Clostridium difficile and its cytotoxin in the stools of young hospitalized children. Influence of antibiotic treatment

Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months. We divided the population in two groups depending on the antibiotic treatment. We have found C difficile in 39 samples (25%). The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months. An environmental sampling yielded once C difficile. Contamination may be related to the environment. 29% of the isolates produced a cytopathic toxin. Toxin titers in infants' stools range from 1/160 to 1/10240. One only of these children had diarrhea. C difficile and its toxin does not seem to infer any signs of enteric illness with infants. The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group. The rate of strains yielding a cytophatic toxin is similar in the 2 groups. It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine.     

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.     

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.     

A Cost-Effective Approach for Detection of Toxigenic Clostridium difficile: Toxigenic Culture Using ChromID Clostridium difficile Agar

We evaluated the performance and the cost of toxigenic culture using a commercial chromogenic medium (CDIF) for 538 stool specimens. Compared with real-time PCR, this method was found to detect an additional 9% of positive specimens and result in 61% reduction in material costs, with a trade-off increase in turnaround time of 1 day.     

Clostridium difficile and cytotoxin in routine faecal specimens.

Over a five-month period 1239 unselected, routine faecal specimens from 856 patients were examined for Clostridium difficile. One hundred specimens representing 69 patients were culture-positive. Toxin was detected in the stool of ten. During the study period, there were 41 Salmonella, 12 Campylobacter and 9 Shigella infections. C difficile was isolated together with Salmonella from 12 patients. No patient required specific treatment for C difficile infection. The significance of these findings is discussed.     

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.     

Correlation between cytotoxin production and sporulation in Clostridium difficile.

Correlation between cytotoxin production and sporulation was demonstrated when a Clostridium difficile culture was inoculated into fresh broth to give an initial count of less than 10 vegetative cells/ml with no spores. Under these conditions, cytotoxin was produced and released during sporulation. Addition of a sporulation inhibitor (acridine orange, 30 mg/L), resulted in a marked decrease in both sporulation and cytotoxin production, despite there being no change in the number of vegetative cells in the culture. These results indicate that sporulation might be closely related to cytotoxin production.     

Multicenter Clinical Evaluation of the Portrait Toxigenic C. difficile Assay for Detection of Toxigenic Clostridium difficile Strains in Clinical Stool Specimens

We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based “gold standard” method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.     

Toxin A of Clostridium difficile is a potent cytotoxin.

Clostridium difficile is the cause of antibiotic-associated colitis in humans. The organism produces toxin A, which is generally known as the enterotoxin, and toxin B, which is known as the cytotoxin. Toxin A has been reported to have slight cytotoxic activity; in this study we show that cell lines (F9, OTF9-63, and P19) which express a carbohydrate to which toxin A binds are more sensitive to the toxin. These cell lines can be used as research tools for determining concentrations of biologically active toxin A and should also prove useful for studies of the mechanism of action of the toxin.     

Purification and characterization of Clostridium sordellii lethal toxin and cross-reactivity with Clostridium difficile cytotoxin.

Lethal toxin (LT) was purified from Clostridium sordellii IP82 by DEAE-Trisacryl, Ultrogel AcA3-4 gel filtration, and hydroxyapatite column chromatography. The molecular weight of purified LT was estimated to be 240,000 to 250,000, and the pI was at pH 4.55. LT was lethal for mice by intraperitoneal injection (3.4 X 10(5) mouse lethal doses per mg of protein), cytotoxic for Vero cells (6.1 X 10(4) cytotoxic units per mg of protein), erythematous and edematous by intradermal injection in guinea pigs, and induced a moderate fluid accumulation in the guinea pig intestinal loop test. The lethal activity was inactivated by N-bromosuccinimide, N-chlorosuccinimide, chloramine-T, and sodium dodecyl sulfate. The data suggest that tryptophan and methionine residues present in the toxin are important for lethal activity. Furthermore, LT was inactivated by oxidized glutathione and activated by dithiothreitol. Inactivation by sulfhydryl-group reagents 5,5'-dithiobis(2-nitrobenzoic acid) and iodoacetamide was only obtained with dithiothreitol-treated LT. Thiol groups which are protected as a disulfide bond(s) seem to be essential for the LT activity. A specific antiserum against LT neutralized the biological activities of LT and also cytotoxic activity and lethal activity of Clostridium difficile toxin B but not of C. difficile toxin A. However, this serum did not recognize antigen from C. difficile culture supernatant by immunoblotting. It was concluded that antibodies prepared from C. sordellii LT that neutralized C. difficile cytotoxic activity recognized a low number of epitopes or tertiary structures of C. difficile cytotoxin.     

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.     

Isolation of Clostridium difficile from hospitalized patients without antibiotic-associated diarrhea or colitis.

Stool samples from 100 hospitalized patients and 21 healthy adults, obtained between March and June 1980, were cultured on a special selective medium containing cefoxitin and cycloserine to detect Clostridium difficile. This organism was isolated from 13 of the hospitalized patients and from 1 healthy subject. None of the patients with positive cultures had received antimicrobial therapy in the 3 preceding months. The observed rate of C. difficile isolation from adults not suffering from antibiotic-associated diarrhea or colitis is higher than previously reported. C. difficile culture is not recommended as a substitute for toxin assay in the evaluation of patients with intestinal disorders after antimicrobial chemotherapy.     

Comparison of the VIDAS Clostridium difficile toxin A immunoassay with C. difficile culture and cytotoxin and latex tests.

The VIDAS Clostridium difficile toxin A immunoassay (CDA) is a new, automated, enzyme-linked fluorescent-antibody assay for detection of C. difficile toxin A antigen in stool specimens. Simultaneous, parallel testing was performed by using the VIDAS CDA, the Culturette brand CDT latex test for C. difficile antigens, and conventional laboratory cell culture tests for C. difficile, cytotoxicity and C. difficile culture. One hundred ninety-four consecutive fresh soft or liquid stool samples submitted for C. difficile testing between July and September 1990 were evaluated. Of the 194 samples tested, 19 (10%) were from 16 patients who met our case definition for C. difficile-associated disease. The in vitro tests were evaluated in relation to two forms of a clinical case definition. In one form, a positive culture for toxin-producing C. difficile or a positive cytotoxin result obtained directly from the stool specimen was required as laboratory evidence of C. difficile. In the other, a positive result of any of the four laboratory tests was accepted for the laboratory portion of the case definition. No significant difference between the sensitivity of the VIDAS CDA and that of the Culturette brand CDT latex test was found (48 to 58% sensitivity for the CDT latex test and 52 to 63% sensitivity for the VIDAS CDA compared with 93 to 100% sensitivity for culture and 70 to 100% sensitivity for cytotoxin testing). The performance of the VIDAS CDA, however, was hampered by a high percentage of tests (19%) which gave an uninterpretable result.     

Comparison of five cultural procedures for isolation of Clostridium difficile from stools.

Several procedures have been described for the culture of Clostridium difficile from stool specimens. The goal of this study was to determine the effectiveness of five of these methods for the isolation of C. difficile from feces of patients suspected of having C. difficile-associated illness. A total of 564 stool specimens were cultured by using heat shock, ethanol treatment (ET), and direct plating on Carr-Scarborough cycloserine-cefoxitin-fructose agar (CCFA) with horse blood (C/S medium), BBL CCFA medium, and Remel C. difficile agar. Cytotoxin assays were performed on all specimens. A total of 113 specimens (20%) were positive for C. difficile by one or more methods. The numbers of positive cultures by using heat shock, ET, and direct plating on C/S medium, BBL CCFA medium, and Remel C. difficile agar were 79 (70%), 89 (79%), 91 (81%), 79 (70%), and 52 (46%), respectively. We concluded that ET and direct plating on C/S medium were the most effective procedures for isolating C. difficile from stool specimens and found significant variation in the performance of modified CCFA from different manufacturers.     

Comparison of typing methods for Clostridium difficile isolates.

A simple discriminative typing method for Clostridium difficile has been developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis. Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.     

Comparison of media for screening of diarrheic stools for the recovery of Clostridium difficile.

Recoveries of Clostridium difficile from stool specimens by using three media, cycloserine-mannitol agar (M-CMA), cycloserine-mannitol-blood agar (M-CMBA), and cycloserine-cefoxitin agar (M-CCA), were compared. Of 321 clinical specimens, 37 yielded C. difficile. Thirty-four were positive on M-CCA, 21 were positive on M-CMA, and 20 were positive on M-CMBA. M-CCA recovered significantly more C. difficile than did M-CMBA or M-CMA.     

Restriction endonuclease analysis of nosocomial isolates of Clostridium difficile.

A total of 110 clinical isolates of Clostridium difficile were analyzed by agarose gel electrophoresis by using both bacterial restriction endonuclease analysis (REA) and plasmid profiles. A total of 72 isolates were divided into 12 groups according to their REA patterns. Some 38 isolates exhibited unique patterns. Pattern A occurred in 20% of isolates. Isolates with patterns B, E, and G were cytotoxin negative. The remaining groups were cytotoxin positive. Multiple isolates obtained from two stool specimens were studied to examine the variation in REA profiles found in single specimens. In these specimens no variation in REA profiles was found. The stability of C. difficile was studied by examining sequential in vitro subcultures of a single isolate and strains isolated over a 4-month period from two long-term carriers. REA patterns were stable over time, both in vitro and in vivo. Because plasmid DNA was observed in 53% of isolates, plasmid profiles alone could not be used to study the spread of C. difficile; however, they were necessary for the interpretation of REA patterns in some instances.     

Clostridium difficile infection in children hospitalized due to diarrhea

The frequency of Clostridium difficile infection (CDI)-related hospitalizations is increasing. The aim of this study was to determine the extent of CDI among children hospitalized with diarrhea, risk factors or predictors for severe CDI, the prevalence of NAP1, and to compare the course of CDI depending on bacteria toxicity profile. A retrospective analysis of case records of 64 children (age range 3 months–16 years, median age 2.12 years) with CDI as defined by diarrheal disease and positive polymerase chain reaction (PCR) test (Xpert C. difficile) was conducted. Modified national adult guidelines were used to assess the severity of CDI. CDIs represented 2.7 % of patients with diarrhea (13.5 cases per 1,000 admissions). Thirty-three CDIs (52 %) were community-associated. Antibacterial use preceded CDI in 61 patients (95 %). Seventeen cases (27 %) were binary toxin-positive (CDT+), 13 of which were NAP1 (20.5 %). Over 75 % of CDIs with NAP1 was hospital-acquired, and more often proceeded with generalized infection (p?<?0.05). Risk factors for severe CDI (34 %) included NAP1 [odds ratio (OR), 4.85; 95 % confidence interval (Cl), 1.23, 21.86) and co-morbidities (OR, 4.25; 95 % Cl, 1.34, 14.38). Diarrhea ≥10 stools daily was associated with severe CDI (p?=?0.01). Recurrence occurred in three patients (4.5 %). There was no mortality. C. difficile is an important factor of antibiotic-associated diarrhea in children. Co-morbidities and NAP1 predispose to severe CDI.     

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.