Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B [TOX-B] test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n ؍ 273) and soft (n ؍ 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80°C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the "gold standard," the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens.
We evaluated a single membrane device assay for simultaneously detecting both Clostridium difficile glutamate dehydrogenase (GDH) and toxin A/B antigens against a standard that combines two PCR assays and cytotoxigenic culture. Results showing dual GDH and toxin A/B antigen positives and negatives can be reported immediately as true positives and negatives, respectively. Specimens with discrepant results for GDH and toxins A/B, which comprised 13.2% of the specimens, need to be retested.Rapid and accurate diagnosis of Clostridium difficile infection (CDI) is crucial for patient care, infection control, and efficient surveillance. The well-accepted standard is cytotoxigenic culture, which is done by culturing C. difficile from the stool and then performing a cytotoxin assay on the isolate (9). The cytotoxigenic culture is labor-intensive, subjective, and time-consuming, which has limited its wide use in the clinical setting. Enzyme immunoassays (EIAs) are the most common diagnostic laboratory methods used for rapid detection of C. difficile-specific glutamate dehydrogenase (GDH) and/or toxin A/B antigens in stool specimens. However, traditional EIAs lack sensitivity and specificity (4,5,11,12,18).Recently, a C. Diff Quik Chek Complete dual-antigen EIA (D-EIA; TechLab, Blacksburg VA) became commercially available; this assay comprises rapid detection of both GDH antigen and toxin A/B with one easy-to-use cartridge (Fig. 1). Previous studies based on two membrane-bound enzyme immunoassays for GDH and toxins A/B indicated that the single GDH testing was more sensitive than that for detection of C. difficile toxins A/B; however, false-positive results were recognized upon comparison with results for culture (10,15). It has been recommended that GDH be used as the first-line screening test, followed by cell culture for toxin testing (2, 15).More than 700,000 patient visits occur each year, with approximately 35,000 patients being admitted at the Vanderbilt University Medical Center (VUMC). Approximately 9,000 stool specimens were submitted for C. difficile testing for the year 2008. Currently, the Premier toxin A and B EIA (A/B EIA; Meridian Bioscience, Inc., Cincinnati, OH) is used in the clinical microbiology laboratory for detection of C. difficile toxin in stool samples: this test uses a 96-well microtiter format to detect both toxins A and B (7,8,11,17). In this study, we validated the D-EIA in comparison to a standard that combines two PCR assays and a cytotoxigenic culture.Patients and specimens. Consecutive stool specimens submitted for C. difficile testing were collected between 2 and 14 May 2009. Liquid or soft stool specimens with sufficient leftover volumes were included. Multiple specimens, up to three, from the same patient may be included during the study period. This study was approved by the VUMC Institutional Review Board. Specimens were stored refrigerated and tested by enzyme immunoassays and molecular assays within 48 h after collection. Specimens were stored at Ϫ80°C and sent out for anaerobic C....
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 l was used for extraction on the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC) for the Prodesse ProGastro Cd assay and 200 l 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 broth (BBL, BD Diagnostics, Sparks, MD). The prevalence of toxin-producing strains of C. difficile was 15.7% (n ؍ 44) as determined by anaerobic toxigenic culture. The sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay compared to the TOX-B test were 83.3%, 95.6%, 69.4%, and 98%, respectively. Compared to toxigenic culture, the sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay were 77.3%, 99.2%, 94.4%, and 95.9%, respectively, and those of the TOX-B test were 63.6%, 99.2%, 93.3%, and 93.6%, respectively. Although no statistical difference (Fisher's exact test) was detected (P ؍ 0.242) between the sensitivities of the Prodesse ProGastro Cd assay and a standard CCCNA compared to anaerobic culture for the detection of toxigenic C. difficile, the Prodesse ProGastro Cd assay did detect more toxigenic C. difficile isolates than the CCCNA.
We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.
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