Pseudomembranous colitis caused by a toxin A(-) B(+) strain of Clostridium difficile.
ABSTRACT We report a case of severe pseudomembranous colitis due to a toxin A(-) B(+) strain of Clostridium difficile in an immunosuppressed patient and discuss the implications for diagnostic testing in suspected C. difficile-associated diarrhea.
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ABSTRACT: Clostridium difficile infection (CDI) is considered to be the main cause of bacterial infectious diarrhea in nosocomial settings. Since the beginning of the new century a continuous rise in the incidence of severe CDI has been observed worldwide. Even though some CDI cases are not associated with previous antibiotic exposure, this remains as the principal risk factor for the development of CDI. The rate of recurrences represents perhaps one the most challenging aspect on the management of CDI. There are several microbiological tests available, but glutamate dehydrogenase antigen test can be selected as the first screening step in a diagnostic algorithm, with positive samples then confirmed using a toxin(s) test, to distinguish toxinogenic from nontoxinogenic CDI. Although metronidazole and vancomycin are and have been the mainstay treatment options for CDI, there are some unmet medical and therapeutical needs. Usually oral metronidazole is recommended for initial treatment of nonsevere CDI and vancomycin for treatment of severe disease. Fidaxomicin may be considered in patients who cannot tolerate vancomycin, although more data are needed. For treatment of a nonsevere initial recurrence of CDI, oral metronidazole should be used, but for treatment of subsequent recurrences or more severe cases fidaxomicin may be helpful.Expert Review of Anticancer Therapy 12/2012; 10(12):1405-23. · 3.22 Impact Factor
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ABSTRACT: The alarming emergence of hypervirulent strains of Clostridium difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. The C. difficile toxins A and B are critical virulence factors, and strains can either be toxin-producing (virulent) or non-toxin-producing (nonvirulent). Strains that are isolated from human infections generally produce either toxin A or toxin B or both. The methods currently available for culturing C. difficile do not differentiate strains that produce active toxins from strains that do not produce toxins or produce inactive toxins. As a result, the identification and isolation of toxin-producing strains from stool is currently a two-step process. First, the stool is plated on a selective medium, and then suspected colonies are analyzed for toxin production or the presence of the toxin genes. We describe here a novel selective and differential culture method, the Cdifftox plate assay, which combines in a single step the specific isolation of C. difficile strains and the detection of active toxin. This assay was developed based on our recent finding that the A and B toxins of C. difficile cleave chromogenic substrates that have stereochemical characteristics similar to their natural substrate, UDP-glucose. The Cdifftox plate assay is shown here to be extremely accurate (99.8% effective) in detecting toxin-producing strains through the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. The Cdifftox plate assay advances and improves the culture approach such that only C. difficile strains will grow on this agar, and virulent strains producing active toxins can be differentiated from nonvirulent strains, which do not produce active toxins. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains.Journal of clinical microbiology 12/2011; 49(12):4219-24. · 4.16 Impact Factor
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ABSTRACT: Abstract Enterocyte turnover along with proper epithelial barrier function are crucial aspects of mucosal defense. Apoptosis is a highly regulated type of programmed cell death that allows for the homeostatic turnover of the epithelial layer. Recent studies have suggested that microbial modulation of enterocyte apoptosis can result in increased epithelial permeability, leading to gastrointestinal pathophysiology. In this review, we highlight key mechanisms and pathways via which various viral, bacterial and parasitic pathogens are able to modulate enterocyte apoptosis. We also discuss how these alterations to enterocyte apoptosis can result in the activation of chronic gastrointestinal disorders, such as allergies, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). The role of proteinase-activated receptors in the pathogenesis of modulated apoptosis-induced pathogenesis is also discussed. Newly discovered processes, through which host epithelial cells may have evolved, rescue mechanisms from microbe-induced apoptosis are discussed. Together, these mechanisms are key to our ever-increasing understanding of host-microbe interactions in the gut.Critical Reviews in Microbiology 01/2013; · 5.07 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Apr. 2000, p. 1696–1697Vol. 38, No. 4
Pseudomembranous Colitis Caused by a Toxin A?B?
Strain of Clostridium difficile
AJIT P. LIMAYE,* DAVID K. TURGEON, BRAD T. COOKSON, AND THOMAS R. FRITSCHE
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?B?strain of Clostridium difficile in
an immunosuppressed patient and discuss the implications for diagnostic testing in suspected C. difficile-
A 60-year-old man was admitted to the hospital for evalua-
tion of crampy abdominal pain and severe diarrhea. The pa-
tient’s underlying medical conditions included chronic immu-
nosuppression following liver transplantation 5 years prior to
admission, chronic hepatitis C infection, end-stage renal dis-
ease requiring hemodialysis, hypothyroidism, and hypertension.
His medications included prednisone, tacrolimus, mycopheno-
late 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 devel-
oped 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 exam-
ination, he was febrile (101.5°F) and appeared ill. Moderate
right-lower abdominal tenderness was noted. Relevant labora-
tory studies included a leukocyte count of 31,000 cells/?l (with
toxic granulation), and a stool gram stain showed many leuko-
cytes and fecal flora. An abdominal computed tomography
scan showed right colon thickening but no abscess. Stool spec-
imens 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 immunocompro-
mised 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 histopatho-
logic 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.
C. difficile was cultured from stool under anaerobic condi-
tions 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 non-
hemolytic, grayish-translucent colonies. The isolate was iden-
tified as C. difficile based on biochemical reactions according to
the An-Ident system (bioMe ´rieux Vitek, Inc., Hazelwood,
Filtrates from BHI dialysis flasks were prepared and ana-
lyzed 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 com-
pletely 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 devel-
oped 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 di-
arrhea 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 clin-
* 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: email@example.com.
ical isolates (3, 4, 5, 7). However, the clinical significance and
pathogenicity of toxin A?B?strains of C. difficile are incom-
pletely 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 hos-
pital (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 mo-
lecular 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 ex-
cluded. It is possible that the patient’s underlying immunosup-
pression 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. Further-
more, it is unknown whether a “mutant” toxin A is produced by
toxin A?B?strains and, if it is, whether such a toxin is enter-
opathogenic. 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 relat-
edness 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, de-
tection 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 sub-
units 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 pre-
sumptive 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 charac-
terization 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 diagnos-
tic methods that rely solely on detection of toxin A need to be
aware that results may be falsely negative. If C. difficile-asso-
ciated diarrhea is clinically suspected and toxin A is not de-
tected, then the possibility of a toxin A?B?strain should be
considered, and further diagnostic testing should be per-
formed. The relative frequency of toxin A?B?clinical strains
and their relative pathogenicity compared to that of toxin A?
B?strains warrant further study.
We thank Limin Zheng, Laurie Neville, and David Lyerly of
TechLab, Inc., Blacksburg, Va., for EIA testing, cytotoxicity testing,
and PCR analysis.
1. al-Barrak, A., J. Embil, B. Dyck, K. Olekson, D. Nicoll, M. Alfa, and A.
Kabani. 1999. An outbreak of toxin A negative, toxin B positive Clostridium
difficile-associated diarrhea in a Canadian tertiary-care hospital. Can. Com-
mun. Dis. Rep. 25:65–69.
2. Bongaerts, G. P. A., and D. M. Lyerly. 1997. Role of bacterial metabolism
and physiology in the pathogenesis of Clostridium difficile disease. Microb.
3. Brazier, J. S. 1998. The diagnosis of Clostridium difficile-associated disease.
J. Antimicrob. Ther. 41(Suppl. C):29–40.
4. Kato, H., N. Kato, S. Katow, T. Maegawa, S. Nakamura, and D. M. Lyerly.
1999. Deletions in the repeating sequences of the toxin A gene of toxin
A-negative, toxin B-positive Clostridium difficile strains. FEMS Microbiol.
5. Kato, H., N. Kato, K. Watanabe, N. Iwai, H. Nakamura, T. Yamamoto, K.
Suzuki, S.-M. Kim, Y. Chong, and E. B. Wasito. 1998. Identification of a
toxin A-negative, toxin B-positive Clostridium difficile by PCR. J. Clin. Mi-
6. Knoop, F. C., M. Owens, and I. C. Crocker. 1993. Clostridium difficile: clinical
disease and diagnosis. Clin. Microbiol. Rev. 6:251–265.
7. Lyerly, D. M., L. A. Barroso, T. D. Wilkins, C. Depitre, and G. Corthier.
1992. Characterization of a toxin A-negative, toxin B-positive strain of Clos-
tridium difficile. Infect. Immun. 60:4633–4639.
8. Lyerly, D. M., H. C. Krivan, and T. D. Wilkins. 1988. Clostridium difficile: its
disease and toxins. Clin. Microbiol. Rev. 1:1–18.
9. Lyerly, D. M., L. M. Neville, D. T. Evans, J. Fill, S. Allen, W. Greene, R.
Sautter, P. Hnatuck, D. J. Torpey, and R. Schwalbe. 1998. Multicenter
evaluation of the Clostridium difficile TOX A/B Test. J. Clin. Microbiol.
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causes and cures. Digestion 60:91–100.
FIG. 1. PMC. Colonoscopy photograph demonstrating multiple yellowish
patches (“pseudomembranes”) and erythematous, friable mucosa.
VOL. 38, 2000 CASE REPORTS1697