Cycloserine Cefoxitin Fructose Research Articles

Antibiotic resistance of enteropathogenic bacteria in a teaching hospital in North Khuzestan during a three-year period.

Gastrointestinal infections affect many people annually. The most common bacterial agents involved in these infections are enteropathogenic bacteria and in the continuation of using broad-spectrum antibiotics, Clostridium difficile-associated diarrhea is involved, especially in hospitalized patients. The aim of the present study was to investigate the pattern of antibiotic resistance among enteropathogenic bacteria. In this cross-sectional study, 163 samples of patients with diarrhea in Dezful Ganjavian Hospital were examined. The samples were cultured in MacConkey, Hektoen enteric agar and GN broth, and cycloserine cefoxitin fructose agar media and incubated under standard conditions. In order to identify enteropathogenic bacteria, biochemical tests and serological confirmatory tests were used. Antibiotic resistance pattern of the isolates was investigated by Kirby-Bauer disk diffusion susceptibility test. The frequency of pathogenic bacteria includes 41.1% of Shigella flexneri, followed by 41.1% of S. sonnei, 6.7% of Enteropathogenic E. coli, 5.5% of Salmonella enterica Serogroup B, and 5.5% of Shigella dysenteriae. The results revealed a total of 46 patients with orders regarding C. difficile culture, no C. difficile was isolated from the samples. The studied isolates showed the highest resistance to trimethoprim-sulfamethoxazole, and ceftriaxone (88.3%), and the most effective antibiotic in the treatment of patients was ciprofloxacin with 86% sensitivity. Susceptibility to antibiotics was different among the isolates, which shows that the early identification of the infection agent and the selection of the correct antibiotic treatment are effective in improving the gastrointestinal infection and preventing the spread of the infection.

A study to assess the prevalence and risk factors for Clostridioides difficile infection in patients with inflammatory bowel disease in a tertiary care hospital in Northern India

Objectives: The prevalence of Clostridioides difficile infection (CDI) is on rise among patients with inflammatory bowel disease (IBD). This study sought to describe the prevalence and risk factors of CDI in patients with IBD as compared to non-IBD controls. Materials and Methods: This was a prospective study conducted at a Department of Microbiology in collaboration with a Department of Gastroenterology. The patients with IBD and controls without IBD presenting with diarrhea were included in the study. The screening test for C. difficile infection was done by glutamate dehydrogenase (GDH) assay and toxin detection by enzyme-linked immunoassay (ELISA). Anaerobic culture for C. difficile was done on a selective cycloserine cefoxitin fructose agar and polymerase chain reaction (PCR) was done for Toxin A (TcdA) and Toxin B (TcdB) gene detection. C. difficile infection was confirmed if GDH and toxin ELISA or PCR were positive. Statistical Analysis: Data were analyzed with the Statistical Package for the Social Sciences version 20.0.The numerical variables were presented by means and standard deviations. Comparison of continuous variables was done using Student’s t-test. Categorical variables were analyzed by Chi square test. P<0.05 was considered to be statistically significant. Results: A total of 160 cases and 112 age- and gender-matched control were included in IBD group and nonIBD group, respectively. Only one culture was positive, 12 and six were positive for GDH ELISA and TcdA and TcdB ELISA, respectively, and 7 were positive by PCR for toxin genes. The factors found significantly associated with CDI were proton-pump inhibitors use (P = 0.001), levofloxacin (P =0.001), and azathioprine (P =0.042). Using PCR as a reference method for C. difficile toxin detection, the sensitivity, and specificity of GDH ELISA and ELISA for toxins were 100%, 96.8% and 85.7%, and 100%, respectively. Conclusions: The prevalence of CDI among patients with IBD has been found to be low, that is (only 4.4%) in this study population.

2410. Molecular Characteristics of Environmental Clostridioides difficile From a Large Texas Hospital

Background Clostridioides difficile is an anaerobic spore-forming, toxin-producing Gram-positive bacillus listed by the CDC as an “urgent threat” pathogen. Epidemiologic studies using whole-genome sequencing (WGS) have found that genetically distinct lineages infections occur in hospitalized patients, in addition to the fact that C. difficile spores persist on hospital surfaces after disinfection. The purposes of this study were to isolate and characterize C. difficile from the environment of a large Texas hospital.MethodsWe collected 330 swab samples of hospital environmental surfaces using sterile cotton gauze. The samples were then anaerobically enriched in brain heart infusion broth for 48–72 hours and plated onto cycloserine–cefoxitin fructose agar (CCFA). Suspected colonies were then genetically characterized using PCR (for tcdA, tcdB, cdtA, cdtB and tpi genes) and genotyped using fluorescent PCR ribotyping techniques.ResultsA total of 90/330 (27.3%) environmental samples were culture positive for C. difficile, of which 75/90 (82.1%) tested were toxigenic C. difficile by the presence of tcdA, tcdB, cdtA or cdtB. A total of 16 distinct ribotypes were identified from 41 C. difficile isolates tested using a fluorescent-ribotyping method. The predominant ribotypes isolated were F078–126 (8/41), F002 (5/41), F106 (4/41), F255 (4/41), and F014-020 (3/41).ConclusionWe found a diversity of C. difficile strain types in various hospital high-touch surface environment in addition to ribotype F027 and F078, suggesting the hospital environment a reservoir and significant source C. difficile infections.Disclosures All authors: No reported disclosures.

Prevalence of Clostridium difficile and its toxins in hospital patients with diarrhoeal diseases in Lusaka, Zambia.

Clostridium difficile infection (CDI) has emerged as an important nosocomial and antibiotic-associated diarrhoeal problem leading to increasing morbidity and mortality, especially in resource-privileged regions. CDI varies in incidence, pathogenicity and risk factors across geographical locations, yet little information is available on CDI in sub-Saharan Africa. This study aimed to determine the prevalence of Clostridium difficile and related toxin expression in stool specimens from patients with diarrhoeal disease at the University Teaching Hospital, Lusaka, Zambia. Between June and September 2017, patients presenting with acute or persistent diarrhoea provided stool samples that were cultured anaerobically on cycloserine cefoxitin fructose agar. Isolates were identified by Gram staining, C. difficile latex agglutination and confirmed by PCR targeting of the tpi housekeeping gene. Toxins A or B were detected by ELISA. Of 135 participants enrolled, 13 (10%) were C. difficile positive, of which four (31%) were toxigenic by ELISA. Among HIV-positive and HIV-negative participants, the frequency of culturable C. difficile (19% vs 12%; p=0.17) and of toxigenic isolates (15% vs 0%,p=0.19) did not differ. We can now revise previous research and confirm that CDI contributes to diarrhoea among hospitalised adult patients irrespective of HIV status.

Development of a rapid-viability PCR method for detection of Clostridioides difficile spores from environmental samples

Clostridioides difficile is a common pathogen that is well known to survive for extended periods of time on environmental healthcare surfaces from fecal contamination. During epidemiological investigations of healthcare-associated infections, it is important to be able to detect whether or not there are viable spores of C. difficile on surfaces. Current methods to detect C. difficile can take up to 7 days for culture and in the case of detection by PCR, viability of the spores cannot be ascertained. Prevention of C. difficile infection in healthcare settings includes adequate cleaning and disinfection of environmental surfaces which increases the likelihood of detecting dead organisms from an environmental sample during an investigation. In this study, we were able to adapt a rapid-viability PCR (RV-PCR) method, first developed for detection of viable Bacillus anthracis spores, for the detection of viable C. difficile spores. RV-PCR uses the change in cycle threshold after incubation to confirm the presence of live organisms. Using this modified method we were able to detect viable C. difficile after 22 h of anaerobic incubation in Cycloserine Cefoxitin Fructose Broth (CCFB). This method also used bead beating combined with the Maxwell 16 Casework kit for DNA extraction and purification and a real-time duplex PCR assay for toxin B and cdd3 genes to confirm the identity of the C. difficile spores. Spiked environmental sponge-wipes with and without added organic load were tested to determine the limit of detection (LOD). The LOD from spiked environmental sponge-wipe samples was 104 spores/mL but after incubation initial spore levels of 101 spores/mL were detected. Use of this method would greatly decrease the amount of time required to detect viable C. difficile spores; incubation of samples is only required for germination (22 h or less) instead of colony formation, which can take up to 7 days. In addition, PCR can then quickly confirm or deny the identity of the organism at the same time it would confirm viability. The presence of viable C. difficile spores could be detected at very low levels within 28 h total compared to the 2 to 10-day process that would be needed for culture, identification and toxin detection.

The rate and importance of Clostridium difficile in colorectal cancer patients.

The aim of this study was to analyze the Clostridium difficile and their toxins in cancerous tissues in comparison to their adjacent healthy tissues in patients with colorectal cancer (CRC) in Iran. Intestinal infection or colonization by microbial pathogens and their released metabolites may have a role in the exacerbation of CRC. A total of 60 biopsy samples from 30 cancerous and 30 adjacent healthy tissues were collected from patients with CRC. Biopsies were homogenized and cultured in cycloserine cefoxitin fructose agar-agar medium to investigate the presence of C. difficile. DNA was extracted, PCR was performed on pure colonies for bacteria detection, and toxin genes were evaluated in each bacterium positive cases. Real-time PCR was performed on extracted DNA for quantitative comparison of Clostridium difficile in healthy and tumor tissues in CRC patients. Clostridium difficile was isolated from 18 of the cancerous tissue (60%) and 6 of their healthy adjacent tissue (20%) in the culture medium, but toxin genes were positive just in one sample in both groups. Real-time PCR showed the colonization in all samples. This study showed a higher prevalence of Clostridium difficile in cancerous lesions in comparison to healthy tissues. We suggest that the investigation of the rate of CD of colorectal cancer patients before surgery is critical for patients. Further studies with more samples size to study the importance of this bacterium and its toxins in the investigation of colorectal cancer patients survey is recommended.

510. First Environmental Investigation of Toxigenic Clostridium difficile Strains in Texas Hospitals

Background Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients in the developed world and an emerging pathogen in developing countries due to increased use of broad-spectrum antibiotics worldwide. Spores of toxigenic C. difficile can survive and disseminate in any environs and act as sources for human colonization or infections. Although likely ubiquitous in any environs, the prevalence of C. difficile spores in the hospital environment of Texas hospitals is poorly understood. The objectives of the study are to isolate and characterize C. difficile from the hospital environs of three hospitals in three cities in Texas.MethodsAs part of a Texas hospital-wide surveillance effort, we collected shoe-bottom swabs samples from hospital employees, patients, and visitors inside three large hospital from three cities. Samples were analyzed for C. difficile using anaerobic enrichment culture and molecular methods. Suspected colonies from cycloserine cefoxitin fructose agar (CCFA) plates were identified by PCR (tcdA, tcdB, cdtA, cdtB, tpi) and genotyped using fluorescent PCR ribotyping.ResultsA total 229 of 1079 (21.2%) surface swab and 81 of 121 (66.9%) shoe swab samples were culture positive for toxigenic C. difficile (tcdA and tcdB). A total of 29 distinct ribotypes were identified from 166 C. difficile isolates tested. Predominant ribotypes were F106, F019, F014-020, F002, and F255. Interestingly, ribotype F027 was not a predominant strain among the swab samples. Each hospital had widely diverse strains. Shoes were the most contaminated item in all the hospitals.ConclusionWe identified a high prevalence of toxigenic C. difficile with diverse ribotypes from hospital environmental shoe-bottom swabs and high touch surface swabs in hospitals in three cities of Texas. Our findings suggest that patients might be at higher risk for C. difficile colonization or infection in these hospitals.Disclosures K. W. Garey, Merck & Co.: Grant Investigator, Grant recipient.

Clostridium difficile Induced Diarrhoea Among Hospitalized Patients of Tertiary Care Hospitals in Dhaka

Clostridium difficile (C. difficile) has become a global public health challenge as C. difficile associated-diarrhea (CDAD) is increasing in incidence and severity of disease in several countries during recent years. This cross sectional study evaluated the frequency of CDAD among 100 adult patients who were clinically diagnosed as nosocomial diarrhoea in various clinical wards of Bangabandhu Shiekh Mujib Medical University (BSMMU) and Dhaka Medical College and Hospital (DMCH). CDAD diagnosis was based on detection of C. difficile along with clinical symptoms of diarrhea. Stool microscopy was done for cytology followed by anaerobic culture in cycloserine cefoxitin fructose agar (CCFA) media, confirmed by latex agglutination of culture isolates. Toxin genes (both A and B) were detected by multiplex Polymerase chain reaction (PCR) from culture isolates. Out of 100 diarrhoeal stool samples collected, 25% samples were pus cell positive in microscopy, culture yielded growth of C. difficile in 10% samples and all isolated C. difficile were confirmed by both latex agglutination and PCR. Out of 10 isolates, 7 were only tpi (triose phosphate isomerase) gene positive which is species-specific for C. difficile indicating the presence of non-toxigenic C. difficile and 3 isolates had both tpi and toxin genes (both tcdA and tcdB gene) on PCR indicative of toxigenic C. difficile respectively. C. difficile toxin gene detection by PCR along with culture is highly specific and sensitive diagnostic modality for CDAD. Differentiation between toxigenic and non-toxigenic strains by PCR may facilitate the appropriate patient management.
 Bangladesh J Med Microbiol 2018; 12 (1): 4-9

First Environmental Investigation of Toxigenic Clostridium difficile at a Large Hospital in Bangladesh

BackgroundToxigenic Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients in the developed world and an emerging pathogen in developing countries due to increased use of broad-spectrum antibiotics. Although likely ubiquitous worldwide, the prevalence of toxigenic C. difficile spores in the hospital environs of developing countries is poorly understood. The objectives of the study are to isolate and characterize C. difficile from the hospital environs of a large hospitals in Dhaka, Bangladesh.MethodsAs part of our environmental surveillance effort, we collected 330 shoe-bottom swab samples from hospital employees, patients, and visitors inside of a large hospital in Dhaka, Bangladesh. Samples were analyzed for C. difficile using anaerobic enrichment culture and molecular methods. Suspected colonies from cycloserine cefoxitin fructose agar (CCFA) plates were identified by PCR (tcdA, tcdB, cdtA, cdtB and tpi genes) and strain typed using fluorescent PCR ribotyping, and MLVA methods.ResultsA total 149 of 333 (44.7%) shoe-bottom swab samples were culture positive for C. difficile of which 19.8% samples were toxigenic (tcdA and tcdB) C. difficile. A total of 11 distinct ribotypes were identified from 58 toxigenic C. difficile isolates tested. Predominant ribotypes were F053-163 (24.1%), F017 (20.7%), F106 (19.0%), F014-020 (17.2%). Other ribotypes were R001, F005, F010, F018, F054, F216, and FP407. No R027 and R078 C. difficile isolated. A broad MLVA diversity has been seen among the tested strains.ConclusionWe identified a high prevalence of toxigenic C. difficile with diverse ribotypes from hospital environmental shoe-bottom swabs in Bangladesh. This is the first hospital environmental report of C. difficile from Bangladesh.Disclosures All authors: No reported disclosures.

Estimation of prevalence and risk factors for clostridium difficile infection: a neglected pathogen in a tertiary care setting in India

Introduction: Clostridium difficile is a Gram positive spore bearing anaerobic bacillus increasingly associated with both community and hospital acquired colitis and diarrhoea. Among all the risk factors, inclusive of the host and the environmental factors, antibiotics are the most important ones, as validated by various studies. Patients receiving antibiotics and other drugs such as immunosuppressives, chemotherapeutics and proton pump inhibitors may also be important risk factors. The present study was planned to find out the prevalence and risk factors for Clostridium difficile associated diarrhoea (CDAD). Material and Methods: After taking approval from ethics committee ,150 patients with antibiotic associated diarrhoea were taken as study group and 50 patients with exposure to antibiotics but who did not develop diarrhoea were taken as controls. Stool specimens were processed for both culture on Cycloserine Cefoxitin Fructose Agar (CCFA) and toxin detection by IVD Tox A+B ELISA. Risk factor analysis was done by calculating odds ratio and significance of p value among various parameters related to drugs and other factors. Result: Prevalence of CDAD in the present study was 8.67%.Third generation cephalosporins, clindamycin, aminoglycosides, quinolones and trimethoprim sulfamethoxazole were significant risk factors for both antibiotic associated diarrhoea (AAD) and Clostridium difficile associated diarrhoea (CDAD).Use of proton pump inhibitors, immunosuppressants and prolonged stay in the hospital were other significant risk factors associated with CDAD. Conclusion: Although CDAD occurs at a lower frequency in this setting, rational antibiotic policy and infection control measures should be followed to prevent its occurrence and nosocomial spread.

Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.

Antimicrobial Susceptibility of Clostridium Difficile Clinical Isolates in Iran

BackgroundClostridium difficile infection (CDI) is major growing problem in hospitals and its high incidence has been reported in recent years.ObjectivesThe aim of this study was to investigate the antimicrobial susceptibility patterns of C. difficile clinical isolates against antibiotics commonly used for treatment CDI in hospitalized patients.Material and MethodsDuring a 12 month study, 75 C. difficile isolates were collected from 390 patients with CDI. All samples were treated with alcohol and yeast extract broth. The treated suspensions were cultured on a selective cycloserine cefoxitin fructose agar (CCFA) supplemented with 5% sheep blood and incubated in anaerobic conditions, at 37 °C for 5 days. Cdd-3, tcdA and tcdB genes were identified using PCR assay.ResultsThe prevalence of A+B+ , A+ B- and A- B+ strains were 64(85.3%), 5(6.7%) and 6(8%) respectively. In vitro susceptibility of 75 clinical isolates of C. difficile to 5 antimicrobial agents, including metronidazole, vancomycin, clindamycin, erythromycin and cefotaxime were investigated by Clinical and Laboratory Standards Institute (CLSI) agar dilution method. Metronidazole and vancomycin had good activity against C. difficile isolates with MIC90s of 2 and 1 µg/ml, respectively. Seventy one (94.6%) of strains was inhibited by concentrations that did not exceed 2µg/ml for metronidazole. Resistant to metronidazole observed in 5.3% of isolates. Forty three (57.3%) of the isolates were resistant to erythromycin. Of 43 resistant strains to erythromycin, 9 (12%) isolates had high-level MIC of more than 64 µg/ml. All strains were resistant to cefotaxime. Sixty seven (89.3%) isolates were resistant to clindamycin (MIC90s > 256 µg/ml) and only 6.7% were sensitive to clindamycin. Multidrug-resistant (three or more antibiotics) was seen in 36(48%) isolates.ConclusionsMetronidazole and vancomycin still seem to be most effective drugs for treatment CDI.

Prevalence and risk factors of Clostridium difficile - associated diarrhea in Iranian hospitalized patients

Context: Clostridium difficile is a frequently identified cause of nosocomial gastrointestinal disease. It has been proved to be a causative agent in antibiotic-associated diarrhea. Aims: This study was aimed to determine the prevalence and risk factors of Clostridium difficile-associated diarrhea (CDAD) in hospitalized patients with nosocomial diarrhea in Shiraz, Iran. Materials and Methods: In this study from June to December 2012, a total of 122 stool samples of patients with nosocomial antibiotic associated diarrhea that were admitted in to the intensive care units (ICUs) (41), surgery (16) and organ transplantation wards (65) in Namazi hospital, Shiraz, Iran were collected. All stool samples were cultured on a selective Cycloserine Cefoxitin Fructose Agar and grew isolates were analyzed by cytotoxicity assay and enzyme immune assay for detection and conformation of toxins. Results: The mean ± standard deviation of age was 49.4 ± 13.8 and 75 (61.5%) of patients were male. Nine (7.4%) cases of nosocomial diarrhea were diagnosed as CDAD that all isolates were toxigenic. Five of 65 organs receive transplant patients and 4/41 hospitalized patients in ICUs ward were developed CDAD. None of samples that obtained from surgery ward infected with C. difficile. Ceftazidime and Ampicilline-Sulbactam were the most common antimicrobial drugs used. Multivariate analysis showed that use of diapers, antibiotic and immunosuppressive therapies were significantly associated with CDAD (P < 0.05). Conclusions: Hospital transmission of C. difficile commonly occurred, supporting infection-appropriate measures directed toward the reduction of CDAD.

Comparison of a Commercial Real-Time PCR Assay for tcdB Detection to a Cell Culture Cytotoxicity Assay and Toxigenic Culture for Direct Detection of Toxin-Producing Clostridium difficile in Clinical Samples

Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B [TOX-B] test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n = 273) and soft (n = 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80 degrees C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the "gold standard," the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens.

'Second-look' cytotoxicity: an evaluation of culture plus cytotoxin assay of Clostridium difficile isolates in the laboratory diagnosis of CDAD

Clostridium difficile is one of the most frequent causes of hospital-acquired diarrhoea. Our objective was to prove that some stool samples with a direct negative cytotoxicity assay may indeed harbour toxigenic C. difficile and that this can be demonstrated by performing a 'second-look' cytotoxicity assay using the isolated C. difficile strains.Over an eight-year period (1992–1999), the 8241 stool samples submitted for direct cell culture from patients with suspected C. difficile -associated diarrhoea (CDAD) were simultaneously plated on cycloserine cefoxitin fructose agar. C. difficile strains isolated from samples with a negative direct cell culture assay were re-tested for toxin production ('second-look' cell culture assay). Using both methods 6423 samples (78%) were negative. Of the remaining 1818 samples, 127 (7%) yielded C. difficile isolates which were confirmed as non-producers of toxin by both methods, 1437 (85%) were positive in direct cell culture assay, and 254 were positive only after the 'second-look' cell culture assay. Thus, our approach allowed us to detect an extra 15% of toxin-producing strains that could have gone undetected otherwise.The combination of direct-cell culture assay, culture for toxigenic C. difficile and 'second-look' cell culture assay enhances the potential for diagnosis of CDAD and enables us to be more efficient with our patient care resources.

Evaluation of Gas-Liquid Chromatography (GLC) for Rapid Detection ofClostridium Difficilein Fecal Specimens.

Clostridium difficile intestinal infection is a major nosocomial hazard in patients receiving antimicrobial therapy. Rationale for rapid diagnosis include lifesaving antimicrobial therapy in patients with severe colitis and early isolation measures for transmission control. We have therefore analysed the sensitivity, specificity and predictive value of GLC identification of isocaproic acid in diarrheic stools from adult hospitalized patients in comparison with selective fecal culture on Cycloserine Cefoxitin Fructose Agar. During the study period, the prevalence of positive culture for C. difficile was 38/595 fecal specimens (6.4%). Compared with culture, GLC had a sensitivity of 24/38 (63%) and a specificity of 524/557 (94%). The predictive value of a positive GLC was 24/57 (42%) and of a negative GLC was 524/538 (97%). Measurement of the isocaproic acid peak height did not allow determination of a cutt-off value improving the test accuracy. The sensitivity of detection of isocaproic acid in stools by GLC is too low to be used as screening test for C. difficile infection. However, in a low prevalence population, a positive GLC test increased the pre-test probability of infection sevenfold.

Comparison of Media for the Isolation ofClostridium difficileFrom Fecal Specimens

Dowell reported in 1980 that two selective media, cycloserine mannitol agar (CMA) and cycloserine mannitol blood agar (CMBA), developed at the Centers for Disease Control (CDC), gave better results than cycloserine fructose egg-yolk agar or cycloserine cefoxitin fructose egg-yolk agar in the isolation of Clostridium difficile from feces and in the quantitative recovery of the organism from pure cultures. In 1983, Bartley and Dowell modified the basal medium for CMA and CMBA by deleting L-leucine and Lserine; since then, the modified media has been used for isolating C difficile from feces. In this study, we compared the modified CMA and CMBA media with cycloserine cefoxitin fructose agar (CCFA) in the quantitative recovery of C difficile isolates and modified CCFA from two vendors in the isolation of C difficile from feces. In addition, all fecal specimens were cultured on CDC anaerobe blood agar (BA) after treatment for 1 hour with 95% ethyl alcohol (BA-ETOH). Of 350 fecal specimens examined using all media, 105 yielded C difficile. Of the 105 specimens, 80 were positive with CMBA (76%), 68 with CMA (65%), 44 with CCFA (vendor A) (42%), 38 with CCFA (vendor B) (36%), and 95 with BA-ETOH (90%). The selective media ranked as follows in quantitative recovery of 24 C difficile strains from pure cultures: CMBA>CMA>CCFA. We conclude that the modified CMBA and CMA media are superior to modified CCFA medium for recovery of C difficile and that culture on BA following alcohol treatment is a superior and costeffective method for detecting C difficile from clinical fecal specimens.

The Clinical Significance of Antibiotic-Associated Pseudomembranous Colitis in the 1990s

Antibiotic-associated pseudomembranous colitis is an uncommon but potentially serious adverse reaction, resulting in acute diarrhoea and characterised by colonic pseudomembranes. A direct relationship between the disease, recent antibiotic therapy and proliferation of Clostridium difficile in the colonic lumen was established in the late 1970s. It is thought that antibiotic therapy may alter the enteric flora, enabling C. difficile to proliferate and produce toxins with cytopathic (toxin B or cytotoxin) and hypersecretory (toxin A or enterotoxin) effects on the mucosa. Apart from clindamycin, the first antibiotic recognised to be clearly associated with pseudomembranous colitis, the antimicrobial agents most commonly responsible are cephalosporins and ampicillin (or amoxicillin). However, virtually all antibiotics except parenterally administered aminoglycosides can cause the disease. Vancomycin and metronidazole, 2 drugs used to treat antibiotic-associated pseudomembranous colitis, have also been reported to be responsible for the complication when used parenterally. Pseudomembranous colitis may develop after perioperative prophylactic antibiotic therapy with cephalosporins. Antibiotic-associated pseudomembranous colitis is most frequent in elderly and debilitated patients and in intensive care units. Nosocomial acquisition of C. difficile has been documented. Therefore it has been recommended that enteric isolation precautions should be taken with patients with this disease. The clinical symptoms include watery diarrhoea, abdominal cramping, and frequently fever, leucocytosis and hypoalbuminaemia. Toxic megacolon and acute peritonitis secondary to perforation of the colon are the most serious complications. The pseudomembranes are usually seen during endoscopic procedures, sigmoidoscopy or, if possible, colonoscopy; the most useful microbiological tests for confirmation of the diagnosis include cycloserine cefoxitin fructose agar (CCFA) stool cultures and stool toxin assays on tissues or by immunological techniques. However, cultures and toxin tests may be positive in patients without pseudomembranous colitis or C. difficile-associated diarrhoea. Mild cases may respond to discontinuation of the drug responsible, but therapy with an anticlostridial antibiotic is often necessary: a 10-day course of oral vancomycin, metronidazole or bacitracin should be given. Relapses are seen in 5 to 50% of patients treated. Antibiotic treatment should avoid sporulation leading to other relapses. 'Biotherapy' (lactobacilli, Saccharomyces) has also been proposed.

Laboratory diagnosis of Clostridium difficile-associated diarrhoea.

This paper reviews the various laboratory procedures available for the isolation and identification of Clostridium difficile and the detection of toxins produced by this organism. Laboratories should be selective in determining which patients require investigation for Clostridium difficile-associated diarrhoea. Transport and storage of stool specimens at 4 degrees C is recommended when delays in processing may occur. Tissue culture techniques are still the best method for detection of cytotoxin and a variety of cell lines can be used. Other methods for detecting cytotoxin, and methods for detecting other toxins are not sufficiently developed yet to warrant introduction into diagnostic laboratories. Culture techniques remain the most sensitive for diagnosis, particularly since the development of a variety of enrichment techniques. Cycloserine cefoxitin fructose agar is still adequate, although reduced concentrations of antimicrobial agents are necessary, and improvements, such as the addition of sodium taurocholate, increase the recovery of spores. Enrichment cultures have markedly increased isolation rates for Clostridium difficile but the significance of these isolates needs to be carefully evaluated. Until simpler and more reliable tests are available in clinical laboratories for the detection of toxins, the isolation of Clostridium difficile from patients with diarrhoeal disease should be considered paramount.

A selective enrichment broth for the isolation of Clostridium difficile.

The cycloserine, cefoxitin, fructose agar medium (CCF agar) devised by George et al' and the modification of it where egg yolk is replaced by horse blood (Oxoid CCF agar), have been widely and successfully used for the isolation of Clostridium diffiwile from faecal specimens.'-4 For epidemiological and environmental work where only small numbers of organisms may be present, it is likely that a more sensitive method of culture is required. Hafiz et al used a broth containing p-cresol in studying vaginal carriage of C diffcile5 whilst Wilson et a16 have reported that the incorporation of sodium taurocholate in CCF agar (in place of egg yolk) enhances the recovery of spore forms of C difficile from solid media. We describe the use of cycloserine cefoxitin fructose broth containing 0 1% sodium taurocholate (CCFT broth) for the isolation of C diffjile from vaginal and faecal specimens. women attending the Department of Genital Medicine (DGM). Groups IVa and IVb were mothers attending the matemity unit. Predelivery high vaginal swabs were taken by the midwife at onset of labour. Post delivery vaginal swabs were taken just prior to discharge. Group V were the babies delivered to mothers in group IV.