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Journal of Clinical Microbiology, April 2008, p. 1386-1390, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02305-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid PCR-Based Diagnosis of Septic Arthritis by Early Gram-Type Classification and Pathogen Identification

Samuel Yang,^1,* Padmini Ramachandran,^1, Andrew Hardick,² Yu-Hsiang Hsieh,¹ Celeste Quianzon,¹ Marcos Kuroki,¹ Justin Hardick,² Aleksandar Kecojevic,¹ Avanthi Abeygunawardena,¹ Jonathan Zenilman,² Johan Melendez,² Vishal Doshi,¹ Charlotte Gaydos,^1,2 and Richard E. Rothman^1,2

Department of Emergency Medicine, Johns Hopkins University, Baltimore, Maryland,¹ Division of Adult Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, Maryland²

Received 30 November 2007/ Returned for modification 26 December 2007/ Accepted 19 February 2008

Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 10¹ CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.

Published ahead of print on 27 February 2008.

Samuel Yang and Padmini Ramachandran both contributed equally to the manuscript.