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Clostridium difficile–Associated Diarrhea
A Review
Eleftherios Mylonakis, MD;
Edward T. Ryan, MD;
Stephen B. Calderwood, MD
Arch Intern Med. 2001;161:525-533.
ABSTRACT
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Clostridium difficile causes 300 000 to
3 000 000 cases of diarrhea and colitis in the United States every
year. Antibiotics most frequently associated with the infection are clindamycin,
ampicillin, amoxicillin, and cephalosporins, but all antibiotics may predispose
patients to C difficile infection. The clinical presentation
varies from asymptomatic colonization to mild diarrhea to severe debilitating
disease, with high fever, severe abdominal pain, paralytic ileus, colonic
dilation (or megacolon), or even perforation. The most sensitive and specific
test available for diagnosis of C difficile infection
is a tissue culture assay for the cytotoxicity of toxin B. However, this test
takes 1 to 3 days to complete and requires tissue culture facilities. Detection
of C difficile toxin by means of enzyme-linked immunoassay
is more rapid and inexpensive. A minority of patients may require more than
1 stool assay to detect toxin. Oral metronidazole or oral vancomycin hydrochloride
for 10 to 14 days are equally effective at resolving clinical symptoms; oral
metronidazole is preferred in most cases because of lowered cost and less
selective pressure for vancomycin-resistant organisms. Approximately 15% of
patients experience relapse after initial therapy and require retreatment,
sometimes with an extended, tapering regimen. Immunity appears to be incomplete
and predominantly mediated by serum IgG to toxin A. Measures for preventing
the spread of the pathogen, appropriate diagnostic testing, and treatment
may avert morbidity and mortality due to C difficile–associated
diarrhea.
INTRODUCTION
Clostridium difficile, a Gram-positive, spore-forming
anaerobic bacillus, was first described in 1935,1
but it was not associated with antibiotic-related diarrhea until the late
1970s.2, 3, 4 Clostridium difficile can lead to severe complications
and currently is the most common cause of nosocomial diarrhea (often adding
up to 2 weeks to the length of the hospitalization, at an additional cost
of $6000-$10 000 per case).5, 6, 7, 8, 9, 10
This report focuses on new information on epidemiological features, pathogenesis,
immunological findings, clinical manifestations, diagnosis, and treatment
of C difficile infection.
EPIDEMIOLOGICAL FEATURES
Clostridium difficile is the cause of approximately
25% of all cases of antibiotic-associated diarrhea.11
In most cases of antibiotic-associated diarrhea in which C difficile is not detected, no etiologic agent is identified, and
diarrhea is usually mild and not accompanied by abdominal pain.11
Symptoms in such cases usually respond to discontinuation of treatment with
the offending antibiotic.
Most cases of C difficile–associated
disease occur in hospitals or long-term care facilities (rate of 25-60 per
100 000 occupied bed-days), causing more than 300 000 cases per
year in the United States alone.12, 13, 14
The incidence of this infection in the outpatient setting (7.7 cases per 100 000
person-years; approximately 20 000 cases per year in the United States)
is lower, but not negligible.15 Overall, the
risk for development of C difficile–associated
diarrhea (CDAD) within 6 weeks of a course of antibiotics in the outpatient
setting is low (6.7 cases per 100 000 exposures).15
Among hospitalized individuals, the risk for CDAD after clindamycin
therapy has been estimated to range from 1 in 10 to 1 in 10 000.16 A recent study demonstrated that clindamycin increases
the risk for CDAD even more in patients colonized with clindamycin-resistant C difficile.17 The risks
of other individual antibiotics for the development of C difficile infection have not been evaluated in large prospective
studies, but the antibiotics most frequently associated with infection in
addition to clindamycin have been ampicillin, amoxicillin, and the cephalosporins.17 However, all antibiotics (even vancomycin and metronidazole)
have been associated with CDAD, and clinicians should be aware that nonessential
antibiotic prescription can lead to morbidity and mortality due to C difficile infection18 (Table 1). Especially during hospital outbreaks, antibiotic restriction
is indicated for antibiotics that have been identified as significant risks
for CDAD. This measure, along with isolation of cases, reducing the environmental
burden with appropriate use of disinfectants,28, 29
and education of hospital personnel, can help control the outbreak.
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Table 1. Antimicrobial and Antineoplastic Agents That Induce Clostridium difficile–Associated Diarrhea and Colitis*
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PATHOGENESIS AND IMMUNOLOGICAL FEATURES
Infants and young children commonly harbor C difficile in fecal flora but have no symptoms related to production of toxin.20, 30 For unclear reasons, the number of
carriers quickly declines as children age, and toxinogenic C difficile is isolated from stool specimens in only 0% to 3% of healthy
adults.20 During hospitalization, however,
colonization frequently occurs (21% of patients in one study, for example).13 Although asymptomatic carriers are an important hidden
reservoir of C difficile, clinical symptoms develop
in only about one third of colonized patients, and asymptomatic colonization
with C difficile may be associated with a decreased
risk for development of CDAD.31
Clostridium difficile forms spores that persist
in the environment for years, and contamination by C difficile is common in hospitals and long-term care facilities, especially in
rooms occupied by an infected individual.13, 32, 33, 34, 35
Patient-to-patient transmission of the organism occurs, and the organism can
be cultured from many environmental surfaces in rooms of infected patients
and on hands, clothing, and stethoscopes of health care workers. Hospital
personnel may carry the bacteria from room to room and promote infection,
but fecal carriage by staff is rare.13, 32, 33
Outbreaks can occur in hospitals, nursing homes, and other extended-care facilities.22
The first step in development of C difficile
colonization is disruption of the normal flora of the colon, usually caused
by antibiotics or, in unusual cases, by antineoplastic or immunosuppressive
drugs.21, 36 Colonization occurs
by the fecal-oral route; ingested spores of C difficile survive the gastric acid barrier and germinate in the colon.20, 22 Symptoms of CDAD may start on the
first day of antibiotic therapy or 6 weeks or longer after antibiotic therapy
is stopped.6
The following 2 factors recently have been shown to increase the probability
of symptomatic disease in patients who acquire C difficile colonization in hospital: the severity of other illnesses, and reduced
levels of serum IgG antibody to toxin A.37
These results suggest that preexisting anti–toxin A antibody may ameliorate
severity of disease and that immunization might be efficacious in preventing
and controlling nosocomial CDAD.
Some strains of C difficile are nontoxinogenic;
these strains do not cause disease. Clinically significant strains produce
2 protein exotoxins, toxin A, a 308-kd protein, and toxin B, an approximately
270-kd protein; these toxins are largely responsible for disease manifestations.
However, rare cases of CDAD caused by toxin A–negative, toxin B–positive
strains have been reported.38, 39
The genes encoding both toxins are located very near each other on a 19–kilobase-pair
pathogenicity island in toxinogenic strains.40
Toxin A binds to a specific receptor on the brush border of the intestinal
epithelium, a glycoprotein with an  -linked galactose; the intestinal
receptor in humans for toxin B is less well characterized.41
After binding to appropriate receptors, the toxins are internalized and act
within the eukaryotic cell to modify proteins covalently in the Rho subfamily,
a group of low-molecular-weight GTP-binding proteins involved in regulation
of the F-actin cytoskeleton.42 Both toxins
modify Rho proteins at a specific threonine residue by addition of a glucose
molecule, leading to inactivation of the protein.43, 44, 45
This is followed by disaggregation of polymerized actin, opening of tight
junctions between cells, cell rounding, and subsequent cell death. The toxins
also induce the release by various cells of proinflammatory mediators and
cytokines, as well as activation of the enteric nervous system, leading to
neutrophil chemotaxis and fluid secretion.46
Toxin B is not enterotoxic in animals (as is toxin A), but it is a much more
potent cytotoxin in tissue culture than toxin A, and full tissue damage requires
the action of both toxins.22, 47
Early histological changes due to C difficile
toxins include patchy epithelial necrosis with an exudate of neutrophils and
fibrin; the exudate may appear to "erupt" into the colonic lumen and, if prominent,
such a histological feature has been termed a summit
or volcano lesion. More prominent histological changes
are characterized by diffuse epithelial necrosis and ulceration with development
of an overlying pseudomembrane containing cellular debris, leukocytes, fibrin,
and mucin (Figure 1).48
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Figure 1. Histological section of colon
infected with Clostridium difficile colitis. Diffuse epithelial
necrosis with development of an overlying pseudomembrane containing cellular
debris, leukocytes, fibrin, and mucin is seen. Vessels in the lamina propria
are engorged and an inflammatory polymorphonuclear cell infiltrate is present
throughout the mucosal surface.
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In animal models, antibodies directed against toxin A prevent toxin
binding, neutralize secretory and inflammatory effects, and limit or prevent
clinical disease.49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59
The immune response to toxin B is less well understood, but anti–toxin
B antibodies also appear to protect against C difficile–associated disease.60, 61
Clinical studies suggest that the level of anti-toxin antibody in serum
is inversely associated with severity of disease and risk for relapse.32, 62, 63, 64, 65
Serum levels of IgG antibody to toxin A are lower in children with recurrent
CDAD than in age-matched controls, whereas rising levels of anti–toxin
A antibodies have been associated with resolution of clinical disease.63, 66, 67 Also, serum samples
of convalescent patients contain IgG and IgA that can neutralize toxin A67; immunization with a toxoid of toxin A has achieved
active protection from clinical disease52, 68;
and passive protection from clinical disease is achieved by the administration
of anti–toxin A antibodies.50, 51, 65
In humans, therefore, systemic anti–toxin IgG and IgA and mucosal IgA
all appear to be involved in protective immunological responses to C difficile infection.
Antibodies against C difficile toxins are present
in most adults and older children (>60%).61, 62
The presence of such antibodies may be due to repetitive, transient presence
of toxigenic C difficile organisms in the normal
intestinal flora. Cellular immunity appears to be of lesser import, but has
not been studied in detail.
CLINICAL MANIFESTATIONS
The clinical presentation of CDAD is variable and includes diarrhea,
colitis without pseudomembranes, pseudomembranous colitis, and fulminant colitis
(Table 2). Some individuals with
toxigenic strains in stool remain totally asymptomatic.31
Mild to moderate CDAD is usually accompanied by lower abdominal cramping pain
but no systemic symptoms or physical findings.22
Moderate or severe colitis usually presents with profuse diarrhea, abdominal
distention with pain, and, in some cases, occult colonic bleeding. Also, systemic
symptoms such as fever, nausea, anorexia, and malaise are usually present.22 A minority of patients have disease primarily in
the cecum and right colon, presenting with marked leukocytosis and abdominal
pain but little or no diarrhea.
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Table 2. Common Signs, Symptoms, and Diagnostic Features of Clostridium difficile– Associated Diarrhea
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Fulminant colitis develops in approximately 1% to 3% of patients, with
ileus, toxic megacolon, perforation, and death.20
Clinicians should be aware that the development of these life-threatening
complications may be accompanied by a decrease in diarrhea due to loss of
colonic muscular tone and ileus.
Other complications of C difficile infection
include hyperpyrexia, chronic diarrhea, and hypoalbuminemia with anasarca.11 Clostridium difficile infection
may occasionally complicate idiopathic inflammatory bowel disease. Also, a
reactive arthritis occurring 1 to 4 weeks after C difficile colitis develops in some patients, resembling that seen after other
enteric infections such as Yersinia, Shigella, and Salmonella disease.69
DIAGNOSIS
Nonspecific laboratory abnormalities in patients with CDAD include leukocytosis
with a shift to the left and fecal leukocytes in about 50% to 60% of cases.
The average peripheral white blood cell count is 12 x 109/L
to 20 x 109/L, but occasionally the peripheral count is higher
and cases of leukemoid reaction have been described.6, 11, 70
Even among patients with the most severe colitis, C difficile constitutes only a small part of the colonic flora, and
its morphologic features are identical to that of other Clostridia species, making Gram staining of fecal specimens of no value
in diagnosing CDAD. Clostridium difficile can be
isolated by means of anaerobic culture of stool, but this test is seldom used
for clinical diagnosis (it takes 2-3 days to complete and does not distinguish
toxinogenic from nontoxinogenic strains; nontoxinogenic strains are not associated
with clinical illness).
The most sensitive and specific test available for diagnosis of C difficile infection is a tissue culture assay for the
cytotoxicity of toxin B, using preincubation with neutralizing antibody to
show the specificity of the cytotoxicity.22, 71
This test can detect as little as 10 pg of toxin in stool and has a high sensitivity
(94%-100%) and specificity (99%).20, 22
However, the test takes 1 to 3 days to complete and requires tissue culture
facilities.
More recently, enzyme-linked immunosorbent assays (ELISAs) have been
developed to detect toxin A and/or B in stool. These assays detect 100 to
1000 pg of either toxin, and have a sensitivity of 71% to 94% and a specificity
of 92% to 98%.71, 72 Because of
the rapidity of testing and ease of performance, ELISAs for toxins A and B
are now used most frequently by clinical laboratories for diagnosis of C difficile infection. Approximately 5% to 20% of patients
may require more than 1 stool assay to detect toxin.23
If CDAD is suspected, a single stool specimen should be sent. If the results
are negative and diarrhea persists, 1 or 2 additional stool samples can be
sent.73, 74 The cytotoxicity assay
for toxin B will detect an additional 5% to 10% of cases missed by ELISA techniques,
and it may be useful to perform this test if the ELISA results are negative
but clinical suspicion is high (Table 3).20 Diagnostic testing during or
at the end of treatment or during follow-up is not needed, unless symptoms
recur.73 Transmission of infection to other
patients is associated with ongoing diarrhea and not with the presence of
the toxin in stool. Enteric precautions can be removed when diarrhea subsides,
without the need for repeated diagnostic testing.73
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Table 3. Diagnostic Tests for Detecting Clostridium difficile*
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Studies have also evaluated other laboratory methods for the diagnosis
of CDAD. The polymerase chain reaction is extremely sensitive but requires
technical expertise and is unable to distinguish between asymptomatic carriage
and symptomatic infection.75 A dot-immunobinding
assay yielded fair initial results several years ago, but this test has had
very limited clinical experience since.76 Counterimmunoelectrophoresis
for detection of C difficile antigens has demonstrated
poor positive predictive value, and a latex agglutination test is less sensitive
than other available tests; newer forms of ELISAs are under evaluation.77, 78
Radiographic imaging studies can be used to assist in making a diagnosis
of CDAD. Abdominal imaging studies may reveal paralytic ileus and a dilated
colon (Figure 2). Diffusely thickened
or edematous colonic mucosa may sometimes be seen by means of abdominal computerized
tomographic scans (Figure 3); such
thickening can present as "thumbprinting" on abdominal plain films.
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Figure 2. Abdominal plain radiograph showing
ileus and mucosal thickening due to Clostridium difficile disease.
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Figure 3. Abdominal computerized axial tomography
findings of an individual with Clostridium difficile colitis.
Thickened wall of the descending colon is seen on the left side.
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Endoscopy for CDAD is reserved for special situations, such as when
other diseases need to be ruled out, rapid diagnosis is necessary, or a stool
sample cannot be obtained because ileus develops.74
The results of sigmoidoscopy may be normal in patients with mild disease.
Endoscopy in more severe colitis without pseudomembrane formation usually
reveals diffuse or patchy, nonspecific colitis.22
In more dramatic cases, endoscopy may demonstrate characteristic, adherent
yellow plaques that vary in diameter from 2 to 10 mm and in some cases, coalesce
to cover large areas of the mucosa (Figure
4). In most cases, the rectum and sigmoid are involved, but 10%
of episodes of colitis involve only the right colon and may not be detected
by flexible sigmoidoscopy.79 Due to the increased
risk for intestinal perforation, endoscopy should be used sparingly as a diagnostic
tool in patients with suspected CDAD.
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Figure 4. Gross appearance of colon with
multiple foci of Clostridium difficile–associated pseudomembranes
with characteristic adherent yellow plaques that vary in diameter from 2 to
10 mm and can coalesce to cover large areas of the mucosa.
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TREATMENT
Practical guidelines for the treatment of CDAD are summarized in Table 4. The inciting antibiotic therapy
should be discontinued if possible, and supportive therapy should be given
with fluids and electrolytes as needed. Education of hospital personnel and
infection control issues are of paramount importance in the management of
CDAD. Education should reinforce the importance of handwashing between patients
and the need for glove use for the handling of bodily substances of all patients.
Hospital personnel should thoroughly disinfect objects contaminated with C difficile and should be familiar with the disease and
its epidemiological features.73 Enteric isolation
precautions are recommended for patients with CDAD, and patients should be
moved to a private room if possible.74, 80
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Table 4. General Guidelines for the Management of Clostridium difficile–Associated Diarrhea*
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Antiperistaltic and opiate drugs should be avoided in patients with
CDAD, because they mask symptoms and may worsen the course of the disease.74, 81 Diarrhea will resolve without specific
antimicrobial therapy in up to one fourth of patients.73, 82, 83
Antimicrobial therapy for CDAD is indicated for patients with moderate
or severe disease or significant underlying conditions. There is no evidence
that treatment of asymptomatic carriers of C difficile
provides any clinical benefit, and such therapy has been associated with a
prolongation of the carrier state.20, 84
In the appropriate clinical setting, antimicrobial therapy should be instituted
even before the laboratory results for C difficile
are available.
Oral metronidazole and vancomycin hydrochloride are the antibiotics
most commonly used, whereas bacitracin methylene disalicylate, teicoplanin,
and fusidic acid also have some clinical efficacy. Clostridium
difficile is uniformly susceptible to vancomycin, but occasional strains
have been reported that are resistant to metronidazole.81, 85
Two studies have directly compared initial therapy consisting of metronidazole
(in an oral dose of 500 mg 3 times per day or 250 mg 4 times per day [qid])
with oral vancomycin hydrochloride (500 mg qid), each given for 10 days, and
have shown that these therapies are equally efficacious in resolving symptoms
as well as in the subsequent risk for relapse.83, 86
Another study examined different doses of vancomycin hydrochloride and showed
that an oral dose of 125 mg qid was equivalent to one of 500 mg qid for 10
days for symptomatic response and risk for subsequent relapse.87
In many series of patients, therapy with metronidazole or vancomycin was effective
in resolving symptoms in more than 95% of patients, although 10% to 20% of
patients subsequently experienced relapse.22, 71, 81
Because of lower cost and avoidance of selective pressure for vancomycin-resistant
organisms such as vancomycin-resistant enterococci, initial therapy with oral
metronidazole (either 250 mg qid or 500 mg 3 times per day) is currently the
preferred initial therapy for C difficile colitis.
However, some authorities prefer initial therapy with vancomycin in the most
severely ill patients, or in women who are pregnant.74, 81
The duration of initial therapy should be 10 to 14 days (or therapy may be
continued until 1 week after completion of the inciting antibiotic therapy,
if it cannot be stopped earlier).
Metronidazole is well absorbed from the small intestine and produces
low or undetectable levels in stool as C difficile
diarrhea subsides, whereas oral vancomycin is not absorbed and produces high
fecal levels even in the absence of diarrhea.84, 88
In the presence of severe ileus or toxic megacolon, intravenous metronidazole
hydrochloride (given as 500 mg every 8 hours) produces fecal concentrations
above the inhibitory concentration for C difficile
and may be used for initial therapy.88 Patients
receiving metronidazole should avoid alcohol, because it may result in disulfiramlike
reactions.74 Metronidazole resistance in C difficile has been reported, but it is probably rare.89
Oral bacitracin methylene disalicylate (20 000 to 25 000 U
qid) has been shown to be as effective as vancomycin in ameliorating clinical
symptoms of C difficile disease, but it was less
effective in eradicating the organism or toxin from stool; patients treated
with bacitracin also had a higher risk for subsequent relapse.90, 91
Bacitracin should be reserved for unusual situations, in which metronidazole
and vancomycin cannot be used.
In critically ill patients who are unable to take oral antimicrobials,
treatment is empirical and may include intravenous metronidazole or administration
of vancomycin by means of rectal enema or through long catheters in the small
intestine. Intravenous treatment can be used with metronidazole but not vancomycin.
Occasionally, surgery (usually subtotal colectomy) is required.5, 73
Despite the high initial response rate to therapy, recurrence of symptoms
develops in 10% to 20% of patients, usually within the first 2 weeks after
initial treatment is discontinued.76 Two studies
attempted to identify possible risk factors for recurrent CDAD. High-risk
groups included patients with chronic renal failure, those with multiple previous
episodes of CDAD, individuals who had to continue other antibiotic therapy,
patients with community-acquired CDAD, those with high white blood cell counts
( 15 x 109/L), and those infected with certain strains
of C difficile.85, 92
About 50% of all recurrences of symptoms are actually due to reinfection and
not to relapse of the primary infection.93, 94
The first relapse or recurrence of C difficile
colitis can be treated with another 10- to 14-day course of oral metronidazole
or vancomycin, either of which produces a response rate of approximately 95%;
mild disease can be managed without further antibiotic treatment.20, 71
A smaller number of patients have multiple relapses. One study showed
a relapse rate of 24% after an initial episode of C difficile colitis, but a relapse rate of 65% after treatment of recurrent disease.85 Antimicrobial resistance as the mechanism for relapse
has not been documented, and one hypothesis is that spores of C difficile persist in the colon or the environment of the patient
and that subsequent germination in the intestine produces vegetative forms
and clinical illness. Therapy for multiple relapses of C difficile colitis has not been examined by randomized, prospective,
controlled clinical trials and the best therapeutic approach is currently
uncertain.74 A tapering course of metronidazole
or vancomycin during a 4- to 6-week period has been used. A 6-week tapering
course of vancomycin, as seen in the following tabulation, may allow spores
to germinate during days between vancomycin therapy, with killing of vegetative
forms on
reexposure20, 95:
In one study, such an approach cured all 22 patients who had had multiple
previous relapses.95
Another approach to relapsing CDAD is the use of vancomycin hydrochloride
with oral cholestyramine resin (4 g twice daily). Cholestyramine binds C difficile toxins and may assist in amelioration of CDAD.
Since cholestyramine also binds vancomycin, patients should be instructed
to stagger their doses of cholestyramine and vancomycin (as well as other
drugs) by at least 3 hours.20 Oral vancomycin
hydrochloride, 125 mg qid, and oral rifampin, 600 mg twice daily for 7 days,
may have some increased efficacy compared with vancomycin alone. Relapsing C difficile disease has also been treated with a combination
of Saccharomyces boulardii, beginning 4 days before
the end of a 10-day course of standard antimicrobial therapy such as vancomycin.
In one study, this combination reduced the relapse rate in patients with previous
relapses compared with use of vancomycin alone.95, 96
However, results with this approach were not as good as published studies
examining the tapering vancomycin regimen above,92, 93
and there is no evidence that the concomitant use of S boulardii with antibiotics alters diarrhea or prevents the appearance of C difficile toxin in the stool. Saccharomyces
boulardii is not available in many countries, including the United
States, but Saccharomyces cerevisiae (brewer's yeast)
is more widely available, and has been used successfully in patients with
refractory CDAD.97, 98 A small
number of patients with refractory C difficile disease
have responded to antimicrobial therapy in conjunction with an infusion of
intravenous immunoglobulin at a dose of 200 to 300 mg/kg,99
or to orally administered bovine colostrum antibody harvested from cows immunized
against C difficile100, 101, 102, 103
or orally administered human IgA,104 but these
are not yet accepted therapies. Similarly, reconstitution of intestinal flora
through bacteriotherapy has occasionally been used in the treatment of individuals
with refractory CDAD.105, 106
CONCLUSIONS
Clostridium difficile is the most common cause
of nosocomial diarrhea and is a leading cause of diarrhea in the elderly.
The antibiotics most frequently associated with infection are clindamycin,
ampicillin and/or amoxicillin, and cephalosporins; however, all antibiotics
have been associated with CDAD, and nonessential antibiotic prescription should
be avoided.107 The clinical presentation may
vary from mild diarrhea to severe debilitating infection. In some cases, life-threatening
complications may be accompanied by a decrease in diarrhea due to ileus. Education
of hospital personnel, appropriate diagnostic testing, early treatment, and
measures for preventing spread of the pathogen may avert morbidity and mortality.
AUTHOR INFORMATION
Accepted for publication August 22, 2000.
This work was partially supported by grants AI01332 (Dr Ryan) and AI40725
(Dr Calderwood) from the National Institute of Allergy and Infectious Diseases,
Bethesda, Md. Dr Mylonakis is the recipient of a postdoctoral research fellowship
for physicians from the Howard Hughes Medical Institute, Chevy Chase, Md.
We thank Rex Neal Smith, MD, for his assistance with pathological specimens.
From the Division of Infectious Diseases, Massachusetts General Hospital
(Drs Mylonakis, Ryan, and Calderwood), and the Department of Microbiology
and Molecular Genetics, Harvard Medical School (Dr Calderwood), Boston, Mass.
Corresponding author and reprints: Stephen B. Calderwood, MD, Division
of Infectious Diseases, Gray-Jackson 504, Massachusetts General Hospital,
55 Fruit St, Boston, MA 02114 (e-mail: scalderwood{at}partners.org).
REFERENCES
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