Objective. Blood cultures and cultures of disc material are required to identify and treat bacterial agents responsible for septic spondylodiscitis, but these methods have limited sensitivities. We undertook this study to compare nonculture amplification-based DNA analysis with conventional culture of disc aspirate.Methods. Nineteen patients with spondylodiscitis, including 11 with a history of spinal surgery, presented with negative blood cultures and underwent percutaneous disc or epidural abscess puncture for bacterial diagnosis. Amplification by polymerase chain reaction was performed on 16S ribosomal DNA universal target genes and femA staphylococci-specific target genes in all patients, and on the upstream p34 mycobacterial gene in 1 patient. Species identification relied on amplicon sequencing and comparison with templates from GenBank. Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene. Further assessment using a staphylococciand methicillin resistance-specific DNA array was performed on 3 samples.Results. Microbiologic and molecular assays identified the causative organism in 14 of 19 patients (74%) and 19 of 19 patients (100%), respectively. In culture-positive patients, DNA-based and microbiologic results were highly correlated. Five agents (Staphylococcus simulans, Staphylococcus sciuri, Brucella species, Actinomyces israelii, and Mycobacterium tuberculosis complex) were identified only by DNA-based methods. In 1 sample, Corynebacterium jeikeium and coagulasenegative Staphylococcus were both cultured, whereas DNA analysis identified only Staphylococcus hominis.Conclusion. DNA-based methods are highly sensitive and specific. They can usefully complement standard microbiologic methods for identifying the cause of infectious spondylodiscitis and contribute to speciesspecific therapeutic orientation in patients with negative blood and disc aspirate cultures.Infectious spondylodiscitis consists of concurrent infection of the intervertebral disc and adjacent vertebral osteomyelitis, observed spontaneously or after invasive spinal procedures or surgery (1-3). Magnetic resonance imaging (MRI) is the diagnostic technique of choice, with sensitivity and specificity of 96% and 94%, respectively, whereas radiographic abnormalities only appear several weeks or months after clinical symptoms (4,5).Given the wide spectrum of potential pathogens (Staphylococcus aureus, coagulase-negative staphylococci [CoNS], streptococci, gram-negative bacilli, Mycobacterium species, fungi), definite identification of the microbial agent is mandatory before initiation of longduration and appropriate parenteral antibiotic therapy. In the absence of bacteremia, percutaneous disc aspiration or biopsy is performed for pathogen identification, but conventional microbiologic techniques fail to identify the causative agent in 30-50% of patients (2,6-9).Nonculture molecular methods based on the amplification and/or sequence analysis of conserved or species-specific genomic ...