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Multiplex Real-Time Polymerase Chain Reaction: A Practical Approach for Rapid Diagnosis of Tuberculous and Brucellar Vertebral Osteomyelitis

Colmenero, Juan D., MD*; Morata, Pilar, PhD; Ruiz-Mesa, Juan D., MD*; Bautista, Dolores, MD; Bermúdez, Pilar, MD§; Bravo, María José, PhD¶∥; Queipo-Ortuño, María Isabel, PhD*∥

doi: 10.1097/BRS.0b013e3181e8eeaf
Diagnostics
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Study Design. Case-control study for assessing a diagnostic test.

Objective. The aim of this study was to analyze the diagnostic yield of a multiplex real-time polymerase chain reaction (PCR) assay in the differential diagnosis of tuberculous vertebral osteomyelitis (TVO) and brucellar vertebral osteomyelitis (BVO).

Summary of Background Data. Vertebral osteomyelitis (VO) is one of commonest osteoarticular complications of tuberculosis and brucellosis. However, the very similar clinical, radiologic, and histologic characteristics of these entities mean that diagnosis requires etiological confirmation, but conventional microbiologic methods have important limitations.

Methods. Fifteen vertebral samples from patients with TVO or BVO and 9 from pyogenic and nontuberculous mycobacteria VO were studied by multiplex PCR and conventional microbiologic techniques. To identify Brucella DNA, we used a fragment of 207 bp from the conserved region of the gene coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and for Mycobacterium tuberculosis complex, a fragment of 164 bp from the intergenic region SenX3-RegX3.

Results. The histopathologic findings were inconclusive in 4 of 14 cases (28.6%) with TVO or BVO and cultures were positive in 11 of 15 cases (73.3%). Multiplex PCR correctly identified 14 of the 15 samples from patients with TVO and BVO and was negative in all the control samples. Thus, the overall sensitivity and specificity of the multiplex PCR were 93.3% and 90%, respectively, with an accuracy of 92% (95% CI, 81.4%–100%).

Conclusion. These results suggest that multiplex real-time PCR is far more sensitive than conventional cultures, and this, together with its speed, makes this technique a very practical approach for the differential diagnosis between TVO and BVO.

The case-control study analyze the diagnostic yield of a multiplex real-time polymerase chain reaction for the differential diagnosis between tuberculous vertebral osteomyelitis and brucellar vertebral osteomyelitis in comparison with conventional microbiological methods. The sensitivity and specificity of the multiplex polymerase chain reaction were 93.3% and 90%, respectively, with an accuracy of 92%. Our results suggest that this multiplex real-time polymerase chain reaction is more sensitive than conventional cultures and can provide results to the clinician within 4 hours.

From the *Infectious Diseases Service, Carlos Haya University Hospital, Málaga, Spain; †Department of Biochemistry and Molecular Biology, University of Malaga, Malaga, Spain; ‡Department of Pathology, Carlos Haya University Hospital, Málaga, Spain; §Microbiology Service, Carlos Haya University Hospital, Málaga, Spain; ¶Immunology Service, Carlos Haya University Hospital, Málaga, Spain; and ∥IMABIS Foundation, Carlos Haya University Hospital, Málaga, Spain.

Acknowledgment date: March 15, 2010. Revision date: May 6, 2010. Acceptance date: May 10, 2010.

The manuscript submitted does not contain information about medical device(s)/drug(s).

Institutional funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

Supported by the Instituto de Salud Carlos III (ISCIII), F.I.S (grant PI06/0495), and the Consejería de Innovación Ciencia y Empresa (grant CTS-276) and Excellence Project (grant P-08 CTS 3969), both of the Junta de Andalucía, Spain.

All patients provided written informed consent prior to the collection of biological samples. The utilization of samples for research purposes was approved by the Ethical Committee of Carlos Haya University Hospital, Malaga, Spain.

Address correspondence and reprint requests to María Isabel Queipo-Ortuño, PhD, Fundación Imabis, Hospital Universitario Carlos Haya, 29010 Malaga, Spain; E-mail: iqueipo@uma.es

© 2010 Lippincott Williams & Wilkins, Inc.