Download The Impact Of Clostridium difficile infection: a review of the literature

The Impact Of
Clostridium difficile infection: a review of the literature
Elham .A. Draa*, Ali. S. Arebi*, Safwan .Alarabi*.
* Department of Medicine, Zawia Teaching Hospital, faculty of medicine
University of Zawia, Zawia, Libya.
Abstract
Clostridium difficile is anaerobic Gram-positive, spore-forming bacterium that now
recognized as the major cause of Antibiotic-associated diarrhea (AAD) worldwide.
C. difficile is being increasingly seen in human’s infections, particularly under conditions
of perturbed microbiota such as antibiotic treatment. The spectrum of gastrointestinal
infections caused by C.difficile is a broad ranging from asymptomatic colonization to severe
pseudomembranous colitis (PMC), resulting in a relatively high morbidity and mortality rate,
and raised health care costs .We reviewed the pathogenesis, epidemiology, risk factor
management and prevention of C. difficile-associated diarrhea (CDAD).
key words
Clostridium difficile, Antibiotic-associated diarrhea (AAD), pseudomembranous colitis
(PMC), Nosocomial infection, C. difficile-associated diarrhea (CDAD).
Introduction
Clostridium difficile is anaerobic Gram-positive, spore-forming bacillus, which has been
recognized as the most frequent cause of hospital-acquired infectious diarrhea worldwide,
accounting for up to 1 of every 200 patients who are admitted to hospital, 20% of cases of
nosocomial diarrhea1 and 15% of all antibiotic treatment associated diarrhea2. The spectrum
of enteric disease caused by C. difficile is a broad ranging from asymptomatic colonization to
mild self-limiting antibiotic-associated diarrhea and can progress to more severe
pseudomembranous colitis (PMC) and life threatening fulminant colitis. Complications
include the development of recurrent C. difficile-associated diarrhea (CDAD) in 20 -30% of
cases, ileus or toxic megacolon colonic perforation and death2, 3. The CDAD is a term used to
describe the symptomatic manifestation of disease therefore excluding the asymptomatic
carrier. The majority of hospital patients with CDAD are elderly, and generally have been on
antimicrobials that decrease the “ colonization resistance” of the colon leading to C. difficile
colonization and production of C. difficile two toxins which contribute to disease
pathogenesis3,4 .
The transmission of C. difficile assisted by its ability to form spores that are resistant to
many disinfectants allowing it to remain viable in the hospital environment for long periods of
time1,5 .
Historical background
C. difficile was first described in 1935 when Hall and O’Toole discovered a new anaerobe
during their study of the development of normal bacterial flora in neonates2,6.The bacterium
was name Bacillus difficilis because of the difficulty in isolating it, the bacterium was found
to be non pathogenic in newborns infants, but pathogenic in experimental animals due to the
presence of a lethal toxin7. C. difficile was presented in the stools of healthy neonates and was
classified as a commensal; Staphylococcus aureus was originally implicated as the causative
organism of (AAD) and PMC. This view did not change until the 1970s when a prospective
study of 200 patients treated with the antibiotic clindamycin, of whom 21% developed
diarrhea and 10% had PMC by endoscopy. Consequent lab testing of stool specimens proved
the presence of C. difficile toxin 1,8.
Over the last 25 years, many studies have been published on the bacterium and the spectrum
of associated diseases. In most patients, C. difficile infection does not progress to PMC,
because of increased awareness and better laboratory diagnosis of CDAD has become a
popular way of describing the disease7.
General Microbiology
The Clostridium difficile is an anaerobic spore bearing (subterminal) bacillus, belongs to the
division Firmicutes, family of the Clostridiaceae and the genus Clostridium.
C. difficile is a bacillus of approximately 3-5 μm in length. The genus Clostridium
(clostridia) present as Gram-positive, rod-shaped, in Gram staining. The clostridia are ancient
bacteria that live in virtually all of the anaerobic habitats of nature where organic compounds
are including soils, aquatic sediments and the intestinal tracts of animals8 .
C. difficile is able to form structures within the vegetative cell which are called endospores or
spores (Figure 1). The non-sporing cell is named as “vegetative”. Spores of C. difficile are
resistant to exposure to heat, chemical disinfection, alcohol, air and drying. Upon dehydration
the resultant spore formed is oval and terminal in position, and can survive for up to 6 months
in the environment 12,13. Recently studies suggest that non-chlorine-based detergents
encourage sporulation in C. difficile strains, indicating the importance of choice of cleaning
agents used in hospitals 14. It has been shown that C. difficile spore might contribute to
recurrent CDAD cases after discontinues treatment, re-infection with new strain occurred in
to one half of recurrent cases while the remaining patients relapsed with the –one third
original strain15
Figure 1: C. difficile endospores appear clear, vegetative cells dark
.
Adapted from C. difficile © 2006, Kenneth Todar University of Wisconsin-Madison
http://textbookofbacteriology.net/clostridia_2.html
(permission taken from Kenneth Todar)
Epidemiology
C. difficile is spore forming bacteria which can be part of the normal intestinal flora as many
as 50% of children under the age of two and less frequently in individuals over two years of
age.
The organism is carried in gastrointestinal tract (GIT) in approximately 3 % of the general
population and 30 % in hospitalized. People requiring prolonged use of antibiotics and the
elderly are at a greater risk of acquiring this disease, and can be affected even by low doses.
Since the bacteria are found in faeces, a patient who has CDAD excretes large numbers of the
spores in their liquid faeces, these can contaminate items or surfaces around the patient’s bed
including keypads, equipment, the toilet areas ,sluices, commodes, bedpan and washers.
Further the spores can survive for a long time and can be a source of hand-to-mouth infection
for others.
Healthcare workers can also spread the bacteria to other patients or contaminate surfaces
through hand contact, in most cases the disease develops after cross-infection from another
patient, either through direct patient to patient contact, via healthcare staff, or via a
contaminated environment.7
Incidence
The reported incidence of CDAD ranges from 0 to 15 cases per 100 patients in health-care
settings during a non-outbreak period, but can raise to 16 and 20 cases per 100 patients during
an outbreak2. The incidence of CDAD has however continued to increase since the 1990s
despite established infection control programs and effective treatments. Variation in healthcare facilities, the patient’s age and condition, and antibiotics use all contribute to the likely
hood of an outbreak2.
Generall, the prevalence of CDAD in community or outpatient settings is typically lower (7–
12 cases per 100,000 person-years), but attention has been focused on community acquired
CDAD as the number of reported cases is increasing. Since 2003, the incidence of CDAD in
many US Canadian and European hospitals has increased markedly, also associated with
increased frequency of severe or fulminant disease and recurrent CDAD2,7.
Moreover, the mortality due to C. difficile infection in England and Wales rose from 3,757 in
2005 to 6,480 in 2006, an increase of 72% . Age-standardised C. difficile mortality rates
were increased by 77% among males, and 66 % among females between 2005 and 2006.
Accuracy of reporting has been believed as one of the causes for the statistical increase. PCR
ribotype 027outbreaks were reported in 7 hospitals in the Netherlands from April 2005 to
February 2006. One hospital detected at the same time a second outbreak due to a toxin A–
negative C. difficile PCR ribotype 017 toxinotype VIII strain. ® VIII. The 017 strain had a
deletion of the toxin A gene, didnot contain genes for binary toxin production, and had a
normal tcdC gene15,16
Clinical features
Gastrointestinal
The spectrum of disease severity ranges from uncomplicated diarrhea to colitis or PMC and
rarely, ileus or toxic megacolon. Symptoms typically occur within 48h of infection with C.
difficile and most patients who are infected in hospital become infected within 3 weeks of
admission. Less frequently, the onset of symptoms can be delayed by up to 2–3 months9 .
The clinical presentation of CDAD typically includes mild or severe diarrhea and lower
abdominal cramping pain but can also present with systemic symptom as fever, nausea,
anorexia and leukocytosis, they may have hypovolemia, shock, and hypoalbuminemia due to
oozing albumin from the blood plasma into the colonic lumen10, acute diarrhea with
hypoalbuminemia is a good indicator of C. difficile intestinal disease. Stools are usually not
grossly bloody, but they can be11.
Occult blood is present in 30% of patients. Fecal leukocytosis is also present in one third of
the patients. Colitis, when present, is usually most severe in the rectum and distal colon but it
can be present on the right side of the colon.
1-3% of patients develop fulminant colitis, in which case the patient may not have diarrhea
but develop signs and symptoms of severe toxicity with fever and diffuse abdominal pain and
distension which might result in fatal disease, due to bowel perforation and peritonitis24.
Physicians rarely suspect C. difficile disease in the absence of diarrhea7, 25.
The presence of PMC suggests an advanced stage of the disease, and although considered
“nonspecific,” it is highly indicative of C. difficile infection; it is almost restricted to the colon
and rectum and is well defined histopathologically. Pseudomembranous can be visualized by
sigmoidoscopy or colonoscopy, as loss of the mucosal folds and plaques with hemorrhagic
spots and deep ulceration. Microscopically, this pseudomembrane appears as fibrinous
exudates containing epithelial cells, mucin and leucocytes25.
Severely ill patients may have no diarrhoea as a characterized feature of toxic megacolon or
paralytic ileus because of loss of colonic muscular tone. Toxic megacolon may be associated
with severe sepsis and multiple organ dysfunctions26. Mortality associated with toxic
megacolon is high, ranging from 25% to 40%. Recurrent diarrhoea is seen in 15–30% of
cases27.
Despite the infection of the immediately adjacent right colon, the involvement of the distal
third of small intestine in infections with C. difficile is rare. Previously reported studies of
patients with small bowel involvement have had their intestine altered surgically, which may
be a predisposing factor of small bowel susceptibility to pseudomembranous enteritis28.
Extra intestinal
Clostridium difficile has rarely been isolated from extra intestinal sites. Apart from a few case
reports and small case series, the clinical features of extra colonic C. difficile infections have
not been studied systematically, probably, the organism has low pathogenic potential outside
the intestinal tract. The manifestations of infection include, bacteremia, visceral abscess
formation subphrenic, splenic, pancreatic and left iliacus muscle abscess)28, infection of
implanted prosthetic devices29, encephalopathy, reactive arthritis, and osteomyelitis30.
Cellulites and necrotizing fasciitis of the thigh31.
C. Difficile Pathogenesis and Toxins
C. difficile infection is commonly related to hospitalization, causes antibiotic associated
diarrhea and more serious intestinal conditions such as colitis and PMC. However, CDAD is
also increasing in occurrence and severity as a community-acquired disease, probably due to
use of proton pump inhibitors36,37. These conditions generally result from overgrowth of C.
difficile in the colon, usually after the normal flora has been disturbed by antimicrobial
chemotherapy allowing C. difficile to flourish in the intestinal tract. C. difficile then
proliferates and produce toxins that causes a watery diarrhea(TcdA and TcdB), if it lacks the
gene for toxin production, no disease develops6.This protective effect of the normal
microflora is often referred to as “colonization resistance” because it can act as a barrier
against infection with C. difficile38.
Table1: Antimicrobial agents that induce C. difficile-associated diarrhea and colitis, Adapted
from Ciaràn Kelly (British Society of Gastroenterology Meeting 2008,10-13 March,
Birmingham oral presentation).
Frequent
Infrequent
Rare
Ampicillin
Chloramphenicol
Metronidazole
amoxicillin
Trimethoprim
Parenteral aminoglycoside
Cephalosporins
Macrolide
Vancomycin
Clindamycin
Sulfonamide
Quinolones I
Tetracycline
Uncolonized patient
Antibiotic exposure +PPI
Disruption of colonic micro flora
C. difficile ingestion and colonization
______________________________________
Good IgG response
Asymptomatic and carrier state
Poor or partial IgG response
Production of C. difficile toxin A and B
Activation of macrophages and mast
Cells, Upregulation of cytokines
and other mediators of inflammation
Clinical disease
Figure 2: Pathogenesis of C. difficile Infection. Modified from reference5.
The carrier proportion of hospital patients can be as high as 16-35% of inpatients39. CDAD is
considered as health-care associated, when diarrhoea starts after at least 48h after admission
to the hospital, or onset in the community within 4 weeks after discharge. A case is
community acquired when symptoms occur within 48h of admission and without previous
hospitalization in the last 12 weeks40.
C. difficile multiply and produces two toxins: Toxin A is referred to as an enterotoxin
because it causes fluid accumulation in the bowel. Toxin B (cytotoxin) is an extremely Lethal
(cytopathic ) toxin. The C. difficile toxins damage colonocytes, impaired cell migration, and
cause apoptosis5. C. difficile does not invade the colonic mucosa, the disease does not develop
if no toxins are produced5.
Even when a toxin is produced, some people became carriers or develop a mild, self-limited
diarrhea while others developed severe colitis and may have recurrence of the disease. Serum
antibody levels in response to the toxin may modulate disease pathogenesis.
Generally, C. difficile disease is caused by genotypes and strains that are resistant to the used
antibiotic. Penicillins, cephalosporins and clindamycin are drugs that may precipitate disease7.
However, even vancomycin- the effective drug in treating C. difficile infection can cause the
disease, this probably happens because vancomycin suppresses both normal flora and the
vegetative forms of C. difficile, but not its spores. When vancomycin treatment is stopped, the
spores germinate and the new vegetative forms, flourish in the altered bacterial environment7.
Risk Factors and Risk Group
Although current antibiotic treatment is one of the most important risk factors, the host and
environmental factors may also play a role. The major risk factors are highly variable
depending on age, duration of hospitalization, and severity of underlying disease23, 41, 42. In
patients older than 60 years, the CDAD incidence amongst 5133 cases was ten-fold higher
than younger patients41. In a study on 73 patients in an outbreak situation, the mean age was
74 years.
Risk factors for CDAD typically include host factors (advanced age, impaired immune status,
co morbidities, etc.), increase in exposure to C. difficile spores (longer length hospital stays,
the health-care environment, infected roommates, contaminated hands, etc.), and factors
causing disruption of protective colonic microflora layer (antibiotic medications, various
procedures, repeated enemas, prolonged nasogastric tube insertion and gastrointestinal tract
surgery increase a person's risk of developing the disease ). Risk factors for CDAD are
shown in Table2.
Table2: Risk factors for health-care associated and community acquired CDAD. The contents
of the table are from references2.
Health-care-associated
Risk factor
CDAD
Community-acquired CDAD
Broad-spectrum antibiotics
Usually no antibiotic
Disruption of normal colonic
Medications
exposure
microflora
Surgery
Some fluoroquinolones
Chronic gastrointestinal
conditions
Hospital environmental
Home surfaces
Exposure to C. difficile
surfaces
Family members
spores
Infected roommates
Pets
Hand carriage by health-care
Personnel
Impaired immune system
Host factors
Young children
Low production of anti-toxin Post-partum women
A and/or B antibodies Use of PPIs
Comorbidities
Advanced age
Strain type (outbreaks)
Microbial factors
Table3: The main C. difficile virulence factors
Sporulation capacity
Two high-molecular-weight toxins:Toxin Aenterotoxin or TcdA
Toxin B cytotoxin or TcdB
Bacterial factors
A binary toxin ( CDT)
Motility/ chemotaxis
Adhesion
Surface layer protein (SLP)
Flagella
Host factors
Cytoskeletal rearrangement
Microtubules
Micro filaments
Tyrosine phosphorylation
Proinflammatory -IL 8 induction
Diagnosis
A diagnosis of CDAD generally requires both clinical and laboratory and exclusion of other
etiologies of diarrhea, such as drugs or chronic intestinal conditions, C. difficile diarrhea is
confirmed by the presence of toxin in stool specimen. A positive culture for C. difficile
without a toxin assay is not sufficient to make the diagnosis of CDAD. Stool cultures may be
complicated by the finding of non-toxigenic strains of the bacterium, so the most reliable and
widely used tests involve detection for the presence of the toxin A and/or toxin B in the stool,
although atypical toxin variant strains (A–B+) can also cause symptoms (Table 4).
Interestingly, some patients have stool assays positive for toxin but they do not exhibit
disease symptoms (asymptomatic carriers). On the other hand, the lack of a toxin-positive
stool assay does not rule out CDAD. The toxins are very unstable. They degrade at room
temperature and cannot be detected two hours after collection of stool specimen, leading to
false negative results (29-56% of cases). Methods to reduce the percentage of false negative
results include the use of rapid enzyme immunoassays that identify both toxin A and toxin B,
repeat stool assays and investigate the isolate for toxin44 .
The most highly sensitive diagnostic test for C. difficile detection is both cell culture
cytotoxin assay as well as anaerobic culture of organisms, although there is no accepted
standard, the cell cytotoxicity assay is considered the “gold standard”44,45, while some
microbiologists used toxinogenic culture as the “gold standard”46 .
Endoscopy, although with poor sensitivity, macroscopic diagnosis of CDAD can be made by
rectoscopy, sigmoidoscopy or colonoscopy for direct visualization of the colonic mucosa and
detection of pseudo membranes44. However, a negative result does not exclude infection
because diarrhoea can develop without pseudomembranes47. Endoscopy is usually
recommended when patients have ileus and are unable to produce watery or loose faecal
samples so it helps to obtain a stool sample for toxin assay48,49. A computed tomography
scaning is often diagnostic of C difficile colitis, provided physicians think of this disease in
the absence of diarrhea.
Table 4: Stool Tests for Diagnosis of C. Difficile Infection. Adapted from reference61
Test
Disadvantages
Detects
Advantages
Polymerase
Toxin A or B
high
requires expertise in
chain reaction
genes in isolates or
sensitivity97%
molecular
directly in faeces
specificity100
diagnostic
techniques
Latex
bacterial enzyme
fast
poor sensitivity and
agglutination
(glutamate
inexpensive
specificity
assay
Dehydrogenase)
easy to perform
Enzyme
Ttoxin A or B
fast
not as sensitive as
easy to perform
cytotoxin assay
immunoassay
high specificity
Cytotoxin assay
requires tissue
Toxin B
GSold standard
culture facility
Highly sensitive
takes 24-48 hours
94%-100%
False-positives
Highly specific
Results vary with
99%-100%
experience
Of the technologist
Culture
Toxigenic and
Non toxigenic C.
sensitive
requires anaerobic
Difficile
allows strain
culture
typing in
not specific for
epidemics
toxin producing
bacteria
takes 2-5 days
Treatment for C. difficile-associated disease
The management of CDAD remains problematic (Table 5). The first approach to the
treatment, as soon as C. difficile disease is diagnosed, is withdrawal or change the
precipitating antibiotic with lower CDAD risk antibiotic50, studies show number of patients
respond well to this approach and additional treatment is required45.
Severe cases may require antibiotic treatment, the most common treatments are oral
vancomycin or oral metronidazole . Additionally another approach for treatment or
prophylaxis is probiotics therapy, where use of non-pathogenic organisms helps to prevent
colonization of C. difficile51,52. Agents that inhibit toxin A binding such as cholestyramine and
immunotherapy with antitoxin A antibodies have also been applied53.
Patients must be monitored for dehydration and electrolyte imbalance after prolonged
diarrhea. Antidiarrheal agents such as Lomotil or Imodium should not be taken as they have
been shown to increase the severity of symptoms and precipitate megacolon. C. difficile
infections usually can be treated successfully with a 10 day course of antibiotics including
metronidazole or vancomycin.
Although, resistance to metronidazole and vancomycin has been reported but so far has not
been a significant clinical problem. It is suggested that patients with C .difficile infection who
require long-term antibiotic therapy (e.g. those with bacterial endocarditis or osteomyelitis)
should maintain metronidazole or vancomycin therapy for 1 week after completion of longterm antibiotic therapy53.
Table 5: Standard therapy for Clostridium difficile infection, Adapted from reference 53
Disease characteristic
Regimen
Metronidazole 250mg orally (P.O) four times
Mild disease (no systemic
dailyv for 500 mg three times a day for 10-14
symptom only mild diarrhea)
days
Moderate disease (fever,
Vancomycin HCl ,125-500 mg P.O four times
profuse diarrhea, abdominal
daily for10-14 days
pain leukocytosis)
Severe disease (Paralytic
Surgical consult and intraluminal
ileus, toxic megacolon sepsis,
vancomycin
dehydration)
Inability to take medication
Intraluminal vancomycin with or without
intravenous Metronidazol
Therapy for recurrent C. difficile infection
The main complication of CDAD treatment is the relapse, of which occurs in about 20 to 25%
of cases. The symptoms usually return after 3 to 21 days (average,6) from withdrawal of
metronidazole or vancomycin. Assays for C. difficile toxin are not immediately necessary
after finishing treatment course, at which the results could be confusing, as about one third of
patients with successful therapy are positive in assays. Most recurrent C.D infection respond
to another course of standard antibiotics doses for 10 days, while 3 to 5 % of patients have
more than six relapses54,58. Management is controversial, and the course may involve
complications and considerable expense57.
For repeated relapses, therapy for four to six weeks is recommended to control C. difficile
infection, these prolonged treatment include the administration of pulsed doses of vancomycin
(125 mg every other day which can maintain C. difficile in the spore form and has minimum
effects on the colonic flora), the use of 4g of cholestyramine three times daily to absorb
C. difficile toxin , or the use of agents such as probiotics Saccharomyces boulardii or
Lactobacillus56,58. The use of intravenous immunoglobulin has been suggested, based on
recent data show that patients with relapses have diminished plasma concentrations of IgG
antibodies against toxin A58. Reconstituting normal flora by enemas and infusion of donor
feces show good response rates but this solution is not ideal, due to possible risk of
transmission of retroviruses or other agents59.
Prevention and control
Prevention and control of CDAD in hospitals basically depend on two factors: limiting use of
antibiotic and interruption of transmission of C. difficile by infection control strategies. In
many cases stoppage of suspected antibiotic is enough, metronidazole is a commonly used
drug with vancomycin for severe cases. Recently, use of probiotics for prevention,
vaccination against C.difficile and colonization with nontoxigenic C.difficile strains has also
been suggested.
The vegetative form of C. difficile is largely found on fecally-contaminated surfaces and may
be killed by EPA- hospital-use disinfectant products or by exposure to air. However, it is
important to note that disinfectants (alcohol solution) or disinfectant-detergents used on
environmental surfaces are not effective against the spore form of C. difficile. Hand hygiene,
washing practice techniques and the proper handling of contaminated wastes (including
diapers) are absolutely effective in preventing the spread of the disease and interruption of the
disease transmission from person to person via the hands. Environmental surfaces
contaminated with C. difficile spores should be cleaned with an effective disinfectant
(bleach), aggressive cleaning and disinfection is effective in reducing the number of C.
difficile positive cultures in the environment60.
In the case of a C. difficile outbreak, isolation of patients with CDAD and education of staff
with attention to traditional hand washing with soap and water for the mechanical removal
and rinsing of spores from the hands is recommended. No sharing electronic thermometers,
clean surfaces in bathrooms, kitchens and other areas on a regular basis with household
detergent/ a chlorine containing disinfectant is recomended60.
Reference List
(1) Tedesco FJ, Barton RW, Alpers DH. Clindamycin-associated colitis. A prospective
study. Ann Intern Med 1974 Oct;81(4):429-33.
(2) Hurley BW, Nguyen CC. The spectrum of pseudomembranous enterocolitis and
antibiotic-associated diarrhea. Arch Intern Med 2002 Oct 28;162(19):2177-84.
(3) Elliott B, Chang BJ, Golledge CL, Riley TV. Clostridium difficile-associated diarrhoea.
Intern Med J 2007 Aug;37(8):561-8.
(4) Loo VG, Bourgault AM, Poirier L, et al. Host and pathogen factors for Clostridium
difficile infection and colonization. N Engl J Med 2011;365:1693-703.
(5) Schroeder MS. Clostridium difficile--associated diarrhea. Am Fam Physician 2005 Mar
1;71(5):921-8.
(6) Gupta A, Khanna S. Community-acquired Clostridium difficile infection: an increasing
public health threat. Infect Drug Resist. 2014;7:63–72.PubMed
(7) McFarland LV. Update on the changing epidemiology of Clostridium difficile associated
disease. Nat Clin Pract Gastroenterol Hepatol 2008 Jan;5(1):40-8.
(8) Jackson S, Calos M, Myers A, Self WT. Analysis of proline reduction in the nosocomial
pathogen Clostridium difficile. J Bacteriol 2006 Dec;188(24):8487-95.
(9) Quirk M. Clostridium difficile epidemic strain far reaching. Lancet Infect Dis 2006
Feb;6(2):74.
(10) Bartlett JG. Clinical practice. Antibiotic-associated diarrhea. N Engl J Med 2002 Jan
31;346(5):334-9.
(11) Monaghan T, Boswell T, Mahida YR. Recent advances in Clostridium difficile
associateddisease. Gut 2008 Jun;57(6):850-60.
(12) Buggy BP, Hawkins CC, Fekety R. Effect of adding sodium taurocholate to selective
media on the recovery of Clostridium difficile from environmental surfaces. J Clin
Microbiol 1985 Apr;21(4):636-7.
(13) Wilson KH, Kennedy MJ, Fekety FR. Use of sodium taurocholate to enhance spore
recovery on a medium selective for Clostridium difficile. J Clin Microbiol 1982
Mar;15(3):443-6.
(14) Wilcox MH, Fawley WN. Hospital disinfectants and spore formation by Clostridium
difficile. Lancet 2000 Oct 14;356(9238):1324.
(15) Tang-Feldman Y, Mayo S, Silva JJ, Jr., Cohen SH. Molecular analysis of Clostridium
difficile strains isolated from 18 cases of recurrent clostridium difficile-associated
diarrhea. J Clin Microbiol 2003 Jul;41(7):3413- 3414
(16) Kuijper EJ, Van den Berg RJ, Debast S, Visser CE, Veenendaal D, Troelstra A, et al.
Clostridium difficile ribotype 027, toxinotype III, the Netherlands. Emerg Infect Dis
2006 May;12(5):827-30.
(17) Johnson S, Sambol SP, Brazier JS, Delmee M, Avesani V, Merrigan MM, et al.
International typing study of toxin A-negative, toxin B-positive Clostridium difficile
variants. J Clin Microbiol 2003 Apr;41(4):1543-7.
(18) Rupnik M, Kato N, Grabnar M, Kato H. New types of toxin A-negative, toxin Bpositive
strains among Clostridium difficile isolates from Asia. J Clin Microbiol 2003
Mar;41(3):1118-25.
(19) Al-Barrak A, Embil J, Dyck B, Olekson K, Nicoll D, Alfa M, et al. An outbreak of
toxin A negative, toxin B positive Clostridium difficile-associated diarrhea in a
Canadian tertiary-care hospital. Can Commun Dis Rep 1999 Apr 1;25(7):65-9.
(20) Alfa MJ, Kabani A, Lyerly D, Moncrief S, Neville LM, Al-Barrak A, et al.
Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile
responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea. J
Clin Microbiol 2000 Jul;38(7):2706-14.
(21) Kato H, Kato N, Watanabe K, Iwai N, Nakamura H, Yamamoto T, et al. Identification
of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol
1998 Aug;36(8):2178-82.
(22) Kuijper EJ, de WJ, Kato H, Kato N, van Dam AP, van d, V, et al. Nosocomial outbreak
of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin
A-negative strain. Eur J Clin Microbiol Infect Dis 2001 Aug;20(8):528-34.
(23) Kyne L, Merry C, O'Connell B, Kelly A, Keane C, O'Neill D. Factors associated with
prolonged symptoms and severe disease due to Clostridium difficile. Age Ageing 1999
Mar;28(2):107-13.
(24) Koss K, Clark MA, Sanders DS, Morton D, Keighley MR, Goh J. The outcome of
surgery in fulminant Clostridium difficile colitis. Colorectal Dis 2006 Feb;8(2):149-54.
(25) Barroso LA, Wang SZ, Phelps CJ, Johnson JL, Wilkins TD. Nucleotide sequence of
Clostridium difficile toxin B gene. Nucleic Acids Res 1990 Jul 11;18(13):4004.
(26) Dobson G, Hickey C, Trinder J. Clostridium difficile colitis causing toxic megacolon,
severe sepsis and multiple organ dysfunction syndrome. Intensive Care Med 2003
Jun;29(6):1030.
(27) Maroo S, LaMont JT. Recurrent clostridium difficile. Gastroenterology 2006
Apr;130(4):1311-6.
(28) Jacobs A, Barnard K, Fishel R, Gradon JD. Extracolonic manifestations of Clostridium
difficile infections. Presentation of 2 cases and review of the literature. Medicine
(Baltimore) 2001 Mar;80(2):88-101.
(29) McCarthy J, Stingemore N. Clostridium difficile infection of a prosthetic joint
presenting 12 months after antibiotic-associated diarrhoea. J Infect 1999 Jul;39(1):94-6.
(30) Riley TV, Karthigasu KT. Chronic osteomyelitis due to Clostridium difficile. Br Med J
(Clin Res Ed) 1982 Apr 24;284(6324):1217-8.
(31) Bhargava A, Sen P, Swaminathan A, Ogbolu C, Chechko S, Stone F. Rapidly
progressive necrotizing fasciitis and gangrene due to Clostridium difficile: case report.
Clin Infect Dis 2000 Jun;30(6):954-5.
(36) Dial S, Delaney JA, Barkun AN, Suissa S. Use of gastric acid-suppressive agents and
the risk of community-acquired Clostridium difficile-associated disease. JAMA 2005
Dec 21;294(23):2989-95.
(37) Dial S, Delaney JA, Schneider V, Suissa S. Proton pump inhibitor use and risk of
community-acquired Clostridium difficile-associated disease defined by prescription for
oral vancomycin therapy. CMAJ 2006 Sep 26;175(7):745-8.
(38) Macdonald TT. The mucosal immune system. Parasite Immunol 2003 May;25(5):23546.
(39) Aslam S, Hamill RJ, Musher DM. Treatment of Clostridium difficile-associated disease:
old therapies and new strategies. Lancet Infect Dis 2005 Sep;5(9):549-57.
(40) Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium difficile-associated disease
in North America and Europe. Clin Microbiol Infect 2006 Oct;12 Suppl 6:2-18.
(41) Karlstrom O, Fryklund B, Tullus K, Burman LG. A prospective nationwide study of
Clostridium difficile-associated diarrhea in Sweden. The Swedish C. difficile Study
Group. Clin Infect Dis 1998 Jan;26(1):141-5.
(42) Kyne L, Sougioultzis S, McFarland LV, Kelly CP. Underlying disease severity as a
major risk factor for nosocomial Clostridium difficile diarrhea. Infect Control Hosp
Epidemiol 2002 Nov;23(11):653-9.
(43) Banno Y, Kobayashi T, Kono H, Watanabe K, Ueno K, Nozawa Y. Biochemical
characterization and biologic actions of two toxins (D-1 and D-2) from Clostridium
difficile. Rev Infect Dis 1984 Mar;6 Suppl 1:S11-S20.
.
(44) Delmee M, Van BJ, Simon A, Janssens M, Avesani V. Laboratory diagnosis of
Clostridium difficile-associated diarrhoea: a plea for culture. J Med Microbiol 2005
Feb;54(Pt 2):187-91.
(45) Bartlett JG. Antibiotic-associated diarrhea. Clin Infect Dis 1992 Oct;15(4):573-81.
(46) Zheng L, Keller SF, Lyerly DM, Carman RJ, Genheimer CW, Gleaves CA, et al.
Multicenter evaluation of a new screening test that detects Clostridium difficile in fecal
specimens. J Clin Microbiol 2004 Aug;42(8):3837-40.
(47) Poutanen SM, Simor AE. Clostridium difficile-associated diarrhea in adults. CMAJ
2004 Jul 6;171(1):51-8.
(48) Fekety R. Guidelines for the diagnosis and management of Clostridium difficile
associated diarrhea and colitis. American College of Gastroenterology, Practice
Parameters Committee. Am J Gastroenterol 1997 May;92(5):739-50.
(49) . Brecher SM, Novak-Weekley SM, Nagy E.Laboratory diagnosis of Clostridium difficile
infections. Clin Infect Dis. 2013;57(8):1175-1181
(50) Riley TV, Cooper M, Bell B, Golledge CL. Community-acquired Clostridium difficile
associated diarrhea. Clin Infect Dis 1995 Jun;20 Suppl 2:S263-S265.
(51) Clayton L Golledge, Thomas V Riley. Natural therapy for infectious diseases. The
Medical Journal of Australia MJA ; 1996;-164.
(52) Lewis SJ, Freedman AR. Review article: the use of biotherapeutic agents in the
prevention and treatment of gastrointestinal disease. Aliment Pharmacol Ther 1998
Sep;12(9):807-22.
(53) Sunenshine RH, McDonald LC. Clostridium difficile-associated disease: new
challenges from an established pathogen. Cleve Clin J Med 2006 Feb;73(2):187-97.
(54) Bartlett JG. Management of Clostridium difficile infection and other antibioticassociated
diarrhoeas. Eur J Gastroenterol Hepatol 1996 Nov;8(11):1054-61.
(55) Bartlett JG. Treatment of Clostridium difficile colitis. Gastroenterology 1985
Nov;89(5):1192-5.
(65) McFarland LV, Surawicz CM, Greenberg RN, Fekety R, Elmer GW, Moyer KA, et al.
A randomized placebo-controlled trial of Saccharomyces boulardii in combination with
standard antibiotics for Clostridium difficile disease.
JAMA 1994 Jun 22;271(24):1913-8.
(57) McFarland LV, Surawicz CM, Rubin M, Fekety R, Elmer GW, Greenberg RN.
Recurrent Clostridium difficile disease: epidemiology and clinical characteristics. Infect
Control Hosp Epidemiol 1999 Jan;20(1):43-50.98
(58) Gorbach SL, Chang TW, Goldin B. Successful treatment of relapsing Clostridium
difficile colitis with Lactobacillus GG. Lancet 1987 Dec 26;2(8574):1519.
(59) van Nood E, Vrieze A, Nieuwdorp M, Fuentes S, Zoetendal EG, et al. (2013)
Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J
Med 368: 407-415.
(60) Siegel JD, Rhinehart E, Jackson M, Chiarello L. 2007 Guideline for Isolation
Precautions: Preventing Transmission of Infectious Agents in Health Care Settings. Am
J Infect Control 2007 Dec;35(10 Suppl 2):S65-164.
(61) Drudy D, O'Donoghue DP, Baird A, Fenelon L, O'farrelly C. Flow cytometric analysis of
Clostridium difficile adherence to human intestinal epithelial cells. J Med Microbiol 2001
Jun;50(6):526-34.