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

J Clin Microbiol. 2012 Aug;50(8):2681-7. doi: 10.1128/JCM.00621-12. Epub 2012 Jun 6.

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.

Publication types

  • Evaluation Study

MeSH terms

  • Automation, Laboratory / methods*
  • Bacterial Proteins / genetics*
  • Bacterial Toxins / genetics*
  • Bacteriological Techniques / methods*
  • Clostridioides difficile / genetics
  • Clostridioides difficile / isolation & purification*
  • Clostridium Infections / diagnosis
  • Clostridium Infections / microbiology
  • Feces / microbiology
  • Humans
  • Microarray Analysis / methods*
  • Molecular Diagnostic Techniques / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • Sensitivity and Specificity
  • Time Factors

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • toxB protein, Clostridium difficile