title: Diagnosing toxigenic Clostridium difficile: new confidence bounds show culturing increases sensitivity of the toxin A/B enzyme immunoassay and refute gold standards.
abstract: OBJECTIVE
  To scrutinize published sensitivity estimates obtained using questionable gold standards by comparing sensitivities of culturing Clostridium difficile in commercially available media followed by enzyme immunoassay (EIA) toxin A or B detection (culture test) with applying the EIA to stool samples alone (direct test).
  
  
  METHODS
  In 2008, consecutive stool samples were cultured on C. difficile selective culture media: (1) medium I: Clostridium difficile-selective agar (CDSA; Becton Dickinson); (2) medium II: CLO agar (BioMérieux); (3) medium III: C. difficile agar according to Brazier (Oxoid). In addition, a direct test was performed (Ridascreen, r-Biopharm), which was also used to confirm toxin A or B production in the cultured C. difficile. New confidence bounds for sensitivities were applied, without assuming any perfect reference test or any conditional independence of the tests compared.
  
  
  RESULTS
  Of 256 liquid stool samples, 18.4% were diagnosed as positive by at least 1 of the 4 tests; 12.8% were positive with culture medium I, 16.4% with medium II, and 13.6% with medium III, and 10.1% were positive by the direct test. Assuming culture tests to be at least as specific as the direct test yields an upper bound of 61% (upper 95% confidence bound (CB) 81%) for the sensitivity of the direct test. Assuming a prevalence of 15% yields sensitivity gains of the culture tests of at least 18% (lower 95% CB--4%) for medium I, 40% (lower 95% CB 21%) for medium II, and 23% (lower 95% CB 2%) for medium III.
  
  
  CONCLUSIONS
  Published high sensitivities of direct toxin A/B EIAs, up to 96%, and the correctness of the cytotoxicity test assumed for their estimation are doubtful. With culture medium II, sensitivity gains of at least about 20% are obtainable. Direct toxin A/B EIAs alone are insufficiently sensitive for the clinical diagnosis of C. difficile infections.
authors.affiliation: Institut für Hygiene, Universitätsklinikum der privaten Universität Witten-Herdecke, Campus Köln-Merheim, Köln, Germany. mattnerf@kliniken-koeln.de
authors.forename: Frauke
authors.initials: F
authors.lastname: Mattner
authors.forename: Ingo
authors.initials: I
authors.lastname: Winterfeld
authors.forename: Lutz
authors.initials: L
authors.lastname: Mattner
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D001427:Bacterial Toxins / Q000032:analysis*; D015373:Bacterial Typing Techniques / Q000379:methods* / Q000592:standards; D016360:Clostridioides difficile / Q000145:classification / Q000302:isolation & purification* / Q000472:pathogenicity; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis / Q000382:microbiology*; D004768:Enterotoxins / Q000032:analysis*; D005243:Feces / Q000382:microbiology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile