Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples.
BACKGROUND
The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile.


METHODS
The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR.


RESULTS
A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 10(3) colony forming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%.


CONCLUSIONS
The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.
Department of Microbiology, Gulhane Military Medical Academy, Etlik, Ankara, Turkey. ; Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA. ; St Luke's Episcopal Hospital, Houston, TX, USA.
Abdullah
A
Kilic
Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA.
Mohammad J
MJ
Alam
St Luke's Episcopal Hospital, Houston, TX, USA.
Naradah L
NL
Tisdel
Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA.
Dhara N
DN
Shah
Department of Microbiology, Gulhane Military Medical Academy, Etlik, Ankara, Turkey.
Mehmet
M
Yapar
St Luke's Episcopal Hospital, Houston, TX, USA.
Todd M
TM
Lasco
Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA. ; St Luke's Episcopal Hospital, Houston, TX, USA.
Kevin W
KW
Garey
D036002:ADP Ribose Transferases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D016360:Clostridioides difficile / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction; D014305:Triose-Phosphate Isomerase / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium; D014305:Triose-Phosphate Isomerase
Binary toxin; Clostridium difficile; Multiplex real-time PCR; Toxin variant strains