Cycloserine Cefoxitin Fructose Agar 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.

Frequency of Clostridioides difficile Infection Among Hospitalized Patients in Kerman City, Iran

Background: Clostridioides difficile is one of the major causes of nosocomial infections, being responsible for 15 to 25% of antibiotic-associated diarrhea. It is important to determine the epidemiology and prevalence of this bacterium at hospitals and healthcare centers. Objectives: This study aims to investigate the prevalence of C. difficile infection (CDI) by identifying toxigenic isolates of C. difficile in different wards of the hospital. Methods: A total of 417 diarrheal stool samples were taken from hospitalized patients in different wards of three educational hospitals in Kerman City, Iran from 2018 to 2020. The samples were cultured on cycloserine-cefoxitin fructose agar and C. difficile suspected colonies were isolated. Identification of the cdd-3 gene for definitive diagnosis of C. difficile and identification of toxin genes in the positive isolates was performed using the PCR method. Results: A total of 68 isolates (16.3%) of C. difficile were isolated from the specimens. Besides, 8.6% (36/417) and 7.6% (32/417) of the isolates were toxigenic and nontoxigenic, respectively; thus, the prevalence of CDI was 8.6%. Most of the toxigenic isolates had the A+B+CDT- toxin phenotype. The highest prevalence of CDI was observed in males, ICU ward, and age group of 41 - 60. Conclusions: A total of 8.6% of hospitalized patients with diarrhea were infected with C. difficile. The prevalence of CDI in Kerman City is lower than that in Europe, East Asia, and other parts of Iran, but it is almost the same as that in the Middle East.

Loop mediated isothermal amplification of Clostridioides difficile isolates in gastrointestinal patients

This study investigated the prevalence of Clostridioides difficile by culture, multiplex polymerase chain reaction (M-PCR), and loop mediated isothermal amplification (LAMP) in patients with suspected C. difficile infections (CDIs). Also, the results of three methods were compared. All stool specimens collected from CDI suspected patients were cultured on selective C. difficile cycloserine-cefoxitin fructose agar and incubated in an anaerobic jar up to 7 days. The bacterial isolates were identified using standard tests. Multiplex-PCR (M-PCR) was performed for detection of tcdA, tcdB, and tpi genes. The LAMP assay was performed to detect the tcdB gene of C. difficile. C. difficile was isolated from 20.0% (n = 10/50) of samples by culture. M-PCR showed that 34.0% (n = 17/50) of the specimens were positive for C. difficile based on the presence of tpi gene. Out of the 17 C. difficile, 13 strains (76.0%) were positive for tcdB gene using M-PCR. However, the LAMP assay showed that 30.0% (15/50) of specimens were positive for the presence of tcdB gene. M-PCR and LAMP methods showed 100.0% sensitivity compared to the culture method. However, the specificity of the LAMP (87.5%) was relatively higher than the M-PCR (82.5%) compared to the culture. Based on the results of this study, the prevalence of toxigenic C. difficile strains was high in suspected CDI patients. So, the differentiation between toxigenic and non-toxigenic strains is necessary. Our data showed that the LAMP assay is a good method for direct detection of toxigenic C. difficile strains from stool specimens.

Screening of Clostridium difficile Isolates for Metallo-Resistant Gene and Extended Spectrum Beta-Lactamases

Clostridium difficile naturally inhabits intestinal tract of animals including humans. Spores of C. difficile are known contaminant in the Environment. Indiscriminate use of antibiotics has led to the emergence of a powerful resistance determinant (known as Metallo resistant genes) in many bacteria worldwide. Multidrug resistant bacteria (MRB) pose threats to health globally. This study aims to determine the distribution of Metallo resistant Clostridium difficile in hospital environment within Yola. A total of 150 surface samples from the hospital wards were collected from Federal Medical Centre, Yola (FMC) and Specialist hospital, Yola (SHY) Adamawa state, Nigeria. Clostridium difficile were recovered by inoculating samples in modified Cycloserine-cefoxitin fructose broth (CCFB) and Cycloserine-cefoxitin fructose agar (CCFA). Screening of isolates for extended spectrum β-lactamase (ESBL) and metallo-β-lactamase enzymes was done by double disc diffusion method and Imipenem-EDTA combined disc test respectively. A total of 18 C. difficile isolates were obtained and found to be extended spectrum β-lactamase (ESBL) positive. 16 (88.89 %) of the Clostridium difficile were found to be metallo-β-lactamase positive. Polymerase chain reaction showed that 11(61.1 %) of the isolates possess bla-IMP metallo resistant gene. This study suggests that multidrug resistant C. difficile with metallo resistant genes are wide spread in hospital settings and there is need for relevant authorities to improve on the sanitization of environment as well as continuous surveillance to hinder C. difficile transmission.

Prevalence of Clostridium difficile in Hospital Environment within Yola Adamawa State Nigeria

Introduction: Clostridium difficile infection (CDI) is an emerging health problem in hospital setting. The ability of the spores to persist in the environment is a key factor in rates of infection. Clostridium difficile has also been described as one of the leading cause of nosocomial diarrhea. It is responsible for an increase in hospital stay with high healthcare and economic repercussions. Objectives: This study was aimed at determining the prevalence and antimicrobial susceptibility profile of Clostridium difficile isolated from surfaces within hospital environment in Yola Adamawa State Nigeria. Materials and Methods: A total of 150 surface samples were collected from different wards of two hospitals (Specialist Hospital Yola (SHY) and Federal Medical Centre Yola (FMCY)) using moistened swabs. Clostridium difficile isolates were obtained by enriching and culturing samples in cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin fructose agar (CCFA) respectively. Susceptibility test (MIC determination) was done by well diffusion technique on Mueller Hinton agar supplemented with 5% sodium taurocholate. Results: A total of 18 (12%) Clostridium difficile were recovered from the two hospitals sampled. FMC had a prevalence rate of 13.3% with table top, toilet, window and bed sheets as sites implicated while SHY had a prevalence rate of 10.7% with bed sheet, bed railing and window as implicated sites. The antibiotic susceptibility test reveals that Clostridium difficile isolates obtained were resistant to the antibiotics tested which includes Ciprofloxacin (MIC >64 µg/ml), Erythromycin (MIC >64 µg/ml), Metronidazole (MIC 64 µg/ml), Tetracycline (MIC > 128 µg/ml) and Clindamycin (MIC > 64 µg/ml). Conclusions: The study depicts that multidrug resistant Clostridium difficile is prevalent in hospital environments within Yola Adamawa State Nigeria. The need for the sensitization of healthcare workers to improve understanding of Clostridium difficile transmission, treatment, management and prevention is of paramount importance.

Aseptic Stethoscope Barriers Prevent C difficile Transmission In Vitro

ObjectiveTo evaluate whether Clostridioides (formerly Clostridium) difficile–contaminated stethoscope diaphragms remained aseptic by the placement of an aseptic diaphragm barrier.MethodsOn November 1, 2019, fresh cultures of C difficile were diluted to 107 colony-forming units (CFU)/mL and used to inoculate 16 stethoscope diaphragms; 8 had an aseptic diaphragm barrier applied and 8 served as nonbarrier controls. Contaminated stethoscopes were anaerobically incubated, then swabbed at 15 and 30 minutes, 2 and 4 hours, and 1, 2, 3, and 7 days after inoculation and subsequently plated onto blood, chocolate, and cycloserine-cefoxitin fructose agar. Plates were incubated for 48 hours and on November 9, 2019, the resulting colonies were manually counted. Statistical analyses (RStudio, version 1.0.153) used analysis of variance with post hoc Tukey honestly significant difference.ResultsOverall, mean colony count was 33 CFU on stethoscopes without barriers vs zero on those with barriers (P≤.05). Growth was greatest at 48 hours, with colony counts as high as 160 CFU. The presence of the barrier resulted in no growth in 100% of stethoscope diaphragms for up to 1 week.ConclusionWe found that stethoscope diaphragm barriers provide an aseptic patient contact point, thus reducing the potential for transmission of C difficile during the physical examination. In critical care environments, in which many hospitals use acoustically inferior disposable stethoscopes, the option of a disposable aseptic stethoscope barrier may allow high-quality auscultation while reducing the potential for pathogen transmission.

816. Effectiveness of Aseptic Stethoscope Barriers in Allowing Clean Contact for Clostridioides Difficile-Contaminated Stethoscopes

BackgroundHealthcare-associated infections (HAIs), such as C. difficile colitis, pose a significant health risk. C. difficile is a spore-forming gram-positive anaerobic bacillus capable of surviving on various surfaces. While a strong emphasis has been placed on hand-washing and environmental cleaning with bleach products to limit the spread of C. difficile, stethoscope contamination has been poorly addressed. Studies have demonstrated that the stethoscope diaphragm harbors similar levels and type of contamination to one’s hands. While a non-alcohol-based solution is recommended for stethoscope hygiene in settings at risk for C. difficile, the use of an aseptic stethoscope diaphragm barrier has not been evaluated. Our purpose is to evaluate whether C. difficile-contaminated stethoscope diaphragms remain aseptic by the placement of an aseptic diaphragm barrier.MethodsFresh cultures of C. difficile were diluted to 107 CFU/mL. After inoculating 16 stethoscope diaphragms with C. difficile, 8 had an aseptic diaphragm barrier applied, and 8 served as non-barrier controls. Contaminated stethoscopes were placed in an anaerobic incubator, then swabbed at 15 and 30 minutes, 2 and 4 hours, and 1, 2, 3, and 7 days after inoculation, and subsequently plated onto blood, chocolate, and cycloserine-cefoxitin fructose agar. These plates were incubated for 48 hours, and resulting colonies were manually counted. Statistical analysis was performed (RStudio version 1.0.153) by ANOVA (Analysis of Variance) with post-hoc Tukey HSD (Honestly Significant Difference).ResultsOverall, mean colony count was 33 CFU on the 8 stethoscope diaphragms without barriers, vs zero on those with barriers (p≤ 0.05). Growth rates were greatest at 48 hours, with colony counts as high as 160. While stethoscope diaphragms without barriers had increasing rates of C. difficile culture growth, the presence of the barrier resulted in no growth in 100% of stethoscope diaphragms for up to 1 week after contamination (Figure 1).C. difficile colony counts from stethoscope diaphragms at time-points up to 1 week. ConclusionAseptic barriers allow C. difficile-contaminated stethoscope diaphragms to remain without bacterial growth for up to a week. Disposable aseptic diaphragm barriers may be effective in preventing the spread of C. difficile.DisclosuresWilliam F. Peacock, MD, AseptiScope Inc. (Board Member)

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.

A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates.

BACKGROUND Clostridium difficile is the major causative agent of nosocomial antibiotic-associated colitis. The gold standard for C. difficile detection is stool culture followed by cytotoxic assay, although it is laborious and time-consuming. We developed a screening test based on a two-step conventional polymerase chain reaction (PCR) approach to detect gluD, the glutamate dehydrogenase (GDH) enzyme gene, which is a marker for screening of C. difficile. Targeting gluD comparing to the conserved stable genetic element of pathogenicity locus (PaLoc), with an accessory gene of Cdd3, was an effective method for the detection of this pathogen from patients with enterocolitis. METHODS Fresh fecal samples of the patients who were clinically suspicious for antibiotic-associated colitis were collected. Stool specimens were cultured on the cycloserine-cefoxitin fructose agar (CCFA) in an anaerobic condition, following alcohol shock treatment and enrichment in Clostridium difficile Brucella broth (CDBB). On confirmed colonies, PCR was carried out for detection of PaLoc subsidiary gene, Cdd3, and toxicogenic genes, tcdA and tcdB. The gluD that is GDH gene detection was performed by conventional PCR on the extracted DNA from 578 fresh stool samples. RESULTS 57 (9.8%) strains of C. difficile were approved by conventional PCR for gluD and Cdd3 genes, in which 37 (6.4%) colonies had tcdA+/tcdB+ genotype, 2 (0.3%) tcdA+/tcdB-, 4 (0.7%) tcdA-/ tcdB+ and the remaining 14 (2.4%) colonies were tcdA and tcdB negative. CONCLUSION These results demonstrate that targeting gluD by PCR is quite promising for rapid detection of C. difficile from fresh fecal samples. Furthermore, the multiple-gene analysis for tcdA and tcdB assay proved a reliable approach for diagnosing of toxigenic strains among clinical samples.

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

Isolation and genotyping of Clostridium perfringens and Clostridium difficile in Capuchin Monkeys (Sapajus spp.)

ABSTRACT: The importance of Clostridium perfringens and C. difficile for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens and C. difficile in stool samples from free-living and captive capuchin monkeys (Sapajus flavius and Sapajus libidinosus) in Brazil. Ten free-living S. flavius and 14 captive S. libidinosus were sampled for this study. To isolate C. difficile, stool samples were inoculated on plates containing cycloserine-cefoxitin fructose agar supplemented with horse blood and sodium taurocholate. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and enrichment in brain heart infusion (BHI) broth followed by plating onto selective agar. C. difficile was not detected in the present study. The results were identical for both protocols tested for isolation of C. perfringens. Four samples (16.7%) were positive for C. perfringens type A, including one sample from a free-living animal (4.2%) and three from captive animals (12.5%), meaning there was no significant difference between these two groups. C. perfringens isolates were negative for all additional virulence factors evaluated, including enterotoxin encoding-gene (cpe) and beta-2 encoding-gene (cpb2). These results suggested that C. perfringens type A is found in the microbiota of capuchin monkeys, although it is less frequent than previously reported in domestic animals.

Use of culture- and ELISA-based toxin assay for detecting Clostridium Difficile, a neglected pathogen: A single-center study from a tertiary care setting.

INTRODUCTION:Clostridium difficile is a Gram-positive spore-bearing anaerobic bacillus increasingly associated with both community- and hospital-acquired colitis and diarrhea. It is the most common identifiable bacterial cause of healthcare-associated diarrhea associated with antibiotic use and one of the most common anaerobic infections. The diagnosis of C. difficile infection includes detection of toxin A/B in stool specimens by direct enzyme immunoassay, culture of pathogen from the stool specimens using a selective agar Cycloserine-Cefoxitin fructose agar (CCFA), tissue culture assay, and detection of glutamate dehydrogenase an enzyme produced by C. difficile. With few reports from India on this disease, the present study was planned to throw more light on the prevalence and utility of laboratory diagnostic methods for C. difficile-associated diarrhea (CDAD).MATERIAL AND METHODS:After taking approval from the Ethics Committee, 150 patients with antibiotic-associated diarrhea were taken as a study group and fifty patients with exposure to antibiotics but who did not develop diarrhea were taken as controls. Stool specimen was processed for both culture on CCFA and toxin detection by IVD Tox A + B ELISA.RESULTS:Only four specimens were culture positive, whereas 13 were ELISA positive. All culture-positive isolates were toxigenic. C. difficile was neither isolated nor its toxin detected in the control group. Culture- and toxin-based assays may not detect all cases of CDAD.CONCLUSION:Based on the results of the present study, culture does not provide any additional yield over toxin assay. Better diagnostic modalities would be required to prove CDAD.

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.

Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting

Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock.

Evaluation of three enzyme immunoassays and toxigenic culture for diagnosis of Clostridium difficile-associated enteritis in piglets

The aim of this study was to compare test performances of three commercial enzyme immunoassays (EIAs) for A and B toxin detection and that of a simple toxigenic culture protocol to the cytotoxicity assay (CTA) as the gold standard for diagnosis of Clostridium difficile-associated enteritis in piglets. A total of 73 piglets submitted to the Veterinary School of Universidade Federal de Minas Gerais were included in this study. Intestinal content was collected from 62 diarrheic and 11 non-diarrheic piglets, 1 to 7 days old. Vero cells were used in the CTA protocol to detect A and B toxins. Fecal samples were inoculated on cycloserine-cefoxitin fructose agar for isolation of C difficile. The EIAs were performed according to the manufacturers’ instructions. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each EIA and for toxigenic culture against CTA. The CTA was positive for 22 of the 73 samples (30.1%). Sensitivities of all EIAs and toxigenic culture for the piglet samples were low (41% to 64%), whereas specificities were 80% to 98%. These results suggest that the EIAs and toxigenic culture protocol tested are not suitable for diagnosis of C difficile infection in individual piglets.

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.