Automated detection of toxigenic Clostridium difficile in clinical samples: Isothermal tcdB amplification coupled to array-based detection

Brian Hicke, Chris Pasko, Benjamin Groves, Edward Ager, Maylene Corpuz, Georges Frech, Denton Munns, Wendy Smith, Ashley Warcup, Gerald Denys, Nathan A. Ledeboer, Wes Lindsey, Charles Owen, Larry Rea, Robert Jenison

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2681-2687
Number of pages7
JournalJournal of clinical microbiology
Volume50
Issue number8
DOIs
StatePublished - Aug 1 2012

Fingerprint

Clostridium difficile
Sensitivity and Specificity
Virulence
Confidence Intervals
Automation
Enterobacteriaceae
Immunoassay
Routine Diagnostic Tests
DNA

ASJC Scopus subject areas

  • Microbiology (medical)

Cite this

Automated detection of toxigenic Clostridium difficile in clinical samples : Isothermal tcdB amplification coupled to array-based detection. / Hicke, Brian; Pasko, Chris; Groves, Benjamin; Ager, Edward; Corpuz, Maylene; Frech, Georges; Munns, Denton; Smith, Wendy; Warcup, Ashley; Denys, Gerald; Ledeboer, Nathan A.; Lindsey, Wes; Owen, Charles; Rea, Larry; Jenison, Robert.

In: Journal of clinical microbiology, Vol. 50, No. 8, 01.08.2012, p. 2681-2687.

Research output: Contribution to journalArticle

Hicke, B, Pasko, C, Groves, B, Ager, E, Corpuz, M, Frech, G, Munns, D, Smith, W, Warcup, A, Denys, G, Ledeboer, NA, Lindsey, W, Owen, C, Rea, L & Jenison, R 2012, 'Automated detection of toxigenic Clostridium difficile in clinical samples: Isothermal tcdB amplification coupled to array-based detection', Journal of clinical microbiology, vol. 50, no. 8, pp. 2681-2687. https://doi.org/10.1128/JCM.00621-12
Hicke, Brian ; Pasko, Chris ; Groves, Benjamin ; Ager, Edward ; Corpuz, Maylene ; Frech, Georges ; Munns, Denton ; Smith, Wendy ; Warcup, Ashley ; Denys, Gerald ; Ledeboer, Nathan A. ; Lindsey, Wes ; Owen, Charles ; Rea, Larry ; Jenison, Robert. / Automated detection of toxigenic Clostridium difficile in clinical samples : Isothermal tcdB amplification coupled to array-based detection. In: Journal of clinical microbiology. 2012 ; Vol. 50, No. 8. pp. 2681-2687.
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