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Clostridium difficile (C.diff): A difficult diagnosis
Recent publications have documented better results with the adoption of a two-step approach: very sensitive detection of the organism itself using an EIA for GDH, followed by a second test for cytotoxin and neutralization5,13. Because a rapid test for GDH will be positive in all stool specimens from patients colonized with C. difficile, regardless of toxin status and not subject to degradation during transport, it is the most sensitive test. A stool found to yield a negative result in this initial test can be reported as negative immediately. Because the first sample is the most valuable9, the caregiver can go on to other diagnostic algorithms and the immediate necessity for a decision to change antibiotic therapy can be forestalled. The laboratory must still continue with a cytotoxin assay, which increases turnaround time, or a culture and toxin test, which is also lengthy. At this time, however, the two step approach is the most effective. A more virulent and fluoroquinolone-resistant strain of Clostridium difficile of restriction endonuclease group B1 has been detected increasingly in many parts of the world7,8. The new strain is characterized by production of a binary toxin (CDT) and partial deletions in the toxin A and B repressor gene locus tcdC. Due to the increased morbidity and mortality associated with the new clone, detection of this pathogen takes on new urgency. Because metronidazole is losing effectiveness and vancomycin use should be avoided if possible, even novel probiotic treatment approaches have been gaining popularity, as reviewed by Sullivan and Nord10,12. But physicians really need a more reliable and timely diagnostic test. Can molecular methods come to the rescue? Recent studies suggests that they can2,11. Peterson et al. evaluated a real-time PCR assay that uses primers targeting a conserved region of the toxin B gene. Since all toxin-producing strains evaluated to date carry toxin B (some have deletions in toxin A), this was viewed as a reasonable surrogate marker for toxigenic strains. The study was carried out in two phases. In the first phase, a common commercial EIA test was compared with PCR testing in an analysis of 618 samples sent to the laboratory for C. difficile testing. Compared with the gold standard of anaerobic culture combined with toxin testing, the EIA had a sensitivity of 67% and a specificity of 92%, whereas PCR had a sensitivity of 94% and a specificity of 97%. During this phase, the investigators also developed a set of criteria required for a definitive diagnosis of C. difficile-associated diarrhea (CDAD), and found that virtually all bona fide CDAD cases had 3 or more loose stools per day. For 370 specimens from patients who met this criterion, sensitivity was still higher for PCR than for the EIA (93% vs. 73%). Both the real-time PCR and anaerobic culture assays were significantly more sensitive than the enzyme immunoassay (P<.01 to P<.05). The authors concluded that compared to EIA, real-time PCR is a more sensitive and equally rapid test and should be considered as an option to replace enzyme immunoassay for toxigenic C. difficile detection in clinical practice. References
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Clostridium difficile is living up to its name. Laboratory testing for this troublesome gastrointestinal pathogen has undergone a number of approaches after the initial breakthrough discovery of this anaerobic spore-forming bacterium as a cause of antibiotic-associated pseudomembranous colitis back in the late 1970’s1,3,6. It was appreciated that the organism was carried asymptomatically by children, especially those <1 year old, and some adults, particularly those in the hospital. For this reason, the case definition has always included diarrhea, defined by unformed stools of varying numbers over a period of time. This definition should be used by the microbiology laboratory to initially limit the specimens tested. Because of the rare (<1%) but serious complication of toxic megacolon, in which patients do not exhibit loose stools, microbiologists should refrigerate and hold any formed stools rejected for testing for at least a day after they have notified the caregiver of the rejection, allowing time for the clinician to call back and explain that the test should be performed because the patient has toxic megacolon4. Another controversial decision for laboratories has been how many specimens to test from a single patient if the first specimen is negative for C. difficile (by toxin test or by culture). Although there are publications on both sides of the question, most microbiologists limit the number of specimens per patient per symptomatic episode to one or at most, two9. Early studies characterizing the disease often included histopathological comparisons of mucosal appearance seen macroscopically on endoscopy (pseudomembranes consisting of white or yellow shaggy material composed of sloughed cells) or microscopic lesions seen on colonic mucosal biopsy. This invasive diagnostic test is rarely used today and laboratory testing is the primary mode of diagnosis. That makes the choice of test and its interpretation of critical importance. 
1. Cytotoxin detection in cell culture assays. In this method, filtered stool extracts are added to a healthy monolayer of cells growing in a microtiter plate and incubated up to 48 hours. If the cytotoxin (known as C. difficile toxin B) is present in the sample, it will cause the cells in the monolayer to round up and slough off the plate (called cytopathic effect, CPE). Differentiation from non-specific cytotoxicity is achieved by adding specific anti-toxin (actually developed against a similar organism, C. sordellii) to a second well containing the patient’s stool extract. Only if the original CPE was due to C. difficile toxin B will the antitoxin prevent CPE in the corresponding well. The cytotoxin neutralization (CTN) assay has traditionally been considered to be the most sensitive and specific for laboratory diagnosis, but it has two major drawbacks: results are not available rapidly enough to influence clinical decisions in a timely manner, and the method requires technical expertise and a continuous supply of tissue culture cells14.
3. Anaerobic culture for C. difficile. Culture is among the most sensitive of assays, but a secondary test for presence of toxin production must be performed, as non-toxigenic strains are not uncommon. This lengthens the turnaround time for results delivery. In addition, the organism’s spores are extremely hardy, but vegetative forms are more fragile and anaerobic conditions must be well maintained to allow recovery. A special medium, cycloserine-cefoxitin fructose agar (CCFA), must be stored anaerobically and inoculated and incubated anaerobically for at least 48 hours for optimal recovery. Because of the technical difficulties with culture and the delay for results reporting, C. difficile cultures are performed in relatively few routine diagnostic laboratories.