title: Rapid molecular characterization of Clostridium difficile and assessment of populations of C. difficile in stool specimens.
abstract: Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12201-2002, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Wroblewski
authors.forename: George E
authors.initials: GE
authors.lastname: Hannett
authors.forename: Dianna J
authors.initials: DJ
authors.lastname: Bopp
authors.forename: Ghinwa K
authors.initials: GK
authors.lastname: Dumyati
authors.forename: Tanya A
authors.initials: TA
authors.lastname: Halse
authors.forename: Nellie B
authors.initials: NB
authors.lastname: Dumas
authors.forename: Kimberlee A
authors.initials: KA
authors.lastname: Musser
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D016360:Clostridioides difficile / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016172:DNA Fingerprinting; D016521:Electrophoresis, Gel, Pulsed-Field; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis* / Q000382:microbiology; D004768:Enterotoxins / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009518:New York; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D037521:Virulence Factors; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium