Simulated Joint Infection Assessment by Rapid Detection of Live Bacteria with Real-Time Reverse Transcription Polymerase Chain Reaction

Birmingham, Patrick; Helm, Jeannine M.; Manner, Paul A.; Tuan, Rocky S.
March 2008
Journal of Bone & Joint Surgery, American Volume;Mar2008, Vol. 90-A Issue 3, p602
Academic Journal
Background: Although microbiological bacterial culture is currently considered the gold standard for diagnosis of septic arthritis, many studies have documented substantial false-negative and false-positive rates. The objective of this study was to determine whether real-time quantitative reverse transcription polymerase chain reaction can be used to detect bacterial messenger RNA (mRNA) in synovial fluid as a way to distinguish live and dead bacteria as an indicator of active infection. Methods: Synovial fluid samples were obtained from twelve consecutive patients who presented with knee pain and effusion but no evidence of infection. Following assurance of sterility with plate cultures, each sample was inoculated with clinically relevant bacteria and incubated for twenty-four hours to simulate septic arthritis. Bacterial viability and load were assessed with cultures. Selected samples were also treated with a single dose of a combination of two antibiotics, vancomycin and gentamicin, and sampled at several time points. Total RNA isolated from each sample was analyzed in triplicate with one-step real-time quantitative reverse transcription polymerase chain reaction to detect mRNA encoding for the genes groEL or femC. Controls included sterile, uninoculated samples and inoculated samples analyzed with quantitative polymerase chain reaction without reverse transcription. mRNA content was estimated on the basis of detection limits as a function of serial dilutions and was expressed as a function of colony number in bacterial cultures and RNA content as determined spectrophotometrically. Results: All synovial fluid samples that had been inoculated with one of the four bacterial species, and analyzed in triplicate, were identified (distinguished from aseptic synovial fluid) with real-time quantitative reverse transcription polymerase chain reaction; there were no false-negative results.


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