title: Indeterminate tcdB using a Clostridium difficile PCR assay: a retrospective cohort study.
abstract: BACKGROUND
  C. difficile (CD) real-time polymerase chain reaction (PCR) for toxin B gene (tcdB) is more sensitive, and reduces turnaround time when compared to toxin immunoassay. We noted typical amplification curves with high tcdB cycle thresholds (Ct) and low endpoints (Ept) that are labeled negative by the Xpert(®) C. difficile assay (Cepheid) and undertook this study to determine their significance.
  
  
  METHODS
  We defined an indeterminate CD assay result as detection of a typical PCR amplification curve with an Ept >10 that was interpreted as negative by the Xpert(®) assay. Samples with indeterminate Xpert(®) result were collected for 5 months and retested by Xpert(®), cultured for toxigenic CD, and isolates subjected to PCR ribotyping, detection of toxin genes and multilocus variable-number tandem repeat analysis (MLVA) typing. Chart reviews were completed to assess if patients met the Society of Healthcare Epidemiology of America and the Infectious Diseases Society of America CD infection (CDI) clinical case definition. Illness severity was compared with tcdB Ct and culture results.
  
  
  RESULTS
  During the 5-month study period, 48/3620 (1%) of specimens were indeterminate and 387/3620 (11%) were positive. Of the 48 patients with indeterminate results, 39 (81%) met the clinical case definition of CDI, and 7 of these (18%) met criteria for severe CDI. Toxigenic stool cultures were positive for 86% (6/7) of patients with severe CDI, 19% (6/32) of patients with non-severe CDI, and 44% (4/9) of patients who did not meet the clinical case definition of CDI (p = 0.002). Lower tcdB Ct and higher Ept were associated with greater likelihood of toxigenic culture positivity (p = 0.03) and more severe symptoms (p = 0.06). Indeterminate results were not associated with a particular technologist or instrument module, or CD strain type.
  
  
  CONCLUSIONS
  A subset of specimens (1%) using the Xpert(®) C. difficile assay have typical amplification curves and are interpreted as negative. At least one-third of these results are associated with positive CD culture. The mechanism of these indeterminate results is not technique-related, equipment-related, or due to particular CD strains. Clinicians should be aware that even PCR testing has the potential to miss CDI cases and further highlights the importance of clinical context when interpreting results.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Health Network/Mount Sinai Hospital, Toronto, ON, Canada. jerome.leis@mail.utoronto.ca
authors.forename: Jerome A
authors.initials: JA
authors.lastname: Leis
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Gold
authors.forename: John
authors.initials: J
authors.lastname: Ng
authors.forename: Zahir
authors.initials: Z
authors.lastname: Hirji
authors.forename: Dylan R
authors.initials: DR
authors.lastname: Pillai
authors.forename: George
authors.initials: G
authors.lastname: Broukhanski
authors.forename: Paula
authors.initials: P
authors.lastname: Raggiunti
authors.forename: Susy
authors.initials: S
authors.lastname: Hota
authors.forename: Allison
authors.initials: A
authors.lastname: McGeer
authors.forename: Susan M
authors.initials: SM
authors.lastname: Poutanen
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D016360:Clostridioides difficile / Q000235:genetics* / Q000302:isolation & purification; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*; D005188:False Negative Reactions; D005243:Feces / Q000382:microbiology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D058889:Molecular Typing; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D021521:Ribotyping
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile