title: Diagnosing periprosthetic infection: false-positive intraoperative Gram stains.
abstract: BACKGROUND
  Intraoperative Gram stains have a reported low sensitivity but high specificity when used to help diagnose periprosthetic infections. In early 2008, we recognized an unexpectedly high frequency of apparent false-positive Gram stains from revision arthroplasties.
  
  
  OBJECTIVE
  The purpose of this report is to describe the cause of these false-positive test results.
  
  
  METHODS
  We calculated the sensitivity and specificity of all intraoperative Gram stains submitted from revision arthroplasty cases during a 3-month interval using microbiologic cultures of the same samples as the gold standard. Methods of specimen harvesting, handling, transport, distribution, specimen processing including tissue grinding/macerating, Gram staining, and interpretation were studied. After a test modification, results of specimens were prospectively collected for a second 3-month interval, and the sensitivity and specificity of intraoperative Gram stains were calculated.
  
  
  RESULTS
  The retrospective review of 269 Gram stains submitted from revision arthroplasties indicated historic sensitivity and specificity values of 23% and 92%, respectively. Systematic analysis of all steps of the procedure identified Gram-stained but nonviable bacteria in commercial broth reagents used as diluents for maceration of periprosthetic membranes before Gram staining and culture. Polymerase chain reaction and sequencing showed mixed bacterial DNA. Evaluation of 390 specimens after initiating standardized Millipore filtering of diluent fluid revealed a reduced number of positive Gram stains, yielding 9% sensitivity and 99% specificity.
  
  
  CONCLUSIONS
  Clusters of false-positive Gram stains have been reported in other clinical conditions. They are apparently rare related to diagnosing periprosthetic infections but have severe consequences if used to guide treatment. Even occasional false-positive Gram stains should prompt review of laboratory methods. Our observations implicate dead bacteria in microbiologic reagents as potential sources of false-positive Gram stains.
authors.affiliation: Department of Clinical Pathology, The Cleveland Clinic, Cleveland, OH, USA.
authors.forename: Margret
authors.initials: M
authors.lastname: Oethinger
authors.forename: Debra K
authors.initials: DK
authors.lastname: Warner
authors.forename: Susan A
authors.initials: SA
authors.lastname: Schindler
authors.forename: Hideo
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Bauer
mesh_terms: D019643:Arthroplasty, Replacement / Q000009:adverse effects* / Q000295:instrumentation; D005189:False Positive Reactions; D005840:Gentian Violet; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology / Q000601:surgery; D006801:Humans; D007430:Intraoperative Care; D007595:Joint Prosthesis / Q000009:adverse effects*; D008828:Microbiological Techniques / Q000592:standards; D009820:Ohio; D010619:Phenazines; D011237:Predictive Value of Tests; D011446:Prospective Studies; D016459:Prosthesis-Related Infections / Q000175:diagnosis* / Q000382:microbiology / Q000601:surgery; D012086:Reoperation; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000592:standards
chemical_list: C035863:Gram's stain; D010619:Phenazines; D005840:Gentian Violet