Spotlight
Last year, clinicians worldwide received a wake-up call on the extent of the Clostridium difficile infection (CDI) problem. The Association for Practitioners in Infection Control and Epidemiology (APIC) announced the results of a one-day prevalence survey conducted between May and August of 2008 [1]. Test data were received from close to 650 hospitals of varying sizes and types. They were primarily acute care, but encompassed pediatric, cancer, chronic care, and cardiac specialties, and ranged from public to private to county facilities. The numbers were astounding and sobering. More than 12 of every 1000 hospital inpatients were infected with C. difficile. 73% of these patients acquired their disease in the healthcare setting and only half of the patients surveyed had resolution of their symptoms within seven days, highlighting the serious morbidity of CDI.
Wake-up Call Clinical, Medical & Diagnostic Products
Approximately 70% of the infected patients were older than 60 years. 67.6% of all patients had co-morbid conditions and nearly 80% had received antibiotics within the previous 30 days. The mortality statistics were also frightening. In the UK, reporting of CDI is mandatory and a percentage of funding to acute health-care trusts in England and Wales is dependent on the institution’s reduction of the prevalence of CDI.
Previous to the APIC study, the mortality rate from CDI had been estimated at 4.2%. One major shortcoming of the survey was that 94.4% of the positive results were based on an enzyme immunoassay performed by the laboratory. As will be further discussed, the point prevalence survey actually underestimated the depth of the problem by at least 50%.
EXPLAINING THE UNDERESTIMATION
Why are the survey results likely to have actually underestimated the true disease burden from C. difficile? Over the years, laboratories had moved from performing culture and testing the isolates for toxin production (known as toxigenic culture, the true gold standard for detection of toxin-producing C. difficile in feces), to cytotoxin neutralisation cell culture assays. It had been assumed that the cell culture cytotoxin neutralisation test was as reliable as toxigenic culture for CDI diagnosis, although technically difficult and slow to generate results (requiring at least overnight incubation for a preliminary result). However, few laboratories had cell culture expertise, and most laboratories quickly moved on to enzyme immunoassays (EIAs) for either toxin A or toxins A and B, with or without simultaneous testing for a protein traditionally thought to be present in all C. difficile isolates, for example, glutamate dehydrogenase (GDH). Experts believe that the EIA tests gained favor because they were so much easier to perform, did not
“Originally advocated by
Dr Dale Gerding, the toxigenic culture method has been improved in recent years by the use of anaerobic chambers and better agar growth media”
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Dr Ellen Jo Baron, for Cepheid
delaying test results for another 24 hours [5].
Alarmingly, a commonly used toxin A and B EIA test used by most laboratories, likely the type of test most used to detect C. difficile infection in the APIC point prevalence study, was only 36% sensitive against a better comparator [4]. Pitfalls with the GDH algorithm included the use of two separate types of tests and the delay (up to three days) in turnaround time for positive results. However, the Hopkins workers claimed that they saved more than $250,000 per year by not testing all samples for cytotoxin. Although their positive predictive value was only 53%, their negative predictive value was 99.7% [4]. Other experienced workers questioned that approach and the scientists at Hopkins revisited the diagnosis of CDI in their 2009 paper [3][5].
TOXIGENIC CULTURE METHOD
Originally advocated by Dr Dale Gerding, the toxigenic culture method has been improved in recent years by the use of anaerobic chambers and better agar growth media. The selective medium cycloserine-cefoxitin fructose agar, developed at the Wadsworth Veterans Administration hospital during the 1970s [6] has been improved with the addition of horse blood and taurocholate, resulting in better recovery of C. difficile from fecal cultures.
Only stools that take the shape of the container, which is how we define true diarrhea, should be tested - unless the physician indicates that the patient has toxic megacolon. To increase yield, fecal specimens are treated with either heat shock (heat a 20% suspension of feces in chopped meat carbohydrate broth at 80°C for 15 minutes) or alcohol shock (suspend the feces 50/50 in 95% or absolute ethanol and mix gently at room temperature for one hour) to kill vegetative cells and allow spores to remain viable.
The resulting suspension is then plated onto selective media and incubated anaerobically. Colonies will grow after 24-48 hours. On blood agar, these colonies fluoresce chartreuse and have a very distinctive horse manure smell. Colonies are inoculated into chopped meat carbohydrate broth, which is further incubated for up to five days. The supernatant is then tested for cytotoxin using the cell culture cytotoxin neutralisation assay or an EIA for toxin B (less effective) [7].
require expertise or cell culture capability, results were available more quickly, and some early studies showed EIA results comparable to cytotoxin results and thus adequate for laboratory diagnosis of CDI.
COMPARING THE TESTS
A recently published comparison conducted at Johns Hopkins Hospital showed that a toxin B cell culture cytotoxin neutralisation test was only 67% sensitive when compared to toxigenic culture assay (the true gold standard) [3]. A PCR assay for toxin B gene sequences (tcdB gene) was 83.6% sensitive compared with toxigenic culture, but increased to 90.9% when the previous ‘standard’ cell culture cytotoxin B assay was used as the comparison.
The Hopkins group had previously published an influential paper advocating a two-step approach, with GDH used as a preliminary screening test and more intense testing performed only for samples positive for GDH [4]. The cell culture cytotoxin neutralisation was the gold standard for this study, and the results showed the GDH two-step algorithm to perform well. A separate study, however, showed that to reach optimal sensitivity and specificity, cell culture cytotoxicity should be performed on GDH-positive samples, again
The cell culture cytotoxin neutralisation assay uses fecal supernatant, usually diluted 1:100 or 1:200, layered over a monolayer of human or other mammalian cells in culture, similar to cell cultures used for recovering viruses. When present in the feces, cytotoxin B causes cytopathic effect (CPE), for example, rounding up and sloughing off of cells from the monolayer. If this effect is specifically inhibited by a C. difficile toxin B neutralising antitoxin (commercially available), the test is considered positive for C. difficile cytotoxin. CPE begins showing up at 12–18 hours of incubation but laboratories usually wait for 48 hours before finalising a negative result.
QUEST FOR IMPROVEMENT
Dr Jon Rosenblatt and colleagues at Mayo Clinic suspected that laboratories were missing important C. difficile cases using the two-step approach [8]. They developed an in-house PCR test for the tcdC gene, and compared their results to EIAs and GDH detection, using toxigenic culture as the gold standard comparator.
The best performance among any of the four separate EIA assays that they evaluated was 48% sensitivity. But to their surprise, the GDH/toxin assay they evaluated detected only 32% of the samples containing toxin-positive C. difficile, and the GDH component alone was only 76% sensitive versus culture for C. difficile. The Mayo group concluded that GDH was neither a sensitive alternative to culture nor an accurate screening method for toxin-positive stools. [8] Even their own PCR for two C. difficile genetic targets was only 86% sensitive compared with toxigenic culture.
When the results of the APIC point prevalence study are re-
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