the London Chest Hospital were examined for the presence of Clostridium difficile. The organism was isolated from 2 (17%) of control patients who were receiving antibiotics and from one (3.6%) of control patients who had no antimicrobial treatment. Thirty two per cent of the patients with cystic fibrosis excreted C difficile, though none of them had diarrhoea. Two of the three isolates from control patients and nine of the 12 isolates from patients with cystic fibrosis produced toxin B (cytotoxin) in vitro. Toxin B was present in the stools of one of the control patients and three of the patients with cystic fibrosis; toxin A (enterotoxin) was not detected in the faeces of the patients with cystic fibrosis. Two cytotoxigenic strains of C difficile isolated from patients with cystic fibrosis were examined in hamsters; both were virulent, and the animals died.     

The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces

AIMS:Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research methods may necessitate storage of faecal specimens for long periods, followed by subsequent re-examination. This study investigated the effects of storage conditions upon the viability of this organism and its toxin. METHODS: Three genotypically distinct strains of C difficile (two clinical isolates including the UK epidemic strain, and an environmental isolate) were grown anaerobically at 37 degrees C for 72 hours in a pool of five faecal emulsions. Aliquots of each emulsion were stored at either -20 degrees C (frozen) or 4 degrees C (refrigerated). Emulsions were assayed for viable cells, spores, and cytotoxin titre before storage and at days 1, 3, 5, 7, 14, 28, and 56. An aliquot of each emulsion was also removed, assayed, and replaced in storage at each time point to investigate the effects of multiple freezing/refrigeration/thawing. RESULTS: Neither storage temperature nor multiple cycles of refrigeration/freezing and thawing adversely affected the viability of C difficile vegetative cells or spores. Single and multiple exposures of samples to 4 degrees C had little effect upon the C difficile toxin titre. Toxin titres of multiply frozen and thawed faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 5 of the experiment in two of the three strains, and in all strains by day 28. Toxin titres of singly frozen faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 56 of the experiment in two of the three strains. CONCLUSION: Storage temperature and multiple cycles of freezing (refrigeration)/thawing had minimal effects upon the viability of C difficile or its spores. Storage at 4 degrees C has no discernible effect on C difficile cytotoxin. However, storage at -20 degrees C has a detrimental effect upon C difficile cytotoxin, and multiple cycles of freezing and thawing may further adversely effect toxin titres.     

Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin

A procedure was devised for routine examination of feces for Clostridium difficile with selective enrichment broth culture containing increased levels of carbohydrates and antibiotics to detect cytotoxin and volatile acids in broths inoculated with fecal samples. C. difficile was detected and identified with a rapidity comparable to that of conventional culture on selective cycloserine-cefoxitin fructose agar. Detection rates for C. difficile in inoculated broths (111/401 or 27%) were significantly higher than for culture on cycloserine-cefoxitin fructose agar (47/401 or 11%, P greater than 0.001). All fecal samples containing C. difficile and cytotoxin were correctly identified by the procedure. Isocaproic acid peak heights greater than 2 mm in selective enrichment broths inoculated with fecal samples indicated that C. difficile was present in the fecal sample examined. Of the positive specimens examined, 58% (64/111) produced peak heights greater than 10 mm. Peak heights less than 2 mm were not associated with C. difficile in the fecal sample. The investigated procedure provided a reliable alternative to the routine processing of feces for detecting C. difficile and associated cytotoxin in feces. Inoculated broths with isocaproic acid peak heights greater than 2 mm, after 24 to 48 h of incubation, and in which cytotoxin was detected, were subcultured to blood agar to obtain isolates of the organism as required. Broths which showed isocaproic acid peak heights less than 2 mm, and in which cytotoxin was not detected, were discarded as negative for C. difficile. The procedure was deemed potentially useful for epidemiological surveys of C. difficile.     

Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions.

Fecal specimens from 223 subjects were evaluated for the presence of Clostridium difficile by use of a selective medium developed in our laboratory and for the presence of C. difficile cytotoxin. C. difficile and cytotoxin were detected in 89 and 83%, respectively, of patients with antimicrobial agent-associated pseudomembranous colitis (PMC). In patients in whom PMC was not documented, C. difficile and cytotoxin were present in only 37 and 21%, respectively. C. difficile and cytotoxin were also recovered from the feces of 6 and 3, respectively, of 13 antimicrobial recipients who did not have diarrhea. Although C. difficile appears to be a major cause of PMC, it is not responsible for at least some two-thirds of cases of antimicrobial agent-associated diarrhea in which PMC is not documented. Neither the recovery of C. difficile nor the detection of its cytotoxin should be considered diagnostic for C. difficile-induced disease.     

Antibiotic-associated diarrhoea: an overlooked aetiology?

Sixty-three faeces samples from hospital in-patients with probable antibiotic-associated diarrhoea (AAD) are investigated. All samples are examined for routine bacterial enteric pathogens, pus cells, red blood cells and parasites. The samples are also screened for Clostridium difficile cytotoxin B (CDT), C. perfringens enterotoxin and Candida spp. (by microscopy and quantitative culture). Faecal samples from two control groups (healthy volunteers and community samples from GP patients) are also screened. A possible pathogen was found in 71% of AAD cases. Candida spp. overgrowth was the most common (44.4%), followed by CDT (34.9%) and Clostridium perfringens enterotoxin (9.5%). There was good agreement between the significant Gram films and quantitative Candida spp. culture (kappa=0.683). Clinical information revealed the majority of patients were on multiple antibiotic regimes, receiving 'high risk' antibiotics. Of community samples, 21% were positive for Clostridium perfringens enterotoxin, indicating that C. perfringens is a problem in the community, not necessarily associated with antibiotic use. The results suggest that quantitative Candida spp. culture should be performed on all specimens requesting AAD investigations.     

Evidence for antibiotic induced Clostridium perfringens diarrhoea

Clostridium difficile is a well documented cause of antibiotic associated diarrhoea in hospitalised patients, but may account for only approximately 20% of all cases. This leader reviews the current knowledge and understanding of the pathogenesis, epidemiology, and diagnosis of non-food borne Clostridium perfringens diarrhoea. Although enterotoxigenic C perfringens has been implicated in some C difficile negative cases of antibiotic associated diarrhoea, C perfringens enterotoxin detection methods are not part of the routine laboratory investigation of such cases. Testing for C perfringens enterotoxin in faecal samples from patients with antibiotic associated diarrhoea and sporadic diarrhoea on a routine basis would have considerable resource implications. Therefore, criteria for initiating investigations and optimum laboratory tests need to be established. In addition, establishing the true burden of C perfringens antibiotic associated diarrhoea is important before optimum control and treatment measures can be defined.     

An evaluation of a rapid latex test for the diagnosis of Clostridium difficile-associated diarrhea

We present here the results of an evaluation of a rapid latex test for detection of Cl. stridium difficile-associated in comparison with our standard cytotoxin assay and culture for C. difficile. Some 515 diarrheal stools were examined. C. difficile was cultured from 70 specimens (13.5%); 53 specimens (10.2%) were positive with the latex test, and 50 (9.6%) by cytotoxin assay. The latex test did not differ significantly from the cytotoxin assay in sensitivity or specificity compared to culture results. There was also no significant difference in the specificity of the latex test compared to cytotoxin assay in patients in whom the diagnosis of C. difficile-associated diarrhea was negative. Positive and negative predictive values of the latex test for C. difficile-associated diarrhea were similar to those of cytotoxin assay. The latex test thus appears to be a rapid and practical test for the laboratory diagnosis of C. difficile-associated diarrhea. To optimize specificity and sensitivity its use should be restricted to patients where the diagnosis is strongly suspected and a rapid answer is required. As it does not distinguish between toxigenic virulent C. difficile strains and non-toxigenic avirulent strains, it would seem prudent to confirm positive results subsequently by demonstrating in-vivo or in-vitro cytotoxin production.     

Analysis of latex agglutination test for Clostridium difficile toxin A (D-1) and differentiation between C difficile toxins A and B and latex reactive protein.

Virulent toxigenic and avirulent non-toxigenic strains of Clostridium difficile gave a positive result in the latex agglutination test (LAT) for C difficile toxin A (D-1). Similar concentrations of latex agglutinating antigen were produced by these strains in vivo. Positive reactions were also given by C sporogenes, proteolytic C botulinum Types A, B, and A/F, and Bacteroides assaccharolyticus. The latex agglutinating antigen was denatured by boiling for 10 minutes, but not by heating at 56 degrees C for 30 minutes. The reaction was abolished by incubation of test material with crude C difficile antitoxin but not with other clostridial antitoxins or specific antitoxin to C difficile toxin A. The latex agglutinating antigen present in C difficile eluted between 0.39% and 0.47% M sodium chloride, and that produced by the other clostridia, between 0.35% and 0.43% M sodium chloride by fast protein liquid chromatography. The latex agglutinating antigen of C difficile was neither cytotoxic nor mouse lethal and was distinct from toxin A and toxin B. In the analysis of faecal specimens from patients with diarrhoea the latex agglutination test correlated better with the presence of C difficile than with toxin B and detected both toxigenic and non-toxigenic strains. The latex agglutination test should only be used in the laboratory as an alternative to culture for C difficile and not as a method for the detection of C difficile toxins.     

Evaluation of a latex agglutination test for Clostridium difficile in two nursing home outbreaks.

The Culturette Brand Clostridium difficile test (CDT; Marion Laboratories, Inc., Kansas City, Mo.) is a latex agglutination test for C. difficile. The recent controversy involving the identity of antigens detected by CDT has made decisions on its use difficult. We compared the test results with those of selective culture and stool cytotoxin assays in investigations of two nursing home outbreaks of C. difficile-associated disease in order to formulate usage recommendations. Selective culture for C. difficile identified 27 (19%) of 142 subjects as carriers. CDT and the stool cytotoxin assay identified only 52 and 48% of these carriers, respectively. Compared with the stool cytotoxin assay, CDT had a high sensitivity (92%) and specificity (89%) for the detection of C. difficile disease, but the positive predictive value of the test was only 17% when the prevalence of disease was 2%. We conclude that the CDT should not be used to identify carriers but that it is a sufficiently sensitive and specific screening test for diagnosing C. difficile disease. However, since the positive predictive value of the CDT is low when the prevalence of disease is low, positive test results should be confirmed by the stool cytotoxin assay.     

Effect of various diets on toxin production by two strains of Clostridium difficile in gnotobiotic mice.

When axenic mice fed a commercial diet were monoassociated with two toxigenic strains of Clostridium difficile, 100% of them died 3 days after inoculation and both enterotoxin and cytotoxin were produced in their intestinal tract. However, when axenic mice were fed various semisynthetic diets before C. difficile challenge, some of them survived and their fecal cytotoxin and enterotoxin productions were highly reduced, whereas the C. difficile population level did not decrease to a great extent. Thus, gnotobiotic mice associated with C. difficile were a good model for the study of modulation by the dietary regimen of intestinal cytotoxin and enterotoxin production.