—  SPECIALTY CONFERENCE  —

Infectious Disease Pathology

Case 5 - Clostridium Difficile Infection

Jeannette Guarner
Emory University
Atlanta, GA





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Clinical History
57 year old man being treated for acute lymphocytic leukemia with persistent diarrhea.

Pertinent Laboratory Data:

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.


Case 5 - Figure 1

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 [1] . 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 ) [2, 3, 4] . 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 [5] . 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 [6] . 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 [7, 8, 9, 10] . In adults with C. difficile-associated diarrhea, pseudomembranes were detected in 51 to 89% of patients by endoscopy and in 63% by histology [6, 7] . 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 [10] . 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 [8] . 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 [11] . 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 [9] . 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 [6].

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 [12] . 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 [13] . The 2 assays that have been used as reference standard include the cell culture cytotoxicity assay (CCA) and the toxigenic culture [14] . 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 results.

The first description of an EIA to detect C. difficile in stool was in 1981 [15] . 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% [14] . 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 [16] .

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 [17] . 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) [18, 19, 20] . Some authors have added a third test that determines severity of inflammation to their algorithm: lactoferrin [18] . 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. difficile [12] . 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 [21] . 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 [13] .

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 [12, 14, 22, 23]
tcdB BD GeneOhm (BD Diagnostics, LaJolla, CA, USA) 83.6 to 100 95.4 to 99.4 [12, 14, 17, 23]
tcdB Xpert (Cepheid, Sunnyvale, CA, USA) 94.4 to 100 93 to 96.7 [12, 14, 19, 24]


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.

References
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