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.Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality (6, 9). Delayed or inadequate treatment of SA can lead to irreversible joint destruction with subsequent disability. Accordingly, prompt diagnosis and early initiation of therapy are critical in improving the outcome (7).The diagnosis of SA in the acute care setting is challenging because of the relatively poor sensitivity and specificity of clinical examination findings and lack of a rapid reliable diagnostic assay. Further, overreliance on conventional laboratory tests for synovial fluid analysis is hindered by the relatively poor performance characteristics of these methods (11,12,16). In particular, the sensitivity of Gram staining has been reported in the range of 29% to 50% (3, 4), and the sensitivity of culture may be only 82% (9). Lack of a rapid and accurate diagnostic tool results in acute care clinicians often choosing the conservative approach of hospital admission and empirical broadspectrum antibiotics for patients with suspected SA. The benefits of this management strategy may be offset, however, by added costs and potential iatrogenic complications associated with unnecessary treatment and hospitalizations, as well ...

Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

“…1,2 However, a major limitation is the lack of differentiation between viable and dead bacteria. 3,4 Several previous studies 5,6 that have evaluated synovial fluid or orthopedic implants after total hip or knee arthroplasty have reported evidence of bacteria in a high proportion of cases.…”

Identification of Bacterial Pathogens in Ascitic Fluids from Patients with Suspected Spontaneous Bacterial Peritonitis by Use of Broad-Range PCR (16S PCR) Coupled with High-Resolution Melt Analysis

“…Identification of the causative agent in ascitic fluid after 16S rRNA gene amplification has relied on either probe-based amplicon analysis, which limits testing to a finite number of anticipated pathogens, or sequencing, which is low throughput. We have previously combined 16S rRNA PCR (16S PCR) with high-resolution melt analysis (HRMA) for rapid broad-range detection and identification of bacterial pathogens (18,(24)(25)(26)(27)(28). HRMA offers a simple, low-cost, closed-tube approach to amplicon analysis with the capacity for single-nucleotide discrimination and easy integration with PCR analysis.…”

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

“…In this study, combining results from both molecular and conventional culture methods, bacteria were only detected in 18/152 (11.8%) specimens. Other similar studies have described composite culture and 16S rDNA PCR detection rates ranging from 18.5% -62.5% in joint specimens [1,2,[17][18][19]. Further investigation revealed a significant proportion of specimens in this study (56%) were sent from patients without a documented suspicion of infection.…”

“…However, as illustrated in this study, it is very useful in identifying bacteria present at higher levels but which are unlikely to grow on conventional culture media due to their fastidious nature or prior antibiotic treatment. Alternative molecular methods, such as performing a battery of species-specific PCR reactions on 16S rDNA assay positive specimens [17], may circumvent the need for sequencing, but only enable detection of the limited number of organisms actively sought.…”