Targeting the tcdA gene: is this appropriate for detection of A and/or B Clostridium difficile toxin-producing strains?

In a recent article (3), Norén et al. reported an extensive evaluation of illumigene Clostridium difficile compared with the cell culture cytotoxin B assay (CTBA) and/or toxigenic culture (TC) of cytotoxin-producing C. difficile isolates. The illumigene assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile. The authors reported a sensitivity of 98% (49/50 isolates) and a specificity of 98% (218/222 isolates) for the illumigene assay, using combined CTBA plus TC as the gold standard. While demonstrating good clinical performance, the authors raised the question of whether the toxin A fragment amplified by the illumigene assay is the optimal target for detecting toxin A-negative/toxin B-positive (A B ) strains. The Clostridium difficile PaLoc segment encodes both the toxin A gene (tcdA) and the toxin B gene (tcdB), has conserved border regions, and is found at the same site on the C. difficile genome for all toxigenic strains. Both the tcdA and the tcdB genes have similar structures consisting of three distinct fragments: a catalytic domain, a putative translocation domain, and a repetitive domain (4). The repetitive regions are prone to homologous recombination resulting in various deletions at the 3 ends of the toxin genes. For example, the well-characterized A B strains (toxinotypes VI and VII) and the most frequently occurring strains (toxinotypes VIII and X) all have various deletions at the 3 end of the tcdA gene. However, the 5 portion of the tcdA gene remains intact for all of these strains. The rationale for the illumigene design is to target the 5 region of the tcdA gene that is present in all known A B strains and seems to be more conserved than the tcdB gene (5). Studies involving well-characterized C. difficile strains were conducted to confirm the primer design. The illumigene assay detected 4 A B and 30 A B strains (2). Couturier and She (1) independently confirmed these findings. Again, the illumigene assay was able to detect toxin A B strains of toxinotypes 0 (16 strains), III (6 strains), XII (1 strain), and IX/XXIII (1 strain). More importantly, it also amplified toxinotypes VIII (3 strains) and X (1 strain), which are toxin A B (40 non-C. difficile isolates were not detected, including 3 strains of Clostridium sordelii). In summary, the illumigene C. difficile assay was designed to detect the genetically intact PaLoc region of toxigenic C. difficile regardless of expression phenotypes.