The etiologic diagnosis of infectious discitis is improved by amplification‐based DNA analysis

Abstract

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 ...

Actinomyces and Related Organisms in Human Infections

“…neuii together with Dermabacter hominis in the calcaneus (222). Spondylodiscitis can rarely be due to Actinomyces organisms, such as A. meyeri (92) and A. israelii (223). A strain representing the novel Actinomyces species A. timonensis was isolated from an os-teoarticular specimen of a 13-year-old girl suffering from chronic sacroiliitis (224).…”

Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples

“…Amplicons obtained from these samples were sequenced as previously described (Lecouvet et al, 2004). Sequences obtained were compared with public available sequence databases for similarity-based species identification, using the BLAST (http://www.ncbi.nlm.nih.gov/blast/ Blast.cgi).…”

“…TRT-PCR was subsequently performed on DNA extracted from 197 faecal specimens. For each negative sample, PCR inhibition was assessed as previously described (Lecouvet et al, 2004).…”

“…Samples with single IS900 positive signal were a priori considered as doubtful until further investigated. For this purpose, the TRT-PCR product of three samples producing a IS900 single positive TRT-PCR signal was cloned in a TOPO-XL vector according to the manufacturer's instructions (Invitrogen, Merelbeke, Belgium) and sequenced as previously described (Lecouvet et al, 2004). Sequences obtained were compared to public available sequence databases for species identification.…”

Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer (Cervus elaphus) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

“…Briefly, 10 4 copies of a pCR-XL-TOPO plasmid carrying the 16S rRNA gene of Staphylococcus aureus was amplified alone and mixed with the sample. Amplification was carried out as described previously (15). PCR inhibition was considered relevant when no amplification or markedly decreased amplification of 10 4 plasmid copies of S. aureus from the mixture was observed.…”