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?
ABSTRACT 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).
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ABSTRACT: This guideline will examine the use of molecular tests and multi-test algorithms for the diagnosis of Clostridium difficile Infections (CDI). These new tests, alone or in a multi-step algorithm consisting of more than one assay, are more expensive than the older EIA assays; however, rapid and accurate testing can save money overall by initiating appropriate treatment and infection control protocols sooner and by possibly reducing length of hospital stay . We recommend testing only unformed stool in patients with clinically significant diarrhea (CSD) by a molecular method or by a two to three step algorithm.Clinical Infectious Diseases 06/2013; · 9.37 Impact Factor
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ABSTRACT: Nucleic acid amplification techniques (NAATs) represent a major advance in the diagnosis of Clostridium difficile (C. difficile) infection. This review analyzes the different options available for a molecular diagnosis of C. difficile infection, as well as the strengths and weaknesses of NAATs. The performances of seven commercials NAATs are compared (BD GeneOhm Cdiff, Illumigene C. difficile, Xpert C. difficile, BD Max Cdiff, Portrait Toxigenic C. difficile, ProGastro Cd, Seeplex Diarrhea ACE). The sensitivity and the rapidity of NAATs are excellent: additional efforts should focus on the discrimination between infection and colonization. Reporting the DNA load of toxigenic C. difficile in the stool sample may represent a solution. Diagnostic algorithms combining immunoassays and NAATs could also improve the specificity and reduce the global cost of this analysis.Expert Review of Molecular Diagnostics 09/2013; 13(7):681-92. · 4.09 Impact Factor
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ABSTRACT: The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8-97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.Expert Review of Molecular Diagnostics 11/2013; · 4.09 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2008, p. 1523–1525
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 4
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??
Peter H. Gilligan*
Clinical Microbiology-Immunology Laboratories, University of North Carolina Hospitals, and Departments of
Microbiology-Immunology and Pathology-Laboratory Medicine, University of North Carolina School of
Medicine, Chapel Hill, North Carolina 27514
Received 31 October 2007/Returned for modification 11 December 2007/Accepted 25 January 2008
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
The recent emergence of a highly virulent strain of Clostrid-
ium difficile, with its associated increased morbidity and in-
creased mortality, has placed a renewed emphasis on the lab-
oratory diagnosis of C. difficile disease (7, 10). For well over a
decade, first the Premier toxin A enzyme immunoassay (EIA)
and then the Premier toxin A and B EIA (A/B EIA; Meridian
Bioscience, Inc., Cincinnati, OH) were used to detect this toxin
(21). The latter is the most widely used test for the detection of
C. difficile toxins in the United States (4). Recent studies (11,
20) have shown that this assay has both excellent sensitivity
(?90%) and excellent specificity (?95%) compared to the
results of a tissue culture cytotoxicity neutralization (CTN)
assay, an assay frequently considered the “gold standard” for
the diagnosis of C. difficile disease (1, 9, 11, 12, 15, 18, 19, 21).
Other data suggest that the detection of toxigenic strains of C.
difficile is actually a more sensitive approach to the diagnosis of
C. difficile disease (5, 6, 13, 14), but culture is infrequently done
for diagnostic purposes in the United States because of its slow
turnaround time and the need for special isolation media and
technical expertise. In addition, the specificity of toxigenic cul-
ture for the diagnosis of C. difficile disease has also been ques-
tioned (1, 14).
Given the emergence of a highly virulent C. difficile strain in
the United States, a recent report by Ticehurst and colleagues
was a cause for concern (18). They showed that another widely
used EIA, the C. difficile Tox A/B II assay (TechLab, Blacks-
burg, VA), which has performance characteristics similar to
those of the A/B EIA (11, 12, 19), had a sensitivity of only 36%
for C. difficile detection when the results were compared to
those obtained by the use of a two-step algorithm in which
specimens were first tested for the presence of the glutamate
dehydrogenase (GDH) antigen by a solid-phase EIA (C. Diff
Chek-60; TechLab) and the positive results were confirmed by
the CTN assay. The test for the GDH antigen proved to be an
excellent screening test, with a sensitivity of ?95% and a neg-
ative predictive value of ?99% compared with the results of
the CTN assay (18). In practical terms, that meant that GDH-
negative specimens could be reported to be negative without
further testing. By using the CTN assay as a reference method,
it was found that the assay for the GDH antigen had a positive
predictive value of 53%, indicating that a confirmatory test was
required to detect specimens containing C. difficile toxins.
I applied the two-step algorithm approach using a modifi-
cation of the GDH antigen assay, which used a recently devel-
oped membrane-bound lateral flow device, C. Diff Quik Chek
(TechLab), and compared its performance characteristics to
those of the A/B EIA with a membrane-bound lateral flow
device (Tox A/B Quik Chek; TechLab) for the detection of
toxins A and B (the LF-A/B assay).
Three hundred sixty-eight clinical specimens submitted be-
tween April and June 2007 for the detection of C. difficile toxin
by the A/B EIA were studied. The inclusion criteria were as
follows: a clinician had to order the detection of C. difficile
toxin in a specimen, the specimen had to be less than 24 h old,
the specimen had to take the form of the specimen container,
and the specimen volume had to be sufficient to allow perfor-
mance of the four assays that were used in this study. All
specimens were stored at 4°C prior to assay. The A/B EIA, the
C. Diff Quik Chek assay (the GDH assay), the LF-A/B assay,
and the CTN assay with the TechLab C. difficile toxin B assay
were all done according to the manufacturer’s instructions,
with the exception that the results for specimens with optical
densities at 450/630 nm of 0.1 to 0.5 by the A/B EIA were
confirmed by the CTN assay. In-house data have shown that
only 30% of specimens with optical density values between 0.1
and 0.5 by the A/B EIA could be confirmed to be positive by
the CTN assay. Positive controls were run on each day of the
assay for all tests. In addition, a negative control was run for
the A/B EIA, while each of the GDH and LF-A/B assays has
* Mailing address: Clinical Microbiology-Immunology Laboratories,
UNC Hospitals, CB 7600, Chapel Hill, NC 27514. Phone: (919) 966-
6313. Fax: (919) 966-0486. E-mail: firstname.lastname@example.org.
?Published ahead of print on 6 February 2008.
an internal negative control. The individual performing the
GDH and the LF-A/B assays was blinded to the A/B EIA
For the purposes of comparisons of the algorithm with the
A/B EIA and the LF-A/B assays, specimens were assigned a
true-positive result by the two-step algorithm if they were both
GDH assay positive and CTN assay positive; they were as-
signed a true-negative result if they GDH assay negative or
GDH assay positive but CTN assay negative. These designa-
tions are based on the high sensitivity and the high negative
predictive value previously reported for this algorithm (18).
Confidence intervals were determined by the use of EP Eval-
uator software (David G. Rhoads Associates, Kennett Square,
PA). This study was approved by the Institutional Review
Board of the University of North Carolina.
Of the 368 specimens studied, 70 were positive and 298 were
negative by the GDH assay.
Of the 70 GDH assay-positive specimens, 37 were confirmed
to contain C. difficile toxin by the CTN assay. Only 22 of the 37
specimens confirmed to have positive results were positive by
the A/B EIA, resulting in an A/B EIA sensitivity of 59.5%. The
sensitivity was even lower for the LF-A/B assay (43%). The
positive predictive values, the negative predictive values, and
the specificities of the EIAs were all very good to excellent
(Table 1). Two specimens were found to be GDH assay neg-
ative but A/B EIA positive. Both specimens were negative by
the CTN assay. The results for five specimens found to be
positive by the LF-A/B assay could not be confirmed by the
CTN assay; two of the five specimens were also positive by the
The use of two-step diagnostic algorithms, in which an easily
performed and highly sensitive but less specific assay is used as
a screening test to eliminate specimens with negative results
and in which the results for specimens with positive results are
confirmed by a more specific but often more complex confir-
matory test, is a well-established diagnostic paradigm. Such
algorithms are widely used for the diagnosis of both syphilis
and human immunodeficiency virus infection (3). In the
present study, I wanted to learn if the two-step algorithm
described by Ticehurst et al. (18) would enhance the ability to
detect C. difficile toxin compared to the ability of the most
commonly used C. difficile toxin A and B EIA, the A/B EIA
(Meridian Bioscience). Although the differential was not as
dramatic as that reported by Ticehurst et al. (18), I found that
the two-step algorithm had an enhanced ability to detect C.
difficile toxin-positive specimens by 40% compared to the re-
sults of the A/B EIA. I also evaluated a novel lateral flow toxin
A and B EIA, the LF-A/B assay, but found it to be both less
sensitive and less specific than the A/B EIA.
What are some of the practical observations made during
this study? First, the LF-A/B assay had performance charac-
teristics very similar to those reported for the C. Diff Chek-60
assay (16, 18) in that it had a positive predictive value of only
53% but a sensitivity of 100% compared to tissue culture
results. Because the results for all negative specimens were not
confirmed by the CTN assay, the specificity and the negative
predictive value of this GDH assay cannot be stated with cer-
tainty. However, 293 of 298 of the GDH assay-negative spec-
imens were also negative by the two other immunoassays that
were studied, with the A/B EIA having been shown to be highly
specific and to have a high negative predictive value in several
studies (9, 11, 12, 15, 19). The seven GDH assay-negative
specimens that were positive by one of the two other EIAs
were found to be negative by the CTN assay. These data
suggest that the LF-A/B assay does have a very high negative
predictive value, a crucial characteristic of any screening test,
but because of the study design, this cannot be proven conclu-
Second, the GDH screening test is easily performed and the
results are easily interpreted. The test takes approximately 20
min to perform and is very simple, and the filters clogged for
less than 5 of 368 specimens tested. There was one failed run
during the study because the reagents were not added in the
proper order. This was determined by failure of the internal
control. I used positive controls on each day of use. Except for
the failed run, the positive control gave an appropriate reac-
tion each time, suggesting that positive controls need to be
tested only once per test lot. My laboratory can currently offer
this testing three times per day, and stat testing is also easily
accomplished. My laboratory can offer testing by the A/B EIA
only once a day, and stat testing is inconvenient and is rarely
Third, this approach is more expensive, and for an estimated
20% of specimens that require the CTN assay, the turnaround
time is delayed compared to the turnaround time for the EIA.
The material costs per specimen for the A/B EIA were approx-
TABLE 1. Performance characteristics of A/B EIA and the LF-A/B assay compared with those of a two-step algorithm using C. Diff Chek-60
and C. difficile toxin B assays
Test and result
No. of specimens with
the following two-step
Sensitivity (%)Specificity (%) NPVa(%)
99.4 (97.8–99.8) 95.6 91.7
43.2 (28.7–59.1)98.5 (96.5–99.4)93.9 76.2
aNPV, negative predictive value (predictive value of a negative test result).
bPPV, positive predictive value (predictive value of a positive test result).
cValues in parentheses are the 95% confidence limits, determined by the “score” method.
1524 NOTES J. CLIN. MICROBIOL.
imately $5.00, while the current per specimen cost for the
LF-A/B assay is $9.00. In addition, 20% of specimens require
the CTN assay, which has an approximate cost of $8.00. These
specimens require both centrifugation and filtration, which are
labor-intensive and which thus add further to the cost of this
algorithm. The overall material cost per specimen for the two-
step algorithm is $10.60. In order to provide the best possible
turnaround time, testing by the CTN assay must be offered 7
A major question concerning this study is the explanation for
the discrepancy between the high sensitivities (?90%) for the
A/B EIA reported recently (11, 20) and the comparatively
modest sensitivity found in this study (59.5%). It is possible
that there were systematic errors in the performance of this
assay during the study period. However, quality control failures
have been rare, and the lack of proficiency test failures since
the institution of this assay in my laboratory makes this type of
error less likely. Alternatively, there may have been a regional
shift in the antigenic nature of the toxin, resulting in the EIA
becoming less sensitive. I did notice that several of the GDH
assay- and CTN assay-positive but A/B EIA-negative speci-
mens were CTN positive only after 48 h of incubation, sug-
gesting the presence of low levels of toxin and perhaps explain-
ing the inability of the EIA to detect the toxin. However, a
similar number of EIA-negative specimens were GDH and
CTN assay positive at 24 h, suggesting that a low toxin level was
not the entire explanation for the relative insensitivity of the
The data presented here and by others (2, 6, 9, 16, 18)
indicate that GDH detection is the most sensitive and conve-
nient method by which C. difficile can be detected in stools. The
assay also has a high negative predictive value. Because of this
test’s modest positive predictive value of approximately 50%, a
rapid, convenient confirmatory test for GDH-positive speci-
mens is needed before this test is likely to gain wide acceptance
in clinical laboratories. The data presented here and by others
(18) indicate that two solid-phase EIAs, the C. difficile Tox A/B
II EIA (TechLab) and the A/B EIA (Meridian Bioscience), as
well as a single-test lateral flow immunochromatographic test,
the LF-A/B assay (TechLab), lack sufficient sensitivity com-
pared with the results of the CTN assay to be used as confir-
matory tests. For laboratories wishing to implement screening
for C. difficile by the GDH assay as part of a testing algorithm,
for now it is necessary to use either the CTN assay or toxigenic
culture (5, 6, 14) as a confirmatory test for GDH assay-positive
I thank Melissa B. Miller for critical assessment of the manuscript
and Gloria Crawford for statistical assistance.
I thank TechLab for providing the kits used in this study.
P.G. has received speaking fees from Remel, Lenexa, KS.
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