Diagnostic Testing of Clostridium difficile
September 1, 2008
Diagnostic Testing of Clostridium difficile
Abstract & Commentary
By Ellen Jo Baron, PhD, D(ABMM), Professor of Pathology and Medicine, Stanford University Medical School Director, Clinical Microbiology Laboratory, Stanford University Medical Center
Dr. Baron reports no financial relationships relevant to this field of study.
Synopsis: CDAD was associated with a significant increase in costs for inpatient care and increased costs at 180 days after the initial hospitilization when the CDAD episode occurred.
Although Enzyme-Immunoassay (EIA) tests have replaced cytotoxin assays for diagnosis of Clostridium difficile-associated diarrhea (CDAD) in most US laboratories, the changing epidemiology of this disease suggests that an adjustment in diagnostic testing algorithms is needed. The move to EIAs was not because the tests were more sensitive, but because they were easier and faster to perform than the gold standard cell culture cytotoxicity with a control neutralization step, now performed in only 1% of US laboratories, according to the most recent College of American Pathologists proficiency testing survey results.
Data compiled by Dr. Cliff McDonald of the CDC, gleaned from US hospital discharge summaries, suggest that there were 100,000 cases of CDAD in 1992 and around 300,000 in 2006. More than half of reported cases occur in long-term care facilities. However, these numbers may underestimate the true numbers of cases.1 Extrapolation from smaller, more accurate studies suggests that there are 500,000 cases/year and 23,000 deaths nationwide.2 Costs are usually estimated for hospitalization only, but if patients are followed for six months after discharge, true costs of disease probably range from $3800-$7200 per patient, totaling greater than $1 billion/year. C. difficile infection can increase length of stay 2.8 days,3 and the subsequent costs due to long-term care facility utilization or home care contribute to the overall figures.
Also fueling the renewed interest in CDAD is the swift spread of a relatively recently recognized and more virulent strain known variously as NAP1 (North American Pulsed-Field type 1), PCR ribotype 027, or restriction endonucleas analysis (REA) type BI ("bee eye"). C. difficile diarrheal cases are currently approximately 80% healthcare-associated and 20% community-associated. Half of all strains studied in the United States in the past two years are of the BI strain, which has also emerged rapidly as a major healthcare-associated pathogen in western Europe. Enhancing the spread of the BI strain is its resistance to fluoroquinolones (FQs) concurrent with expanding utilization of FQs worldwide. Outbreaks are unlikely to be controlled by switching one FQ for another (from levofloxacin to gatifloxacin, for example).4 However, in at least one institution after FQs were removed from the formulary completely, the rates of CDAD decreased.5 Authorities agree that both infection control measures and antibiotic restrictions are needed for optimal control.
Several factors are implicated in the rapid emergence of this hypervirulent epidemic strain. Increased spore production, allowing better survival in the environment, particularly in an institutional setting, helps to explain the rapid spread and persistence of BI. Enhanced adherence to human intestinal epithelial cells mediated by a surface protein A appears to be another major virulence factor for this strain.6 Yet another is its increased toxin output. The toxin genes are governed by regulatory proteins, all residing on a pathogenicity locus (PaLoc). Increased toxin production in the hypervirulent strains may be due to a point mutation in the regulatory gene tcdC. Toxin production in most C. difficile strains begins when cells enter stationary phase after they run short of nutrients. However, BI strains produce 16x higher levels of Toxin A and 23 times the levels of Toxin B throughout all growth stages. Toxin B has now been shown to be the primary toxin.7 NAP1 strains also produce a characteristic binary toxin, although the contribution of this toxin to severity of disease is not known.
So why should laboratories change their testing strategies? Apparently, recent studies show that currently popular EIA tests are not as sensitive as previously thought.8 Given the severity of CDAD disease and the need for rapid diagnosis, the other well-established test, initial stool culture on cycloserine-cefoxitin fructose agar (with taurocholate to enhance the production of vegetative cells from spores), followed by toxin testing, does not yield a turnaround time rapid enough for meaningful clinical decisions. One possible solution is the use of a rapid and very sensitive screening test, such as an immunochromatographic test, for glutamate-dehydrogenase, a protein present in all C. difficile strains, whether toxigenic or not. This protein is not exclusive to C. difficile, however, so GDH assay specificity is not sufficient for a stand-alone test. A study from Johns Hopkins showed that a GDH screen had a sensitivity of 100%, so no potential cases were missed with this system.8 Many laboratories are thus moving to a strategy of offering the GDH test as a rapid screen and reflecting all positives to a reference test such as cyto-toxin. GDH test sensitivities vary, so microbiologists need to study the literature to choose the appropriate commercial product.
The ultimate diagnostic test will likely prove to be molecular. Two pre-clinical trials of one PCR-based test have shown better sensitivity than the GDH screening methods. Once commercially produced rapid molecular assays are widely available, which no doubt will transpire within the next year, they will surely replace all three of the current diagnostic strategies: the two-test algorithm, culture followed by cytotoxin assay, and cell culture cytotoxicity.
Reference
- Gould CV, McDonald LC. Bench-to-bedside review: Clostridium difficile colitis." Crit Care. 2008;12:203.
- Redelings MD, et al. Increase in Clostridium difficile-related mortality rates, United States, 1999-2004. Emerg Infect Dis. 2007;13:1417-1419.
- Dubberke ER, et al. Short- and long-term attributable costs of Clostridium difficile-associated disease in nonsurgical inpatients. Clin Infect Dis. 2008;46:497-504.
- Gaynes R, et al. Outbreak of Clostridium difficile infection in a long-term care facility: association with gatifloxacin use. Clin Infect Dis. 2004;38:640-645.
- Dubberke ER, et al. Clostridium difficile-associated disease in a setting of endemicity: identification of novel risk factors. Clin Infect Dis. 2007;45:1543-1549.
- Waligora AJ, et al. Characterization of a cell surface protein of Clostridium difficile with adhesive properties. Infect Immun. 2001;69:2144-2153.
- Lyras D, et al. Are toxin A and toxin B essential virulence factors of Clostridium difficile? Abstracts of the 9th Biennial Congress of the Anaerobe Society of the Americas , 75. 2008. Ref Type: Abstract
- Ticehurst JR, et al. Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin. J Clin Microbiol. 2006;44:1145-1149.
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