Toxigenic Clostridium Difficile Research Articles

Infections caused by Clostridium difficile bacterium

Toxigenic strains of Clostridium difficile are the major cause of intestinal tract diseases of hospitalized patients. For a long time, Clostridium difficile was considered to produce both types of toxins (A+/B+ strain). However, the investigations conducted in the last ten years point to the existence of clinically significant isolates which produce only toxin B, i.e. toxin A negative. Toxigenic strains Clostridium difficile are present in our country so this bacterium sort has to be considered in differential causal diagnosis of diarrhea syndrome. Considering that it can cause a difficult form of the disease, it is an obligation to establish the presence of some toxins in stool samples of patients and/or production of some toxins in liquid culture of isolates.

Molecular and Susceptibility Analysis of Toxigenic <i>Clostridium difficile</i> Obtained from Adult Patients Suspected of CDI in Trinidad

Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits; however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.

Isolation of toxigenic Clostridium difficile from ready-to-eat salads by multiplex polymerase chain reaction in Isfahan, Iran.

Background:Since 2003, the incidence of community associated Clostridium difficile infection (CA-CDI) has increased; different types of food have been supposed to be the vectors of C. difficile strains. The purpose of this study is to investigate the occurrence of C. difficile strains in ready-to-eat salads distributed in food services.Materials and Methods:A total of 106 ready-made salad specimens were sampled from different restaurants and food services located in Isfahan, in the center of Iran. Positive isolates of C. difficile were identified and confirmed for the existence of three genes including tpi, tcdA and tcdB by multiplex PCR.Results:A total of six (5.66%) samples were positive for C. difficile strains. Of which, one strain (16.6%) was positive for A and B toxins.Conclusion:The existence of toxigenic C. difficile in ready-made salads could be a caution for public health. Further investigation is required to assess the relationship between the isolated strains in our study and those from diarrheic patients through molecular typing.

Prevalence and characterization of enterotoxigenic Bacteroides fragilis and toxigenic Clostridium difficile in a Taipei emergency department.

Enterotoxigenic Bacteroides fragilis (ETBF) and toxin-encoding Clostridium difficile (TXCD) are associated with gastroenteritis. Routine anaerobic blood culture for recovery of these anaerobic pathogens is not used for the detection of their toxins, especially for toxin-variant TXCD. The aim of this study was to investigate the prevalence and risk factors of the genotypes of these anaerobes in patients with acute diarrheal illnesses. The data and samples of 513 patients with gastroenteritis were collected in a Taipei emergency department from March 1, 2006 to December 31, 2009. Nonenterotoxigenic B. fragilis (NTBF) and ETBF and the toxin genotypes of TXCD were detected by molecular methods. The prevalence rates of NTBF, ETBF, and TXCD infections were 33.14%, 1.56%, and 2.34%, respectively. ETBF infections often occurred in the elderly (average age=67.13 years) and during the cold, dry winters. TXCD infections were widely distributed in age and often occurred in the warm, wet springs and summers. The symptoms of ETBF-infected patients were significantly more severe than those of NTBF-infected patients. This study identified and analyzed the prevalence, risk factors, and clinical presentations of these anaerobic infections. Future epidemiologic and clinical studies are needed to understand the role of ETBF and TXCD in human gastroenteritis.

1645Do asymptomatic carriers of toxigenic Clostridium difficile identified through inappropriate testing represent a significant risk for transmission?

1645Do asymptomatic carriers of toxigenic Clostridium difficile identified through inappropriate testing represent a significant risk for transmission?

Clostridium difficile isolates with high linezolid MICs harbor the multiresistance gene cfr.

We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC50 and MIC90 of linezolid were 0.75 μg/ml and 1.5 μg/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 μg/ml to 16 μg/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variable-number tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance.

Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

Background Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.MethodsTaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).ResultsThe analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA−TcdB+, and TcdA−TcdB− types), TcdB-producing strains (TcdA+TcdB+ and TcdA−TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA−TcdB− strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.ConclusionOur qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

Performance assessment of the BD MAX Cdiff assay in comparison to Xpert C. difficile assay in a setting with very low prevalence of toxigenic Clostridium difficile PCR ribotype 027

In a clinical setting with low prevalence of ‘epidemic’ PCR ribotype 027, the BD MAX Cdiff assay was found to be a suitable alternative to the Xpert C. difficile assay for the detection of toxigenic Clostridium difficile in samples which are reflex PCR tested after obtaining a discrepant immunoassay result. There was no significant difference between the sensitivities and specificities of both commercial molecular assays.

Clostridium Difficile Colitis: Epidemiology, Diagnosis, Treatment, and Modalities

Clostridium difficile infection (CDI) or Clostridium difficile colitis is an infection that causes significant morbidity and mortality. It accounts for 20-30% of antibiotic associated diarrhea and is occurring more often in the absence of antibiotics. It is the leading cause of hospital associated diarrhea. The organism has a remarkable ability to thrive outside of the colon, making hand washing and other preventative measures particularly important. The diagnosis of CDI involves obtaining a stool specimen positive for toxigenic Clostridium difficile or Clostridium difficile toxins. Pseudomembranous colitis findings during colonoscopy may also be utilized to make the diagnosis. Polymerase chain reaction (PCR) test is the ideal testing method because it is highly sensitive and specific and results may be obtained in approximately one hour. The treatment of CDI involves terminating the offending agent followed by antimicrobial therapy dictated by these classifications: 1) mild, moderate or severe 2) complicated vs. non-complicated 3) first episode, first recurrence or second recurrence Metronidazole and vancomycin remain the initial treatment of choice, but other considerations such as possible offending agents, severity of disease and number of previous episodes are also of great importance. There are several other treatments available including fecal microbiota transplant although this is reserved for severe cases or third recurrences. However the best way to combat the disease remains through preventative measures such as proper hand sanitation.

Epidemiology and clinical features of toxigenic culture-confirmed hospital-onset Clostridium difficile infection: a multicentre prospective study in tertiary hospitals of South Korea.

Hypervirulent Clostridium difficile strains, most notably BI/NAP1/027, have been increasingly emerging in Western countries as local epidemics. We performed a prospective multicentre observational study from December 2011 to May 2012 to identify recent incidences of toxigenic culture-confirmed hospital-onset C. difficile infections (CDI) and their associated clinical characteristics in South Korea. Patients suspected of having been suffering from CDI more than 48 h after admission and aged ≥20 years were prospectively enrolled and provided loose stool specimens. Toxigenic C. difficile culture (anaerobic culture+toxin A/B/binary gene PCR) and PCR ribotyping were performed in one central laboratory. We enrolled 98 toxigenic culture-confirmed CDI-infected patients and 250 toxigenic culture-negative participants from three hospitals. The incidence of toxigenic culture-confirmed hospital-onset CDI cases was 2.7 per 10,000 patient-days. The percentage of severe CDI cases was relatively low at only 3.1%. UK ribotype 018 was the predominant type (48.1%). There were no hypervirulent BI/NAP1/027 isolates identified. The independent risk factors for toxigenic culture-confirmed hospital-onset CDI were invasive procedure (odds ratio (OR) 7.3, P=0.003) and past CDI history within 3 months (OR 28.5, P=0.003). In conclusion, the incidence and severity of CDI in our study were not higher than reported in Western countries.

Evaluation of the illumigene C. difficile assay for toxigenic Clostridium difficile detection: a prospective study of 302 consecutive clinical fecal samples

Toxigenic Clostridium difficile is a major pathogen causing nosocomial diarrhea. Consequently, rapid detection of toxigenic C. difficile is very important in clinical laboratories. The illumigene C. difficile DNA amplification assay (illumigene; Meridian Bioscience, Inc.) is a rapid method that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification. In the present study, we evaluated the diagnostic performance of the illumigene assay using 302 consecutive stool specimens in comparison with the VIDAS C. difficile A&B enzyme-linked fluorescent immunoassay (VIDAS-CDAB; bioMérieux). Toxigenic culture was used as the reference method. The sensitivity, specificity, positive predictive value, and negative predictive value of the illumigene assay were 88.1%, 96.7%, 86.7%, and 97.1%, respectively, while those of the VIDAS-CDAB assay were 40.4%, 98.8%, 87.5%, and 88.5%, respectively. It is of note that use of a combination of the illumigene and VIDAS-CDAB assays did not improve any of the 4 evaluated parameters (88.1%, 95.5%, 82.5%, and 97.1%, respectively). The illumigene assay showed limits of detection of 250 and 11,467 CFU/mL for ATCC 9688 (tcdA+, tcdB+, cdtB−) and ATCC 43598 (tcdA−, tcdB+, cdtB−) reference strains, respectively, and there was no cross-reactivity with 8 frequently isolated bacterial species. In conclusion, the illumigene assay might be a useful method for rapid detection of toxigenic C. difficile in clinical laboratories. Additionally, the VIDAS-CDAB assay seems unnecessary if the illumigene assay is used.

In vitro antibiotic susceptibility profile of Clostridium difficile excluding PCR ribotype 027 outbreak strain in Hungary

Our study showed the antibiotic susceptibility profile of toxigenic Clostridium difficile isolated from nosocomial and community-acquired CDI between 2008 and 2010. MICs of 200 C. difficile strains were determined using E®test method in the case of erythromycin, clindamycin, moxifloxacin, rifampicin, and metronidazole. All strains were susceptible to metronidazole in the study period. Resistance rates to erythromycin, clindamycin and moxifloxacin were 31%, 29.5%, and 21.5%, respectively. In the case of rifampicin, the MIC range was quite wide, 11.5% of the tested strains proved to be highly resistant (MIC ≥32 μg/ml) to rifampicin. When we compared these results with our earlier findings from 2006 to 2007, only minor changes in susceptibility over the time-periods could be observed in the case of erythromycin, clindamycin, moxifloxacin, and rifampicin, but metronidazole susceptibility did not show changes.

Detection of toxigenic Clostridium difficile by loop-mediated isothermal amplification (LAMP)

Detection of toxigenic Clostridium difficile by loop-mediated isothermal amplification (LAMP)

Survey of Clostridium difficile in retail seafood in College Station, Texas

The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America with the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer to humans. The objective of the present study was to investigate the prevalence of C. difficile in retail seafood from grocery stores in College Station, Texas. C. difficile was found in 4.5% (3/67) of shellfish and finfish samples. The positive samples included one each from fresh mussel, frozen salmon and frozen shrimp. The mussel and salmon isolates were characterized as toxinotype V and pulsed-field gel electrophoresis (PFGE) type-NAP7. The shrimp isolate was identified as toxinotype XII, but had an unknown PFGE type. Susceptibilities to 11 antimicrobial agents were identical for the mussel and salmon isolates and were sensitive to eight of 11 antimicrobials (including ampicillin) and intermediate to clindamycin. However, the shrimp isolate was resistant to clindamycin and ampicillin. This study demonstrates that seafood, like other food commodities, can be contaminated by C. difficile.

Prevalence and duration of asymptomatic Clostridium difficile carriage among healthy subjects in Pittsburgh, Pennsylvania.

Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens.

Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP)

Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP)

Kibdelomycin Is a Potent and Selective Agent against Toxigenic Clostridium difficile

Clostridium difficile is the causative agent of C. difficile-associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates (n=168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms (n=598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC90=0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC50>32 μg/ml; n=270). Potent anti-C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti-C. difficile agent.

Portrait Toxigenic Clostridium difficile assay, an isothermal amplification assay detects toxigenic C. difficile in clinical stool specimens

The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8–97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.

Strongyloides stercoralisInfection in a Non-Endemic Area

Patient: 75-year-old Caucasian male. Chief Complaint: Frequent diarrhea. History of Present Illness: The patient had a history of Crohn’s disease, initially diagnosed in 1976 with a positive test for toxigenic Clostridium difficile ( C. difficile ) in August 2011, successfully treated with methronidazole (500 mg PO bid ×10 d). There was a concern that diarrhea may be a result of C. difficile recurrence. Past Medical History: The patient had an extensive medical history, including eosinophilia (8%, normal 0%–7%) in October 2011, gastroesophageal reflux disease, anemia secondary to Crohn’s disease, coronary artery disease, hypertension, osteoarthritis, and Type II diabetes mellitus. Additionally, the patient has severe Crohn’s disease which involves the terminal ileum and colon, and has undergone multiple small bowel resections as well as a colectomy. Medications included immunomodulatory therapy with balsalazide (Colazal), azathioprine (Imuran), infliximab (Remicade), ranitidine, and prednisone for both Crohn’s disease and osteoarthritis. Previous biopsies of the small intestine and colon (before the current presentation) indicated changes consistent with Crohn’s disease. Travel History: The patient was originally from Louisiana, and briefly lived in Las Vegas, NV. For approximately 40 years, he has lived in western Iowa. The patient did not have a history of foreign travel nor had he recently traveled outside of Iowa or Nebraska. He drives a bus for a local business. Principle Laboratory Findings: Loose brown stool was collected in December 2011 and submitted for laboratory testing for toxigenic C. difficile and cultured for enteric pathogens. An ova and parasites exam was not ordered at that time. Salmonella, Shigella , and Campylobacter species, as well as Shiga toxin producing- Escherichia coli , were not detected; the toxigenic C. difficile assay was negative. A laboratory technologist noted the presence of small trails of displaced bacteria on the blood agar plate from the original stool culture (Image 1). From …

Utility of a Commercial PCR Assay and a Clinical Prediction Rule for Detection of Toxigenic Clostridium difficile in Asymptomatic Carriers

A commercial PCR assay of perirectal swab specimens detected 17 (68%) of 25 asymptomatic carriers of toxigenic Clostridium difficile, including 93% with skin and/or environmental contamination. A clinical prediction rule, followed by PCR screening, could be used to identify carriers at high risk of C. difficile shedding.