Evaluation of a commercial enzyme immunoassay kit for the detection ofClostridium difficile toxin A

A new enzyme immunoassay (EIA) kit developed for the rapid detection ofClostridium difficile toxin A in faecal specimens, Premier (Meridian Diagnostics), was evaluated using 101 faecal specimens. Sixty-nine specimens were positive forClostridium difficile by isolation of the organism and by cytotoxicity in tissue culture. The EIA for toxin A was positive in 49 of these 69 cases. No specimen that was negative for cytotoxicity was positive by EIA. Eight of the 32 specimens negative by both EIA and cytotoxicity assay yieldedClostridium difficile by culture. In five of these cases the cytotoxigenic status of the isolate was determined, and four were positive. There was no direct relationship between cytotoxin titre and EIA reading.     

Impaired detection of faecal verocytotoxin in the presence ofClostridium difficile cytotoxin in patients with haemolytic uraemic syndrome

Three cases of haemolytic uraemic syndrome associated with infection with verocytotoxin producingEscherichia coli are described. The concomitant presence ofClostridium difficile cytotoxin in the patients' stool impaired the detection of free faecal verocytotoxin. Stool specimens containingClostridium difficile cytotoxin should thus be considered negative for verocytotoxin only after neutralisation of theClostridium difficile cytotoxin with antitoxin.     

Laboratory diagnosis ofClostridium difficile-associated diarrhoea

This paper reviews the various laboratory procedures available for the isolation and identification ofClostridium difficile and the detection of toxins produced by this organism. Laboratories should be selective in determining which patients require investigation forClostridium difficile-associated diarrhoea. Transport and storage of stool specimens at 4 °C is recommended when delays in processing may occur. Tissue culture techniques are still the best method for detection of cytotoxin and a variety of cell lines can be used. Other methods for detecting cytotoxin, and methods for detecting other toxins are not sufficiently developed yet to warrant introduction into diagnostic laboratories. Culture techniques remain the most sensitive for diagnosis, particularly since the development of a variety of enrichment techniques. Cycloserine cefoxitin fructose agar is still adequate, although reduced concentrations of antimicrobial agents are necessary, and improvements, such as the addition of sodium taurocholate, increase the recovery of spores. Enrichment cultures have markedly increased isolation rates forClostridium difficile but the significance of these isolates needs to be carefully evaluated. Until simpler and more reliable tests are available in clinical laboratories for the detection of toxins, the isolation ofClostridium difficile from patients with diarrhoeal disease should be considered paramount.     

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.     

Nosocomial Outbreak of Clostridium difficile-Associated Diarrhoea due to a Clindamycin-Resistant Enterotoxin A-Negative Strain

A clindamycin-resistant toxin A-negative, toxin B-positive Clostridium difficile strain caused an outbreak among 24 hospitalized patients at the Department of Surgery, the Intensive Care unit, and the Department of Internal Medicine of an 800-bed academic hospital. Nineteen patients had undergone a surgical intervention and all 24 patients received at least one dose of antibiotics prior to the development of Clostridium difficile-associated diarrhoea. Twenty-seven episodes of Clostridium difficile-associated diarrhoea in 24 patients were categorized as mild (n=19), severe (n=7), or fatal (n=1). Relapses occurred in three patients. Nineteen of the 27 episodes required anti-Clostridium difficile treatment. Molecular typing performed by arbitrary primer polymerase chain reaction (PCR) and PCR amplification of rRNA intergenic spacer regions revealed that the outbreak strains recovered from culture were identical. The outbreak strain belonged to serogroup F and was resistant to erythromycin, clindamycin, and tetracycline, whereas susceptibility to chloramphenicol varied. No phenotypic activity of enterotoxin A was detected. A deletion of approximately 1.7 kb was found in the toxin A gene. Cytotoxin B had an unusual effect on cell culture assays that, at first, was not recognized as Clostridium difficile specific but could be neutralized with anti-Clostridium difficile B cytotoxin. Electronic Publication     

Usefulness of semi-quantitative cultures in the diagnosis ofClostridium difficile associated disease

Semi-quantitative stool cultures on CCFA were compared to cytotoxic assays for the diagnosis ofClostridium difficile associated disease (CAD). There was a significant correlation between the amount ofClostridium difficile growth on CCFA, the presence of cytotoxin and a clinical diagnosis of CAD in the 541 initial stool specimens tested.     

Usefulness of Simultaneous Detection of Toxin A and Glutamate Dehydrogenase for the Diagnosis of Clostridium difficile-Associated Diseases

 The aim of this study was to evaluate an immunoassay (Triage; Biosite Diagnostics, BMD, France) for detecting both a specific antigen of Clostridium difficile (glutamate dehydrogenase [GDH]) and toxin A. Evaluation of the test was carried out in 304 fecal samples from patients suspected of having Clostridium difficile-associated diseases. The results with GDH and toxin A were compared with those of a culture and cytotoxicity assay (toxin B). The prevalence rates for toxin B-positive and culture-positive fecal samples were 11.2% and 25%, respectively. The sensitivity of the Triage immunoassay was 90.8% for GDH and 79.4% for toxin A. A negative result with both toxin A and GDH was very reliably able to eliminate a diagnosis of Clostridium difficile-associated disease (negative predictive value 99.6%). Triage is a very rapid (20 min) and easy-to-perform test. It could be useful for diagnostic purposes and also for detecting nontoxigenic strains in epidemiogical studies.     

Evaluation of a new commercialClostridium difficile toxin A enzyme immunoassay using diarrhoeal stools

A new, commercially available enzyme immunoassay for the detection of toxin A in stool specimens, the PremierClostridium difficile Toxin A test (Meridian Diagnostics), was evaluated using 228 diarrhoeal stool specimens. Using a cytotoxin assay on HeLa cells as the reference method, this new test resulted in a sensitivity of 88 % and a specificity of 95 %. Using the presence or absence of a toxigenic strain in the stools as the reference method, the sensitivity was similar to that of the cytotoxin assay (71.7 % versus 70.5 %) and the overall correlation was even better (89.4 % versus 82 %). The PremierClostridium difficile Toxin A assay is rapid and easy to perform and is an excellent alternative to the usual toxin B assay.     

Impact of imipenem/cilastatin therapy on faecal flora

To evaluate the effects of parenteral imipenem/cilastatin therapy upon human faecal microflora, stool specimens obtained from ten patients before, during and after therapy were cultured quantitatively for aerobic and anaerobic microorganisms. The patients received 500 mg imipenem combined with 500 mg cilastatin every 6 h for 6–11 days. The antimicrobial therapy was associated with a small decrease in the numbers of enterobacteria, anaerobic cocci and bacteroides during treatment but afterward the microflora normalized in all patients. None of the patients was colonized with new imipenem-resistant bacteria, hadClostridium difficile or cytotoxin in the stools, or developed diarrhoea.     

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.     

Comparison of enzyme immunoassays and rapid diagnostic tests for <Emphasis Type="Italic">clostridium difficile</Emphasis> glutamate dehydrogenase and toxin a + B to toxinogenic culture on a highly selective chromogenic medium

To compare Clostridium. (C.) difficile toxin A/B and glutamate dehydrogenase (GDH) enzyme immunoassays or rapid diagnostic tests to toxinogenic culture on recently described highly selective agar plates. Five hundred consecutive samples sent in for C. difficile diagnostics were tested by toxin A/B enzyme immunoassay (EIA) and rapid diagnostic test (RDT), GDH EIA and RDT, and culture on chromID C. difficile plates for 48 hrs, with toxin testing from culture if the toxin EIA from feces was negative. Samples with discordant results from EIA and RDT were submitted to C. difficile-specific 16S rRNA gene and tcdB PCR. Ninety-two, 88, 31, and 37 samples were positive by GDH EIA, GDH RDT, toxin A/B EIA, and toxin A/B RDT respectively. Seventy-four samples were positive by culture, 54 culture-positive samples were subjected to repeat toxin testing, with an additional 29 samples positive. Thus, there were 60 C. difficile toxin A/B positive samples in total (12 %). Single-step screening with GDH EIA, GDH RDT, toxin A/B EIA, and toxin A/B RDT would have missed seven (12 %), 11 (18 %), 29 (48 %) or 27 (45 %) of all positive samples respectively. Single-step screening with GDH or toxin A/B tests from feces misses a significant proportion of patients compared to toxinogenic culture, putting these patients at risk from undiagnosed C. difficile infection. More data are needed to establish the clinical significance of a positive toxinogenic culture result in the absence of detectable toxin A/B in feces.     

Enzyme immunoassay for detection ofClostridium difficile toxins a and b in patients with antibiotic-associated diarrhoea and colitis

A sandwich enzyme immunoassay (ELISA) was developed to detectClostridium difficile toxins A and B in stools from patients with antibiotic associated diarrhoea and colitis. Immune serum to crudeClostridium difficile toxin and non-immune serum were coated onto polystyrene microtiter plates to act as capture antibodies; toxins A and B in human stools were detected by antibodies from rabbits immunized with purified toxins A and B. The ELISA for toxin B showed cross-reactions withClostridium bifermentans andClostridium sordellii and lacked diagnostic sensitivity in clinical samples. The ELISA for toxin A showed no cross-reactions with other clostridiae investigated and was positive in 33 % (62/189) of patients with antibiotic associated diarrhoea. This compared with 38 % (71/189) positive in the tissue culture assay forClostridium difficile cytotoxin. With a predictive value of 96 % in clinical specimens, the ELISA for toxin A constitutes a sensitive and specific tool for diagnosis ofClostridium difficile associated diarrhoea and colitis.     

Evaluation of an oligonucleotide probe and an immunological test for direct detection of toxigenicClostridium difficile in stool samples

A 33 basepair oligonucleotide probe, designed from the sequence of theClostridium difficile toxin B gene, was evaluated for its ability to detect toxigenicClostridium difficile directly in stool samples, without culture or DNA isolation. Two different labelling techniques were investigated: radiolabelling and digoxigenin-labelling. One hundred ninety-six stools were tested, with a good correlation (96 %) obtained between the oligonucleotide probe and the gold standard, the cytotoxicity tissue culture assay. The sensitivity and specificity were 83 % and 100 %, respectively. In parallel, a new commercially available enzyme immunoassay for the detection ofClostridium difficile toxin A in stool specimens was investigated. In 162 samples tested, a sensitivity of 80 % and a specificity of 98 % were obtained.     

Presence ofClostridium difficile toxin in guinea pigs with penicillin-associated colitis

Cecal filtrates from guinea pigs treated with penicillin contained a toxin which produced cytotoxic changes in HeLa cell cultures and was lethal to guinea pigs when administered intracecally. The cytotoxicity could be neutralized byClostridium difficile andC.sordellii antitoxins,but not by other clostridial antitoxins. Rabbit immunization with toxic cecal extracts produced antibody which neutralized the cytotoxicity of guinea pig cecal extracts, of stool extracts from humans with antibiotic-associated colitis and of culture supernatant fluids ofC.difficile. Treatment with vancomycin reduced the number of deaths and increased the survival time of penicillin-treated animals. No cytotoxin was present in cecal extracts from these guinea pigs. Gram-negative bacteremia was present in half the penicillin-treated animals, the sick ones as well as the healthy ones. Treatment with vancomycin did not decrease the incidence of bacteremia. Gram-negative bacteremia and changes in fecal flora were observed in some antibiotic-treated guinea pigs; all diseased animals, however, contained this cytotoxin.C.difficile was isolated from cecal contents of sick animals and these isolates produced the cytotoxinin vitro. The results suggest thatC. difficile toxin can cause antibiotic-associated colitis in guinea pigs.     

Role of culture and toxin detection in laboratory testing for diagnosis ofClostridium difficile-associated diarrhea

Two variations of an egg yolk agar base medium containing cycloserine, cefoxitin, and fructose (CCFA), one with 250 g and the other with 500 g of cycloserine/ml of agar medium were compared to study the effect of the cycloserine concentration on recovery ofClostridium difficile from stool samples. In addition, the role of prior anaerobic reduction of these media in the detection ofClostridium difficile-associated diarrhea (CDAD) was tested. Each medium was studied over a two-month period, with outcome compared between the testing periods and to historical data from our institution. Clinical correlation of test results was performed. The use of the originally described formulation of CCFA with 500 g of cycloserine/ml of agar combined with 4 h of anaerobic reduction prior to specimen inoculation increased the rate of isolation of toxigenicClostridium difficile from clinical specimens from 6 to 17% (p < 0.001). Combining direct detection of stool toxin and properly performed culture for toxigenicClostridium difficile enhances the potential for diagnosis of CDAD. For optimal performance the culture medium should contain the originally proposed cycloserine concentration of 500 g/ml of agar and should be anaerobically reduced at least 4 h prior to specimen inoculation.     

Stronger correlation between antibiotic use and the incidence of Clostridium difficile determined by culture results instead of faecal toxin detection only

The detection of Clostridium difficile in previous studies evaluating antibiotic use as a risk factor was limited to toxin assay tests. The reported associations may have been misleading due to the low sensitivity of toxin assay tests compared to culture results. Antibiotic use and the incidence of C. difficile of 19 units (wards) over 5 years were analysed. Stool samples were tested for toxin A/B and cultured. The correlation of antibiotic use with the incidence of C. difficile determined by culture results was compared to the correlation determined by toxin assay results. Additionally, single antibiotics were analysed as risk factors. Of 5,772 faecal samples tested for C. difficile, 154 single-first cases were detected by the toxin assay and 251 additional single-first cases by culture. Antibiotic use was a significantly stronger risk factor in the correlation based on the culture results (R ² = 0.63) versus toxin assay results (R ² = 0.40). Multivariate analysis did not improve the correlation significantly and only the group of broad-spectrum beta-lactams was identified as an independent risk factor. The correlation between antibiotic use and C. difficile incidence rates significantly improves if detection is not limited to faecal toxin assays. Therefore, antibiotic pressure was previously underestimated as a risk factor.     

Comparison of four laboratory tests for diagnosis ofClostridium difficile-associated diarrhea

Four different laboratory tests for diagnosis ofClostridium difficile-associated diarrhea were compared to determine the optimal one for management of patients with hospital-acquired diarrhea. Stool samples from 231 patients with diarrhea were tested by the following methods: culture forClostridium difficile with subsequent determination of exotoxin production, with a toxigenicClostridium difficile positive (TCP) result considered truly positive; enzyme immunoassay (EIA); latex agglutination test; and an immunobinding blot assay. The rates of positive results were as follows: EIA 5.5%, TCP 7.3%, latex agglutination 16.7%, and immunobinding blot assay 26.1%. Compared to the TCP results, the sensitivity and specificity were, respectively, 61 and 98% for EIA, 47 and 85% for latex agglutination, and 60 and 76% for the immunobinding blot assay. Samples from patients with 6 stools/day were TCP and EIA positive in 27 and 17% of cases, respectively, whereas in patients with < 6 stools/day, these percentages decreased to 2 and 3%, respectively (p < 0.001). In hospitalized patients with 6 stools/day, EIA appears to be the optimal test for diagnosis ofClostridium difficile-associated diarrhea, with a 73% positive predictive value and a 97% negative predictive value. However, in patients with < 6 stools/day, the prevalence ofClostridium difficile is low, and laboratory detection of this organism remains problematic.     

Utility of a rapid latex test for the detection of Clostridium difficile in fecal specimens

Currently, the method of choice for the laboratory diagnosis of Clostridium difficile disease is the detection of cytotoxin in stool filtrates by tissue culture. Since many hospital laboratories do not have tissue culture facilities, there is a need for a rapid test which is both sensitive and specific to diagnose C. difficile disease. A commercial latex agglutination was compared with the conventional cytotoxin tissue culture assay for the detection of C. difficile or its toxin(s) in fecal specimens. Of the 574 specimens evaluated, 111 were cytotoxin positive while 97 were positive by the latex agglutination test. There were 17 specimens positive by latex agglutination but negative by tissue culture assay. The overall sensitivity and specificity of the CDT latex test was 86.1 percent and 95.3 percent respectively. This rapid latex test can serve as an excellent screening procedure for the presence of C. difficile. Those specimens positive by the latex test should be further evaluated for the presence of cytotoxin by tissue culture.     

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.     

Epidemiology and prevention ofClostridium difficile infections in a leukemia unit

A 29-month prospective study was carried out in a leukemia unit with the aim of investigating the epidemiology of Clostridium difficile infections and limiting their spread. Systematic cultures of stools and assays for cytotoxin were performed on patient admission and at weekly intervals, yielding 1,355 cultures and assays. The study period was divided in period A, before total unit renovation, and period B, afterwards. During period B all patient carriers of Clostridium difficile received vancomycin. A comparison of the two periods showed that the percentage of positive cultures fell from 16.6 % to 3.6 % and the positive toxin assays from 9.9% to 1.2%. It was concluded that colonization by Clostridium difficile can be prevented in hospital wards with generally high rates of infection by a combination of decontamination of the environment, introduction of preventive measures and treatment of Clostridium difficile carriage with vancomycin.