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Journal of Clinical Microbiology, January 1998, p. 184-190, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multicenter Evaluation of the Clostridium difficile
TOX A/B TEST
D. M.
Lyerly,1,*
L. M.
Neville,1
D. T.
Evans,1
J.
Fill,2
S.
Allen,2
W.
Greene,3
R.
Sautter,4
P.
Hnatuck,4
D. J.
Torpey,5 and
R.
Schwalbe5
TechLab, Inc., Corporate Research Center,
Blacksburg, Virginia 240601;
Anaerobe
Laboratory, Indiana University Hospital, Indianapolis, Indiana
462022;
Clinical Pathology, Hershey
Medical Center, Hershey, Pennsylvania
170333;
Department of Microbiology,
PinnacleHealth System, Harrisburg, Pennsylvania
17105-87004; and
Department of Medical
and Research Technology, School of Medicine, University of
Maryland, Baltimore, Maryland 21201-10825
Received 3 July 1997/Returned for modification 8 September
1997/Accepted 8 October 1997
 |
ABSTRACT |
Clostridium difficile, the primary cause of nosocomial
diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause
severe intestinal disease leading to complications such as relapses and
infections due to vancomycin-resistant enterococci. The disease results
from two toxins, toxins A and B, produced by this pathogen. In this
study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay
(EIA) that detects toxins A and B. We compared the test with the tissue
culture assay, which is recognized as the "gold standard" for
C. difficile testing. Evaluations were performed
in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four
clinical laboratories. Of 1,152 specimens tested, 165 were positive by
the TOX A/B TEST and tissue culture and 973 were negative by both
tests. The sensitivity and specificity were 92.2 and 100%,
respectively. The positive and negative predictive values were 100 and
98.6%, respectively, and the correlation of the TOX A/B TEST with
tissue culture was 98.8%. When discrepant samples were resolved by
culture, the sensitivity and specificity were 93.2 and 98.9%,
respectively. The positive and negative predictive values were 100 and
98.8%, respectively, with a correlation of 99.0%. There were no
specimens that were positive by the TOX A/B TEST and negative by tissue
culture. Fourteen specimens were negative by the TOX A/B TEST but
positive by tissue culture. Of these, two were negative by toxigenic
culture, five were positive by toxigenic culture, and seven were not
available for further testing. There were no indeterminate results,
since the test does not have an indeterminant zone. In a separate
study, 102 specimens that were positive by tissue culture and the TOX
A/B TEST were examined in toxin A-specific EIAs. Two specimens that
presumptively contained toxin A-negative, toxin B-positive
(toxA
/toxB+) isolates were identified. One specimen was from a
patient with a clinical history consistent with C. difficile infection. Isolates obtained from these specimens by
selective culture on solid media and in broth tested
toxA/toxB+ when grown in brain heart infusion dialysis flasks,
which stimulate in vitro production of both toxins. Our findings show
that the TOX A/B TEST is suitable as a diagnostic aid for C. difficile disease because it correlates well with tissue culture
and detects isolates that may be missed with toxin A-specific EIAs.
| |
INTRODUCTION |
Clostridium difficile,
which causes virtually all cases of pseudomembranous colitis,
is now recognized as the primary cause of nosocomial diarrhea in the
United States and many other industrialized countries (3,
21). Health care costs for this disease, which run into the
hundreds of millions of dollars each year in the United States, are
continuing to rise. Relapses, which can be extremely difficult to
treat, occur in 10 to 20% of patients with C. difficile
disease. Fortunately, most relapse patients can be effectively treated
with metronidazole and vancomycin, but these treatments have triggered
new problems, such as the emergence of vancomycin-resistant
enterococci. New therapeutic approaches are being investigated for
C. difficile disease, but these therapies are still
under development. The strategy used by the health care profession in
the management of C. difficile disease is not limited simply
to the treatment of hospitalized patients with diarrhea. Many
hospitalized patients acquire C. difficile asymptomatically as carriers, raising the question of whether these persons should be
treated to minimize their chances of disease and spread of the organism
(6, 24). In some instances, asymptomatic elderly persons who
are positive for C. difficile are denied entrance into
facilities such as nursing homes because of the potential risk of
outbreaks (4).
Although C. difficile disease continues to be a major
challenge for the health care profession, our basic understanding of the toxins of this organism and significant advances in new and improved diagnostic testing are leading to better diagnosis of and
therapy against this opportunistic pathogen. The two toxins that it
produces, toxins A and B, have been well characterized (9, 20, 21,
28-30). Toxins A and B are the largest bacterial toxins known,
with molecular weights of 308,000 and 279,000, respectively. Both
toxins have contiguous repeating units at the COOH terminus. The
repeating units on toxin A represent the portion that binds to
galactose receptors (15, 18). These repeating units also represent the portion of the toxin recognized by the monoclonal antibody used in most of the diagnostic enzyme immunoassays (EIAs) on
the market (10). Overall, the two toxins exhibit 45%
homology at the amino acid level, strongly suggesting that the
toxA and toxB genes resulted from gene
duplication (2, 9, 29, 30). This possibility is
substantiated further by the recent finding that both toxins are
glucosyltransferases which glucosylate factor rho, a small
GTP-binding protein involved in the regulation of the cytoskeletal
system in mammalian cells (1, 13). Toxin A is a potent
enterotoxin that is lethal and cytotoxic, and it probably causes most
of the clinical symptoms. Toxin B also is lethal and is much more
cytotoxic than toxin A. In the present study, we developed a new
EIA, the TOX A/B TEST, designed to detect both toxin A and toxin B and
examined its potential use as an in vitro diagnostic aid in C. difficile disease. The test incorporates highly specific
affinity-purified polyclonal and monoclonal antibodies against toxin A
and highly specific polyclonal antibodies against toxin B. The test was
evaluated through a multicenter study comparing its performance to the
tissue culture test, considered the "gold standard" in C. difficile testing. During this study, we identified two isolates
that tested negative for toxin A and positive for toxin B
(toxA/toxB+) and showed that these isolates are not detected by toxin
A-specific EIAs.
| |
MATERIALS AND METHODS |
Study sites and stool specimens.
Six separate studies were
performed in the TOX A/B TEST evaluation: (i) study 1, performed
in-house at TechLab with stool specimens from the in-house fecal log;
(ii) study 2, performed in-house at TechLab with specimens supplied by
an outside clinical laboratory; (iii) study 3, performed at Indiana
University Hospital, Indianapolis; (iv) study 4, performed at the
Hershey Medical Center, Hershey, Pa.; (v) study 5, performed at
PinnacleHealth, Harrisburg, Pa.; and (vi) study 6, performed at the
School of Medicine at the University of Maryland, Baltimore. All
specimens used in the study were stored either at 2°C to 8°C for
72 hours before assay or frozen at 20°C or 70°C. Specimens
included in the study were submitted to the laboratory for routine
testing and included specimens from patients who had recently received
antibiotics and from patients not on antibiotics.
Bacterial strains and toxin reagents.
C. difficile and
Clostridium sordellii VPI strains were obtained from the
anaerobe collection at Virginia Polytechnic Institute and State
University (Blacksburg, Va.). Strain CCUG 8864 was kindly supplied by
Peter Borriello (PHLS Central Public Health Laboratory, London,
England). Serogroup type F strains IS37 and IS73 were kindly provided
by Jon Brazier (Public Health Laboratory Service, Health Park, Cardiff,
Wales). Normal intestinal bacteria and enteric pathogens were obtained
from the American Type Culture Collection (Rockville, Md.). Toxins A
and B were purified from C. difficile VPI 10463 as
previously described (19).
EIAs.
The TOX A/B TEST (TechLab, Inc.) is a microwell EIA
that utilizes affinity-purified mouse monoclonal antibody against toxin A and affinity-purified goat polyclonal antibody against toxins A and
B. Microwells provided with the test are coated with affinity-purified goat polyclonal antibodies against toxins A and B. The detecting antibody consists of monoclonal antibody against toxin A and
affinity-purified goat polyclonal antibody against toxin B, each
conjugated to horseradish peroxidase. The test was performed according
to the manufacturer's instructions. Briefly, stool specimens were
diluted 1:5 in kit diluent. One drop of conjugate per microwell was
added, followed by the addition of 2 drops of diluted stool. Microwells
were incubated at 37°C for 50 min and washed five times with 1× wash
solution. One drop of substrate A was added to each well followed by 1 drop of substrate B. After 10 min at room temperature, reactions were stopped by the addition of 1 drop of stop solution. The positive control consisted of the addition of positive control reagent to a well
containing conjugate. The negative control consisted of the addition of
diluent to a well also containing conjugate. Test results were
interpreted as follows: (i) for visual readings, a colorless result was
considered negative and any yellow color was considered positive; (ii)
for a spectrophotometric single wavelength at 450 nm, negative was
<0.120 and positive was 
0.120; and (iii) for a spectrophotometric
dual wavelength at 450/620 nm or 450/550 nm, negative was <0.080 and
positive was 0.080. EIA titers were determined for C. difficile strains for a more accurate assessment of the
sensitivity of the TOX A/B TEST compared with tissue culture results.
For determination of EIA titers, serial 10-fold dilutions of test
samples were prepared and assayed as described in the package insert.
EIA titers were expressed as the reciprocal of the highest dilution
giving a positive reaction.
The Tox-A Test (TechLab, Inc.) and the Premier C. difficile
Toxin A (Meridian Diagnostics, Inc., Cincinnati, Ohio), and Cytoclone A+B (Cambridge Biotech Corp., Worcester, Mass.) tests were used according to each manufacturer's instructions. The Tox-A Test and the
Premier test detect toxin A; the Cytoclone A+B test detects both toxins
A and B. Specimens were prepared as directed by each manufacturer, and
positive and negative results were determined as specified by each
manufacturer.
Tissue culture assay.
Tissue culture assays were performed
according to individual in-house testing procedures: (i) Indiana
University Hospital used the C. difficile Tox-B Test
(TechLab, Inc.) with human foreskin monolayers, (ii) Hershey Medical
Center used the C. difficile Toxin/Antitoxin Kit (TechLab,
Inc.) with MRC-5 cells, (iii) PinnacleHealth used the Toxi-Titer
(Bartels Immunodiagnostics) test with human foreskin monolayers, (iv)
the University of Maryland School of Medicine used the C. difficile Tox-B Test with human foreskin monolayers, and (v)
TechLab used the C. difficile Tox-B Test with Chinese
hamster ovary K-1 cells. At each site, a standardized dilution of toxin
B was used as the positive control as recommended with each kit.
Specimens were diluted and prepared according to each manufacturer's
instructions, and results were determined at 24 and 48 h. For the
Tox-B Test, a 1/50 dilution (final concentration in the tissue culture
well) of stool specimen was tested, and for the Toxi-Titer a 1/40
dilution was tested. With the C. difficile Toxin/Antitoxin
Kit, the final dilution was 1/100. Cytotoxicity titers were determined
for individual C. difficile strains for a more accurate
assessment of the sensitivity of the TOX A/B TEST compared to tissue
culture. For determination of cytotoxicity titers, serial 10-fold
dilutions of test samples were prepared and assayed as previously
described (19). Cytotoxic titers were expressed as the
reciprocal of the highest dilution giving a positive reaction.
Culture.
Prereduced anaerobically sterilized brain heart
infusion broth (Carr-Scarborough, Stone Mountain, Ga.) was supplemented
with cycloserine and cefoxitin (CC-BHI) (Oxoid, Ogdensburg, N.Y.). The
final concentrations of cycloserine and cefoxitin were 500 µg/ml and
16 µg/ml, respectively. For inoculation of broth media, 1 drop of
stool specimen was added to a tube containing 5.0 ml of supplemented
broth. Inoculated media were incubated at 37°C for 3 to 4 days.
Selective cycloserine-cefoxitin-fructose agar (11) (CCFA)
(Oxoid) was prepared as recommended by the manufacturer. Presumptive
colonies were characterized by a yellowish color, flat morphology,
yellow-green fluorescence, and a horsey smell. CCFA plates were
incubated anaerobically at 37°C for a minimum of 3 days. Selected
strains were grown in brain heart infusion dialysis flasks as
previously described (19).
PAGE and crossed immunoelectrophoresis.
Isolates were grown
at 37°C for 96 h in brain heart infusion dialysis flasks as
previously described (19). After incubation, cultures were
collected and centrifuged to remove cells and debris, and the
supernatant fluids were passed through 0.2-µm-pore-size membranes.
Filtrates were stored at 2°C to 8°C. Polyacrylamide gel
electrophoresis (PAGE) was performed in nondenaturing 3 to 27%
polyacrylamide gradient gels (Jule Biotechnologies, Inc., New Haven,
Conn.). Samples (20 µl) of filtrate were mixed with bromphenol
blue-glycerol (5 µl) and loaded into sample wells. Electrophoresis
was performed in Tris-borate-EDTA buffer, pH 8.3, at a constant voltage
and 40 mA. After electrophoresis, gels were stained with Coomassie blue
R-250 and destained. Crossed immunoelectrophoresis was performed in
1.2% agarose gels as previously described (19).
Gas chromatography.
Isolates were characterized according to
their cellular fatty acid profiles with the Microbial Identification
System (Microbial ID, Inc.), as previously described (25).
| |
RESULTS |
Reactions of toxins A and B and C. difficile strains in
the TOX A/B TEST.
The reactions of highly purified toxins A and B
in the TOX A/B TEST were compared with those in the Cytoclone A+B test,
the Tox-A Test, and the Premier C. difficile Toxin A
test. All four tests detected toxin A at approximately 1 ng/ml. The
minimum level of toxin B detected by the TOX A/B TEST was 2 to 5 ng/ml,
whereas the minimum level detected by Cytoclone A+B was 5 to 10 ng/ml. Toxin B did not react in either the Tox-A Test or the Premier C. difficile Toxin A test.
The reactivity of eight
C. difficile strains which varied in
their in vitro production of toxins A and B was examined. Included
were
seven toxigenic strains ranging from 10
1 to 10
6
in cytotoxic titer when grown anaerobically in prereduced brain
heart
infusion broth. Six of the toxigenic strains were toxA+/toxB+.
The
other toxigenic strain, CCUG 8864, was toxA
/toxB+ and carried
a
truncated
toxA gene that lacked the repeating units (
5,
17,
27). All seven toxigenic strains, including CCUG 8864, reacted
strongly (
A450 > 2.500) in the TOX A/B
TEST and the Cytoclone
A+B test. The six toxA+/toxB+ strains reacted in
the Tox-A Test
and the Premier test, whereas strain CCUG 8864 was
negative in
these tests. Also included in the analysis was VPI 11186, a
nontoxigenic
strain that lacks the
toxA and
toxB
genes but which cross-reacts
extensively with nontoxic antigens from
VPI 10463. VPI 11186 was
negative in all of the EIAs and noncytotoxic
in the tissue culture
test.
Further analyses were done to examine the reactions of VPI 10463 and
VPI 11186 in stool specimens of different consistencies.
For the
analyses, cultures of each strain were prepared and centrifuged
and the
supernatant fluids were used to spike negative stool specimens
of
differing consistencies. Filtrates from VPI 10463 (toxA+/toxB+)
were
positive at dilutions of 5 × 10
3 or lower in liquid,
semisolid, and solid stool specimens. VPI
11186 filtrates
(toxA
/toxB
) were consistently negative when
tested in specimens of
different consistencies.
Forty species of organisms representing members of the normal
intestinal flora and various enteric pathogens were tested in
the TOX
A/B TEST. The only organisms that reacted in the test
were toxigenic
C. difficile and
C. sordellii. The
C. sordellii strain that was positive, VPI 9048, produces toxin HT (hemorrhagic
toxin) and toxin LT (lethal toxin),
which have extensive identity
with toxin A and toxin B, respectively,
at the gene and protein
levels. Toxins HT and LT have been shown
previously to cross-react
extensively with antibodies against toxins A
and B (
22,
23,
26). A nontoxigenic strain of
C. sordellii did not react in
the TOX A/B TEST.
Performance characteristics.
A total of 1,152 stool specimens
submitted to the clinical laboratory were assayed in the TOX A/B TEST
and tissue culture assay. Included were 177 specimens positive by
tissue culture. Of these, 165 were positive in the TOX A/B TEST. A
total of 973 specimens were negative in both tests. Included in the
analysis were 10 specimens that were initially identified as tissue
culture positive but were subsequently confirmed as tissue culture
negative and ruled false positive. Three of these were neutralized by
neutral goat serum, and seven caused atypical stretching of the cells uncharacteristic of C. difficile toxin. The seven
specimens that caused atypical reactions were all from one test
location, and the reactions were observed with human foreskin cells.
For the purposes of this study, these samples were considered negative. When compared to tissue culture, the TOX A/B TEST exhibited sensitivity and specificity of 92.2 and 100%, respectively, with positive and
negative predictive values of 100 and 98.6%, respectively. The
correlation between the TOX A/B TEST and tissue culture was 98.6%. Of
the 14 specimens that were tissue culture positive but negative in the
TOX A/B TEST, 2 were negative by toxigenic culture, 5 were positive by
toxigenic culture, and 7 were not available for further testing. The 2 specimens that were negative by toxigenic culture were ruled false
positive. No specimens tested positive in the TOX A/B TEST but negative
by tissue culture. When discrepancies were resolved by toxigenic
culture, the TOX A/B TEST exhibited sensitivity and specificity of
93.2% and 100%, respectively. The positive and negative predictive
values were 100 and 98.8%, respectively, with a correlation of 99.0%.
Table 1 shows the results from each individual study.
Comparative studies of centrifuged versus noncentrifuged specimens and
visual versus spectrophotometric readings (
A450
and
A450/620) were done. For the analysis, 337 specimens that included
30 TOX A/B TEST-positive specimens and 307 TOX
A/B TEST-negative
specimens were tested. Initially, specimens were
prepared and
a portion of each diluted specimen was tested according to
the
directions in the package insert. The remaining portion of each
diluted specimen was centrifuged (5,000 ×
g) for 5 min, and the
supernatant fluid was assayed in the TOX A/B TEST
according to
the directions. The results showed a 100% correlation
between
centrifuged and noncentrifuged specimens and between visual and
spectrophotometric readings.
Identification of toxA/toxB+ isolates.
A total of 102 specimens that were positive by tissue culture and TOX A/B TEST were
examined further in the Tox-A Test for toxA/toxB+ isolates. Included
in the evaluation were specimens from each of the studies listed in
Table 1. Ten of the 102 specimens tested negative in the Tox-A Test.
Eight of these gave low readings (A450 values in
the range of 0.120 to 0.260 in the TOX A/B TEST), were only weakly
cytotoxic, and tested negative in both the Premier and Cytoclone A+B
tests, suggesting to us that these specimens contained only trace
levels of toxins A and B. These specimens were subcultured into
selective CC-BHI broth and incubated at 37°C for 96 h, and the
cultures were tested. Six of the eight cultures reacted positively in
both the Tox-A Test and the TOX A/B TEST and were cytotoxic,
demonstrating that they were true positives for toxigenic C. difficile. The other two cultures were negative.
The remaining two stool specimens that were positive in the TOX A/B
TEST and negative in the Tox-A Test gave
A450
values of
>0.500. One of the specimens, no. 23454, from Indiana
University
Hospital, was from a patient on antibiotic therapy. The
other
specimen, no. 457, was from our in-house fecal log and was
originally
obtained from the Hershey Medical Center. When tested in the
Cytoclone
A+B test, both specimens were positive. In the Premier
C. difficile Toxin A test, both specimens were
negative. These results suggested
to us the possibility that the
specimens contained toxA
/toxB+
isolates. Isolates from each specimen
were obtained by selective
culture in CC-BHI broth followed by further
selection on CCFA.
For CCFA selection, approximately 5 to 10 colonies
exhibiting
typical
C. difficile morphology were picked
for analysis and identification
by gas chromatography. Isolates were
identified by their cellular
fatty acid profiles. Each was subsequently
grown in brain heart
infusion dialysis flasks, which enhances toxin
production approximately
10- to 100-fold compared to toxin levels in
tube broth cultures.
An analysis of the dialysis sac filtrates from
each isolate in
each of the EIAs is shown in Fig.
1. Filtrates from the two isolates
were
strongly positive in the TOX A/B TEST and the Cytoclone A+B
test
(
A450 > 0.500) and negative in the Tox-A Test
and the Premier
C. difficile Toxin A test. Further
quantitative analysis of the
toxin A and toxin B titers was performed
with the Tox-A Test and
the TOX A/B TEST and by tissue culture assay
(Table
2). Again,
the results showed the
absence of toxin A.
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FIG. 1.
Reactions of C. difficile strains in the
TechLab TOX A/B TEST, TechLab Tox-A Test, Meridian Premier
C. difficile Toxin A test, and Cambridge Cytoclone A+B
test. For the analysis, strains were grown at 37°C for 72 h in
brain heart infusion dialysis flasks and culture filtrates were assayed
in each EIA. Strains VPI 10463, VPI 11186, and CCUG 8864 have been
described previously (19, 20). Isolates 23454 and 457 were
obtained in this study.
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|
Further analyses were done by crossed immunoelectrophoresis to examine
the antigenic profiles of these strains (Fig.
2). Isolates
23454 and 457 cross-reacted
extensively with antiserum against
VPI 10463. Filtrates from both
isolates tested weakly positive
for toxin B, as shown by the presence
of a small precipitin arc
in the toxin B location. No precipitin arcs
were detected for
either isolate with affinity-purified toxin A
antibody.
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FIG. 2.
Antigenic profiles of C. difficile
isolates. Filtrates were subjected to crossed immunoelectrophoresis
with antiserum against VPI 10463 or affinity-purified antibody against
toxin A or toxin B. Strains VPI 10463, VPI 11186, and CCUG 8864 have
been described previously (19, 20). Isolates 23454 and 457 were obtained in this study. Toxin A and toxin B arcs were observed
with VPI 10463. Neither toxin was detected with VPI 11186, which does
not carry either the toxA or the toxB gene. This
strain produces an antigen that migrates similarly to toxin A, but the
antigen is distinct from toxin A. Toxin B arcs were observed with CCUG
8864 and isolates 457 and 23454. The 457 and 23454 toxin B arcs
(designated by arrows) were considerably smaller than the toxin B arcs
from VPI 10463 and CCUG 8864. The double precipitin arc observed with
toxin B from CCUG 8864 and isolate 23454 are occasionally observed with
toxin B from VPI 10463. Toxin A arcs were not detected with CCUG 8864, isolate 457, or isolate 23454.
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|
Filtrates from isolates 23454 and 457 were prepared from brain heart
infusion dialysis flask cultures and analyzed by gradient
PAGE to
determine their protein profiles (Fig.
3). We have used
this method in
other studies to type
C. difficile isolates and
examine
their prevalence in outbreaks of antibiotic-associated
diarrhea
(
21a). The two isolates gave protein profiles distinct
from
each other and from CCUG 8864, VPI 10463, and VPI 11186 (Fig.
3).
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FIG. 3.
Protein profiles of C. difficile
isolates. Filtrates were analyzed by PAGE in a 3 to 27% gradient gel
under nondenaturing conditions. Strains VPI 10463, VPI 11186, and CCUG
8864 have been described previously (19, 20). Isolates 457 and 23454 were identified in this study.
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|
Two
C. difficile strains from serogroup type F, strains
IS37 and IS73, were grown in brain heart infusion dialysis flasks,
and
culture filtrates from each strain were analyzed in the TOX
A/B TEST,
the Tox-A Test, and the Tox-B Test. The filtrates from
these strains
were negative in the Tox-A Test. When tested in
the TOX A/B TEST, the
filtrates had EIA titers of 10
2 and undiluted test samples
from each strain gave
A450 values
of >2.0. In
the Tox-B Test, the cytotoxic titers of filtrates
from each strain were
consistently 10
4 or higher, and the cytotoxic activity was
neutralized by
C. difficile antitoxin.
| |
DISCUSSION |
Our study was undertaken to evaluate the performance of the TOX
A/B TEST, a new EIA that detects C. difficile toxins A
and B. Our goal in developing the TOX A/B TEST was to produce an EIA that closely matched the performance of the tissue culture test, which
represents the most sensitive test on the market. The test was designed
to be simple, by using the microwell EIA format of the Tox-A Test, and
rapid, by maintaining the 1-h turnaround time. The interpretation of
test results was simplified by removal of the indeterminant zone, which
is present in most of the C. difficile EIAs now on the
market. Our results show that the TOX A/B TEST detects toxin A at
levels similar to other EIAs on the market and toxin B at levels lower
than the Cytoclone A+B test. The TOX A/B TEST was highly specific for
toxigenic C. difficile strains and detected toxins A
and B produced in vitro in broth cultures by weakly cytotoxigenic
strains. The only non-C. difficile organism found to
cross-react was a toxigenic strain of C. sordellii.
This finding was not surprising, because certain strains of
C. sordellii produce toxins that are highly related
immunologically to toxins A and B. These C. sordellii
strains react positively in other C. difficile EIAs and
by tissue culture. In our clinical evaluations involving 1,152 stool
specimens in six different studies, the TOX A/B TEST exhibited a
correlation of 98.8% with tissue culture.
During our evaluation, we identified two specimens, no. 23454 and no.
457, that exhibited properties consistent with a toxA/toxB+ phenotype. These specimens gave positive reactions in the TOX A/B TEST,
the Cytoclone A+B test, and tissue culture but were negative in toxin
A-specific EIAs. At least one of the specimens was from a patient who
had a clinical history consistent with C. difficile
disease. We subsequently isolated toxigenic C. difficile from both specimens and showed that the isolates
continued to test toxA/toxB+ when grown under conditions optimal for
toxin production. The reaction of toxin B from these isolates with
polyclonal antibody against toxin B was detectable by crossed
immunoelectrophoresis, though it was weak. The weak reactions likely
resulted from lower levels of toxin, since the isolates have cytotoxic
titers 10- to 100-fold lower than that of VPI 10463. The negative
reaction of these isolates in the toxin A-specific EIAs did not appear to result from insufficient levels of toxin A, since other isolates with cytotoxic titers lower than either no. 457 or no. 23454 were highly reactive in toxin A-specific EIAs. These isolates appear to be
phenotypically distinct from each other and from VPI 10463, VPI 11186, and CCUG 8864, based on their different protein profiles.
The identification of these isolates presents a new challenge in the
diagnosis of C. difficile disease. The incidence of
toxA/toxB+ isolates in our study was low (0.2%), and most
C. difficile isolates from symptomatic patients are
toxA+/toxB+. However, the presence of a toxA/toxB+ isolate in a stool
specimen from a patient with a clinical history consistent with
C. difficile disease suggests to us the possibility
that toxA/toxB+ isolates may spread through wards and remain
undetected in hospitals that use toxin A-specific EIAs. The only
toxA/toxB+ isolate that has been carefully characterized at the
molecular level is CCUG 8864, which carries a truncated toxA
gene (5, 17, 27). Type F isolates from asymptomatic neonates
and infants also have been reported to be toxA/toxB+, but they have
not been as carefully studied (7, 8). Our results, which
showed two representative serogroup type F strains to be negative in
the Tox-A Test but positive in the TOX A/B TEST and the Tox-B Test,
support the possibility that serogroup type F strains are toxA/toxB+.
The toxigenic phenotype of these isolates is still unclear, however,
because Von Eichel-Streiber et al. showed that under certain
conditions, the serogroup type F reference strain (strain 1470)
produces both toxins in vitro (29). The ability of type F
strains to cause disease also remains unclear. Previous studies
(7, 8) described type F strains from asymptomatic infants,
but recent results by Kato et al. (14) showed that they are
present in C. difficile disease patients in Japan.
Therefore, additional studies are needed to further characterize these
isolates and their role in disease. Interestingly, the toxA/toxB+
phenotype also has been reported with toxigenic C. sordellii, which produces a toxin A-like enterotoxin (HT) and a
toxin B-like cytotoxin (LT) (12). Like CCUG 8864, this
isolate lacks the repeating units of the enterotoxin gene. Therefore,
there may be a specific genetic mechanism by which these strains arise.
How toxA/toxB+ isolates are involved in C. difficile
disease is not clear. Current data support the idea that toxin A causes most of the clinical symptoms associated with C. difficile disease. This is based on the potent enterotoxic
activity of toxin A, the lack of activity of toxin B in the intestine,
and the ability of toxin A antibodies to protect experimental animals
against C. difficile disease (3, 16, 18, 20,
28). However, toxA/toxB+ isolates may cause disease due to a
more active toxin B. This possibility is based on our findings that
toxin B from CCUG 8864 is 10-fold more cytotoxic and lethal than toxin
B from toxA+/toxB+ isolates (17). In addition, unlike toxin
B from toxA+/toxB+ isolates, CCUG 8864 toxin B is weakly enterotoxic. There also is the possible role of the truncated toxA gene
product. Additional studies are needed to characterize the toxins from these atypical isolates at the molecular level and to continue surveillance and identification of new C. difficile
isolates that produce altered toxins. It is important, however, that
these types of studies are carefully controlled. Improper assay
conditions, such as incomplete washing of EIA plates, may lead to
misidentification of strains as toxA/toxB+ or toxA+/toxB. DNA
probes or PCR primers used to characterize these isolates also must be
selected carefully. For example, DNA probes against the repeating units
of toxin A would indicate the absence of the toxA gene in CCUG 8864 when, in fact, this strain carries the upstream portion of the gene. In
addition, the interpretation of toxA/toxB+ results may be more
complicated than just determining the presence or absence of the
toxA gene, as suggested by results with type F strains that
appear to carry the toxA and toxB genes but for
which only toxin B is detectable in stool specimens. Concomitant
expression of toxins A and B, for example, may exhibit strain-to-strain
variation. Characterization of these isolates will be challenging, but
the results will lead to improved diagnostic testing for C. difficile disease and new information on how the toxins damage the
intestine.
In our clinical evaluations, we identified several specimens that gave
results in the tissue culture test not typical of the C. difficile toxins. Three specimens (0.2% of those tested) caused cell rounding that was not neutralized by C. difficile
antitoxin. Even at higher dilutions, the specimens continued to cause
cell rounding that was not neutralized with antitoxin. We were unable to isolate toxigenic C. difficile from these specimens,
indicating that these likely were not true C. difficile
positives. There also were seven specimens (0.6% of those tested) that
caused cell stretching, all of which were neutralized by C. difficile antitoxin (Fig. 4).
Although cell stretching is not typical of toxigenic C. difficile, neutralization by antitoxin resulted in confusion in
the interpretation of test results. Upon further examination we found
that the activity was neutralized by normal goat serum, and we were
unable to isolate toxigenic C. difficile from the specimens. The incidence of these unusual specimens was low and, for
the purposes of our study, these specimens were considered C. difficile-negative. These findings illustrate,
however, that caution should be taken to avoid "overinterpretation"
of test results with specimens that cause atypical stretching or cell rounding that is not neutralized by C. difficile
antitoxin.
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|
FIG. 4.
Cell-rounding reaction caused in Chinese hamster ovary
K-1 cells by C. difficile toxins A and B. (A)
Cell-rounding activity typical of both toxins. Toxin B masks the
activity of toxin A because of its high specific activity in the assay.
(B) Neutralization of cell-rounding activity with specific
C. difficile antitoxin. The activity is not neutralized
with nonimmune serum. (C) Cell-stretching that may be confused with the
cytotoxic activity of C. difficile toxins A and B. In
our studies, the cell-stretching activity was neutralized by specific
C. difficile antitoxin and by nonimmune serum.
|
|
In conclusion, the TOX A/B TEST is a new in vitro diagnostic test that
can be used as an aid in the diagnosis of C. difficile disease. It is simple and rapid and exhibits high correlation with the
tissue culture test. The TOX A/B TEST also detects C. difficile isolates that may be missed with toxin A-specific EIAs. Therefore, the test should be useful as a tool for identifying unusual
isolates of C. difficile.
| |
ACKNOWLEDGMENTS |
We thank Mary Alice Woodburn and Earl Petzold for assistance in
isolation and characterization of the isolates obtained in this study.
We thank Carlyn Bruce for help in maintaining monolayer cells for the
tissue culture test.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: TechLab, Inc.,
1861 Pratt Dr., Corporate Research Center, Blacksburg, VA 24060. Phone: (540) 231-3943. Fax: (540) 231-3942. E-mail:
techlab{at}bev.net.
| |
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Abstract
Introduction
