Clostridioides ; Clostridioides difficile/isolation & purification* ; Clostridium Infections/epidemiology* ; Guidelines as Topic ; Humans

Clostridium difficile is a significant nosocomial and community-acquired pathogen, and is the leading cause of antibiotic-induced diarrhea associated with high morbidity and mortality. Given that the treatment outcome depends on the severity of C. difficile infection (CDI), we aimed to establish an efficient method of assessing severity, and focused on the stool biomarker fecal calprotectin (FC). FC directly reflects the intestinal inflammation status of a patient, and can aid in interpreting the current guidelines, which requires the integration of indirect laboratory parameters. The distinction of 80 patients with CDI versus 71 healthy controls and 30 severe infection cases versus 50 mild cases was possible using FC as a marker. The area under the receiver operating characteristic curves were 0.821 and 0.746 with a sensitivity of 75% and 70% and specificity of 79% and 80%, for severe versus mild cases, respectively. We suggest FC as a predictive marker for assessing CDI severity, which is expected to improve the clinical management of CDI.
Aged ; Area Under Curve ; Biomarkers/analysis ; Clostridium difficile/*isolation & purification ; Enterocolitis, Pseudomembranous/diagnosis/microbiology/*pathology ; Enzyme-Linked Immunosorbent Assay ; Feces/*chemistry ; Female ; Humans ; Leukocyte L1 Antigen Complex/*analysis ; Male ; Middle Aged ; ROC Curve ; Severity of Illness Index

BACKGROUND: We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. METHODS: We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). RESULTS: Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). CONCLUSIONS: Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.
Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/*genetics/isolation & purification ; DNA, Bacterial/*analysis/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; *Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

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 non-clostridial 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.
ADP Ribose Transferases/genetics ; Bacterial Proteins/*genetics ; Bacterial Toxins/*genetics ; Clostridium difficile/isolation & purification/*metabolism ; DNA, Bacterial/genetics/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Prospective Studies ; Real-Time Polymerase Chain Reaction ; Triose-Phosphate Isomerase/genetics

PURPOSE: Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI. MATERIALS AND METHODS: In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection. RESULTS: ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h. CONCLUSION: Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.
Chromogenic Compounds/chemistry ; Clostridium difficile/chemistry/*isolation & purification ; Culture Media/*chemistry

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.
Bacterial Proteins/*analysis ; Bacterial Toxins/*analysis ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/enzymology/*isolation & purification/metabolism ; Enterotoxins/*analysis ; Feces/microbiology ; Glutamate Dehydrogenase/*analysis ; Humans ; *Immunoenzyme Techniques ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.
Agar/chemistry ; Bacterial Proteins/genetics ; Bacteriological Techniques/*methods ; Chromogenic Compounds/chemistry/metabolism ; Clostridium difficile/genetics/*isolation & purification ; Culture Media/chemistry ; DNA, Bacterial/analysis/metabolism ; Diarrhea/microbiology/pathology ; Feces/*microbiology ; Humans ; Polymerase Chain Reaction ; Time Factors

BACKGROUND: ChromID Clostridium difficile agar (IDCd; bioMerieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). METHODS: A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMerieux SA), and multiplex PCR for tcdA, tcdB, and tpi. RESULTS: The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. CONCLUSIONS: The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.
Agar/*chemistry ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium difficile/genetics/*isolation & purification ; DNA, Bacterial/analysis ; Enterocolitis, Pseudomembranous/diagnosis/microbiology ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Triose-Phosphate Isomerase/genetics

OBJECTIVETo develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin.

METHODSTaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi) of C. difficile strains and the toxins A(TcdA), B(TcdB) and truncated toxin A(TcdAT). Sensitivity, specificity and anti-interference ability of these methods were estimated, as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay.

RESULTSThe detection limits of tpi were 6×10⁻² CFU/µl and 6 × 10⁻¹ CFU/µl in the non-oxin producing and toxin producing strains, respectively. The coefficients of variability(CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3% . The CVs of intra-assay and inter-assay for the detection limit of tpi, tcdA, tcdB and tcdAT in the toxin producing strain were 3.0% and 3.4%, 2.9% and 3.2%, 5.3% and 5.7%, 2.7% and 2.8%, respectively. No interference was detected from other genus or species in clostridium. From 50 clinical samples, thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique, in which 3 were dubious and 2 were negative under VIDAS.

CONCLUSIONThe newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis, clinically.

Bacterial Proteins ; isolation & purification ; Bacterial Toxins ; isolation & purification ; Clostridium difficile ; isolation & purification ; Enterotoxins ; isolation & purification ; Real-Time Polymerase Chain Reaction ; methods

BACKGROUND: Antimicrobial susceptibility testing (AST) of Clostridium difficile is increasingly important because of the rise in resistant strains. The standard medium for the AST of C. difficile is supplemented Brucella agar (sBA), but we found that the growth of C. difficile on sBA was not optimal. Because active growth is critical for reliable AST, we developed a new, modified C. difficile (mCD) agar. C. difficile grew better on mCD agar than on sBA. METHODS: C. difficile isolates were collected from patients with healthcare-associated diarrhea. sBA medium was prepared according to the CLSI guidelines. Homemade mCD agar containing taurocholate, L-cysteine hydrochloride, and 7% horse blood was used. For 171 C. difficile isolates, we compared the agar dilution AST results from mCD agar with those from sBA. RESULTS: No significant differences were observed in the 50% minimal inhibitory concentration (MIC50) and 90% minimal inhibitory concentration (MIC90) of clindamycin (CLI), metronidazole (MTZ), moxifloxacin (MXF), piperacillin-tazobactam (PTZ), and rifaximin (RIX), but the values for vancomycin (VAN) were two-fold higher on mCD agar than on sBA. The MICs of CLI, MXF, and RIX were in 100% agreement within two-fold dilutions, but for MTZ, VAN, and PTZ, 13.7%, 0.6%, and 3.1% of the isolates, respectively, were outside the acceptable range. CONCLUSIONS: The MIC ranges, MIC50 and MIC90, were acceptable when AST was performed on mCD agar. Thus, mCD agar could be used as a substitute medium for the AST of C. difficile.
Anti-Infective Agents/*pharmacology ; Clostridium Infections/microbiology ; Clostridium difficile/*drug effects/isolation & purification ; Diarrhea/*microbiology ; Humans ; Microbial Sensitivity Tests/*methods