tcdAAs a Diagnostic Target in a Loop-Mediated Amplification Assay for Detecting ToxigenicClostridium difficile

Abstract

Background The illumigene® (Meridian Bioscience, Inc., Cincinnati, OH) and GeneOhm® (BD Diagnostics, La Jolla, CA) Clostridium difficile assays target the tcdA gene and tcdB gene, respectively. We assessed the use of tcdA as the molecular target in the illumigene® C. difficile loop‐mediated amplification assay in detecting a wide variety of C. difficile strains including those with tcdA deletions. Methods We tested 38 C. difficile strains and 108 patient stool specimens using the illumigene® assay. The GeneOhm® real‐time polymerase chain reaction (PCR) assay served as the reference method. Discordant results were resolved by repeat testing, anaerobic culture, and a laboratory‐developed real‐time PCR targeting tcdA and tcdB. Results Both illumigene® and GeneOhm® assays detected all 37 C. difficile toxin B+ strains representing seven toxinotypes and including four toxin A−B+ isolates. No cross‐reactivity with 20 other Clostridium species or toxin‐negative C. difficile was observed in either assay. Among patient stool specimens, agreement was 94.4% (102/108). After discordant result resolution, agreement was 96.3% (104/108). Specimens with initially discordant results had target concentrations approaching the limit of detection for the two commercial assays. Discordance appeared unrelated to whether tcdA or tcdB was the amplification target. Conclusion The tcdA 5′ region used by the illumigene® assay is a practical target for toxigenic C. difficile detection.

Accuracy of loop-mediated isothermal amplification for the diagnosis of Clostridium difficile infection: a systematic review

“…While some variant C. difficile strains have deletions at the 3' end of the tcdA gene, the 5' portion remains intact for these strains [12]. A recent study confirmed the ability of the Illumigene assay to detect these Toxin A-/B+ strains [13]. The…”

A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection

“…Firstly, the assays are performed in stool containing a large amount of extraneous DNA which may interfere with the detection [ 11 ]. Recent studies however have not shown much cross reactivity [ 19 , 20 ]. Secondly, the mere presence of toxin genes is not proof of toxin expression.…”

Evaluation of the Cepheid Xpert C. difficile/ Epi and Meridian Bioscience illumi gene C. difficile Assays for Detecting Clostridium difficile Ribotype 033 Strains

“…The illumigene C. difficile assay did not detect the remnant fragments of the tcdA gene in any of the RT033 isolates tested, consistent with a deletion in the 5= end region (4, 5). The illumigene C. difficile assay was designed to target a "conserved" region at the 5= end of tcdA present in all known A ϩ B ϩ and A Ϫ B ϩ toxinotypes (13). This was supported by Couturier et al, who reported detection of the tcdA target sequence in A Ϫ B ϩ strains belonging to toxinotypes VIII and X using the illumigene C. difficile assay (13), and in the present study for RT017 (toxinotype VIII) strains which have a truncated tcdA gene retaining only the A1 fragment of the gene (4).…”

“…The illumigene C. difficile assay was designed to target a "conserved" region at the 5= end of tcdA present in all known A ϩ B ϩ and A Ϫ B ϩ toxinotypes (13). This was supported by Couturier et al, who reported detection of the tcdA target sequence in A Ϫ B ϩ strains belonging to toxinotypes VIII and X using the illumigene C. difficile assay (13), and in the present study for RT017 (toxinotype VIII) strains which have a truncated tcdA gene retaining only the A1 fragment of the gene (4). Other A Ϫ B ϩ ribotypes tested (RT237, RT280, and RT281) that were known to contain a truncated tcdA region (14,15) were not detected by the illumigene C. difficile assay.…”