We evaluated a genus-and group-specific PCR assay panel using 284 prosthetic knee synovial fluid samples collected from patients presenting to our institution with implant failure. Using the Musculoskeletal Infection Society diagnostic criteria, 88 and 196 samples were classified as showing prosthetic joint infection (PJI) and aseptic failure (AF), respectively. Sensitivities of the synovial fluid PCR panel and culture were 55.6% and 76.1% (P < 0.001), respectively, and specificities were 91.8% and 97.4% (P ؍ 0.016), respectively. Among the 70 subjects who had received antibiotics within the month preceding synovial fluid aspiration (48 of whom had PJI), PCR panel and synovial fluid culture sensitivities were 64.5% and 85.4%, respectively (P < 0.0001). In this group, the PCR panel detected Staphylococcus aureus in two culture-negative PJI cases. Overall, the evaluated molecular diagnostic tool had low sensitivity when applied to synovial fluid.A s a result of the increase in joint arthroplasty placement in the recent past, we are witnessing increasing numbers of prosthetic joint infections, creating an economic burden on our health system (1, 2). The diagnosis and management of these infections are complex (3-6), with outcome contingent on detection and identification of the causative microorganism(s) (6, 7). The gold standard, culture of synovial fluid, periprosthetic tissue, and/or the implant itself, has been associated with a range of sensitivities (8-14) and has various turnaround times of up to 2 weeks.In recent years, several nucleic acid amplification tests (e.g., PCR) have been evaluated in an attempt to improve the microbiologic diagnosis of prosthetic joint infection (PJI). The potential benefits of these assays include improved turnaround time compared to culture and increased microbiological detection, especially in the setting of prior antibiotic administration, when culture sensitivity may be low (15-21). Results to date have been conflicting, likely due to the various methodologies and specimen types evaluated. While PCR has achieved satisfactory results in tests of prosthesis sonication fluid (19, 21), its value is less clear in tests of synovial fluid.To date, most studies that have evaluated PCR of synovial fluid have been based on detection of the 16S rRNA gene (16)(17)(18)(22)(23)(24)(25)(26)(27). The advantage of this approach is the lack of required specific knowledge of the bacterial group in anticipation of testing (22). Among the major drawbacks of 16S rRNA-based assays are a requirement for the extra step of DNA sequencing to characterize the detected organism, challenges in detecting multiple organisms in polymicrobial infections, and the potential for false-positive results because of the ubiquity of bacterial DNA in containers and reagents used for testing. Studies have shown various sensitivities and specificities (16,17); while false-positive results are not infrequent (18), lack of amplification due to a small amount of bacterial DNA in synovial fluid is also common (25,27).Our...