title: Rapid PCR-based diagnosis of septic arthritis by early Gram-type classification and pathogen identification.
abstract: 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 10(1) 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.
authors.affiliation: Department of Emergency Medicine, Johns Hopkins University, 5801 Smith Avenue, Davis Building, Suite 3220, Baltimore, MD 21209, USA. syang10@jhmi.edu
authors.forename: Samuel
authors.initials: S
authors.lastname: Yang
authors.forename: Padmini
authors.initials: P
authors.lastname: Ramachandran
authors.forename: Andrew
authors.initials: A
authors.lastname: Hardick
authors.forename: Yu-Hsiang
authors.initials: YH
authors.lastname: Hsieh
authors.forename: Celeste
authors.initials: C
authors.lastname: Quianzon
authors.forename: Marcos
authors.initials: M
authors.lastname: Kuroki
authors.forename: Justin
authors.initials: J
authors.lastname: Hardick
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Kecojevic
authors.forename: Avanthi
authors.initials: A
authors.lastname: Abeygunawardena
authors.forename: Jonathan
authors.initials: J
authors.lastname: Zenilman
authors.forename: Johan
authors.initials: J
authors.lastname: Melendez
authors.forename: Vishal
authors.initials: V
authors.lastname: Doshi
authors.forename: Charlotte
authors.initials: C
authors.lastname: Gaydos
authors.forename: Richard E
authors.initials: RE
authors.lastname: Rothman
mesh_terms: D001170:Arthritis, Infectious / Q000175:diagnosis* / Q000382:microbiology*; D015373:Bacterial Typing Techniques; D001431:Bacteriological Techniques; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D006090:Gram-Negative Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D006094:Gram-Positive Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013582:Synovial Fluid / Q000382:microbiology; D013997:Time Factors
chemical_list: D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S