Infectious Disease Pathology
Case 5 -
Clostridium Difficile Infection
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57 year old man being treated for acute lymphocytic leukemia with persistent diarrhea.
Stool cultures negative; ova and parasites negative; 4 stoool specimens obtained on different days 1 week prior to the biopsy were negative for Clostridium difficile toxin by ELISA.
Pathology of antibiotic-associated colitis
Pseudomembranous colitis is characterized by discrete yellow plaques in the mucosa of the colon and
rectum. Histologically the colonic crypts are dilated due to abundant inflammatory cells and debris and
as they discharge this exudate into the colonic lumen they appear as erupting volcanoes  . The great
majority of cases with pseudomembranous colitis are associated with Clostridium
difficile infection. However, other infections agents have been found to show similar macroscopic
and microscopic features including viruses (cytomegalovirus), parasites (Entamoeba
histolytica), and other bacteria ( enterotoxin-producing Clostridium
perfringens, Staphylococcus aureus, Shigella
dysenteriae , Escherichia coli O157:H7,
and Klebsiella oxytoca )
. It is interesting to note that before C.
difficile was associated with diarrhea due to antibiotics, S. aureus
was thought to be the culprit of antibiotic-associated diarrhea and presence of pseudomembranes in the
colon and rectum  . Some authors have commented that when non-C.
difficile pseudomembranes occur these are in reality ulcerated lesions covered by inflammatory
membranes. In addition, in the case of cytomegalovirus and amoeba, the presence of viral inclusions or
the protozoan should alert the pathologist to the appropriate diagnosis.
It is calculated that only 25% of hospitalized patients with antibiotic-associated diarrhea have
C. difficile infections  . The spectrum of C.
difficile-associated disease varies from mild diarrhea to pseudomembranous colitis and toxic
megacolon. There have been few studies that correlate the evidence of presence of C. difficile or its toxins in stools and endoscopic and histopathologic findings
. In adults with C. difficile-associated diarrhea, pseudomembranes
were detected in 51 to 89% of patients by endoscopy and in 63% by histology
. Patients with
evidence of C. difficile toxins in their stool that do not show
pseudomembranes either have normal endoscopies or erythema. Mogg et al showed that only 31% of patients
with diarrhea have pseudomembranous colitis histologically and presence of C.
difficile toxin in stool, 27% have positive histology and negative C.
difficile toxin, while 42% have suspicious histology with positive toxin in stool  . In severe
cases of C. difficile-associated diarrhea, the presence of pseudomembranes
may not occur if the patient is neutropenic or immunosuppressed thus unable to produce the inflammatory
response that creates the characteristic pseudomembranes  . The lack of pseudomembrane formation with
evidence of C. difficile toxins has been described in patients that have
received hematopoietic stem cell transplantation and those with ulcerative colitis.
In children, C. difficile toxin is frequently detected in their stools
and the association with antibiotic use is not always present  . In eleven children with positive
C. difficile toxin in stools, the histopathology of endoscopic biopsies (8
cases), resections (2 cases), or autopsy material (one case) demonstrated pseudomembranous colitis in the
autopsy sample, intestinal necrosis in two samples, granulomatous inflammation in one, moderate colitis
in one, and mild to minimal pathology in seven  . In this pediatric series there were 3 cases with
clinical syndromes associated with C. difficile: a patient with acute
lymphocytic leukemia and pseudomembranous colitis, a 4-week old with necrotizing enterocolitis, and a
12-week old with Hirschsprung disease. Immunohistochemistry and PCR testing of the pediatric tissues
only showed evidence of clostridia the patient with pseudomembranes. The findings in adults and children
suggest that the correlation between the presence of C, difficile toxin in
stool and typical pseudomembranous colitis is not always present probably due to the broad spectrum of
disease associated with C. difficile and the poor sensitivity of the toxin
assays in stool.
While validating commercially available PCR assays in Emory Medical Laboratories, we identified 7
patients that had positive C. difficile toxin in stool and in whom an
endoscopy with biopsies were performed. The biopsies were obtained 70 days before to 25 days after the
first stool sample was sent for C. difficile testing. Pathology ranged from
ulcers covered by pseudomembranes (3 cases) to no significant pathology (one case). In only three of
these patients we detected the tcdB gene by PCR in stool and the pathology
in these patients included one with ulcer and pseudomembranes, one with a fistula and active colitis, and
a third with focal active colitis. Further correlation studies are needed to better define the role of
C. difficile PCR testing in stool specimens.
Risk factors and pathobiology of C. difficile colitis
Patients at risk of C. difficile infections are those receiving multiple
antibiotics for long periods of time. Rarely the disease has been observed in patients that receive one
dose of antibiotic prophylaxis before surgery. Clindamycin and lincomycin were the original antibiotics
associated with C. difficile infections, nowadays use of these antibiotics
is limited and fluoroquinolones and cephalosporins have now replaced them as the cause of
antibiotic-associated colitis. In addition to having received an antibiotic, patients living in long
term facilities, people over 65 years old, pregnant women, and cancer patients are at risk of the
infection. The risk of acquiring the infection is related to being in contact with spores of C. difficile which survive in the environment and can be transmitted by fomites
(thermometers, commodes, bed pans) or the hands of healthcare workers .
The 2 toxins responsible for the secretory diarrhea and inflammation are toxin A and toxin B, both
encoded in the pathogenicity locus (PaLoc} of C. difficile
 . It was
originally thought that both toxins acted synergistically to disassemble actin filaments, disrupt tight
junctions, and produce cell death; however, knockout mice have shown that only toxin B is necessary to
produce diarrhea. The production of the toxins only occurs during the late-logarithmic growth of
wild-type C. difficile strains and toxin production is under control of 2
other proteins encoded in the PaLoc: TcdR and TcdC which serve as positive and negative regulators
respectively. The C. difficile strains responsible for the increased
incidence and severity of disease beginning 2001 (NAP1/BI/027), have been found to have mutations in
tcdC gene which leads to production of toxin B at all phases of growth as
well as increased production of the toxin.
Laboratory diagnosis of C. difficile colitis
Diagnosis of the specific organism that is causing the antibiotic-associated diarrhea is important
for treatment and prevention purposes. Regrettably C. difficile laboratory
diagnosis has been controversial since the technologies available have been problematic  . The 2
assays that have been used as reference standard include the cell culture cytotoxicity assay (CCA) and
the toxigenic culture  . In the CCA 2 cell monolayers are incubated one with a stool filtrate and the
second with the stool filtrate and an anti-toxin antibody (neutralization) and are observed for toxin
cytopathic effect. The assay is called positive if the culture with the neutralizing antibody does not
show the cytopathic effect present in the culture incubated with only the stool filtrate. In the
toxigenic culture, the bacteria are selectively cultured and any C.
difficile colonies are tested for the presence of toxin using either CCA, enzyme immunoassay (EIA)
or PCR. Both methods are labor intensive and provide specific diagnosis several days after the patient
has been ill. Although treatment can be started empirically as soon as the specimens are obtained, this
is problematic because the patient has to be placed in isolation precautions while waiting for test
The first description of an EIA to detect C. difficile in
stool was in 1981  . The EIAs commercially available are of 2 varieties, those that detect a
constitutive enzyme present in all C. difficile strains, glutamate
dehydrogenase (GDH), and those that detect the toxins which may only detect toxin A or a combination of
toxin A and B. The most popular strategy used by hospitals laboratories has been to use one EIA that
detects the toxins because these tests target the presence of C. difficile
toxin in stool, are easy to perform, and give results the same day. Nevertheless, the sensitivity of
these assays varies between 31 to 99% and the specificity ranges from 65 to 100%  . The high
specificity of these EIAs indicate that a patient with a positive result has high probability of C. difficile antibiotic-associated disease; however, because of the lack of
sensitivity, physicians tend to repeat the test multiple times hoping to find the toxin. Yet, Cardona
and Rand have shown that repeating EIA testing within two days does not increase the possibility of
positive results  .
There are EIAs that combine in one cartridge testing for GDH and toxins A/B (C. Diff Quik Chek
Complete dual-antigen EIA, TechLab, Blacksburg, VA). Quinn et al showed that this test has a sensitivity
of 78.3% with a specificity of 100% and provide an interesting comparison of this test with commercial
and home-brewed PCR assays regarding turn-around time, hands-on, and costs  . They show that the cost
of this dual EIA test is similar to their home-brewed PCR assay, lower than commercially available PCR
assays, but has a much shorter turn-around time and is less technically demanding.
Several authors have advocated the use of algorithms where the GDH test is performed first and all
positive samples are then tested using more specific tests (either EIA for the toxins, one of the culture
techniques, or PCR)
. Some authors have added a third test that determines severity of
inflammation to their algorithm: lactoferrin  . Lactoferrin is an iron-binding glycoprotein that is
released when neturophils degranulate and has been used to assess the degree of inflammation in patients
with inflammatory bowel disease. Wren et al suggest that all GDH positive samples should be tested with
lactoferrin to differentiate mild disease from moderate to severe disease.
In recent years, FDA approved PCR assays have come to the forefront in clinical
laboratories. These assays are performed in stool specimens and have usually targeted the tcdB or tcdC toxin genes of C.
 . The sensitivity of PCR assays varies from 77 to 100% and the specificity from 93
to 99% (table of commercially available PCR assays). Due to the higher sensitivity of PCR assays
compared to EIA toxin detection, repeat testing of stool specimens within seven days is not needed  .
However, detection of these C. difficile genes does not indicate viability
of the bacteria nor can PCR assess severity of disease or bacteria present in the stool but not causing
disease. Thus, the clinical value of C. difficile PCR assays still needs to
be determined  .
Sensitivities and Specificities for commercially available C. difficile PCR assays
|Gene target ||Manufacturer ||Sensitivity ||Specificity ||Reference|
|tcdB ||ProGastro (Gen-Probe Prodesse, Waukesha, WI, USA) ||77.3 to 91.9 ||99 to 99.2 ||
|tcdB ||BD GeneOhm (BD Diagnostics, LaJolla, CA, USA) ||83.6 to 100 ||95.4 to 99.4 ||
|tcdB ||Xpert (Cepheid, Sunnyvale, CA, USA) ||94.4 to 100 ||93 to 96.7 ||
2010 Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) guidelines for diagnosis of C. difficile infection (6)
What is the best testing strategy to diagnose C. difficile infection in
the clinical laboratory and what are acceptable options?
1. Testing for C. difficile or its toxins should be performed only on
diarrheal (unformed) stool, unless ileus due to C. difficile is suspected.
2. Testing of stool from asymptomatic patients is not clinically useful, including use as a test of
cure. It is not recommended, except for epidemiological studies.
3. Stool culture is the most sensitive test and is essential for epidemiological studies.
4. Although stool culture is not clinically practical because of its slow turnaround time, the
sensitivity and specificity of stool culture followed by identification of a toxigenic isolate (ie,
toxigenic culture), as performed by an experienced laboratory, provides the standard against which other
clinical test results should be compared.
5. Enzyme immunoassay (EIA) testing for C. difficile toxin A and B is
rapid but is less sensitive than the cell cytotoxin assay, and it is thus a suboptimal alternative
approach for diagnosis.
6. Toxin testing is most important clinically, but is hampered by its lack of sensitivity. One
potential strategy to overcome this problem is a 2-step method that uses EIA detection of glutamate
dehydrogenase (GDH) as initial screening and then uses the cell cytotoxicity assay or toxigenic culture
as the confirmatory test for GDH-positive stool specimens only. Results appear to differ based on the
GDH kit used; therefore, until more data are available on the sensitivity of GDH testing, this approach
remains an interim recommendation.
7. Polymerase chain reaction (PCR) testing appears to be rapid, sensitive, and specific and may
ultimately address testing concerns. More data on utility are necessary before this methodology can be
recommended for routine testing.
8. Repeat testing during the same episode of diarrhea is of limited value and should be discouraged.
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