Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile.
A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A-B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A-B-, and A-B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A-B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A-B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.
Lemee
Ludovic
L
Groupe de Recherche sur les Antimicrobiens et les Microorganismes, Université de Rouen, UFR Médecine-Pharmacie, and Service de Bactériologie, Centre Hospitalier Universitaire, France.
Dhalluin
Anne
A
Testelin
Sabrina
S
Mattrat
Marie-Andre
MA
Maillard
Karine
K
Lemeland
Jean-François
JF
Pons
Jean-Louis
JL
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D016360:Clostridioides difficile / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D003470:Culture Media; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis* / Q000382:microbiology; D004768:Enterotoxins / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction; D014305:Triose-Phosphate Isomerase / Q000235:genetics*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003470:Culture Media; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D014305:Triose-Phosphate Isomerase