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Indeterminate tcdB using a Clostridium difficile PCR assay: a retrospective cohort study.

Jerome A Leis1, Wayne L Gold, John Ng

  • 1Division of Infectious Diseases, Department of Medicine, University Health Network/Mount Sinai Hospital, Toronto, ON, Canada. jerome.leis@mail.utoronto.ca

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Summary

A small percentage of C. difficile (CD) PCR tests yield indeterminate results, often indicating actual infection. Clinicians must consider clinical context alongside PCR results for accurate diagnosis of CD infection (CDI).

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Area of Science:

  • Clinical microbiology and infectious diseases.
  • Molecular diagnostics and assay interpretation.

Background:

  • Real-time polymerase chain reaction (PCR) for Clostridioides difficile (C. difficile) toxin B gene (tcdB) offers improved sensitivity and reduced turnaround time compared to toxin immunoassays.
  • The Xpert® C. difficile assay can yield negative results despite typical amplification curves with high tcdB cycle thresholds (Ct) and low endpoints (Ept), necessitating investigation into their clinical significance.

Purpose of the Study:

  • To determine the clinical significance of indeterminate results from the Xpert® C. difficile assay, characterized by typical amplification curves but negative interpretation.
  • To evaluate the association between indeterminate results, clinical case definitions of C. difficile infection (CDI), and illness severity.

Main Methods:

  • Defined indeterminate Xpert® C. difficile assay results as typical PCR amplification curves with an Ept >10, interpreted as negative.
  • Collected and retested indeterminate samples using Xpert®, cultured for toxigenic C. difficile, and performed PCR ribotyping, toxin gene detection, and MLVA typing.
  • Reviewed patient charts for adherence to CDI clinical case definitions and compared illness severity with tcdB Ct and culture results.

Main Results:

  • During the study, 1% of specimens yielded indeterminate results, while 11% were positive for C. difficile.
  • Of patients with indeterminate results, 81% met the clinical case definition for CDI, with 18% experiencing severe CDI.
  • Lower tcdB Ct and higher Ept were associated with increased likelihood of toxigenic culture positivity and more severe symptoms; indeterminate results were not linked to specific strains, technologists, or equipment.

Conclusions:

  • A small subset of Xpert® C. difficile assay results (1%) are falsely negative despite typical amplification curves, with at least one-third indicating positive C. difficile culture.
  • The mechanism behind these indeterminate results is independent of technical factors or specific C. difficile strains.
  • Clinicians must recognize that PCR testing can miss CDI cases and emphasize the importance of integrating clinical context for accurate interpretation of C. difficile diagnostic results.