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Bone and joint infections pose significant clinical challenges due to their complexity and the associated high morbidity. Traditional microbiological culture is the gold standard, but has several limitations, including slow time to results and low sensitivity (i.e., culture produces false negative results, especially in patients on antibiotics before sample collection). In recent years, molecular diagnostic techniques, such as the BIOFIRE Joint Infection (JI) Panel, have been developed to overcome these challenges. This study aims to evaluate the diagnostic yield of tissue biopsies (TBs) compared to synovial fluid (SF) specimens in detecting pathogens responsible for these infections using the BIOFIRE JI Panel. The study included 151 SF specimens and 324 paired TBs from 151 subjects with suspected septic arthritis or prosthetic joint infection. Results showed that the BIOFIRE JI Panel had a high diagnostic yield, detecting pathogens in 31.7% of SF specimens and 36.4% of TB specimens, with a high concordance (Kappa = 0.77) between SF BIOFIRE JI Panel and SF culture results, and even better concordance (Kappa = 0.80) between TB BIOFIRE JI Panel and TB culture results. Notably, the BIOFIRE JI Panel demonstrated a higher detection rate in polymicrobial infections, identifying additional microorganisms compared to culture. The study concluded that the BIOFIRE JI Panel improves the diagnostic yield compared to conventional culture, particularly in cases of polymicrobial infections, and that testing TBs can provide valuable information when SF samples yield negative or inconclusive results. These findings suggest that PCR testing on TBs could be a useful adjunct to improve the diagnosis and management of bone and joint infections, especially when a prompt result is required.IMPORTANCEBone and joint infections are difficult to diagnose and treat, often leading to prolonged illness and serious complications. Traditional culture methods, while widely used, can be slow and sometimes miss the true cause of infection, especially if patients are already receiving antibiotics. This study shows that a rapid molecular test, the BIOFIRE Joint Infection Panel, can improve the detection of bacteria in both joint fluid and tissue samples. Importantly, the test was able to identify more infections, including those caused by different species of bacteria, compared to conventional methods. These findings highlight the potential of this approach to provide faster and more accurate results, which could help clinicians choose the right treatment earlier. By supporting quicker and more reliable diagnoses, this method may ultimately improve outcomes for patients with complicated bone and joint infections.