Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population

We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.     

Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays

Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.     

Laboratory diagnosis of Clostridium difficile-associated diarrhea and colitis: usefulness of Premier Cytoclone A+B enzyme immunoassay for combined detection of stool toxins and toxigenic C. difficile strains

Detection of Clostridium difficile toxins A and B in stools by Premier Cytoclone A+B enzyme immunoassay (EIA) was compared with detection by stool culture for C. difficile followed by detection of toxigenic isolates using the same EIA. Chart reviews were performed to evaluate the likelihood of C. difficile-associated diarrhea and colitis (CADC) for all patients with at least one positive toxin assay. While the toxins were detected in 58 of 85 consecutive CADC patients by both assays, CADC in 5 patients was detected only by stool toxin assay, and in 22 patients CADC was detected only by toxigenic culture. Our results suggest that for laboratories using a rapid toxin A+B EIA, direct toxin detection in stools should be combined with toxigenic culture in cases in which there is a negative stool toxin assay.     

Detection of nosocomial Clostridium difficile infections with toxigenic strains despite negative toxin A and B testing on stool samples

A two-step diagnostic algorithm is recommended to detect Clostridium difficile infections; however, samples are regularly found that are glutamate dehydrogenase (GDH) positive but stool toxin negative. In the present single-centre prospective study we focused on these ‘difficult-to-interpret’ samples and characterized them by anaerobic culture, toxigenic culture, slpA sequence typing and multiplex PCR (GenoType CDiff). The majority of stool toxin A and B-negative samples have been caused by toxigenic strains including ribotype 027. The multiplex PCR was faster and more sensitive compared with culture and allowed preliminary identification of hypervirulent strains in stool samples on the same day.     

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.     

Specific detection of toxigenic strains of Clostridium difficile in stool specimens.

Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone.     

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.     

Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin

We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were > or = 99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by approximately 75 to 80% and two-step testing was complete in < or = 3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US dollar 143,000 less than if CCNA alone had been performed on all 5,887 specimens.     

Evaluation of the Cepheid Xpert Clostridium difficile Epi assay for diagnosis of Clostridium difficile infection and typing of the NAP1 strain at a cancer hospital

Clostridium difficile is the most common cause of health care-associated diarrhea. Accurate and rapid diagnosis is essential to improve patient outcome and prevent disease spread. We compared our two-step diagnostic algorithm, an enzyme immunoassay for glutamate dehydrogenase (GDH) followed by the cytotoxin neutralization test (CYT) with a turnaround time of 24 to 48 h, versus the Cepheid Xpert C. difficile Epi assay, a PCR-based assay with a turnaround time of <1 h. In the first phase of the study, only GDH-positive stool samples were tested by both CYT and Xpert PCR. Discordant results were resolved by toxigenic culture. In the second phase, all stool samples were tested by GDH and Xpert PCR. Only GDH-positive stools were further tested by CYT. Genotypic characterization of 45 Xpert PCR-positive stools was performed by sequencing of the tcdC gene and PCR ribotyping. In phase 1, the agreement between the GDH-CYT and the GDH-Xpert PCR was 72%. The sensitivities and specificities of GDH-CYT and GDH-Xpert PCR were 57% and 97% and 100% and 97%, respectively. In phase 2, the agreement between GDH-CYT and Xpert PCR alone was 95%. As in phase 1, sensitivity of the Xpert PCR was higher than that of the GDH-CYT. The correlation between PCR-ribotyping, sequencing, and Xpert PCR for detection of NAP1 strains was excellent (>90%). The excellent sensitivity and specificity and the rapid turnaround time of the Xpert PCR assay as well as its strain-typing capability make it an attractive option for diagnosis of C. difficile infection.     

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.     

Novel one-step method for detection and isolation of active-toxin-producing Clostridium difficile strains directly from stool samples

The alarming emergence of hypervirulent strains of Clostridium difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. The C. difficile toxins A and B are critical virulence factors, and strains can either be toxin-producing (virulent) or non-toxin-producing (nonvirulent). Strains that are isolated from human infections generally produce either toxin A or toxin B or both. The methods currently available for culturing C. difficile do not differentiate strains that produce active toxins from strains that do not produce toxins or produce inactive toxins. As a result, the identification and isolation of toxin-producing strains from stool is currently a two-step process. First, the stool is plated on a selective medium, and then suspected colonies are analyzed for toxin production or the presence of the toxin genes. We describe here a novel selective and differential culture method, the Cdifftox plate assay, which combines in a single step the specific isolation of C. difficile strains and the detection of active toxin. This assay was developed based on our recent finding that the A and B toxins of C. difficile cleave chromogenic substrates that have stereochemical characteristics similar to their natural substrate, UDP-glucose. The Cdifftox plate assay is shown here to be extremely accurate (99.8% effective) in detecting toxin-producing strains through the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. The Cdifftox plate assay advances and improves the culture approach such that only C. difficile strains will grow on this agar, and virulent strains producing active toxins can be differentiated from nonvirulent strains, which do not produce active toxins. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains.     

Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus.     

Comparison of Simplexa Universal Direct PCR with Cytotoxicity Assay for Diagnosis of Clostridium difficile Infection: Performance,Cost, and Correlation with Disease

Simplexa Clostridium difficile universal direct PCR, a real-time PCR assay for the detection of the C. difficile toxin B (tcdB) gene using the 3M integrated cycler, was compared with a two-step algorithm which includes the C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Three hundred forty-two liquid or semisolid stools submitted for diagnostic C. difficile testing, 171 GDH antigen positive and 171 GDH antigen negative, were selected for the study. All samples were tested by the C. Diff Chek-60 GDH antigen assay, cytotoxin neutralization, and Simplexa direct PCR. Of 171 GDH-positive samples, 4 were excluded (from patients on therapy or from whom duplicate samples were obtained) and 88 were determined to be true positives for toxigenic C. difficile. Of the 88, 67 (76.1%) were positive by the two-step method and 86 (97.7%) were positive by PCR. Seventy-nine were positive by the GDH antigen assay only. Of 171 GDH antigen-negative samples, none were positive by PCR. One antigen-negative sample positive by the cytotoxin assay only was deemed a false positive based on chart review. Simplexa C. difficile universal direct PCR was significantly more sensitive for detecting toxigenic C. difficile bacteria than cytotoxin neutralization (P = 0.0002). However, most PCR-positive/cytotoxin-negative patients did not have clear C. difficile disease. The estimated cost avoidance provided by a more rapid molecular diagnosis was outweighed by the cost of isolating and treating PCR-positive/cytotoxin-negative patients. The costs, clinical consequences, and impact on nosocomial transmission of treating and/or isolating patients positive for toxigenic C. difficile by PCR but negative for in vivo toxin production merit further study.     

Multicenter Evaluation of the Quidel Lyra Direct C. difficile Nucleic Acid Amplification Assay

Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods.     

Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens.

Rapid (2.5- to 3.5-h) enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxins have been developed. We report the results of simultaneous testing of 700 fresh stool specimens by the tissue culture cytotoxin assay and four EIAs (Bartels Prima System C. difficile Toxin A EIA, Cambridge Biotech Cytoclone A+B EIA, Meridian Diagnostics Premier C. difficile Toxin A EIA, and TechLab C. difficile Tox-A Test EIA). In cases of disagreement, culturing for toxigenic C. difficile was performed. A total of 61 (8.7%) specimens from 46 patients were positive for C. difficile toxin. The sensitivity of the cytotoxin assay was 87%, and that of culture was 93%. In comparison with the cytotoxin assay results, the sensitivity and specificity of the EIAs were as follows: Bartels, 87 and 96%; Cambridge, 89 and 99%; Meridian, 87 and 98%; and TechLab, 87 and 95%, respectively. In comparison with the cytotoxin assay plus toxigenic culture results, the sensitivity and specificity of the EIAs were as follows: Bartels, 84 and 97%; Cambridge, 85 and 99%; Meridian, 79 and 98%; and TechLab, 80 and 96%, respectively. The EIAs varied in positive predictive values (PPVs). A high PPV was seen with the Cambridge EIA (96%); lower PPVs were seen with the TechLab (64%), Bartels (72%), and Meridian (80%) EIAs because of high false-positive rates. The negative predictive values (98 to 99%) were excellent with all EIAs. Results were indeterminant with 0.3% of the samples by the Meridian EIA and 3% by all the other EIAs. Although the EIAs were less sensitive than the cytotoxin assay, they provide same-day results and may be useful in laboratories without tissue culture facilities.     

Evolution of Testing Algorithms at a University Hospital for Detection of Clostridium difficile Infections

We present the evolution of testing algorithms at our institution in which the C. Diff Quik Chek Complete immunochromatographic cartridge assay determines the presence of both glutamate dehydrogenase and Clostridium difficile toxins A and B as a primary screen for C. difficile infection and indeterminate results (glutamate dehydrogenase positive, toxin A and B negative) are confirmed by the GeneXpert C. difficile PCR assay. This two-step algorithm is a cost-effective method for highly sensitive detection of toxigenic C. difficile.     

European Society of Clinical Microbiology and Infectious Diseases (ESCMID): Data review and recommendations for diagnosing Clostridium difficile-infection (CDI)

The aim of the present systematic review was to evaluate the available evidence on laboratory diagnosis of CDI and to formulate recommendations to optimize CDI testing. In comparison with cell culture cytotoxicity assay (CCA) and toxigenic culture (TC) of stools, we analyzed the test characteristics of 13 commercial available enzyme immunoasssays (EIA) detecting toxins A and/or B, 4 EIAs detecting Clostridium difficile glutamate dehydrogenase (GDH), and a real-time PCR for C. difficile toxin B gene. In comparison with CCA and TCA and assuming a prevalence of CDI of 5%, PPV and NPV varied between 0.28–0.77, 0.12–0.65 and 0.98–1.00, 0.97–1.00, respectively. Only if the tests were performed in a population with a CDI prevalence of 50 percent, would PPVs be acceptable (ranging from 0.71 to 1.00).To overcome the problem of a low PPV, we propose a two step approach, with a second test or a reference method in case of a positive first test. Further reducing the number of false negative results would require either retesting of all subjects with a negative first test, or re-testing all subjects with a negative second test, after an initially positive test. This approach resulted in non-significant improvements, and emphasizes the need for better diagnostic tests. Further studies to validate the applicability of two-step testing, including assessment of clinical features, are required.     

Clostridium difficile infection diagnosis in a paediatric population: comparison of methodologies

The increasing incidence of Clostridium difficile infection (CDI) in paediatric hospitalised populations, combined with the emergence of hypervirulent strains, community-acquired CDI and the need for prompt treatment and infection control, makes the rapid, accurate diagnosis of CDI crucial. We validated commonly used C. difficile diagnostic tests in a paediatric hospital population. From October 2011 to January 2012, 150 consecutive stools were collected from 75 patients at a tertiary paediatric hospital in Perth, Western Australia. Stools were tested using: C. Diff Quik Chek Complete, Illumigene C. difficile, GeneOhm Cdiff, cycloserine cefoxitin fructose agar (CCFA) culture, and cell culture cytotoxin neutralisation assay (CCNA). The reference standard was growth on CCFA or Cdiff Chromagar and PCR on isolates to detect tcdA, tcdB, cdtA, and cdtB. Isolates were PCR ribotyped. The prevalence of CDI was high (43 % of patients). Quik Chek Complete glutamate dehydrogenase (GDH) demonstrated a low negative predictive value (NPV) (93 %). Both CCNA and Quik Chek Complete toxin A/B had poor sensitivity (33 % and 29 % respectively). Molecular methods both had 89 % sensitivity. Algorithms using GDH + Illumigene or GeneOhm reduced the sensitivity to 85 % and 83 % respectively. Ribotype UK014/20 predominated. GDH NPV and GeneOhm and Illumigene sensitivities were reduced compared with adult studies. Quik Chek Complete and CCNA cannot reliably detect toxigenic CDI. A GDH first algorithm showed reduced sensitivity. In a high prevalence paediatric population, molecular methods alone are recommended over the use of GDH algorithm or culture and CCNA, as they demonstrate the best test performance characteristics.     

Reference assays for Clostridium difficile infection: one or two gold standards?

Accurate diagnosis of Clostridium difficile infection (CDI) is essential for optimal treatment, prevention and control. There are two reference assays for CDI diagnosis: the cell cytotoxicity assay (CCTA) and toxigenic culture (TC). Importantly, these tests actually detect different targets: CCTA detects the presence of C difficile toxins (primarily toxin B, but also toxin A), whereas TC detects the presence in the stool of C difficile with the potential to produce toxin. Not surprisingly studies comparing the results of these assays show imperfect agreement. Thus, a faecal sample may be CCTA negative but TC positive, and this raises the crucial question about the clinical significance of the presence of C difficile with the capacity to produce toxin but no actual detectable free toxin. A positive TC result indicates that a patient with diarrhoea is potentially infectious. TC also has the advantage that the cultured isolate is available for typing and for susceptibility testing. In general, however, CCTA has been shown to be a better test for the laboratory confirmation of CDI, although additional culture may be needed to optimise sensitivity. Crucially, when these reference assays are used to determine the accuracy of alternative diagnostic tests, care should be taken to compare methods with their appropriate standard (ie, compare tests that target equivalent end-points). Such issues have contributed to the variable and often suboptimal performance of rapid diagnostic tests for CDI. Further research is urgently needed to improve knowledge of the utility of routine diagnostic tests in CDI and the factors that influence their performance.     

Fluorescent-antibody test for detection of Clostridium difficile in stool specimens.

We evaluated a direct fluorescent-antibody test to detect Clostridium difficile, the most frequent cause of antibiotic-associated colitis. C. difficile organisms were injected into the ear veins of New Zealand White rabbits to induce antibodies, and the globulin fractions of their sera were conjugated to fluorescein isothiocyanate. The resulting conjugate strongly stained all 40 isolates of C. difficile tested. It also stained isolates of C. sordellii, C. bifermentans, C. chauvoei, and C. sporogenes, but not 20 other clostridial isolates or 10 isolates from other species. Results of testing fecal smears with the direct fluorescent-antibody method were compared with results of testing stools for C. difficile toxin and of culturing for C. difficile on a selective medium. A total of 158 fecal specimens from patients with antibiotic-associated diarrhea were tested. In these patients, the fluorescent-antibody test agreed with culture and toxin testing in 93% of the specimens. However, in normal adults, 62% of the fecal specimens from which C. difficile could not be cultured were positive by the fluorescent-antibody test. Absorption of the conjugate with C. sordellii led to a loss of reactivity to other clostridia as well as to 18 of 20 isolates of C. difficile.