Selective and differential medium for isolation of Clostridium difficile.

Clostridium difficile is a recognized cause of pseudomembranous (antimicrobial agent-associated) colitis and may be one of the causes of antimicrobial agent-induced diarrhea. A selective and differential agar medium that contains cycloserine, cefoxitin, fructose, and egg yolk (CCFA) was developed to facilitate the isolation of C. difficile from fecal specimens. Quantitative cultures of 16 stock strains of C. difficile on this medium (and on a medium containing cycloserine, fructose, and egg yolk) yielded counts equivalent to those obtained on blood agar; other media selective for clostridia, including Clostrisel agar, reinforced clostridial agar plus 0.2% para-cresol, and egg yolk-neomycin agar (the latter was inoculated with cultures subjected to prior heat shocking), were also tested and found to be inhibitory to the growth of C. difficile. Of 28 fecal or colostomy effluent specimens cultured on the above media, 14 yielded C. difficile. CCFA was found to be the most sensitive and selective of these media for the recovery of C. difficile. Colonies of C. difficile growing on CCFA had distinctive morphological and fluorescent properties which were sufficient for presumptive identification. CCFA should provide a rapid method for the screening of fecal specimens from patients with antimicrobial agent-associated diarrhea or colitis for C. difficile.     

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.     

Incidence and origin of Clostridium difficile in neonates

The stools of 65 of 92 (71%) infants in a special care nursery yielded Clostridium difficile on culture. Ninety percent of stools collected after 6 to 35 days in the unit were positive, and 36% of these also contained toxin. When tested in vitro, 94% of the isolates produced toxin. Of 110 swabs collected from the environment of the unit, 9% were positive for C. difficile, but the stools of 12 nurses working on the unit were negative. Thirty-five vaginal swabs collected from mothers just before delivery were negative for C. difficile on culture, but 16 of their infants had C. difficile in their stools. It was concluded that there is a high carriage rate in the stools of neonates of C. difficile acquired progressively during the course of their stay in the special care unit. Infection is mainly from environmental sources rather than maternal transmission.     

Evaluation of gas-liquid chromatography for the rapid diagnosis of Clostridium difficile associated disease.

Direct gas-liquid chromatography of faecal specimens with isocaproic acid as a marker was used for the rapid diagnosis of Clostridium difficile associated diarrhoeal diseases. Ninety stools were examined and results were compared with conventional culture on selective medium and cytotoxin assay in tissue culture. Using a combined analysis of isocaproic acid and butyric acid peak heights we defined three categories: positive, negative, and indeterminate. When the indeterminate group was excluded, the positive and negative predictive values of gas-liquid chromatography analysis were 86.9% and 85% respectively compared with culture and 71.4% and 95% respectively compared with cytotoxin assay.     

Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing?

Direct tests for Clostridium difficile are 30–50?% more sensitive than tests for C. difficile toxins but the reasons for this discrepancy are incompletely understood. In addition to toxin degradation and strain differences, we hypothesized that C. difficile concentration could be important in determining whether toxins are detected in fecal samples. We performed standard curves on an FDA-approved real-time PCR test for the C. difficile tcdB gene (Xpert C. difficile/Epi, Cepheid) during a prospective comparison of a toxin immunoassay (Meridian Premier), PCR and toxigenic culture. Immunoassay-negative, PCR-positive samples were retested with a cell cytotoxin assay (TechLab). Among 107 PCR-positive samples, 46 (43.0?%) had toxins detected by immunoassay and an additional 18 (16.8?%) had toxin detected by the cytotoxin assay yielding 64 (59.8?%) toxin-positive and 43 (40.2?%) toxin-negative samples. Overall, toxin-negative samples with C. difficile had 10¹–10⁴ fewer DNA copies than toxin-positive samples and most discrepancies between toxin tests and PCR were associated with a significant difference in C. difficile quantity. Of the toxin-positive samples, 95?% had ≥4.1 log₁₀ C. difficile tcdB DNA copies/mL; 52?% of immunoassay-negative samples and 70?% of immunoassay and cytotoxin negative samples had <4.1 log₁₀ C. difficile tcdB DNA copies/mL. These findings suggest that fecal C. difficile concentration is a major determinant of toxin detection and C. difficile quantitation may add to the diagnostic value of existing test methods. Future studies are needed to validate the utility of quantitation and determine the significance of low concentrations of C. difficile in the absence of detectable toxin.     

Rapid identification of Clostridium difficile by toxin detection.

Rapid identification of Clostridium difficile in a stool specimen could be accomplished within 24 h by detection of toxin elaborated in an agar or broth culture containing cycloserine and cefoxitin. Broth culture seemed to give a more rapid and sensitive result than the agar plate culture. For cultivation of C. difficile in stool, we recommend the use of chopped meat broth and blood agar plate, the former for toxin detection in 1 to 2 days and the latter for colonial morphology and isolation of a pure culture.     

Novel one-step method for detection and isolation of active-toxin-producing Clostridium difficile strains directly from stool samples

The alarming emergence of hypervirulent strains of Clostridium difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. The C. difficile toxins A and B are critical virulence factors, and strains can either be toxin-producing (virulent) or non-toxin-producing (nonvirulent). Strains that are isolated from human infections generally produce either toxin A or toxin B or both. The methods currently available for culturing C. difficile do not differentiate strains that produce active toxins from strains that do not produce toxins or produce inactive toxins. As a result, the identification and isolation of toxin-producing strains from stool is currently a two-step process. First, the stool is plated on a selective medium, and then suspected colonies are analyzed for toxin production or the presence of the toxin genes. We describe here a novel selective and differential culture method, the Cdifftox plate assay, which combines in a single step the specific isolation of C. difficile strains and the detection of active toxin. This assay was developed based on our recent finding that the A and B toxins of C. difficile cleave chromogenic substrates that have stereochemical characteristics similar to their natural substrate, UDP-glucose. The Cdifftox plate assay is shown here to be extremely accurate (99.8% effective) in detecting toxin-producing strains through the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. The Cdifftox plate assay advances and improves the culture approach such that only C. difficile strains will grow on this agar, and virulent strains producing active toxins can be differentiated from nonvirulent strains, which do not produce active toxins. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains.     

Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile.

The PRO Disc (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) can be used to screen for L-proline-aminopeptidase produced by Clostridium difficile grown on cycloserine-cefoxitin-fructose agar (CCFA). Fifty stored isolates of C. difficile (48 toxin-positive and 2 toxin-negative isolates) and 47 fresh C. difficile isolates (39 toxin-positive and 8 toxin-negative isolates) were all PRO Disc positive. Other Clostridium species that were PRO Disc positive could be differentiated from C. difficile by failure to grow on CCFA, different colonial morphology on CCFA, or morphology upon Gram staining.     

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.     

Laboratory diagnosis of Clostridium difficile-associated diarrhea and colitis: usefulness of Premier Cytoclone A+B enzyme immunoassay for combined detection of stool toxins and toxigenic C. difficile strains

Detection of Clostridium difficile toxins A and B in stools by Premier Cytoclone A+B enzyme immunoassay (EIA) was compared with detection by stool culture for C. difficile followed by detection of toxigenic isolates using the same EIA. Chart reviews were performed to evaluate the likelihood of C. difficile-associated diarrhea and colitis (CADC) for all patients with at least one positive toxin assay. While the toxins were detected in 58 of 85 consecutive CADC patients by both assays, CADC in 5 patients was detected only by stool toxin assay, and in 22 patients CADC was detected only by toxigenic culture. Our results suggest that for laboratories using a rapid toxin A+B EIA, direct toxin detection in stools should be combined with toxigenic culture in cases in which there is a negative stool toxin assay.     

Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridium difficile

We examined the incremental yield of stool culture (with toxin testing on isolates) versus our two-step algorithm for optimal detection of toxigenic Clostridium difficile. Per the two-step algorithm, stools were screened for C. difficile-associated glutamate dehydrogenase (GDH) antigen and, if positive, tested for toxin by a direct (stool) cell culture cytotoxicity neutralization assay (CCNA). In parallel, stools were cultured for C. difficile and tested for toxin by both indirect (isolate) CCNA and conventional PCR if the direct CCNA was negative. The "gold standard" for toxigenic C. difficile was detection of C. difficile by the GDH screen or by culture and toxin production by direct or indirect CCNA. We tested 439 specimens from 439 patients. GDH screening detected all culture-positive specimens. The sensitivity of the two-step algorithm was 77% (95% confidence interval [CI], 70 to 84%), and that of culture was 87% (95% CI, 80 to 92%). PCR results correlated completely with those of CCNA testing on isolates (29/29 positive and 32/32 negative, respectively). We conclude that GDH is an excellent screening test and that culture with isolate CCNA testing detects an additional 23% of toxigenic C. difficile missed by direct CCNA. Since culture is tedious and also detects nontoxigenic C. difficile, we conclude that culture is most useful (i) when the direct CCNA is negative but a high clinical suspicion of toxigenic C. difficile remains, (ii) in the evaluation of new diagnostic tests for toxigenic C. difficile (where the best reference standard is essential), and (iii) in epidemiologic studies (where the availability of an isolate allows for strain typing and antimicrobial susceptibility testing).     

Enzyme immunoassay (ELISA) for detection of Clostridium difficile toxin B in specimens of faeces

Antisera against Clostridium difficile toxin B were prepared in sheep and rabbit and were used in indirect and sandwich enzyme-linked immunosorbent assays (ELISA) for the detection of toxin B. Polyvinyl chloride and polystyrene microtitration plates were tested as solid phases for the assay. Both assays had a lower limit of detection for toxin B of 1 ng/ml. They were used to detect the presence of toxin B in 210 human faecal specimens and also in the culture supernatant fluids of C. difficile strains isolated from the faecal samples. There was a close correlation between the results of sandwich ELISA and those of cytotoxicity tests and isolation of C. difficile. Our sandwich ELISA method seems to be useful as a presumptive test for detection of C. difficile toxin B.     

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.     

Cultivation of Mycobacterium paratuberculosis from bovine fecal samples by using elements of the Roche MB Check system.

Components of a commercially available, nonradiometric, biphasic (liquid medium then solid medium) system for the detection of Mycobacterial species, Roche MB Check, were adapted for the isolation of Mycobacterium paratuberculosis from bovine fecal specimens. A two-stage culture procedure was developed in which processed fecal samples were incubated in modified commercial liquid medium and then subcultured onto Herrold's egg yolk medium with mycobactin. By using known culture-positive samples and/or samples from animals clinically affected with paratuberculosis, it was found that visible colonial growth on solid media could be obtained after 4 weeks of incubation in liquid medium containing egg yolk and mycobactin followed by 8 weeks of incubation on Herrold's egg yolk medium. In the second part of the study, conventional fecal culture (sample sedimentation in hexadecylcetylpyridinium chloride followed by incubation on Herrold's egg yolk medium) was compared with the two-stage system using a two-step centrifugation technique for sample preparation. One hundred fecal samples from clinically normal but absorbed-enzyme immunoassay-positive cattle were used for the comparison. Conventional culture yielded a sensitivity of 16.5%, whereas the sensitivity of the two-stage system was 29.4%. When used in parallel, the tests detected 36.5% of the samples. There was no significant difference between the two methods in the time taken to obtain visible colonies. These results indicate that the two-stage method is a sensitive method for isolation of M. paratuberculosis from fecal samples obtained from cattle with clinical paratuberculosis. In addition, the two-stage system is more sensitive than conventional culture for the isolation of M. paratuberculosis from subclinically infected cattle.     

Fluorescent-antibody test for detection of Clostridium difficile in stool specimens.

We evaluated a direct fluorescent-antibody test to detect Clostridium difficile, the most frequent cause of antibiotic-associated colitis. C. difficile organisms were injected into the ear veins of New Zealand White rabbits to induce antibodies, and the globulin fractions of their sera were conjugated to fluorescein isothiocyanate. The resulting conjugate strongly stained all 40 isolates of C. difficile tested. It also stained isolates of C. sordellii, C. bifermentans, C. chauvoei, and C. sporogenes, but not 20 other clostridial isolates or 10 isolates from other species. Results of testing fecal smears with the direct fluorescent-antibody method were compared with results of testing stools for C. difficile toxin and of culturing for C. difficile on a selective medium. A total of 158 fecal specimens from patients with antibiotic-associated diarrhea were tested. In these patients, the fluorescent-antibody test agreed with culture and toxin testing in 93% of the specimens. However, in normal adults, 62% of the fecal specimens from which C. difficile could not be cultured were positive by the fluorescent-antibody test. Absorption of the conjugate with C. sordellii led to a loss of reactivity to other clostridia as well as to 18 of 20 isolates of C. difficile.     

Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays

Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.     

Isolation and enumeration of Clostridium botulinum by direct inoculation of infant fecal specimens on egg yolk agar and Clostridium botulinum isolation media

Direct plating of stool specimens on selective (Clostridium botulinum isolation) and nonselective (egg yolk agar) media was evaluated as an aid in confirming infant botulism. C. botulinum was isolated from 13 of 14 culture-positive specimens with C. botulinum isolation and from 8 of 14 egg yolk agar. No lipase reaction was seen on plates of 31 culture-negative specimens.     

Laboratory diagnosis of Clostridium difficile disease

The laboratory diagnosis of Clostridium difficile -associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.     

Effect of adding sodium taurocholate to selective media on the recovery of Clostridium difficile from environmental surfaces.

The recovery of Clostridium difficile on a medium containing cefoxitin, cycloserine, fructose, and egg yolk was compared with that on media containing one of three preparations of sodium taurocholate. In aerobic environments contaminated with C. difficile, media containing either crude taurocholate from Mann Research Laboratories, New York, N.Y., or pure taurocholate from Sigma Chemical Co., St. Louis, Mo., recovered organisms significantly more often than did cefoxitin-cycloserine-fructose-egg yolk agar.     

Selective medium for isolation of Clostridium botulinum from human feces.

A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 microgram/ml), sulfamethoxazole (76 microgram/ml), and trimethoprim (4 microgram/ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.