This in vitro study was approved by the Institutional Review Board of the Konkuk University Medical Center, Seoul, Korea (a tertiary referral hospital with 900 beds). From January 2015 to April 2015, we collected 271 consecutive, remnant diarrheal stool samples submitted to the clinical microbiology laboratory from patients admitted to our hospital.They included 258 adults and 13 children (male 148, female 123). Since we used remnant samples after routine tests and the data were analyzed anonymously, informed consent was exempted. As a routine practice of C. difficile testing in our hospital, we performed toxigenic culture and VIDAS C. difficile toxin A&B (bioMérieux) simultaneously. The VIDAS C. difficile GDH (bioMérieux) and Xpert C. difficile (Cepheid) were additionally performed in the same samples. Duplicated samples from the same patients and from patients on treatment for CDI were excluded. The samples were tested within 2 hours of collection; otherwise, they were kept at 2–8°C for up to 2 days.
Vidas c difficile toxin a b
Manufactured by bioMérieux
Sourced in France
The VIDAS C. difficile Toxin A&B is a lab equipment product designed for the detection of Clostridium difficile toxins A and B. It provides a quantitative result for the identification of Clostridium difficile infection.
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8 protocols using vidas c difficile toxin a b
1
Comparative Evaluation of C. difficile Diagnostic Tests
2
Diagnostic Evaluation of Clostridium difficile
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Consecutive, non-repetitive fecal specimens (n = 416) were collected from patients presenting with diarrheal symptoms at the Peking Union Medical College Hospital, from August 2012 to July 2014. All the fecal specimens were stored at 4°C before testing, and were simultaneously tested by VIDAS C. difficile GDH assay (bioMérieux, Marcy l'Etoile, France), VIDAS C. difficile Toxin A&B (bioMérieux, Marcy l'Etoile, France), and culture-based method followed by molecular detection of toxin genes (see below for details).
3
C. difficile Detection in Chinese Hospitals
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Stool specimens from suspected CDI cases are routinely sent to the PUMCH laboratory in Beijing for C. difficile detection. The available tests for C. difficile detection at this hospital are toxin A and B detection using a commercial enzyme immunoassay (EIA) (VIDAS C. difficile Toxin A&B, bioMerieux, Marcy l’Etiole, France). However, C. difficile culture and molecular testing are rarely performed in most Chinese hospitals.
4
Characterization of Toxigenic C. difficile Isolates
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A total of 116 non-duplicate toxigenic C. difficile isolates were recovered from patients with suspected CDI in PUMCH between August 2012 and July 2015. The majority of the isolates (69.0%; 80/116) were from the medical wards, followed by outpatient or emergency department (22.4%; 26/116), surgical department (6.0%; 7/116), and finally intensive care units (2.6%; 3/116).
All specimens were initially tested for toxin A/B using enzyme immunoassay (EIA; VIDAS C. difficile Toxin A&B, bioMérieux, Marcy l’Etiole, France) and cultured on selective cycloserine–cefoxitin–fructose agar (CCFA) plates. Typical colonies on CCFA were identified as C. difficile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics GmbH, Bremen, Germany). Only C. difficile isolates obtained from stool specimens with positive EIA results, and confirmed by MALDI-TOF MS, were included in the study.
All specimens were initially tested for toxin A/B using enzyme immunoassay (EIA; VIDAS C. difficile Toxin A&B, bioMérieux, Marcy l’Etiole, France) and cultured on selective cycloserine–cefoxitin–fructose agar (CCFA) plates. Typical colonies on CCFA were identified as C. difficile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics GmbH, Bremen, Germany). Only C. difficile isolates obtained from stool specimens with positive EIA results, and confirmed by MALDI-TOF MS, were included in the study.
5
Diagnostic Criteria for Clostridium difficile Infection
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Diarrhea was defined as unformed stools ≥ 3 times per day on two consecutive days, and CDI was confirmed when toxigenic culture was positive in diarrheal patients, or the toxin assay A&B (VIDAS®C. difficile Toxin A & B; BioMerieux SA, Marcy l’Etoile, France) yielded positive results, and/or pseudomembranes were seen by endoscopy or histology [10 (link)]. Healthcare-associated CDI (HA-CDI) was diagnosed in patients who developed diarrhea at least 72 hour after hospitalization or within two months of their last discharge from hospital [15 (link)].
Clinical cure was defined as resolution of diarrhea within the treatment period. This required conversion to no more than two semi-formed or formed stools per day [15 (link)]. Recurrence was defined as growth of C. difficile with toxin genes, positive toxin assay A&B, or pseudomembranes, with recurrence of symptoms between the end of treatment and 30 days later.
Clinical cure was defined as resolution of diarrhea within the treatment period. This required conversion to no more than two semi-formed or formed stools per day [15 (link)]. Recurrence was defined as growth of C. difficile with toxin genes, positive toxin assay A&B, or pseudomembranes, with recurrence of symptoms between the end of treatment and 30 days later.
6
Rapid ELFA Assay for C. difficile
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Stool samples were analyzed using an enzyme-linked fluorescent (ELFA) assay, VIDAS®C. difficile Toxin A & B (BioMérieux, France). Stools were mixed with 200 μL distilled water and centrifuged at 12,000 × g. Supernatant (300 μL) was loaded in to the test well. After 75 min, test results were evaluated as <0.13-negative, ≥0.13-<0.37-intermedate, ≥0.37 positive.
7
Detecting C. difficile from Stool Samples
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The stool samples were pre-treated with 99% ethanol for 30 min at room temperature. The samples were inoculated on selective media with antibiotics (Clostridium difficile Moxalactam Norfloxacin), and cultured anaerobically for 24-48 h at 36℃. Suspected C. difficile colonies were identified with an API Rapid ID 32A system (bioMérieux SA, Lyon, France). C. difficile toxin A and B were detected by VIDAS C. difficile Toxin A&B (bioMérieux SA) kits.
8
Rapid C. difficile Toxin and GDH Detection
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Stool samples were tested for toxin and GDH using VIDAS C. difficile toxin A&B and VIDAS C. difficile GDH kit (bioMérieux), respectively, according to the manufacturer's instructions. The assay principle combines a two-step enzyme immunoassay sandwich method with a final fluorescent detection (enzyme-linked fluorescence immunoassay, ELFA). An aliquot of liquid stool was added in the specific diluent, and supernatant after centrifugation was tested. The VIDAS C. difficile toxin A&B is completed within 75 minutes, and results are reported as negative, equivocal, or positive with cut-offs of 0.13 and 0.37 test value (TV). There were 10 equivocal results in our study and they were considered negative for the performance calculations in this study. The VIDAS C. difficile GDH is completed within 50 minutes, and results are reported as negative or positive with a cut-off of 0.10 of TV.
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