A low complexity rapid molecular method for detection of Clostridium difficile in stool

PLoS One. 2014 Jan 8;9(1):e83808. doi: 10.1371/journal.pone.0083808. eCollection 2014.

Abstract

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+), n = 111; tcdB(-), n= 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+), n = 47; tcdB(-), n= 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+), n = 25; tcdB(-), n= 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

MeSH terms

  • Clostridioides difficile / genetics*
  • Clostridioides difficile / isolation & purification*
  • Feces / microbiology*
  • Gene Dosage
  • Humans
  • Models, Biological
  • Molecular Diagnostic Techniques / methods*
  • Reagent Kits, Diagnostic
  • Real-Time Polymerase Chain Reaction
  • Reference Standards
  • Sensitivity and Specificity

Substances

  • Reagent Kits, Diagnostic

Grants and funding

The authors have no support or funding to report.