Comparative Evaluation of Luminex xTAG® Gastrointestinal Pathogen Panel and Direct-From-Stool Real-Time PCR for Detection of C. difficile Toxin tcdB in Stool Samples from a Pediatric Population (original) (raw)
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Rapid detection of Clostridium difficile toxins from stool samples using real-time multiplex PCR
Journal of Medical Microbiology, 2013
In this study, a total of 650 stool samples were tested to show that our method is capable of detecting four Clostridium difficile genes; tcdA, tcdB, encoding toxin A (TcdA) and toxin B (TcdB), and the binary toxin C. difficile transferase genes (cdtA and/or cdtB) encoding CDT toxin. Besides detecting the targeted C. difficile genes, our method can be used to detect the presence of any inhibitory components in the PCR. This assay, combined with a selective culture medium, such as the chromID TM C. difficile, can be applied directly for screening C. difficile-associated disease. The PCR-based assay developed here is rapid (4 h per 21 stool samples) and accurate in diagnosing C. difficile infection, 100 % assay sensitivity and negative predictive value (NPV) were obtained. However, the assay specificity of 99.1 % and positive predictive value (PPV) of 94.9 % were slightly lower than the optimal value of 100 %. The assay protocol outlined here can be used as a rapid screening tool to assist infection control units and in managing infected patients by reducing the number of patients requiring isolation and extended hospitalization. Rapid detection can prevent unnecessary antibiotic therapy and potentially reduce the spread of infection by emerging hypervirulent C. difficile strains.
Diyala Journal of Medicine
Background: Clostridium difficile is a gram positive anaerobic spore forming bacteria. C. difficile-associated disease is a critical clinical issue that is accepted to happen mainly after hospitalization and used of expansive range anti-infection agents. Objective:To define the rate of C. difficile infections isolated from children patients suffering from diarrhea, detection profile toxigenicity of C. difficile strains for toxin A and toxin B by using of PCR, and revise different risk factors of C. difficile infections. Patients and Methods: This cross-sectional study included 50 patients who hospitalized for at least 2 days before the appearance of three or more unformed or liquid stools for 24h, genomic DNA was extracted by using 10% fecal supernatant and a ready kit was used for extraction according to manufacturer instructions. Molecular detection of toxigenic C. difficile done by using the specific primer sequences in polymerase chain reaction. Results: Current study showed diarrhea was the most prominent complain among the study population accounting for 41(82%), of whom 39(78%) presented with watery diarrhea. 38(76%) patients had no fever. The most comorbid disease was inflammatory bowel disease (IBD) with 7 (14%) patients. Forty-six (92%) cases had no history of hospitalization in the last 3 months versus only 8% had such history. PCR revealed that 16 (32%) samples were positive for tcdB gene, while all samples were negative for genes tcdA.
Annals of Laboratory Medicine, 2015
Background: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and nonclostridial reference strains was 100%. Conclusions: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.
Gastroenterology, 1998
Background & Aims:Clostridium difficile is an important cause of symptomatic diarrhea in pediatric patients. The bacterium produces two toxins, although many laboratories assay for only one. We questioned this diagnostic approach when patients had positive results for C. difficile at our institution, but initially had tested negative at outside laboratories. Methods: We retrospectively analyzed relative frequencies of C. difficile toxin A alone, toxin B alone, and toxins A and B from pediatric patients with diarrhea. Results were stratified according to toxin detection and patient age. Results: Of 1061 specimens, 276 (26.8%) were positive for C. difficile toxin(s). Fifty-one (18.5%) were positive for toxin A alone, 133 (48.2%) for toxin B alone, and 92 (33.3%) for both toxins. Assaying for toxin B identified C. difficile infection more frequently than did assaying for toxin A (P < 0.0001). The frequency of toxin B detection was significantly higher for older children but not for infants. Conclusions: Testing for C. difficile toxin A or toxin B alone will result in more frequent misdiagnosis than testing for both toxins. This practice may lead to inappropriate further invasive investigations in children, although this finding may not be applicable to adults.GASTROENTEROLOGY 1998;115:1329-1334
Rapid Detection of Clostridium difficile in Feces by Real-Time PCR
Journal of Clinical Microbiology, 2003
Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is closely related to the production of toxins A and B. Toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the "gold standard." However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficile toxin genes tcdA and tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficile strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than C. difficile (n ؍ 14), including Clostridium sordellii for which the lethal toxin gene sequence is closely related to the toxin genes of C. difficile. Following a rapid (15 min) and simple fecal sample preparation protocol, both tcdA and tcdB were efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of C. difficile. It is rapid, sensitive, and specific and allows detection of C. difficile directly from feces samples. Clostridium difficile, the main etiological agent of antibioticassociated diarrhea and pseudomembranous colitis, is also the major recognized cause of nosocomial diarrhea (5, 27). The use of antibiotics such as clindamycin, cephalosporins, and ampicillin disrupts the normal intestinal flora, predisposing patients to colonization by C. difficile, which is encountered mainly in health care centers (13, 21, 28, 34). This organism is carried asymptomatically in about 50% of neonates and 20% of hospitalized patients but only in 2% of healthy adults (20, 26). In fact, asymptomatic carriers usually outnumber symptomatic patients (14). Therefore, the high level of healthy carriers among hospitalized patients coupled with the presence of patients under antibiotic treatment explains the high rate of nosocomial diarrhea associated with C. difficile. The pathogenicity of this organism is associated with the production of two large toxins, toxin A (TcdA) and toxin B (TcdB), both implicated in mucosal damage (21). Nontoxigenic strains are not pathogenic. Most strains produce both toxins, but pathogenic strains of C. difficile producing toxin B only have been reported (16, 17, 29, 31). Variability in the toxin genes of C. difficile has been investigated by Rupnik et al. (32, 33) by using restriction fragment length polymorphism analysis. The majority of strains studied had the same toxinotype as the reference strain VPI 10463 (toxinotype 0), whereas approximately 8% of the strains were variants distributed in toxinotypes I to XV. The detection of toxin B by the tissue culture cytotoxicity assay is considered to be the "gold standard" for the detection of C. difficile from fecal samples (9, 20).
Clinical Infectious Diseases, 2007
Background. 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. Methods. 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. Results. 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.
Journal of Clinical Microbiology, 2009
The ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) is a new commercial TaqMan PCR assay that detects tcdB . The ProGastro Cd assay was compared to the Wampole Clostridium difficile toxin B test (TOX-B test; TechLab, Blacksburg, VA), a cell culture cytotoxicity neutralization assay (CCCNA), and to anaerobic toxigenic bacterial culture, as the “gold standard,” for 285 clinical stool specimens. Assays were independently performed according to manufacturers' directions. A 1.0-ml sample was removed from the stool specimen, of which 20 μl was used for extraction on the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC) for the Prodesse ProGastro Cd assay and 200 μl of the stool filtrate was used for the TOX-B CCCNA. Anaerobic toxigenic culture was done by heating an additional 1.0 ml of the stool sample to 80°C for 10 min before inoculation onto modified cycloserine, cefoxitin, and fructose agar with horse blood (Remel, Lenexa, KS) and into a prereduced chopped meat glucose...