Clostridium Difficile In Stool Samples Research Articles

Aetiology of hospital-acquired diarrhoea in under-five children from an urban hospital in East Delhi, India.

Majority of the studies of hospital-acquired diarrhoea conducted in Western countries have focused on the detection of Clostridium difficile in stool samples. Limited Asian and Indian literature is available on hospital-acquired diarrhoea. This study was aimed to describe the aetiological profile for hospital-acquired diarrhoea in children aged below five years. One hundred children aged one month to five years who developed diarrhoea (≥3 loose stools for >12 h) after hospitalization for at least 72 h were enrolled. Children who were prescribed purgatives or undergoing procedures such as enema and endoscopy or those with underlying chronic gastrointestinal disorders such as celiac disease and inflammatory bowel disease were excluded from the study. Stool samples from the enrolled children were subjected to routine microscopic examination, modified Ziel-Nielson (ZN) staining for Cryptosporidium and culture for various enteropathogens. Multiplex PCR was used to identify the strains of diarrhoeagenic Escherichia coli. Rotavirus detection was done using rapid antigen kit. Toxins (A and B) of C. difficile were detected using enzyme immunoassay. Of the 100 samples of hospital-acquired diarrhoea analysed, diarrhoeagenic E. coli (DEC) was found to be the most common organism, detected in 37 per cent of cases (enteropathogenic E. coli-18%, enterotoxigenic E. coli-8%, enteroaggregative E. coli-4% and mixed infections-7%). Cryptosporidium was detected in 10 per cent of cases. Rotavirus was detected in six per cent and C. difficile in four per cent of cases. The findings of this study suggest that the aetiological profile of hospital-acquired diarrhoea appears to be similar to that of community-acquired diarrhoea, with DEC and Cryptosporidium being the most common causes. The efforts for the prevention and management of hospital-acquired diarrhoea should, thus, be directed towards these organisms.

Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification.

It is challenging to employ nucleic acid-based diagnostics for the in situ detection of Clostridium difficile from complex fecal samples because essential sample preparation and amplification procedures require various experimental resources. In this study, a simple and effective on-site nucleic acid-based detection system was used to detect C. difficile in stool samples. Two columns containing different microbeads, namely, glass and functionalized graphene oxide-coated microbeads, were designed to remove relatively large impurities by filtration and concentrate bacteria, including C. difficile, from stool samples by adsorption. The bacterial nucleic acids were effectively extracted using a small bead beater. The effectiveness of enzyme inhibitors remaining in the sample was efficiently reduced by the direct buffer developed in this study. This sample preparation kit consisting of two simple columns showed better performance in real-time polymerase chain reaction (PCR) and equivalent performance in loop-mediated isothermal amplification (LAMP) than other sample preparation kits, despite 90% simplification of the process. The amplification-ready samples were introduced into two microtubes containing LAMP pre-mixtures (one each for E. coli as an external positive control and C. difficile) by a simple sample loader, which was operated using a syringe. LAMP, which indicates amplification based on color change, was performed at 65 °C in a small water bath. The limit of detection (L.O.D) and analytical sensitivity/specificity of our simple and effective kit were compared with those of a commercial kit. C. difficile in stool samples could be detected within 1 h with 103 cfu/10 mg using LAMP combined simple on-site detection kit.

The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay

This research compares the performance and diagnostic accuracy of three molecular tests for the detection of Clostridium difficile in stool samples, with the Xpert® C. difficile assay.Fifty-nine positive and twenty-five stool negative samples were analyzed by the BDmax™ Cdiff, the Simplexa C. difficile Direct, and the GenomEra™ C. difficile, and compared with the Xpert® C. difficile routinely used in our laboratory.The highest sensitivity was 94.9% for the BDmax™ Cdiff, followed by GenomEra™ C. difficile with 93.2%, and Simplexa™ C. difficile Direct with 89.8%. The specificities of all assays were 100%. GenomEra™ C. difficile had the highest retesting rate (12%). Simplexa™ C. difficile Direct benefits from both short hands-on time and total-turnaround time and 0% retesting.The differences in performance and accuracy between these three molecular assays are insignificant and all may be used as part of the routine algorithm for detection of C. difficile.

Diagnostic accuracy of loop-mediated isothermal amplification in detection of Clostridium difficile in stool samples: a meta-analysis.

IntroductionClostridium difficile infection (CDI) remains a diagnostic challenge for clinicians. More recently, loop-mediated isothermal amplification (LAMP) has become readily available for the diagnosis of CDI, and many studies have investigated the usefulness of LAMP for rapid and accurate diagnosis of CDI. However, the overall diagnostic accuracy of LAMP for CDI remains unclear. In this meta-analysis, our aim was to establish the overall diagnostic accuracy of LAMP in detection of Clostridium difficile (CD) in stool samples.Material and methodsA search was done in PubMed, MEDLINE, EMBASE and Cochrane Library databases up to February 2014 to identify published studies that evaluated the diagnostic role of LAMP for CD. Methodological quality was assessed according to the quality assessment for studies of diagnostic accuracy (QUADAS) instrument. The sensitivities (SEN), specificities (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) were pooled statistically using random effects models. Statistical analysis was performed by employing Meta-Disc 1.4 software. Summary receiver operating characteristic (SROC) curves were used to summarize overall test performance. Funnel plots were used to test the potential publication bias.ResultA total of 9 studies met inclusion criteria for the present meta-analysis. The pooled SEN and SPE for diagnosing CD were 0.93 (95% CI: 0.91–0.95) and 0.98 (95% CI: 0.98–0.99), respectively. The PLR was 47.72 (95% CI: 15.10–150.82), NLR was 0.07 (95% CI: 0.04–0.14) and DOR was 745.19 (95% CI: 229.30−2421.72). The area under the ROC was 0.98. Meta-regression indicated that the total number of samples was a source of heterogeneity for LAMP in detection of CD. The funnel plots suggested no publication bias.ConclusionsThe LAMP meets the minimum desirable characteristics of a diagnostic test of SEN, SPE and other measures of accuracy in the diagnosis of CD, and it is suitable as a rapid, effective and reliable stand-alone diagnostic test for diagnosis of CDI, potentially decreasing morbidity and nosocomial spread of CD.

Clostridium difficileassociated diarrhea - ′suspect, inspect, treat, and prevent′

Context: Clostridium difficile is a fastidious, gram-positive, spore-forming bacterium responsible for infectious diarrhea and pseudomembranous colitis associated with significant morbidity and mortality. Only toxigenic strains produce disease in humans. Pathogenicity is dependent on the presence of diarrhea-producing toxins, named toxin A (TcdA) and toxin B (TcdB). Risk factors include depletion of protective gut flora by antibiotics and diminished immune response to C. difficile due to age and medical co morbidities; and increased use of proton-pump inhibitors (PPI). Treatment includes the stoppage of inciting antibiotics if possible, to allow regeneration of the normal gut microflora, and starting an antibiotic with activity against C. difficile. A good clinical suspicion in patient with co morbidities developing diarrhea during hospital stay can help reduce the burden of this treatable morbid infection. Our aim of doing this study was to determine the prevalence of this infection in our tertiary care hospital, so as to monitor its burden in future. Aims: To determine the prevalence of this infection in our tertiary care hospital. Settings and Design: A retrospective study was conducted in the department of Microbiology in a tertiary care hospital in North India. Materials and Methods: A total of 195 stool samples received over a period of 2 years were included in the study. An enzyme immunoassay was performed for the qualitative determination of toxins A and B from Clostridium difficile in stool samples. Results: A total of 13 (6.67%) stool samples out of 195 samples processed were positive for the presence of Clostridium difficile toxins A/B. Conclusions: CDI has become a global public health challenge today. Various studies show a prevalence rate between 11% - 22%. Lower prevalence rate revealed from our study (6.7%), makes it imperative to maintain a strict surveillance in our patients to ensure opportune detection and treatment

Using a dog’s superior olfactory sensitivity to identify Clostridium difficile in stools and patients: proof of principle study

Objective To investigate whether a dog’s superior olfactory sensitivity can be used to detect Clostridium difficile in stool samples and hospital patients. Design Proof of principle study, using a case-control...

Clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile cultivated from stool samples of hospitalized patients.

The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group.Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow’s medium), and to selective cycloserine-cefoxitin-fructose agar (CCFA) (Biomedics, Parg qe tehnicologico, Madrid, Spain) for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany) and ColorPAC ToxinA test (Becton Dickinson, USA). Examination of stool specimens for the presence of parasites (causing diarrhea) was done using standard methods (conventional microscopy), commercial concentration test Paraprep S Gold kit (Dia Mondial, France) and RIDA®QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany). Examination of stool specimens for the presence of fungi (causing diarrhea) was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA – ridascreen (R-Biopharm AG, Germany).In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25%) of patients with diarrhea were positive for toxins A and B, one (1.25%) were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5%) patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients. Examination of cultivated colonies revealed that most of cultivated species belonged to genera of Campylobacter spp., Salmonella spp., and Candida spp.. In control group, toxigenic Clostridium difficile cultivated from stool samples of two patients (2%) and non-toxigenic Clostridium difficile from samples of five patients (5%).This research confirmed clinical importance of toxigenic Clostridium difficile found in liquid stool samples of hospitalized patient, and the possibility of asymptomatic carriage in 2% of patients with formed stool.

Vibrio cholerae Non-O139 and Non-139 Infection in Hepatitis C Cirrhosis: A Rare Event

Purpose: A 37 year-old Portuguese male, with genotype 4 hepatitis C cirrhosis (Child-Pugh class B), was submitted to antiviral treatment with Peg-interferon α-2a and ribavirin in 2006, with no response. At that time he had no complications of portal hypertension. In December 2010, he was admitted in our Gastroenterology Ward due to fever, abdominal pain and watery profuse diarrhea starting 72 hours prior to admission. Laboratory workup revealed hypokalemia (2.8 mEq/L) and an elevated C-reactive protein (165 mg/L). He received fluids reposition and antibiotherapy (third-generation cephalosporin and metronidazol). Abdominal X-ray was normal; Human Immunodeficiency Virus 1 and 2, Hepatitis B, Cytomegalovirus, Herpes 1 and 2 serologies were negative. Salmonella, Shigella, Campylobacter, parasitology exam, virology exam, micobacterology exam and toxin Clostridium difficile in stool samples were negative. Blood culture yielded curved gram-negative bacillus (fig. 1), identified as Vibrio cholerae and confirmed by molecular methods. Vibrio cholerae serogroups non O1 and non O139 were excluded by serology. He was discharged, asymptomatic, without ascites or encephalopathy. He lived in an urban area and there were no similar complaints in his family members or recent travels. He was not exposed to seawater, but he had consumed cooked shrimps, 72 hours prior to admission. V. cholerae strains not agglutinating with O1 or O139 antisera are referred to as non-O1 and non-O139. Only O1 and O139 serogroups of V. cholerae are responsible for the epidemic or pandemic cholera by secreting a non-invasive enterotoxin, bacteraemia is rare. V. cholerae non-O1 and non-139 may cause sporadic cases of diarrhea but may also lead to invasive extraintestinal illness and bacteraemia. This is a rare condition and is potentially fatal. The majority of reported cases occurred in patients with cirrhosis or haematological maligancies associated with seafood consumption, as in this case.Figure: No Caption available.

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5'-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.

Diarrhoea caused by Clostridium difficile in patients with postoperative subhepatic abscess

Toxigenic strains of Clostridium difficile in the majority of cases cause disease of the intestinal tract 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/toxin B positive (A-/B+ strain) Clostridium difficile. We presented the case of a patient admitted to the Surgery Clinic, Clinical Center Nis due to the presence of calculus in the ductus choledochus. Twenty-four hours after the surgical intervention for calculus removal, the first signs of the operative wound infection began to appear. In the course of infection treatment, different antibiotics were administered (cefuroxine, ciprofloxacin, vancomycin, imipenem). After making etiological microbiological diagnosis and application of antibiotics according to antibiogram results, the signs of the operative wound infection began to withdraw, but the patient reported the abdominal pain and liquid stools with traces of blood (up to 17 stools per day). By microbiological examination, Clostridium diffidile was cultivated and the presence of toxin B was detected in the stool samples. The patient was sent to the Clinic for Infectious Diseases, where the causal therapy of mitronidazol was administered. Liquid and electrolytes were made up by substitution therapy. After the eight-day-treatment, the patient felt much better, and diarrheas stopped on the 10th day of the therapy application. Our results have shown that toxingen strains Clostridium difficile are present in our country so this bacterium sort have to be considered in differential causal diagnosis of diarrhoea syndrome. Considering that it can cause difficult form of the disease, it is an obligation to establish the presence of some toxins of Clostridium difficile in stool samples of patients and/or production of some toxins in liquid culturate of isolates to provide data for the presence of strains which produce only toxin B.

Detection of Clostridium difficile in Stool Samples From Patients in the Early Period After Liver Transplantation

Objective We examined the frequency of detection of Clostridium difficile (CD) toxins compared with the recovery of C. difficile in stool specimen cultures among orthotopic liver transplant (OLT) patients with nosocomial diarrhea in the early period. Materials and Methods The study included stool samples obtained during the first 30 days after OLT in adults who were suspected of CD-associated diseases. The identification of cultured CD strains was performed by standard microbiological methods. The presence of CD toxins was assayed using a commercial immunoassay. Results All patients were followed prospectively for CD infections from the date of OLT for the first 4 weeks after surgery. Among 54 samples, 16.7% were culture-positive for CD. CD toxins were tested on 54 samples, yielding 63% toxin-positive samples and 30% toxin- and culture-negative results. In the first week after OLT, samples from 19 patients were subjected to CD investigation. Among 19 samples positive for toxin, 52.6% of all samples were culture-negative. We analyzed 35 samples from the second to the fourth week after OLT in 31 recipients. Among 35 samples, 68.6% and 25.7% were positive for CD toxin and for culture, while 20% of samples were negative for toxin and culture. Conclusion In our study, 63% of samples were toxin-positive with 16.7% yielding growth of CD and 30% being negative for toxins and cultures.

Detection of Toxigenic Clostridium difficile in Stool Samples by Real-Time Polymerase Chain Reaction for the Diagnosis of C. difficile-Associated Diarrhea

Clostridium difficile-associated diarrhea (CDAD) is the major cause of health care-associated infectious diarrhea. Current laboratory testing lacks a single assay that is sensitive, specific, and rapid. The purpose of this work was to design and validate a sensitive and specific real-time polymerase chain reaction (PCR) diagnostic test for CDAD. This observational validation study of a new real-time PCR assay occurred from July 2004 through April 2006 and involved the testing of 1368 stool samples. As the final validation portion of the investigation, 350 inpatients were prospectively interviewed for clinical findings for 365 episodes of diarrheal illness. Test results and clinical criteria were used to assess the performance of 4 assays. Using clinical criteria requiring at least 3 loose stools in 1 day as part of the reference standard for a positive test result supporting CDAD, the sensitivity, specificity, and positive and negative predictive values were 73.3%, 97.6%, 73.3%, and 97.6%, respectively, for enzyme immunoassay; 93.3%, 97.4%, 75.7%, and 99.4%, respectively, for real-time PCR; 76.7%, 97.1%, 69.7%, and 97.9%, respectively, for cell culture cytotoxin assay; and 100.0%, 95.9%, 68.2%, and 100.0%, respectively, for anaerobic culture (for toxigenic C. difficile strains). The real-time PCR and anaerobic culture assays were significantly more sensitive than the enzyme immunoassay (P<.01 to P<.05). With an assay turnaround time of <4 h, real-time PCR is a more sensitive and equally rapid test, compared with enzyme immunoassay, and is a feasible laboratory option to replace enzyme immunoassay for toxigenic C. difficile detection in clinical practice, as well as for use during the development of new therapeutic agents.