Evaluation of three enzyme immunoassays and a loop-mediated isothermal amplification test for the laboratory diagnosis of Clostridium difficile infection

The laboratory diagnosis of Clostridium difficile infection (CDI) consists of the detection of toxigenic Clostridium difficile, and/or its toxins A or B in stool preferably in a two-step algorithm. In a prospective study, we compared the performance of three toxin enzyme immunoassays (EIAs)—ImmunoCard Toxins A & B, Premier Toxins A & B and C. diff Quik Chek Complete, which combines a toxins test and a glutamate dehydrogenase (GDH) antigen EIA in one device —and the loop-mediated isothermal amplification assay Illumigene C. difficile. In total 986 stool samples were analyzed. Compared with toxigenic culture as the gold standard, sensitivities, specificities, PPV and NPV values of the toxin EIAs were 41.1–54.8?%, 98.9–100?%, 75.0–100?% and 95.5–96.5?% respectively, of the Illumigene assay 93.3?%, 99.7?%, 95.8?% and 99.5?%. Illumigene assays performed significantly better for non-014/020 PCR-ribotypes than for C. difficile isolates belonging to 014/020. Discrepant analysis of three culture-negative, but Illumigene-positive samples, revealed the presence of toxin genes using real-time PCRs. In addition to the GDH EIA (NPV of 99.8?%), the performance of Illumigene allows this test to be introduced as a first screening test for CDI- or as a confirmation test for GDH -positive samples, although the initial invalid Illumigene result of 4.4?% is a point of concern.     

Comparison of a Commercial Real-Time PCR Assay for tcdB Detection to a Cell Culture Cytotoxicity Assay and Toxigenic Culture for Direct Detection of Toxin-Producing Clostridium difficile in Clinical Samples

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.     

Comparison of the premier toxin A and B assay and the TOX A/B II assay for diagnosis of Clostridium difficile infection

Clostridium difficile causes nosocomial diarrhea and is responsible for complications such as pseudomembranous colitis, megacolon, and perforation. Using 442 stool specimens, we compared the sensitivities and specificities of the Premier toxin A and B (Meridian Bioscience, Inc.) and C. difficile TOX A/B II (TechLab, Inc., Blacksburg, VA) immunoassays in the Virology Department of the Kaiser Permanente Regional Reference Laboratories. The Premier toxin A and B assay demonstrated a higher sensitivity (97.44%) and a higher positive predictive value (79.17%) than the C. difficile TOX A/B II assay (87.18% and 75.56%, respectively), while assay specificities and negative predictive values were similar. We also performed experiments using serially diluted, purified toxin A and B antigens to understand the basis for assay differences. The two assays’ toxin A antibodies detected toxin A at comparable levels. Preliminary results indicated that the toxin B antibody in the Premier toxin A and B assay could detect toxin B at a concentration of 125 pg/100 μl, while the toxin B antibody in the C. difficile TOX A/B II assay could not detect toxin B below a concentration of 250 pg/100 μl. Therefore, the Premier toxin A and B assay provides greater sensitivity than the C. difficile TOX A/B II assay, perhaps due to a superior detection ability of its toxin B antibody.     

Rapid and reliable diagnostic algorithm for detection of Clostridium difficile

We evaluated a two-step algorithm for detection of Clostridium difficile in 1,468 stool specimens. First, specimens were screened by an immunoassay for C. difficile glutamate dehydrogenase antigen (C.DIFF CHEK-60). Second, screen-positive specimens underwent toxin testing by a rapid toxin A/B assay (TOX A/B QUIK CHEK); toxin-negative specimens were subjected to stool culture. This algorithm allowed final results for 92% of specimens with a turnaround time of 4 h.     

Multicenter Evaluation of the Clostridium difficile TOX A/B TEST

Clostridium difficile, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the “gold standard” for C. difficile testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA−/toxB+) isolates were identified. One specimen was from a patient with a clinical history consistent with C. difficile infection. Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA−/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins. Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for C. difficile disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs.     

Evaluation of tcdB Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic Clostridium difficile

Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified “gold standard” that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.Clostridium difficile is the leading cause of infectious diarrhea in hospitalized patients. New challenges have been posed by the emergence of highly virulent C. difficile strains that may be refractory to standard treatment and cause disease even in immunocompetent individuals without prior antibiotic exposure (11, 28). Rapid and accurate laboratory diagnosis is critical to reduce the morbidity from C. difficile infection (CDI) and allow the implementation of specific infection control measures.Methods for the detection of toxigenic C. difficile have long been unsatisfactory. The tissue-culture assay for cytotoxin B (CYT) is often considered the “gold standard” for diagnosis (10, 29), but many reports have documented the failure of CYT to detect symptomatic and even life-threatening cases of CDI (14, 33, 44) and the poor sensitivity of CYT in comparison to toxigenic culture (18, 22, 39, 43). The technical complexity of CYT, as well as a requirement for 24 to 48 h of incubation, has resulted in the widespread replacement of CYT with toxin immunoassays that provide results within minutes. In a 2008 College of American Pathologists report of proficiency results, 95% of laboratories reported using an EIA kit to detect C. difficile toxin (6). C. difficile immunoassays are often adopted as stand-alone assays based on validation against CYT, but studies that have employed more sensitive gold standards have documented that rapid toxin EIAs have unacceptably low sensitivities ranging from 32 to 79% (3, 20, 23, 44, 50).Toxigenic culture, when performed under optimal culture conditions and combined with a sensitive and specific toxin detection method, is regarded as the most sensitive method of toxigenic C. difficile detection (18, 44, 50), but complexity and a prolonged turnaround time have discouraged its routine use. An alternative approach has been to test for the glutamate dehydrogenase (GDH) antigen of C. difficile as a surrogate for culture. GDH EIAs have been reported to be highly sensitive for C. difficile detection, allowing same-day reporting of negative results, but positive results must be followed by a sensitive and specific test to differentiate between toxigenic and nontoxigenic strains (5, 57, 58). In this study, we used the Wampole C Diff Quik Chek EIA (GDH-Q), a rapid GDH assay that requires less technical time and expertise in comparison to microtiter plate immunoassays, followed by the Tox A/B Quik Chek (AB-Q) on GDH-Q-negative specimens. The choices for a second-step assay to detect toxigenic C. difficile in GDH-positive specimens have included toxigenic culture, CYT, or toxin EIAs (20, 23, 46, 57), but the insensitivity of CYT and toxin EIAs potentially leaves many cases of CDI undetected.An alternative, highly sensitive method to detect toxigenic C. difficile is real-time PCR (12, 44, 50, 54, 60), with sensitivity values ranging from 83.6% to 93.4% and specificity from 93.9% to 98.2%, respectively, when compared to toxigenic culture (50, 54, 60). Real-time PCR can be completed on the day of specimen submission, thus providing same-day results. However, PCR techniques have not been not widely used for stool specimens, due primarily to budgetary issues, as well as the challenge of extracting nucleic acids from feces and separating template DNA from potentially interfering substances.We have developed a sensitive and specific real-time PCR assay (direct stool PCR [DPCR]) for the C. difficile toxin B-encoding tcdB gene, which can be performed directly on stool specimens. Nucleic acids can be efficiently extracted from feces with the NucliSENS miniMAG system, a semiautomated magnetic silica bead extraction system to remove inhibitors and purify nucleic acids. Results were compared with the total yield of positive specimens detected by CYT and DPCR. Our observations indicate that a multistep algorithm consisting of GDH EIA, toxin EIA, and selective DPCR can provide a specific and cost-effective approach to the laboratory detection of toxigenic C. difficile that is more sensitive than CYT or EIA alone but not as sensitive as toxigenic culture or DPCR.     

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.Clostridium difficile is an important nosocomial pathogen, causing antimicrobial-associated diarrhea and pseudomembranous colitis. Toxins A (TcdA) and B (TcdB) mediate the pathogenesis of C. difficile infection (CDI), and toxin detection is an important part of diagnosis. A cytotoxicity neutralization assay (CNA) is the reference method for toxin detection, but it is expensive and time-consuming and requires tissue culture facilities (34, 35). Most laboratories now use a commercial enzyme immunoassay (EIA) to detect TcdA and/or TcdB, with the benefits of rapid turnaround time and ease of use (3, 21, 22, 23, 26, 27, 33, 35). The putative >90% sensitivity of toxin EIAs is not often realized in practice, but EIA is the only toxin detection method available to many routine medical laboratories. The demand for EIA kits detecting both TcdA and TcdB has increased due to increased worldwide prevalence of TcdA-negative, TcdB-positive (TcdA− TcdB+) strains (1, 12, 24, 29, 32).A two-step algorithm, based upon EIA-based detection of species-specific antigen glutamate dehydrogenase (GDH-Ag) and toxin detection via CNA, was suggested to have improved sensitivity and specificity in the detection of toxigenic C. difficile (34). However, the GDH-Ag assay detects both nontoxigenic and toxigenic strains, and the aforementioned shortcomings of the CNA assay make it unavailable to many routine laboratories.Bacteriologic culture can be time-consuming, but it is more straightforward and sensitive than CNA for the detection of toxigenic C. difficile. Furthermore, it provides isolates for characterization, yielding information about CDI epidemiology and antimicrobial susceptibility (11, 28, 36). We evaluated the combination of bacteriologic culture and EIA-based detection of TcdA and TcdB as a new strategy for diagnosis of CDI, especially in areas where TcdA− TcdB+ strains are prevalent.     

Effective and Reduced-Cost Modified Selective Medium for Isolation of Clostridium difficile

Both for epidemiologic studies and for diagnostic testing, there is a need for effective, economical, and readily available selective media for the culture of Clostridium difficile. We have developed a reduced-cost substitute for cycloserine-cefoxitin-fructose agar (CCFA), which is an effective but expensive selective medium for C. difficile. The modified medium, called C. difficile brucella agar (CDBA), includes an enriched brucella base as a substitute for proteose peptone no. 2, and the concentration of sodium taurocholate has been reduced from 0.1% to 0.05%. To compare the sensitivities and selectivities of CDBA and CCFA, cultures for C. difficile were performed using stool samples from patients with C. difficile-associated disease. CDBA was as sensitive as CCFA for the recovery of C. difficile, with a similar frequency of breakthrough growth of stool microflora (25% versus 31%, respectively). A liquid formulation of the modified medium, termed C. difficile brucella broth (CDBB), stimulated rapid germination and outgrowth of C. difficile spores, at a rate comparable to that in cycloserine-cefoxitin-fructose broth. Our results suggest that CDBA and CDBB are sensitive, selective, and reduced-cost media for the recovery of C. difficile from stool samples.     

Evaluation of three rapid assays for detection of Clostridium difficile toxin A and toxin B in stool specimens

Diagnosis of Clostridium difficile-associated disease continues to be difficult for clinical microbiology laboratories. The aim of this study was to evaluate the performance of three enzyme immunoassays for detection of C. difficile toxins A and B: the recently marketed rapid enzyme immunoassay Ridascreen Clostridium difficile Toxin A/B (R-Biopharm, Darmstadt, Germany) and two established enzyme immunoassays, the C. difficile Tox A/B II Assay (TechLab, Blacksburg, VA, USA) and the ProSpecT C. difficile Toxin A/B Microplate Assay (Remel, Lenexa, KS, USA). Stool specimens (n = 383) from patients with a clinical diagnosis of antibiotic-associated diarrhea were examined by these three enzyme immunoassays and were additionally cultured for C. difficile on selective agar. Samples giving discordant enzyme immunoassay results underwent confirmatory testing by tissue culture cytotoxin B assay and by PCR for toxin A (tcdA) and toxin B (tcdB) genes from C. difficile. Using the criteria adopted for this study, 60 (15.7%) samples tested positive for toxins A and/or B. Sensitivity and specificity of the enzyme immunoassays were, respectively, 88.3 and 100% for the TechLab enzyme immunoassay, 91.7 and 100% for the R-Biopharm enzyme immunoassay, and 93.3 and 100% for the Remel enzyme immunoassay. The differences between these results are statistically not significant (p > 0.05). The results show that all three enzyme immunoassays are acceptable tests for the detection of C. difficile toxins A and B directly in fecal specimens or in toxigenic cultures.     

Comparison of a Rapid Molecular Method,the BD GeneOhm Cdiff Assay,to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing Clostridium difficile in Diarrheal Feces

Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.Toxin-producing Clostridium difficile strains are important pathogens among patients who are treated with antibiotics or chemotherapeutic agents not only in the hospital environment but also in the community (3, 6, 10). Since the recognition of outbreaks of C. difficile infection (CDI) caused by C. difficile PCR ribotype 027 in Canada, the United States, and several European countries, rapid and accurate diagnosis of CDI is very important to stop the spread of these strains (7, 8, 19). In addition, the increasing morbidity and mortality rates associated with CDI and the increasing number of recurrences and therapeutic failures also highlight the need for the development of a rapid and reliable detection method for toxigenic C. difficile in diarrheal feces (12).Only a few laboratories routinely use the tissue culture cytotoxicity and toxin neutralization assays for the detection of toxigenic C. difficile in feces, because they are labor-intensive and time-consuming and standardization is very difficult. Due to their rapid turnaround time, enzyme immunoassays (EIAs) that detect toxin A and/or toxin B in stool are used in most laboratories (11, 16). To increase the sensitivity of these tests and in some instances to facilitate epidemiological investigations, culture of C. difficile has become essential. In spite of this, most laboratories use a single toxin detection test on feces for detection of toxigenic C. difficile (4). In the last 10 years, in-house PCR and real-time PCR assays have been developed to detect C. difficile toxin genes. These assays have shown very good sensitivity and specificity and short turnaround times (1, 17). However, widespread use of PCR methods in routine clinical microbiology is limited because these tests require special DNA extraction procedures to eliminate PCR inhibitors from fecal specimens and they cost more than do traditional testing methods.The BD GeneOhm Cdiff assay provides a rapid method for the qualitative detection of the C. difficile toxin B gene (tcdB) in diarrheal specimens from patients suspected of having CDI. This test is based on the amplification of the tcdB gene and the detection of the amplified DNA using fluorogen-labeled probes. Amplification, detection, and interpretation of the results are done automatically by the SmartCycler instrument (Cepheid, Sunnyvale, CA).Our aims were to compare the performance of the BD GeneOhm Cdiff assay to those of the tissue culture cytotoxicity assay and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux, Marcy-l''Etoile, France), for the direct detection of toxins A and B from fecal samples.     

Comparison of Nine Commercially Available Clostridium difficile Toxin Detection Assays,a Real-Time PCR Assay for C. difficile tcdB,and a Glutamate Dehydrogenase Detection Assay to Cytotoxin Testing and Cytotoxigenic Culture Methods

The continuing rise in the incidence of Clostridium difficile infection is a cause for concern, with implications for patients and health care systems. Laboratory diagnosis largely relies on rapid toxin detection kits, although assays detecting alternative targets, including glutamate dehydrogenase (GDH) and toxin genes, are now available. Six hundred routine diagnostic diarrheal samples were tested prospectively using nine commercial toxin detection assays, cytotoxin assay (CYT), and cytotoxigenic culture (CYTGC) and retrospectively using a GDH detection assay and PCR for the toxin B gene. The mean sensitivity and specificity for toxin detection assays were 82.8% (range, 66.7 to 91.7%) and 95.4% (range, 90.9 to 98.8%), respectively, in comparison with CYT and 75.0% (range, 60.0 to 86.4%) and 96.1% (91.4 to 99.4%), respectively, in comparison with CYTGC. The sensitivity and specificity of the GDH assay were 90.1% and 92.9%, respectively, compared to CYT and 87.6% and 94.3%, respectively, compared to CYTGC. The PCR assay had the highest sensitivity of all the tests in comparison with CYT (92.2%) and CYTGC (88.5%), and the specificities of the PCR assay were 94.0% and 95.4% compared to CYT and CYTGC, respectively. All kits had low positive predictive values (range, 48.6 to 86.8%) compared with CYT, assuming a positive sample prevalence of 10% (representing the hospital setting), which compromises the clinical utility of single tests for the laboratory diagnosis of C. difficile infection. The optimum rapid single test was PCR for toxin B gene, as this had the highest negative predictive value. Diagnostic algorithms that optimize test combinations for the laboratory diagnosis of C. difficile infection need to be defined.Clostridium difficile is a major nosocomial pathogen causing a range of symptoms from mild to severe diarrhea and is the etiological agent of pseudomembranous colitis. The incidence of C. difficile infection has increased markedly in many countries, notably associated with the epidemic spread of PCR ribotype 027 (NAP1) since its recognition in the United States and Canada (6, 7, 13). It is essential to have accurate laboratory diagnosis of C. difficile infection to ensure patients receive appropriate treatment and that correct infection control measures are put in place. Also, inaccurate testing will potentially lead to poor quality surveillance data that may lead to inappropriate infection prevention measures.The cytotoxin assay (CYT), first described by Chang et al., detects the toxins produced by C. difficile in the supernatants of patient feces, using both antitoxin-protected and nonprotected cell monolayers (2). This assay is commonly used as the gold standard method for comparison in toxin kit evaluations, although its use in routine microbiology laboratories has largely been superseded. Cytotoxogenic culture (CYTGC) has been used as an alternative gold standard method to CYT testing, i.e., where CYT testing is performed using culture supernatants instead of directly from the fecal sample (1). These are lengthy assays, however, with results delayed for 24 to 48 h for the CYT and for more than 72 h for the CYTGC assay.Rapid, commercially available, toxin detection kits removed the need for laboratories to maintain the cell lines necessary for CYT testing. Although originally designed to detect either toxin A or toxin B, the kits currently available detect both toxins to enable detection of toxin A-negative, toxin B-positive strains. Alternative detection methods have now been developed, including an assay that detects a surface-associated enzyme of C. difficile, glutamate dehydrogenase (GDH). Zheng et al. reported that the Techlab C. diff Chek-60 GDH assay had good sensitivity compared to CYT testing of 92%, but it had a low specificity of 89.1% and poor positive predictive value (PPV) of 57.7% (21). Commercial molecular diagnostic tests, such as the BD GeneOhm C. difficile PCR assay, which detects the tcdB toxin gene of C. difficile, are now available. A recent study compared this assay to CYT testing and found a sensitivity and specificity of 90.9% and 95.2%, respectively (15). The PPVs of the BD GeneOhm C. difficile PCR assay were only 70.2% compared with CYT testing and 89.5% compared with CYTGC (15), with a prevalence of toxin-positive fecal samples of 15.2%.Despite numerous evaluations of C. difficile testing methods, no evaluation has compared all methods on the same sample set. This study compared six commercially available enzyme immunoassays (EIAs) and three lateral-flow assays for detection of C. difficile toxins A and B, a PCR assay for detection of the tcdB gene of C. difficile, and an assay for detection of C. difficile-specific GDH, with CYT testing and CYTGC.     

Evaluation of repeat Clostridium difficile enzyme immunoassay testing

Clostridium difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis, which have significant morbidity and mortality. Accurate and timely diagnosis is critical. Repeat enzyme immunoassay testing for C. difficile toxin has been recommended because of <100% sensitivity. All C. difficile tests between 1 January 2006 and 31 December 2006 were retrospectively analyzed for results and testing patterns. The Wampole C. difficile Tox A/B II enzyme immunoassay kit was used. There were a total of 8,256 tests from 3,112 patients; 49% of tests were repeated. Of the 3,749 initially negative patient tests, 96 were positive upon repeat testing within 10 days of the first test. Of repeat tests, 0.9% repeated on day 0 (same day as the first test), 1.8% on day 1, 3.8% on day 2, 2.6% on day 3, 5.4% on days 4 to 6, and 10.6% on days 7 to 10 were positive. Thirty-eight patients had a positive test within 48 h of an initial negative test, and based on chart review, 18 patients were treated empirically while 16 were treated following the new result. None had evidence of medical complications. Of initially positive patients, 91% were positive upon repeat testing on day 0, 75% on day 1, and 58% on day 2, to a low of 14% on days 7 to 10. Depending on the clinical setting, these data support not repeating C. difficile tests within 2 days of a negative result and limiting repeat testing to ≥1 week of a positive result.     

Evaluation of a New Commercial TaqMan PCR Assay for Direct Detection of the Clostridium difficile Toxin B Gene in Clinical Stool Specimens

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 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.The severity of disease associated with Clostridium difficile infection (CDI) can vary from asymptomatic colonization or mild gastroenteritis to severe manifestations, such as colitis, pseudomembrane formation, and toxic megacolon (2, 3, 7). The increased recognition of CDI-associated morbidity and mortality with the apparent rise of hypervirulent C. difficile epidemic strains (BI/NAP1/027) necessitates a dependable diagnostic assay for the detection of toxigenic C. difficile as fast as possible (7, 8).A large number of diagnostic methods are available for the detection of toxigenic C. difficile in stool samples. Individual laboratories must balance the performance characteristics with test complexity, costs, and time to results. The cell culture cytotoxicity neutralization assay (CCCNA), once considered a “gold standard,” has been replaced in most laboratories by more rapid technologies. Rapid traditional methods for detection of toxins A and B, i.e., lateral flow devices and enzyme immunoassay methods, are quicker, less complex, and less expensive, but their performances differ with regard to sensitivity and specificity (1, 10, 12, 13, 15-17, 21). Although time-consuming, the most sensitive and specific method is anaerobic culture with selective media for C. difficile followed by testing of recovered isolates for cytotoxin production (1, 6, 12, 15-17, 21). A few real-time PCR assays have been evaluated in routine clinical laboratories for direct detection of toxin A and/or toxin B directly in stool, but only three PCR assays have been directly compared to anaerobic toxigenic culture (1, 4, 12, 15, 17-20). The only consensus on testing methods is that a rapid, sensitive, and specific assay that differentiates between toxigenic and nontoxigenic C. difficile isolates is the preferred tool to assist clinicians in their decision-making process.The real-time PCR assay manufactured by Prodesse, Inc. (Waukesha, WI), is performed on stool after extraction with the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC). The PCR assay uses TaqMan chemistry and consists of proprietary primers specific to the toxin B gene (tcdB) as well as an internal control (IC), incorporated into every reaction, that is amplified on the Cepheid SmartCycler II (Sunnyvale, CA). The IC, added on initial processing of the stool sample, acts as a general process control and monitors for the presence of PCR inhibitors. In this study, we compared the ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) to the Wampole C. difficile toxin B test (TOX-B test; TechLab, Blacksburg, VA) for a Prodesse (Waukesha, WI)-sponsored clinical trial. In addition, both assays were also compared to an anaerobic toxigenic culture method that was not a component of the sponsored clinical trial.(This research was presented in part at the 25th Annual Clinical Virology Symposium, Daytona Beach, FL, 19 to 22 April 2009.)     

Nonutility of repeat laboratory testing for detection of Clostridium difficile by use of PCR or enzyme immunoassay

The diagnostic gains of repeat testing for Clostridium difficile by enzyme immunoassay and PCR (i.e., initial negative result followed by positive result) within a 7-day period were 1.9 and 1.7%, respectively. There is little value of repeat testing for C. difficile by enzyme immunoassay or PCR.     

Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile

Purpose: To evaluate usefulness of applying either the two-step algorithm (Ag-EIAs and CCNA) or the three-step algorithm (all three assays) for better confirmation of toxigenic Clostridium difficile. The antigen enzyme immunoassays (Ag-EIAs) can accurately identify the glutamate dehydrogenase antigen of toxigenic and nontoxigenic Clostridium difficile. Therefore, it is used in combination with a toxin-detecting assay [cell line culture neutralization assay (CCNA), or the enzyme immunoassays for toxins A and B (TOX-A/BII EIA)] to provide specific evidence of Clostridium difficile-associated diarrhoea. Materials and Methods: A total of 151 nonformed stool specimens were tested by Ag-EIAs, TOX-A/BII EIA, and CCNA. All tests were performed according to the manufacturer’s instructions and the results of Ag-EIAs and TOX-A/BII EIA were read using a spectrophotometer at a wavelength of 450 nm. Results: A total of 61 (40.7%), 38 (25.3%), and 52 (34.7%) specimens tested positive with Ag-EIA, TOX-A/BII EIA, and CCNA, respectively. Overall, the sensitivity, specificity, negative predictive value, and positive predictive value for Ag-EIA were 94%, 87%, 96.6%, and 80.3%, respectively. Whereas for TOX-A/BII EIA, the sensitivity, specificity, negative predictive value, and positive predictive value were 73.1%, 100%, 87.5%, and 100%, respectively. With the two-step algorithm, all 61 Ag-EIAs-positive cases required 2 days for confirmation. With the three-step algorithm, 37 (60.7%) cases were reported immediately, and the remaining 24 (39.3%) required further testing by CCNA. By applying the two-step algorithm, the workload and cost could be reduced by 28.2% compared with the three-step algorithm. Conclusions: The two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but it is time-consuming.     

Multicenter Clinical Evaluation of the Portrait Toxigenic C. difficile Assay for Detection of Toxigenic Clostridium difficile Strains in Clinical Stool Specimens

We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based “gold standard” method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.     

Characterization of Clostridium difficile strains isolated from patients in Ontario, Canada, from 2004 to 2006

Clostridium difficile is the bacterium most commonly surmised to cause antimicrobial- and hospital-associated diarrhea in developed countries worldwide, and such infections are thought to be increasing in frequency and severity. A laboratory-based study was carried out to characterize C. difficile strains isolated from persons in Ontario, Canada, during 2004 to 2006 according to toxin type (enterotoxin A, cytotoxin B, and binary toxin [CDT]), tcdC gene characterization, ribotyping, pulsed-field gel electrophoresis, and toxinotyping. Clostridium difficile was isolated from 1,080/1,152 (94%) samples from 21 diagnostic laboratories. Isolates with toxin profiles A⁺ B⁺ CDT⁻, A⁺ B⁺ CDT⁺, A⁻ B⁺ CDT⁻, and A⁻ B⁺ CDT⁺ accounted for 63%, 34%, 2.4%, and 0.6% of isolates, respectively. Alterations in tcdC were detected in six different ribotypes, including ribotype 027. A total of 39 different ribotypes were identified, with ribotype 027/North American pulsotype 1 (NAP1), an internationally recognized outbreak strain associated with severe disease, being the second most common ribotype (19% of isolates). Transient resistance to metronidazole was identified in 19 (1.8%) isolates. While a large number of ribotypes were found, a few predominated across the province. The high prevalence and wide distribution of ribotype 027/NAP1 are disconcerting in view of the severity of disease associated with it.     

Prevalence of Astroviruses in a Children’s Hospital

An enzyme immunoassay for astrovirus was used to screen 357 stool samples from 267 symptomatic inpatients at a tertiary-care children’s hospital. Thirty stool samples from 26 patients contained astrovirus antigen, while rotavirus was found in 34 samples and Clostridium difficile toxin was found in 40. Half of the astrovirus infections were nosocomial. Additional pathogens were identified in six of the astrovirus antigen-positive stool samples. Most (80%) of the astroviruses recovered were of serotype 1. Astrovirus infections were significantly more common than rotavirus or C. difficile infections in very young infants and in those with surgical short-bowel syndrome.     

Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer''s instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.     

Evaluation of a New Automated Homogeneous PCR Assay,GenomEra C. difficile,for Rapid Detection of Toxigenic Clostridium difficile in Fecal Specimens

We evaluated a new automated homogeneous PCR assay to detect toxigenic Clostridium difficile, the GenomEra C. difficile assay (Abacus Diagnostica, Finland), with 310 diarrheal stool specimens and with a collection of 33 known clostridial and nonclostridial isolates. Results were compared with toxigenic culture results, with discrepancies being resolved by the GeneXpert C. difficile PCR assay (Cepheid). Among the 80 toxigenic culture-positive or GeneXpert C. difficile assay-positive fecal specimens, 79 were also positive with the GenomEra C. difficile assay. Additionally, one specimen was positive with the GenomEra assay but negative with the confirmatory methods. Thus, the sensitivity and specificity were 98.8% and 99.6%, respectively. With the culture collection, no false-positive or -negative results were observed. The analytical sensitivity of the GenomEra C. difficile assay was approximately 5 CFU per PCR test. The short hands-on (<5 min for 1 to 4 samples) and total turnaround (<1 h) times, together with the high positive and negative predictive values (98.8% and 99.6%, respectively), make the GenomEra C. difficile assay an excellent option for toxigenic C. difficile detection in fecal specimens.