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.     

Laboratory diagnosis of Clostridium perfringens antibiotic-associated diarrhoea

Clostridium perfringens has been reported as the cause of up to 15% of cases of antibiotic-associated diarrhoea (AAD) and may be diagnosed by detection of enterotoxin (CPEnt) in faeces. The performance of a commercial ELISA method for CPEnt, with culture and PCR methods to confirm the presence of enterotoxigenic C. perfringens, was evaluated in 200 consecutive specimens from patients with clinical details suggestive of AAD: 8% of the specimens were positive for CPEnt, 16% were positive for C. difficile cytotoxin and 2% gave positive test results for both C. perfringens and C. difficile toxins. Culture and PCR results confirmed the majority of ELISA results, although 2 (12.5%) reactive specimens were only weakly positive. C. perfringens is a potentially important cause of infective AAD and can be detected with the C. perfringens enterotoxin ELISA kit, although weak positive results should be considered with caution.     

Clostridium perfringens enterotoxin in antibiotic-associated diarrhea

Clostridium perfringens type A is associated with 5-20% cases of antibiotic-associated diarrhea (AAD) even though Clostridium difficile is implicated in the most severe cases. Fecal specimens from one hundred hospitalized patients, who developed diarrhea regardless of antibiotic intake and who were negative for C. difficile toxin assay, were investigated for C. perfringens enterotoxin (CPE). Simultaneously, cultures were set up for other possible aetiological factors. Ten healthy controls were also similarly investigated. CPE was positive in 2/100 (2%) of the patients and the samples were also positive for the organism in culture. Other organisms isolated were non-toxigenic C. difficile (4%), staphylococci (6%), Candida (18%) and Klebsiella pneumoniae (1%). Stool samples from healthy controls grew mixed growth of no significance and CPE was negative in all of them. Detection of CPE is not part of routine laboratory investigation due to resource implication. Criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens-associated AAD by further research.     

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.     

The influence of drugs on the response of a cell culture preparation to bacterial toxins

The influence was studied of lanthanum chloride, chlorpromazine hydrochloride, indomethacin and sodium cromoglycate on the morphological changes induced in Vero cells by the action of the cholera toxin, the thermolabile enterotoxin (LT) and the Vero cell cytotoxin (VT) of Escherichia coli, the enterotoxin of Clostridium perfringens, and the cytotoxin of Clostridium difficile. These drugs were able to inhibit the effects produced by C. difficile cytotoxin but not by the other toxins examined.     

Correlation of immunoblot type, enterotoxin production, and cytotoxin production with clinical manifestations of Clostridium difficile infection in a cohort of hospitalized patients.

To determine whether strain-specific differences in immunoblot type, enterotoxin production, or cytotoxin production correlated with clinical presentation of Clostridium difficile infection, we evaluated isolates obtained from 428 prospectively studied hospitalized patients. Of 99 isolates available for immunoblot typing, 61 were recovered from asymptomatic carriers and 38 were from patients with C. difficile-associated diarrhea. Of 17 immunoblot types, the seven types comprising the majority of isolates (82 of 99; 83%) were variably associated with disease. Neither the presence of cytotoxin in the stool nor the production of cytotoxin or enterotoxin by isolates in vitro was significantly different for symptomatic versus asymptomatic patients. Selected host factors were more predictive of symptomatic disease than was the specific infecting C. difficile strain. These results suggest that variations in the clinical severity of C. difficile infection in different patients are not solely strain-specific phenomena related to immunoblot type or to the production of cytotoxin or enterotoxin.     

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.     

Antibiotic associated diarrhoea: infectious causes

Nearly 25% of antibiotic associated diarrhoeas (AAD) is caused by Clostridium difficile, making it the commonest identified and treatable pathogen. Other pathogens implicated infrequently include Clostridium perfringens, Staphylococcus aureus, Klebsiella oxytoca, Candida spp. and Salmonella spp. Most mild cases of AAD are due to non-infectious causes which include reduced break down of primary bile acids and decrease metabolism of carbohydrates, allergic or toxic effects of antibiotic on intestinal mucosa and pharmacological effect on gut motility. The antibiotics most frequently associated with C. difficile associated diarrhoea are clindamycin, cephalosporin, ampicillin and amoxicillin. Clinical presentation may vary from mild diarrhoea to severe colitis and pseudomembranous colitis associated with high morbidity and mortality. The most sensitive and specific diagnostic test for C. difficile infection is tissue culture assay for cytotoxicity of toxin B. Commercial ELISA kits are available. Though less sensitive, they are easy to perform and are rapid. Withdrawal of precipitating antibiotic is all that is needed for control of mild to moderate cases. For severe cases of AAD, oral metronidazole is the first line of treatment, and oral vancomycin is the second choice. Probiotics have been used for recurrent cases.     

Antitoxin activity of human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes (PMNL) inactivate Clostridium difficile cytotoxin and C. perfringens phospholipase C, but not C. perfringens enterotoxin. Both whole cells and sonicated suspensions possess activity, but mononuclear cell fractions of peripheral blood do not. Antitoxin activity closely correlates with cell concentration. The highest cell concentrations tested completely inactivated C. difficile cytotoxin by 2 min. Sucrose density gradient fractionation of PMNL showed antitoxin activity to be associated with myeloperoxidase, locating it in the primary or azurophil granules. Toxin inactivation was prevented by protease inhibitors suggesting that it is due to one of the neutral proteases present in these granules. PMNL are more active against C. difficile cytotoxin than purified chymotrypsin. PMNL may be a primary defence against certain bacterial exotoxins.     

Antitoxin activity of human polymorphonuclear leucocytes

Human polymorphonuclear leucocytes (PMNL) inactivate Clostridium difficile cytotoxin and C. perfringens phospholipase C, but not C. perfringens enterotoxin. Both whole cells and sonicated suspensions possess activity, but mononuclear cell fractions of peripheral blood do not. Antitoxin activity closely correlates with cell concentration. The highest cell concentrations tested completely inactivated C. difficile cytotoxin by 2 min. Sucrose density gradient fractionation of PMNL showed antitoxin activity to be associated with myeloperoxidase, locating it in the primary or azurophil granules. Toxin inactivation was prevented by protease inhibitors suggesting that it is due to one of the neutral proteases present in these granules. PMNL are more active against C. difficile cytotoxin than purified chymotrypsin. PMNL may be a primary defence against certain bacterial exotoxins.     

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.     

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.     

Epidemiology of Clostridium difficile-associated infections

Clostridium difficile is responsible for 15–25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudomembranous colitis (PMC). This anaerobic bacterium has been identified as the leading cause of nosocomial infectious diarrhea in adults and can be responsible for large outbreaks. Nosocomial C. difficile infection results in an increased length of stay in hospital ranging from 8 to 21 days. Risk factors for C. difficile -associated diarrhea include antimicrobial therapy, older age (>65 years), antineoplastic chemotherapy and length of hospital stay. Other interventions with high risk associations are enemas, nasogastric tubes, gastrointestinal surgery and antiperistaltic drugs. Prospective studies have shown that nosocomial transmission of C. difficile is frequent but often remains asymptomatic. Patients can be contaminated from environnemental surfaces, shared instrumentation, hospital personnel hands and infected roommates. Once an outbreak starts, C. difficile may be spread rapidly throughout the hospital environment where spores may persist for months. Measures that are effective in reducing incidence of C. difficile infections and cross-infection include: (i) an accurate and rapid diagnosis, (ii) appropriate treatment, (iii) implementation of enteric precautions for symptomatic patients, (iv) reinforcement of hand-washing, (v) daily environmental disinfection, and (vi) a restrictive antibiotic policy. C. difficile is a common cause of infectious diarrhea and should be therefore systematically investigated in patients with nosocomial diarrhea.     

Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhea and food poisoning in North America

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest that C. perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal cpe gene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpe isolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive, C. perfringens fecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpe gene, but the North American food poisoning isolates all carry a chromosomal cpe gene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpe isolates cause most CPE-associated AAD cases and chromosomal cpe isolates cause most C. perfringens type A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.     

Epidemiology, risk factors and prevention of Clostridium difficile nosocomial infections

Clostridium difficile is responsible for 10-25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudo-membranous colitis (PMC). This anaerobic spore-forming bacterium has been identified as the leading cause of nosocomial infectious diarrhea in adults. Pathogenesis relies on a disruption of the normal bacterial flora of the colon, a colonization by C. difficile and the release of toxins A and B that cause mucosal damage and inflammation. Incidence of C. difficile intestinal disorders usually varies from one to 40 per thousand patient admissions. Risk factors for C. difficile-associated diarrhea include antimicrobial therapy, older age (> 65 years), antineoplastic chemotherapy, and length of hospital stay. Nosocomial transmission of C. difficile via oro-fecal route occurs in 3-30% of total patient admissions but it remains asymptomatic in more than 66% of cases. Persistent environmental contamination and carrying of the organism on the hands of hospital staff are common. Measures that are effective in reducing cross-infection consist of an accurate and rapid diagnosis, an appropriate treatment, an implementation of enteric precautions for symptomatic patients, a reinforcement of hand-washing and a daily environmental disinfection. C. difficile is a common cause of infectious diarrhea and should be therefore systematically investigated in patients with nosocomial diarrhea.     

Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin.

Stools from patients with antibiotic-associated diarrhea or colitis were cultured to detect the presence of Clostridium difficile. All specimens contained a cytotoxin which was neutralized by Clostridium sordellii antitoxin. Initial testing employed several methods with comparative merits in recovering this organism. These included the use of nonselective media, antibiotic-incorporated media, alcohol shock, and paracresol-containing broth. Optimal results were achieved with primary plating of serial dilutions onto a selective agar containing cycloserine and cefoxitin. This technique was then employed in a large number of specimens. The overall results showed that C. difficile was recovered in specimens from 71 of 73 patients. All isolates of C. difficile produced a cytotoxin which was neutralized by C. sordellii antitoxin in vitro. These results verify the utility of this medium and support the concept that C. difficile accounts for the cytotoxin found in stools in nearly all cases.     

Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C. difficile, cytotoxin assay, culture, and latex agglutination.

A three-center study was undertaken to compare several test methods for the detection of Clostridium difficile, associated toxin, or related markers by using 927 stool specimens. Methods included direct assay of cytotoxin in stool by tissue culture, C. difficile bacterial culture followed by cytotoxin assay, bacterial culture alone, latex agglutination assay, and the ImmunoCard C. difficile test (Meridian Diagnostics, Inc.). The sensitivities, as determined against direct cytotoxin assay results, of the ImmunoCard C. difficile and latex agglutination assays were 84 and 67%, respectively (92 and 77%, respectively, when adjusted for bacterial culture outcomes). Evaluation for C. difficile-associated disease (CDAD) among 864 patients was based on clinical criteria for antibiotic-associated diarrhea combined with laboratory evidence of toxin or toxin-producing C. difficile in stool specimens. The sensitivity of each test method for screening of CDAD was as follows: bacterial culture, 95%; culture with cytotoxin assay of isolates, 90%; ImmunoCard C. difficile test, 83%; cytotoxin assay 82%; and latex agglutination assay, 67% (P < or = 0.05 versus all other methods). The standard deviations of the test sensitivity statistics between study sites were ranked as follows: cytotoxin assay (+/- 3.1%) < ImmunoCard C. difficile test (+/- 5.7%) < latex agglutination assay (+/- 12.3%) < culture (+/- 24.7%) < culture with cytotoxin assay (+/- 28.0%). The data support the use of the ImmunoCard C. difficile test as an adjunct for the diagnosis of CDAD.     

Detection of Clostridium difficile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens. Evaluation of a latex agglutination test

A new latex test, Culturette Brand Rapid Latex Test for detection of Clostridium difficile toxin A, was tested on 408 stool samples. In 247 frozen tissue culture supernate specimens previously obtained from patients with C. difficile-associated diarrhea (CAD), the latex test (enterotoxin) was positive in 182 (74%) as compared with 194 (79%) for the repeat tissue culture (P greater than 0.1) cytotoxin (toxin B) test. Testing of 161 fresh stool samples found the latex test superior to tissue culture (P less than 0.05) in cases of CAD (90% positivity vs. 70%), with the two tests being equal in both non-CAD diarrheal and non-diarrheal control groups. In vitro evaluation of 61 C. difficile isolates found all (100%) to be producers of enterotoxin A, while only 53 (87%) produced toxin B. The latex test for C. difficile toxin detection is a rapid, simple test for use in the diagnosis in CAD.     

Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhoea.

AIMS: To determine the incidence of sporadic and apparently non-food related diarrhoea associated with Clostridium perfringens enterotoxin. METHODS: Enzyme linked immunosorbent assay (ELISA) and reversed phase latex agglutination (RPLA) were used to detect C perfringens enterotoxin in faecal specimens from 818 sporadic cases of diarrhoea. RESULTS: C perfringens enterotoxin was identified as a cause of sporadic diarrhoea in 56 of 818 (6.8%) cases. Diarrhoea was prolonged (three days or more) in most cases. Ages ranged from 3 months to 89 years, although most patients were over 60 years of age. CONCLUSIONS: These results suggest that C perfringens may be a cause of sporadic cases of diarrhoea when causes such as food consumption or cross-infection are absent, particularly in the elderly.     

Evaluation of methods for detection of toxins in specimens of feces submitted for diagnosis of Clostridium difficile-associated diarrhea

Clostridium difficile is the principal pathogen associated with hospital-acquired acute diarrheal disease. We have evaluated the performances of six approaches for diagnosis of C. difficile-associated diarrhea (CDAD). Consecutive stool specimens (n = 200) from 133 patients were examined by cytotoxin assay, by culture of C. difficile on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test). A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens). The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture. The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.