New Test for Clostridium difficile Allows Rapid, Accurate Diagnosis

November 23, 2010 (San Jose, California) — A self-contained molecular assay for Clostridium difficile allows sensitive and specific identification within 24 hours, and detects the toxin A gene (tcdA) in addition to the toxin B gene (tcdB).

C difficile–associated diarrhea is on the rise in both hospital and community settings, and with it comes an increased need for a rapid diagnostic test to identify the presence of C difficile and help guide treatment, noted Brianne Couturier, PhD, from the ARUP Institute for Clinical Experimental Pathology at the University of Utah School of Medicine, Salt Lake City, during her poster presentation here at the Association for Molecular Pathology 2010 Annual Meeting.

Dr. Couturier presented the results of a new molecular assay that rapidly detects tcdA, along with the traditional tcdB of C difficile, which is considered "essential for virulence," Dr. Couturier said.

She said the results of her study might reopen the debate on the importance of toxin A in clinical disease that many thought had been settled a decade ago.

The study involved 40 clinical isolates from 20 species of C difficile and non–C difficile samples. The researchers compared the abilities of 2 assays to recognize various C difficile strain types and toxin classes and to determine crossreactivity with other Clostridium spp.: the illumigene C difficile assay (Meridian Biosciences), which targets the highly conserved 5′ region of tcdA, using LAMP (loop-mediated isothermal amplification), and tcdB; and the GeneOhm C difficile assay (BD Diagnostics).

The technology had not been cleared by the US Food and Drug Administration at the time the Utah team submitted their study abstract, but subsequently was cleared.

Dr. Couturier explained to Medscape Medical News that although tcdA is present, it is not necessarily upregulated to produce all of the toxin proteins. "The majority of toxins in [toxin] A strains are due to large deletions in the 3′ end of the gene; however, the 5′ end is highly conserved," which is where the assay primer attaches. "Even if it is a toxin A–negative strain, the illumigene assay is able to pick it up."

She said the 2 assays were 100% concordant in identifying the B⁺ strains of C difficile. The illumigene assay was able to detect toxin A⁺B⁺ strains of toxinotypes 0 (16 strains), III (6 strains), V (6 strains), XII (1 strain), and IX/XXIII (1 strain). It also amplified A^–B⁺ strains of toxinotypes VIII (3 strains) and X (1 strain).

As a negative control, neither assay was able to amplify 34 non–C difficile, Clostridium spp. isolates, including the closely related Clostridium sordelii (n = 3).

One of the samples was positive on the illumigene assay and negative on the GeneOhm assay. When she looked more closely, Dr. Couturier found that the sample contained 2 polytypes. "When I isolated them out, one was C difficile A⁺B⁺ and the other was Clostridium symbiosum."

Dr. Couturier prefers the illumigene assay because it is self-contained and a bit quicker. "The BD GeneOhm has the same license mechanism; however, you have to make up the master mix separately. If you don't make full runs, you're wasting reagent, whereas with the illumigene [assay], you don't waste reagents." The workflow is improved and technician time is saved.

Donna Wolk, PhD, D(ABMM), a microbiologist at the University of Arizona, Tucson, pointed out that evidence emerged several years ago to suggest that toxin B is the essential virulence factor for clinical disease caused by C difficile. Toxin A is thought to act in synergy with toxin B. The role of toxin A is still evolving, and reports are conflicting. This study suggests that "maybe the story is not over," Dr. Wolk noted.

"Researchers made an interesting disease association, and these results will require further confirmation to determine whether toxin A alone is sufficient to cause disease. The assay may be a useful tool for exploring those disease connections," she said.

The study was underwritten by Meridian Biosciences. Dr. Couturier is employed by the ARUP Institute for Clinical and Experimental Pathology at the University of Utah. Dr. Wolk has disclosed no relevant financial relationships.

Association for Molecular Pathology (AMP) 2010 Annual Meeting: Abstract ID55. Presented November 18, 2010.