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Journal of Clinical Microbiology, April 2000, p. 1696-1697, Vol. 38, No. 4
Department of Laboratory Medicine, University
of Washington, Seattle, Washington 98195
Received 29 November 1999/Returned for modification 22 January
2000/Accepted 25 January 2000
We report a case of severe pseudomembranous colitis due to a toxin
A A 60-year-old man was admitted to the hospital for evaluation of
crampy abdominal pain and severe diarrhea. The patient's underlying
medical conditions included chronic immunosuppression following liver
transplantation 5 years prior to admission, chronic hepatitis C
infection, end-stage renal disease requiring hemodialysis, hypothyroidism, and hypertension. His medications included
prednisone, tacrolimus, mycophenolate mofetil, levothyroxine,
isradipine, ranitidine, cisapride, metoprolol, and clonidine.
He was well until 3 weeks prior to admission, when sinusitis was
diagnosed, and he was treated with a 10-day course of oral trimethoprim-sulfamethoxazole. Several days after completion of trimethoprim-sulfamethoxazole therapy, the patient developed crampy abdominal pain accompanied by fever and 10 to 15 watery stools per day
without blood or mucus. The patient was admitted to the hospital for
evaluation. On physical examination, he was febrile (101.5°F) and
appeared ill. Moderate right-lower abdominal tenderness was noted.
Relevant laboratory studies included a leukocyte count of 31,000 cells/µl (with toxic granulation), and a stool gram stain showed many
leukocytes and fecal flora. An abdominal computed tomography scan
showed right colon thickening but no abscess. Stool specimens for
enteric pathogens and Clostridium difficile toxin were
obtained, and empiric therapy with 500 mg of metronidazole orally every
6 h and 250 mg of levofloxacin intravenously every 24 h was
begun. A colonoscopy to evaluate for cytomegalovirus or other
opportunistic causes of colitis in this immunocompromised patient was
also done, and it showed numerous whitish plaques (Fig.
1) and friable erythematous mucosa
consistent with pseudomembranous colitis. All subsequent stool cultures
for enteric pathogens (including Salmonella spp.,
Shigella spp., Yersinia spp.,
Campylobacter spp., Vibrio spp.,
Escherichia coli O157:H7, Aeromonas spp., and
Plesiomonas spp.) remained negative. Likewise, three
separate stool ovum and parasite exams were negative. Colon biopsies
showed no histopathologic evidence of viral inclusions, and viral
cultures remained negative. Although the stool toxin A enzyme
immunoassay (EIA) (TechLab Tox A Test; TechLab, Blacksburg, Va.) was
negative, C. difficile toxin was detected in stool by
cytotoxicity assay (confirmed by neutralization with polyclonal
antibody against C. difficile toxin) and by culture. On the
basis of his severe clinical symptoms, detection of C. difficile toxin from stool, endoscopic findings of
pseudomembranous colitis (PMC), and negative studies for other
pathogens, a definitive diagnosis of C. difficile PMC was
made, and therapy was switched from oral metronidazole to 125 mg of
vancomycin orally every 6 h. The patient responded to therapy,
with a decrease in leukocytosis and resolution of his abdominal pain
and diarrhea and was discharged on the 4th hospital day in good
condition. He has had no evidence of recurrence after 5 months of
follow-up.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Pseudomembranous Colitis Caused by a Toxin
A
B+ Strain of Clostridium
difficile
ABSTRACT
Top
Abstract
Case Report
References
B+ strain of Clostridium
difficile in an immunosuppressed patient and discuss the
implications for diagnostic testing in suspected C. difficile-associated diarrhea.
CASE REPORT
Top
Abstract
Case Report
References
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FIG. 1.
PMC. Colonoscopy photograph demonstrating multiple
yellowish patches ("pseudomembranes") and erythematous, friable
mucosa.
C. difficile was cultured from stool under anaerobic conditions in brain heart infusion broth (BHI) supplemented with cefoxitin (30 mg/liter) and incubated at 37°C for 3 to 5 days. Colonial morphology on blood agar was characterized by nonhemolytic, grayish-translucent colonies. The isolate was identified as C. difficile based on biochemical reactions according to the An-Ident system (bioMérieux Vitek, Inc., Hazelwood, Mo.).
Filtrates from BHI dialysis flasks were prepared and analyzed by enzyme-linked immunosorbent assay (ELISA) and tissue culture as previously described (5). The filtrates were negative by the TechLab Tox A test. In the Tox A/B test, the filtrate was strongly positive (A450 >3.00), with an ELISA titer of 103. The filtrate was positive in the tissue culture assay and had a cytotoxic titer of 104. The cytotoxic activity was completely neutralized by polyclonal C. difficile antitoxin.
PCR using primers flanking the repeating units of the toxin A gene was
done according to an in-house procedure developed at TechLab, Inc. The
results indicated that the C. difficile strain from our
patient contained a deletion of approximately 1.7 kb in the toxin A
gene. This is the portion of the gene that encodes the epitope that
reacts with the monoclonal antibody used in the diagnostic EIA kits for
detection of toxin A (PCG-4 epitope). This most likely explains the
toxin A B+ nature of our patient's C. difficile isolate.
C. difficile is an important cause of antibiotic-associated
diarrhea and is the causative agent of PMC (2, 6, 10). It
was previously thought that toxigenic strains of C. difficile always produced both toxin A and toxin B, but recent
studies have documented the presence of toxin A
B+ strains among clinical isolates (3, 4, 5, 7).
However, the clinical significance and pathogenicity of toxin
A B+ strains of C. difficile are
incompletely understood (5). In a recent report from Canada,
al-Barrak and colleagues documented an outbreak of diarrhea associated
with toxin A B+ C. difficile at a
tertiary care hospital (1). However, the molecular
characterization of the C. difficile strains from these patients was not reported. In the present report, we have reported the
detailed clinical course of an immunocompromised patient with severe
PMC due to a toxin A B+ strain of C. difficile and have described the molecular characterization of the strain.
The patient described herein had severe PMC due to a toxin
A B+ strain of C. difficile, with
other potential causes excluded. It is possible that the patient's
underlying immunosuppression allowed for severe disease to occur even
with a toxin A strain of C. difficile.
Although the definitive roles of toxins A and B in C. difficile diarrhea are unknown, it is generally believed that
toxin A plays the major role in C. difficile diarrhea (8). Consistent with this hypothesis, in a rabbit ileal loop assay, clinical strains of toxin A B+
C. difficile do not demonstrate enteropathogenicity
(5). Since the C. difficile strain from our
patient was not tested in such an assay, it is possible that either
toxin B alone was capable of causing diarrhea or the strain contained
other factors capable of causing diarrhea. Furthermore, it is unknown
whether a "mutant" toxin A is produced by toxin A
B+ strains and, if it is, whether such a toxin is
enteropathogenic. Since previous reports have also described toxin
A B+ strains of C. difficile from
symptomatic patients, it would be of interest to specifically determine
whether the natures of the toxin A deletion are similar among such
strains. Further analysis of such phenotypically similar strains by
either additional sequencing or other methods for determining
relatedness may help to address the possibility that such isolates
represent a particular pathogenic clone.
Current diagnostic methods for C. difficile include culture,
detection of organism-specific glutamate dehydrogenase, detection of
toxin B by cell culture or cytotoxicity assay, and detection of toxin A
and/or B from stool by immunoassay. Several of the widely used
diagnostic tests for C. difficile rely on the detection of
toxin A. The monoclonal antibody used in these assays (PCG-4) reacts
with an epitope encoded by a portion of the toxin A gene that is
deleted in toxin A B+ strains (9).
PCR with primers specific for the repeating subunits of toxin A
demonstrated a 1.7-kb deletion in the portion of the toxin A gene that
encodes the PCG-4-reacting epitope in our patient's C. difficile isolate, and this most likely explains the toxin A B+ nature of our isolate.
Although the clinical significance of toxin A
B+ strains of C. difficile has not been well
defined, an outbreak of toxin A B+ C. difficile-associated diarrhea at a Canadian tertiary care hospital
has recently been reported (1). In this report, a presumptive case was defined when stool studies were negative for toxin
A by a toxin A EIA (Prima System EIA; Bartels, Inc.), but positive by
either cytotoxicity assay or a combination toxin A-toxin B EIA (Tox A/B
test; TechLab). If additional characterization of C. difficile strains from this outbreak confirms their toxin A B+ nature, this report will add strong
support to the hypothesis that such strains are fully pathogenic in humans.
Given our findings that a toxin A B+ C. difficile strain is capable of causing PMC, clinical laboratories
that use diagnostic methods that rely solely on detection of toxin A
need to be aware that results may be falsely negative. If C. difficile-associated diarrhea is clinically suspected and toxin A
is not detected, then the possibility of a toxin A
B+ strain should be considered, and further diagnostic
testing should be performed. The relative frequency of toxin
A B+ clinical strains and their relative
pathogenicity compared to that of toxin A+ B+
strains warrant further study.
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ACKNOWLEDGMENTS |
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We thank Limin Zheng, Laurie Neville, and David Lyerly of TechLab, Inc., Blacksburg, Va., for EIA testing, cytotoxicity testing, and PCR analysis.
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FOOTNOTES |
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* Corresponding author. Mailing address: University of Washington Medical Center, Department of Laboratory Medicine, Box 357110, 1959 N.E. Pacific St., Seattle, WA 98195-7110. Phone: (206) 598-6131. Fax: (206) 598-6189. E-mail: limaye{at}u.washington.edu.
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REFERENCES |
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Abstract
Case Report
References
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Journal of Clinical Microbiology, April 2000, p. 1696-1697, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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