Comparison of two commercially available enzyme immunoassays for detection of Clostridium difficile in stool specimens.

Clostridium difficile is the cause of most cases of pseudomembranous colitis, the most severe form of antibiotic-associated diarrhea. Rapid diagnosis guides both the treatment and the control of nosocomial spread of infection. Two enzyme immunoassay (EIA) kits developed for the rapid detection of C. difficile toxin A in fecal specimens, Premier (Meridian Diagnostics, Cincinnati, Ohio) and Tox-A test (TechLab, Virginia Polytechnic Institute Research Park, Blacksburg), were evaluated by using 410 fecal specimens. Seventy-six specimens were positive for C. difficile toxin B by the cytotoxin assay (prevalence rate, 19%). The Meridian EIA was positive for 71 of the 76 samples, yielding a sensitivity of 93%. The TechLab EIA detected 75 of the 76 positive samples, yielding a sensitivity of 99%. The Meridian and TechLab EIAs had specificities of 100 and 93%, respectively. These data indicate that both EIAs are suitable alternatives to the cytotoxin assay in routine diagnostic laboratories. However, confirmation of TechLab EIA-positive test results by the cytotoxin assay remains necessary.     

Evaluation of methods for detection of toxins in specimens of feces submitted for diagnosis of Clostridium difficile-associated diarrhea

Clostridium difficile is the principal pathogen associated with hospital-acquired acute diarrheal disease. We have evaluated the performances of six approaches for diagnosis of C. difficile-associated diarrhea (CDAD). Consecutive stool specimens (n = 200) from 133 patients were examined by cytotoxin assay, by culture of C. difficile on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test). A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens). The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture. The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.     

Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea.

Enzyme immunoassays (EIAs) based on monoclonal antibodies for the detection of Clostridium difficile toxins have recently been developed for clinical use. The aim of this study was to compare three commercially available EIAs, two for toxin A (Premier C. difficile Toxin A; Meridian, Osi, Elancourt, France; and Vidas C. difficile Toxin A; bioMérieux, Marcy l'Etoile, France) and one for toxins A and B (Cytoclone A + B EIA; Cambridge Biotech Corp., Codiapharm, Evian, France), with a cytotoxicity assay and toxigenic culture for the diagnosis of C. difficile-associated diarrhea (CDAD). The study was performed with 285 fresh stools from 285 patients with suspected CDAD. In case of disagreement, the tests were repeated on a frozen aliquot of the same stool sample, and the patient's chart was reviewed. CDAD diagnosis was established in 55 cases (incidence, 19.3%). The sensitivities and specificities of the methods were, respectively, 92.7 and 100% for the cytotoxicity assay, 96.4 and 99.1% for toxigenic culture, 75.5 and 97.8% for Cytoclone, 65.4 and 99.6% for Premier, and 65.4 and 100% for Vidas. The results were uninterpretable in 3.2% of cases with Cytoclone, 0.3% with Premier, and 2.5% with Vidas. We conclude that the cytotoxicity assay and toxigenic culture remain the best methods for the diagnosis of CDAD even though they lack standardization and require 48 to 96 h to obtain the result. Despite their rapidity and simplicity, EIAs are not sensitive enough to be relied on as the sole laboratory test.     

Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases.

Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.). The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C. difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens. Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay. However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs. The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay. Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%). These data indicate that the four EIAs all have specific shortcomings. When using these EIAs, testing strategies that take these shortcomings into consideration should be developed.     

Rapid detection of Clostridium difficile toxin A in stool specimens

Objective: To evaluate a rapid (15-min) enzyme immunoassay in the format of an individual cassette (ImmunoCard toxin A, Meridian, BMD, Marne-la-Vallée, France) for the detection of Clostridium difficile toxin A in stool specimens.
Methods: We compared this new test with the cytotoxicity assay using MRC-5 cells, the ToxA test (TechLab, BioWhittaker, Fontenay-sous-bois, France) and toxigenic culture for the diagnosis of C. difficile -associated diseases (CDAD). A total of 236 stool specimens collected from 220 patients was simultaneously tested with the four methods. Discordant results were resolved by reviewing patients' clinical records.
Results: The prevalence of CDAD was 13.9%. Test sensitivities and specificities were 100% and 99% respectively for the cytotoxicity assay, 87.5% and 100% for ImmunoCard toxin A, 77.4% and 100% for the ToxA test and 100% and 98% for toxigenic culture.
Conclusions: The ImmunoCard Toxin A is a very rapid, individual and easy-to-perform test for the diagnosis of CDAD. It provides same-day results and may be useful for both guiding appropriate treatment and controlling nosocomial spread of C. difficile.     

Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays.

A total of 320 stool specimens obtained from 262 patients suspected of having Clostridium difficile-associated gastrointestinal disease were examined with two cytotoxicity assays (CTAs) and two commercially available enzyme immunoassays (EIAs). The CTAs were an in-house-developed procedure (University of Massachusetts Medical Center [UMMC], Worcester, Mass.) and a commercial test (Bartels CTA; Baxter Healthcare Corp., West Sacramento, Calif.). One EIA was a monoclonal antibody-based assay for C. difficile toxins A and B (Cambridge Biotech Corp. [CBC], Worcester, Mass.). The other EIA employed monoclonal antibodies directed against only toxin A (Meridian Diagnostics, Cincinnati, Ohio). True-positive and true-negative results were defined on the basis of the results of the four assays, clinical assessments of patients, and the results of other laboratory tests. The sensitivities of the four assays were as follows: Bartels CTA, 100%; UMMC CTA, 97.2%; CBC EIA, 84.5%; and Meridian EIA, 69.0%. The Bartels CTA demonstrated a specificity of 99.2%. The other three assays had a specificity of 100%.     

Comparison of real-time PCR for detection of the tcdC gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection

We have developed a rapid real-time PCR method using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics), which will detect the presence of the tcdC gene of Clostridium difficile in stool samples. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin-hyperproducing strains. We compared the results of this PCR with those of three C. difficile toxin-detecting enzyme immunoassays (EIAs), an EIA for the detection of glutamate dehydrogenase (GDH), and culture of C. difficile. A total of 200 stool specimens were studied by the methods under comparison. C. difficile was isolated from 49 specimens by culture, and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 48%, 98%, 88%, and 87% for the Premier toxin A and B test; 48%, 99%, 91%, and 87% for the ImmunoCard toxin A & B test; 48%, 84%, 46%, and 85% for the Xpect C. difficile toxin A/B test; 32%, 100%, 100%, and 84% for the Triage C. difficile panel (for toxin A); and 86%, 97%, 90%, and 96% for the LightCycler PCR. Thus, in comparison to the sensitivity of toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low, while the LightCycler real-time PCR assay for the detection of the tcdC gene of C. difficile is sensitive and specific.     

Laboratory diagnosis of Clostridium difficile disease

The laboratory diagnosis of Clostridium difficile -associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.     

Is a Two-Step Glutamate Dehyrogenase Antigen-Cytotoxicity Neutralization Assay Algorithm Superior to the Premier Toxin A and B Enzyme Immunoassay for Laboratory Detection of Clostridium difficile?

A two-step algorithm for the detection of Clostridium difficile by the use of C. Diff Quik Chek (TechLab, Blacksburg, VA) and a tissue culture cytotoxicity neutralization assay was found to be more sensitive than the widely used solid-phase enzyme immunoassay (EIA), the Premier toxin A and B EIA (Meridian Bioscience, Cincinnati, OH), and a newly developed, rapid single-test EIA for C. difficile toxins A and B (Tox A/B Quik Chek; TechLab).     

Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease.

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.     

Evaluation of three commercial enzyme immunoassay kits for detecting faecal Clostridium difficile toxins.

The detection of faecal cytotoxicity using tissue culture was compared with three commercial Clostridium difficile enzyme immunoassay (EIA) kits; Premier C difficile toxin A (Meridian Diagnostic, Inc.); CD-TOX C difficile toxin A (Porton Cambridge); and Cytoclone A+B EIA (Cambridge Biotech Corporation). Of 160 faecal samples examined by all four methods, 52 (32.5%) were cytotoxic, 44 (27.5%) were positive by Premier, 48 (30%) by CD-TOX EIA, and 50 (31.3%) with Cytoclone. When compared with detection of cytotoxicity by tissue culture assay, the following performance indices were obtained: Premier, sensitivity 84.1%, specificity 99.1%, positive predictive value (PPV) 97.8%, negative predictive value (NPV) 93%; CD-TOX, sensitivity 92.3%, specificity 88.0%, PPV 78.7%, NPV 95.9%; Cytoclone, sensitivity 96.2%, specificity 93.5%, PPV 87.7%, NPV 98.1%. EIA results were available within three hours, whereas the results of the cytotoxin assay were available after 24-48 hours. All three kits provided satisfactory results and, although relatively expensive, all could be used in the laboratory effectively to screen for diarrhoeal disease associated with C difficile.     

Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens.

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of interpretation of results (color, intensity of fluorescence), equipment, available personnel, and number of tests ordered are also important considerations prior to kit selection.     

Loop-mediated isothermal amplification compared to real-time PCR and enzyme immunoassay for toxigenic Clostridium difficile detection

Clostridium difficile infection is the primary cause of health care-associated diarrhea. While most laboratories have been using rapid antigen tests for detecting C. difficile toxins, they have poor sensitivity; newer molecular methods offer rapid results with high test sensitivity and specificity. This study was designed to compare the performances of two molecular assays (Meridian illumigene and BD GeneOhm) and two antigen assays (Wampole Quik Chek Complete and TechLab Tox A/B II) to detect toxigenic C. difficile. Fecal specimens from hospitalized patients (n = 139) suspected of having C. difficile infection were tested by the four assays. Nine specimens were positive and 109 were negative by all four methods. After discrepant analysis by toxigenic culture (n = 21), the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 21 (15%) and 118 (85%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: GeneOhm (95.2%, 100%, 100%, and 99.2%), illumigene (95.2%, 96.6%, 83.3%, and 99.2%), Tox A/B II (52.4%, 97.5%, 78.6%, and 92.4%), and Quik Chek Complete (47.6%, 100%, 100%, and 91.9%). The illumigene assay performed comparably to the GeneOhm assay with a slight decrease in test specificity; the sensitivities of both far exceeded those of the antigen assays. The clinical characteristics of the concordant and discrepant study patients were similar, including stool consistency and frequency. In the era of rapid molecular-based tests for toxigenic C. difficile, toxin enzyme immunoassays (EIAs) should no longer be considered the standard of care.     

Evaluation of the Clearview Clostridium difficile Toxin A Test and various selective culture media in comparison with the cytotoxin assay for the diagnosis of Clostridium difficile-associated diarrhoea

AIM: Clostridium difficile is the major pathogen associated with nosocomial diarrhoea. We evaluated the performances of a commercially available toxin A enzyme immunoassay (EIA; Clearview C. difficile Toxin A Test), culture and tissue culture cytotoxin assay in the diagnosis of C. difficile-associated diarrhoea. METHODS: Comparative test performance was determined from data obtained from 166 faecal samples. The initial analysis compared the performance of toxin A EIA and culture with that of cytotoxin assay, this being defined as a 'laboratory gold standard'. A second analysis compared the individual performance of the toxin A EIA, culture and cytotoxin assay using a combined clinical and laboratory diagnostic assessment as a 'clinical gold standard'. In a parallel, study three selective culture media were compared. RESULTS: From the initial analysis, the sensitivity and specificity of the methods were, respectively, 84.6 and 65.4% for the toxin A EIA, and 38.5 and 93.5% for culture. From the second analysis, the sensitivity and specificity of the methods were, respectively, 100 and 67.5% for the toxin A EIA, 63.6 and 96.7% for culture and 72.7 and 98.0% for cytotoxin assay. Media containing d-cycloserine 250mg/L and cefoxitin 8mg/L performed best, growing 88.2% of the isolates. CONCLUSION: The toxin A EIA we evaluated had poor specificity in the diagnosis of C. difficile-associated diarrhoea. We conclude that in our laboratory the combination of culture and cytotoxin assay is a preferred approach to the diagnosis of C. difficile-associated diarrhoea.     

Clinical and laboratory evaluation of a real-time PCR for Clostridium difficile toxin A and B genes

Many laboratories use enzyme immunoassays (EIAs) for the diagnosis of Clostridium difficile infection (CDI). More recently, polymerase chain reaction (PCR)-based diagnosis has been described as a sensitive test. Real-time PCR for the detection of C. difficile toxin A and B genes was evaluated. A prospective evaluation was performed on stool samples from 150 hospitalized adult patients and 141 healthy volunteers. PCR was compared to toxigenic culture (TC), direct cytotoxicity test (CTT), ImmunoCard? Toxin A and B (Meridian Bioscience), and enzyme-linked immunosorbent assay (ELISA) (Vidas). The results were correlated with clinical data using a standardized questionnaire. The diagnostic yield of the PCR was further evaluated after implementation. Using toxigenic culture as the gold standard, the sensitivity and specificity of PCR were 100 and 99.2%, respectively. Patients were categorized as follows: TC/PCR-positive (n?=?17) and negative TC (n?=?133). The differences in these groups were more frequent use of antibiotics and leukocytosis (p?相似文献   

Evaluation of Two Rapid Assays for Detection of Clostridium difficile Toxin A in Stool Specimens

Rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment. We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B. A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays. With the "gold standard" of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods. Total agreement for all assays was 90.5% (592 of 654). The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA. While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.     

Rapid and sensitive loop-mediated isothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and culture as gold standard

Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings.     

Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C. difficile, cytotoxin assay, culture, and latex agglutination.

A three-center study was undertaken to compare several test methods for the detection of Clostridium difficile, associated toxin, or related markers by using 927 stool specimens. Methods included direct assay of cytotoxin in stool by tissue culture, C. difficile bacterial culture followed by cytotoxin assay, bacterial culture alone, latex agglutination assay, and the ImmunoCard C. difficile test (Meridian Diagnostics, Inc.). The sensitivities, as determined against direct cytotoxin assay results, of the ImmunoCard C. difficile and latex agglutination assays were 84 and 67%, respectively (92 and 77%, respectively, when adjusted for bacterial culture outcomes). Evaluation for C. difficile-associated disease (CDAD) among 864 patients was based on clinical criteria for antibiotic-associated diarrhea combined with laboratory evidence of toxin or toxin-producing C. difficile in stool specimens. The sensitivity of each test method for screening of CDAD was as follows: bacterial culture, 95%; culture with cytotoxin assay of isolates, 90%; ImmunoCard C. difficile test, 83%; cytotoxin assay 82%; and latex agglutination assay, 67% (P < or = 0.05 versus all other methods). The standard deviations of the test sensitivity statistics between study sites were ranked as follows: cytotoxin assay (+/- 3.1%) < ImmunoCard C. difficile test (+/- 5.7%) < latex agglutination assay (+/- 12.3%) < culture (+/- 24.7%) < culture with cytotoxin assay (+/- 28.0%). The data support the use of the ImmunoCard C. difficile test as an adjunct for the diagnosis of CDAD.     

Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing?

Direct tests for Clostridium difficile are 30–50?% more sensitive than tests for C. difficile toxins but the reasons for this discrepancy are incompletely understood. In addition to toxin degradation and strain differences, we hypothesized that C. difficile concentration could be important in determining whether toxins are detected in fecal samples. We performed standard curves on an FDA-approved real-time PCR test for the C. difficile tcdB gene (Xpert C. difficile/Epi, Cepheid) during a prospective comparison of a toxin immunoassay (Meridian Premier), PCR and toxigenic culture. Immunoassay-negative, PCR-positive samples were retested with a cell cytotoxin assay (TechLab). Among 107 PCR-positive samples, 46 (43.0?%) had toxins detected by immunoassay and an additional 18 (16.8?%) had toxin detected by the cytotoxin assay yielding 64 (59.8?%) toxin-positive and 43 (40.2?%) toxin-negative samples. Overall, toxin-negative samples with C. difficile had 10¹–10⁴ fewer DNA copies than toxin-positive samples and most discrepancies between toxin tests and PCR were associated with a significant difference in C. difficile quantity. Of the toxin-positive samples, 95?% had ≥4.1 log₁₀ C. difficile tcdB DNA copies/mL; 52?% of immunoassay-negative samples and 70?% of immunoassay and cytotoxin negative samples had <4.1 log₁₀ C. difficile tcdB DNA copies/mL. These findings suggest that fecal C. difficile concentration is a major determinant of toxin detection and C. difficile quantitation may add to the diagnostic value of existing test methods. Future studies are needed to validate the utility of quantitation and determine the significance of low concentrations of C. difficile in the absence of detectable toxin.     

Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children's hospital

Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdA and tcdB. Stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively cultured on cycloserine-cefoxitin-fructose agar following alcohol shock. Six testing modalities were evaluated, including stool EIA, culture EIA, and real-time PCR (tcdA and tcdB) of cultured isolates and stool samples. Real-time PCR detection was performed with tcdA and tcdB gene-specific primers and hydrolysis probes using the LightCycler platforms (Roche Diagnostics, Indianapolis, IN). A total of 157 samples from 96 pediatric patients were analyzed. The sensitivities of stool real-time PCR and stool EIA were 95% and 35%, respectively, with a specificity of 100% for both methods. The lower limit of detection of the stool real-time PCR was 30 CFU/ml of stool sample per reaction for tcdA and tcdB. This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection of C. difficile toxin genes in stool samples by real-time PCR showed sensitivity superior to that of stool and culture EIAs and performance comparable to that of real-time PCR assay of cultured isolates. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for children that should facilitate appropriate patient management and halt the practice of serial testing by EIA.