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

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 staphylococci- and 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 coagulase-negative 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 species-specific therapeutic orientation in patients with negative blood and disc aspirate cultures.

DNA microarray to monitor the expression of MAGE-A genes.

BACKGROUND: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors. METHODS: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers. The assay involved reverse transcription of total RNA with oligo(dT) primer, followed by PCR amplification and hybridization on a microarray. Amplification in the presence of Biotin-16-dUTP allowed subsequent detection of the amplicons on the microarray carrying 12 capture probes, each being specific for a MAGE-A gene. Probe-amplicon hybrids were detected by a streptavidin-based method. RESULTS: PCR conditions were optimized for low detection limits and comparable amplification efficiencies among all MAGE-A nucleotide sequences. The microarray assay was validated with a panel of 32 samples, by comparison with well-established reverse transcription-PCR assays relying on amplification with primers specific for each gene. Virtually identical results were obtained with both methods, except for MAGE-A3 and MAGE-A5. Detection of MAGE-A5 was more sensitive with the microarray assay. Detection of MAGE-A3 was hampered by the presence of MAGE-A6, which is 98% identical: the MAGE-A3 capture probe cross-hybridized with MAGE-A6 amplicons because these sequences differed by only a single base. CONCLUSIONS: This post-PCR microarray assay could be useful to evaluate MAGE expression in tumors before therapeutic vaccinations with MAGE-A gene products.