Microbiology and Infectious Disease / C difficile Toxin Testing From Culture

Evaluation of Enzyme Immunoassays to Detect

Clostridium difficile Toxin From Anaerobic Stool Culture
Rosemary C. She, MD,1 Robert J. Durrant, MT,2 and Cathy A. Petti, MD1,3
Key Words: Clostridium difficile; Enzyme immunoassay; Cytotoxin; Stool culture
DOI: 10.1309/AJCPMM2E7VSPHNPG

Abstract
Stool culture for Clostridium difficile, while
necessary for strain typing and antimicrobial
surveillance, cannot determine toxin production.
We prospectively tested in triplicate 91 C difficile
cultured isolates for toxin production by 2 enzyme
immunoassays (EIAs) (Meridian Premier Toxins
A&B, Meridian Bioscience, Cincinnati, OH; and
TechLab Tox A/B II, TechLab, Blacksburg, VA) and
cytotoxin neutralization bioassay (CTN). By CTN,
88% (80/91) were toxigenic. Reproducibility was 93%
(85/91) for CTN, 80% (73/91) for Meridian EIA, and
79% (72/91) for TechLab EIA. Compared with CTN,
sensitivities were 87.1% and 89.2% for the Meridian
and TechLab EIAs, respectively. In an additional 115
stool specimens, CTN detected toxin more frequently
from cultured isolates (96/115) than stool (84/115).
For C difficile toxin detection from isolates, EIA was
less reproducible than CTN. EIA methods can be
falsely negative in 10% to 12% of isolates, and these
should be tested by CTN or polymerase chain reaction.
When positive, EIA is fast and reliable for detecting C
difficile toxin from culture.

American Society for Clinical Pathology

Diagnostic uncertainty exists about the optimal approach

to diagnose Clostridium difficileassociated disease.1-3
Physicians seek the most sensitive and specific test to detect
C difficile toxin that also yields a rapid result, but the emergence of more virulent strains has promoted a need for cultured isolates, which has complicated testing algorithms.4,5
Historically, enzyme immunoassays (EIAs) for toxins A and
B have been popular for laboratory practice and clinical care
because the tests are simple to perform and specific, and
results are provided within 24 hours. Today, anaerobic stool
culture has reemerged as an important test in the diagnosis
and management of C difficile infection, remaining the most
sensitive test available6 with the added benefit of providing
a clinical isolate that can be used to monitor antimicrobial
susceptibility patterns, develop vaccines, and perform strain
typing. Culturing stool, however, is not specific because
it does not distinguish toxin-producing from nontoxinproducing strains. Some laboratories that have the technical
expertise offer a cell culture cytotoxin neutralization bioassay
(CTN), a highly specific test to confirm the presence of toxin
Bproducing strains from direct specimen or culture, but this
method is labor-intensive, and results can be delayed up to
48 hours.
During the last 5 years, we observed a more than 60%
increase in test requests specifically to recover C difficile
from anaerobic stool culture with slightly decreased numbers of requests for CTN. This observation prompted us to
explore an efficient, inexpensive, and accurate testing algorithm that incorporates the ability to recover C difficile from
stool culture and determine toxin production. While a few
laboratories have reported using EIA directly from cultured
isolates,7-10 to our knowledge, no studies have specifically

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examined its performance in this setting. We compared the

performance characteristics of 2 commercial enzyme-labeled
immunosorbent assays with cell cytotoxin neutralization bioassay to detect C difficile toxin from culture.

Materials and Methods

Clinical Specimens
C difficile isolates that were recovered from stool samples submitted to ARUP Laboratories, Salt Lake City, UT,
specifically for C difficile culture were collected prospectively. Specimens were collected for comparative analysis
by EIA and CTN between May and September 2006, and for
comparative analysis by CTN on stool vs CTN on cultured
isolates between November 2007 and March 2008. Fecal
specimens were received frozen, or, if refrigerated, the specimen was processed if received within 48 hours of collection.
Specimens were stored at 2C to 8C after initial processing.
Culture
All stool samples were plated to cycloserine-cefoxitinfructose agar with horse blood media by a standard laboratory protocol. C difficile was identified based on growth and
appearance on cycloserine-cefoxitin-fructose agar with horse
blood, Gram stain, fluorescence under a Wood lamp, and the
characteristic horse stable odor.
Enzyme Immunoassay
We used 4 to 6 mature colonies of C difficile to prepare
suspensions with each EIA kit-specific diluent, and they
were tested for toxin with Premier Toxins A&B (Meridian
Bioscience, Cincinnati, OH) and C difficile Tox A/B II
(TechLab, Blacksburg, VA) kits using the manufacturers
protocols for direct specimen testing. Toxin A and toxin B
were not distinguished with these test methods.
Cytotoxin Neutralization Bioassay
CTN was performed on all C difficile isolates in this study.
Colony suspensions were prepared with phosphate-buffered
saline. For testing of fecal specimens, patient stool was
diluted 1:10, centrifuged, and, if necessary, filtered to clarify
the supernatant. C difficile suspensions or fecal supernatants
from each patient were inoculated into 2 wells of human
foreskin fibroblast plate in 2% minimum essential medium
diluent containing penicillin, streptomycin, and amphotericin
B (Fungizone). C difficile goat antitoxin (TechLab) was added
to one of the wells. Plates were incubated at 37C in 5%
carbon dioxide and examined at 4, 24, and 48 hours for cytopathic effect (CPE) including rounding of cells and disruption
of cell monolayer. The presence of CPE in the sample-only
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well and the absence of CPE in the sample well with antitoxin
was defined as a positive result.
Data Analysis
For comparison of EIA and CTN, all toxin testing was
performed in triplicate for each test method evaluated. A positive toxin result for each test method was defined as having
at least 2 positive test results by that method with each isolate. When all results tested in triplicate within a test method
agreed, the test result for that isolate was considered reproducible. For comparative analysis, CTN was considered the gold
standard. Sensitivity, specificity, negative predictive value,
and positive predictive value with 95% confidence intervals
for EIA were determined. Statistical analyses were performed
with Bayesian calculations. The binomial test was used for
comparison of reproducibility between EIA and CTN.

Results
For comparative analysis of EIA and CTN on C difficile
isolates, 91 C difficile isolates were recovered from stool
culture representing 91 patients. CTN, EIA-Meridian, and
EIA-TechLab identified 80 and 11, 70 and 21, 71 and 20 toxin-producing and nontoxin-producing strains, respectively.
The performance characteristics of the EIAs using CTN as the
gold standard are given in zTable 1z. Both EIA methods performed similarly with less than 90% sensitivity and specificity
of 97% or more. Sensitivity, specificity, negative predictive
value, and positive predictive value were not significantly different between the 2 EIAs. When we evaluated the reliability
of each test method, CTN was significantly more reproducible
than the EIA-Meridian and EIA-TechLab methods, with a P
value of less than .05 compared with either EIA zTable 2z.
We obtained 115 samples for comparison of CTN on
stool and CTN on colonies. Both assays were positive in 82
(71.3%) of 115 cases and negative in 17 (14.8%). CTN on
colonies was positive in 14 cases in which stool CTN was
negative. CTN on stool was positive in 2 cases in which CTN
on colonies was negative.

Discussion
Standardization for diagnosis of C difficileassociated
enteric disease is essential for hospital institutions and epidemiologists to accurately conduct surveillance and effectively
implement infection control measures. Anaerobic stool culture for C difficile remains a valuable laboratory test. With the
emergence of epidemic, highly virulent strains,11 the role of
culture has increased in importance because it provides an isolate for further analysis such as strain typing or susceptibility
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Microbiology and Infectious Disease / Original Article

zTable 1z
Test Performance of EIA Methods Compared With CTN for 91 Isolates Tested in Triplicate

No. of Tests

Test Method*
Positive Negative

CTN
EIA (Meridian)
EIA (TechLab)

240
209
214

33
33
32

False-
Positive

0
0
1

Performance (95% Confidence Interval)

FalseNegative

0
31
26

Sensitivity (%)

87.1 (82.9-91.3)
89.2 (85.3-93.1)

Specificity (%)

100 (100-100)
97.0 (91.2-100)

PPV (%)

NPV (%)

100 (100-100)
99.5 (98.6-100)

51.6 (45.1-56.9)
55.2 (42.4-68.0)

CTN, cytotoxin neutralization bioassay; EIA, enzyme immunoassay; NPV, negative predictive value; PPV, positive predictive value.
* CTN served as the gold standard. Meridian Bioscience, Cincinnati, OH; TechLab, Blacksburg, VA.

testing. EIA can serve as a fast, inexpensive, and preliminary

method to detect C difficile toxin from culture, and, when
positive, the likelihood of having a toxin-producing strain is
high in settings with high disease prevalence.
The performance of EIA on cultured isolates in this
study was within the range of previously reported results
from testing directly on stool specimens. The TechLab EIA
has reported sensitivities of 84% to 96% and specificities
of 87% to 100%8,12-15 while the Premier Toxins A&B EIA
has reported sensitivities of 60% to 97% and specificities of
93% to 100%.3,14,16,17 In our study, both EIA kits tested had
specificities of 97% or more and positive predictive values
of 99.5% or more when using the CTN assay on cultured
isolates as the gold standard. These assays, therefore, would
be convenient as screening tools for toxin detection in C difficile isolates. A systematic examination of the reproducibility
of EIA has not been performed before this study, and clinical
microbiologists should be aware that EIA testing from culture
is only 80% reproducible. Based on these data, we propose
a 2-step algorithm for the detection of toxin from C difficile
isolates recovered from anaerobic stool culture. When the EIA
result is positive, the result can be interpreted as a true positive. When the EIA is negative, C difficile isolates should be
tested by CTN or nucleic-acid amplification methods (when
available) to more accurately and reliably exclude C difficile
associated enteric disease.
Lability of C difficile toxin may offer an explanation
for the approximately 10% false-negative EIA results in
which previous data suggest that the microorganism might
require certain environmental conditions, such as stool, to
elaborate toxin.18 Isolates were not serially subcultured, and
suspensions were prepared specifically to provide the optimal
environment to detect toxin. Despite our efforts to minimize
these variables, EIA reproducibility from cultured isolates still
remained poor, suggesting that testing algorithms with EIA
may need revision. We also confirm previous findings that
toxin production is not diminished after culture of C difficile
isolates on solid media, but rather was enhanced in a number
of isolates. In fact, in vitro testing of C difficile isolates for
toxin production may be more sensitive than toxin testing
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zTable 2z
Reproducibility of Triplicate Testing by Method

No. of Isolates (n = 91)

Test Method

Reproducibility (%)

CTN
EIA (Meridian Bioscience,
Cincinnati, OH)
EIA (TechLab, Blacksburg, VA)

72 (79)
85 (93)
73 (80)

.016
.01

CTN, cytotoxin neutralization bioassay.

directly from stool,7,10,19,20 a finding that may be related to

higher organism load enhancing toxin detection.7
We acknowledge that this study had a few limitations.
Our observation that EIA had poor reproducibility and yet
retained high positive predictive value reflects the large
numbers of toxin-producing strains compared with nontoxinproducing strains in this study. In addition, no attempt was
made to discriminate between toxin A and toxin B. We do
not believe this limitation impacted the results of our study
because there were no cases in which the CTN result was
negative and the EIA result was positive. Also, human disease
from toxin Apositive/toxin Bnegative C difficile has not
been documented. Another potential limitation is that we did
not use a third method, specifically nucleic-acid amplification,
to analyze discrepant results between EIA and CTN.
EIA can serve as a quick screening tool for toxin production from cultured isolates. A negative result, however, cannot
reliably rule out the presence of toxin. Also, EIA was poorly
reproducible, and a single EIA should not be solely relied on
for confirmation of toxin production from anaerobic culture,
particularly in clinical contexts with low prevalence of toxinproducing strains.
From the Departments of 1Pathology and 3Medicine, University
of Utah School of Medicine, Salt Lake City; and 2Associated
Regional and University Pathologists Laboratories, Salt Lake
City.
Address reprint requests to Dr She: ARUP Laboratories, 500
Chipeta Way, Salt Lake City, UT 84108.

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Acknowledgments: We thank ARUP fecal testing,

bacteriology, and virology laboratories, Casey Rasch, and Brandi
Limbago, PhD (Centers for Disease Control and Prevention) for
technical assistance.
TechLab provided kits for testing.

References
1. Barbut F, Delme M, Brazier JS, et al. A European survey
of diagnostic methods and testing protocols for Clostridium
difficile. Clin Microbiol Infect. 2003;9:989-996.
2. Ticehurst JR, Aird DZ, Dam LM, et al. Effective detection
of toxigenic Clostridium difficile by a two-step algorithm
including tests for antigen and cytotoxin. J Clin Microbiol.
2006;44:1145-1149.
3. van den Berg RJ, Bruijnesteijn van Coppenraet LS, Gerritsen
HJ, et al. Prospective multicenter evaluation of a new
immunoassay and real-time PCR for rapid diagnosis of
Clostridium difficileassociated diarrhea in hospitalized patients.
J Clin Microbiol. 2005;43:5338-5340.
4. McDonald LC, Coignard B, Dubberke E, et al.
Recommendations for surveillance of Clostridium
difficileassociated disease. Infect Control Hosp Epidemiol.
2007;28:140-145.
5. Bartlett JG. Narrative review: the new epidemic of
Clostridium difficileassociated enteric disease. Ann Intern Med.
2006;145:758-764.
6. Gerding DN, Johnson S, Peterson LR, et al. Clostridium
difficileassociated diarrhea and colitis. Infect Control Hosp
Epidemiol. 1995;16:459-477.
7. Delme M, Van Broeck J, Simon A, et al. Laboratory diagnosis
of Clostridium difficileassociated diarrhoea: a plea for culture.
J Med Microbiol. 2005;54(pt 2):187-191.
8. Russmann H, Panthel K, Bader RC, et al. Evaluation of three
rapid assays for detection of Clostridium difficile toxin A and
toxin B in stool specimens. Eur J Clin Microbiol Infect Dis.
2007;26:115-119.
9. Lozniewski A, Rabaud C, Dotto E, et al. Laboratory diagnosis
of Clostridium difficileassociated diarrhea and colitis: usefulness
of Premier Cytoclone A+B enzyme immunoassay for combined
detection of stool toxins and toxigenic C difficile strains. J Clin
Microbiol. 2001;39:1996-1998.
10. Reller ME, Lema CA, Perl TM, et al. Yield of stool culture with
isolate toxin testing versus a two-step algorithm including stool
toxin testing for detection of toxigenic Clostridium difficile.
J Clin Microbiol. 2007;45:3601-3605.

84
84

Am J Clin Pathol 2009;131:81-84

DOI: 10.1309/AJCPMM2E7VSPHNPG

11. Hubert B, Loo VG, Bourgault AM, et al. A portrait of the

geographic dissemination of the Clostridium difficile North
American pulsed-field type 1 strain and the epidemiology
of C difficileassociated disease in Quebec. Clin Infect Dis.
2007;44:238-244.
12. Turgeon DK, Novicki TJ, Quick J, et al. Six rapid tests for
direct detection of Clostridium difficile and its toxins in fecal
samples compared with the fibroblast cytotoxicity assay. J Clin
Microbiol. 2003;41:667-670.
13. Lyerly DM, Neville LM, Evans DT, et al. Multicenter
evaluation of the Clostridium difficile TOX A/B TEST. J Clin
Microbiol. 1998;36:184-190.
14. Musher DM, Manhas A, Jain P, et al. Detection of Clostridium
difficile toxin: comparison of enzyme immunoassay results
with results obtained by cytotoxicity assay. J Clin Microbiol.
2007;45:2737-2739.
15. Snell H, Ramos M, Longo S, et al. Performance of the TechLab
C. DIFF CHEK-60 enzyme immunoassay (EIA) in combination
with the C difficile Tox A/B II EIA kit, the Triage C difficile
panel immunoassay, and a cytotoxin assay for diagnosis
of Clostridium difficileassociated diarrhea. J Clin Microbiol.
2004;42:4863-4865.
16. Gilligan PH. Is a two-step glutamate dehydrogenase
antigen-cytotoxicity neutralization assay algorithm superior
to the premier toxin A and B enzyme immunoassay for
laboratory detection of Clostridium difficile? J Clin Microbiol.
2008;46:1523-1525.
17. Novak-Weekley SM, Hollingsworth MH. Comparison of the
Premier toxin A and B assay and the TOX A/B II assay for
diagnosis of Clostridium difficile infection. Clin Vaccine Immunol.
2008;15:575-578.
18. Brazier JS. Role of the laboratory in investigations of
Clostridium difficile diarrhea. Clin Infect Dis. 1993;16(suppl
4):S228-S233.
19. Peterson LR, Kelly PJ, Nordbrock HA. Role of culture and
toxin detection in laboratory testing for diagnosis of Clostridium
difficileassociated diarrhea. Eur J Clin Microbiol Infect Dis.
1996;15:330-336.
20. Bouza E, Pelez T, Alonso R, et al. Second-look cytotoxicity:
an evaluation of culture plus cytotoxin assay of Clostridium
difficile isolates in the laboratory diagnosis of CDAD. J Hosp
Infect. 2001;48:233-237.

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