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: SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile, now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade.Microbiology and molecular biology reviews: MMBR 12/2013; 77(4):567-81. · 12.59 Impact Factor
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ABSTRACT: In this study, the prevalence of C. difficile, from patients with gastrointestinal complaints and its association with other enteropathogen microbes were investigated. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. Enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile on the patient's stool samples were detected by a double sandwich enzyme-linked Immunosorbant assay technique using a commercial kit (Premier toxins A & B; Generic Assays, Inc., Germany). The microbial isolation and examination was done, according to the standard identification methods. Out of 356 individuals (57.6% male and 42.4% female) the results of C. difficile were positive for 19 patients (5.3%) and negative for 337 patients (94.6%) according to the results of C. difficile antigen kit. There was no association between the existence of C. difficile toxin and microbial population or antibiotic usage. This prevalence study clearly supports the hypothesis of a probable role of C.difficile in developing gastrointestinal complaints in patients with diarrhea. More studies are needed to evaluate the role of C. difficile in these diseases.Gastroenterology and hepatology from bed to bench. 01/2011; 4(4):210-3.
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ABSTRACT: The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed.Expert Review of Anticancer Therapy 01/2014; 12(1):131-50. · 3.22 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: firstname.lastname@example.org.
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.
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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.
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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.
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Suzuki, S.-M. Kim, Y. Chong, and E. B. Wasito. 1998. Identification of a
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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
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FIG. 1. PMC. Colonoscopy photograph demonstrating multiple yellowish
patches (“pseudomembranes”) and erythematous, friable mucosa.
VOL. 38, 2000 CASE REPORTS1697