Automated detection of toxigenic Clostridium difficile in clinical samples: isothermal tcdB amplification coupled to array-based detection.
Clostridium difficile can carry a genetically variable pathogenicity locus (PaLoc), which encodes clostridial toxins A and B. In hospitals and in the community at large, this organism is increasingly identified as a pathogen. To develop a diagnostic test that combines the strengths of immunoassays (cost) and DNA amplification assays (sensitivity/specificity), we targeted a genetically stable PaLoc region, amplifying tcdB sequences and detecting them by hybridization capture. The assay employs a hot-start isothermal method coupled to a multiplexed chip-based readout, creating a manual assay that detects toxigenic C. difficile with high sensitivity and specificity within 1 h. Assay automation on an electromechanical instrument produced an analytical sensitivity of 10 CFU (95% probability of detection) of C. difficile in fecal samples, along with discrimination against other enteric bacteria. To verify automated assay function, 130 patient samples were tested: 31/32 positive samples (97% sensitive; 95% confidence interval [CI], 82 to 99%) and 98/98 negative samples (100% specific; 95% CI, 95 to 100%) were scored correctly. Large-scale clinical studies are now planned to determine clinical sensitivity and specificity.
Great Basin Scientific, Salt Lake City, Utah, USA. bhicke@gbscience.com
Brian
B
Hicke
Chris
C
Pasko
Benjamin
B
Groves
Edward
E
Ager
Maylene
M
Corpuz
Georges
G
Frech
Denton
D
Munns
Wendy
W
Smith
Ashley
A
Warcup
Gerald
G
Denys
Nathan A
NA
Ledeboer
Wes
W
Lindsey
Charles
C
Owen
Larry
L
Rea
Robert
R
Jenison
D057205:Automation, Laboratory / Q000379:methods*; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000175:diagnosis / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile