2002 Abstract – 1 – Tarkin

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Title: MecA PCR for the Rapid Detection of Methicillin-Resistant Staphylococci Causing Musculoskeletal Infections

Authors: Ivan S. Tarkin, MD *, Travis J.Henry, BS **, Steven H. Hinrichs, MD **, Kevin L. Garvin, MD *, University of Nebraska Medical Center

Department of Orthopedics (*) Department of Pathology and Microbiology

Purpose: A PCR based assay was investigated for the rapid genomic detection of methicillin-resistant staphylococci involved in musculoskeletal infections. These infections are becoming increasingly common, especially in the setting of total joint arthroplasty. Culture, the current gold standard for identifying these organisms, takes 2­3 days for processing and false negative results have been reported, particularly with patients receiving antibiotic therapy.

Method: The mecA gene served as the genetic target for identifying methicillin­resistant staphylococci. This gene is required for the production of penicillin binding protein 2a, which is responsible for the resistant phenotype. The feasibility of the approach was first validated using a septic arthritis model consisting of 73 synovial fluid samples inoculated with methicillin-resistant staphylococci and 4 negative controls. Sixty clinical samples were then studied that included intraoperative samples from revision arthroplasty for suspected infection, methicillin-resistant staphylococcal infected tissue and blood.

Results: The molecular protocol described was processed in less than 5 hours. The limit of detection for the mecA PCR assay was determined to be dependent on the number amplification cycles in the PCR assay with the levels of 104, 103, 102 CFU/ml corresponding with 25, 35, 45 cycles respectively. In the septic arthritis model, 73 samples prepared with methicillin-resistant staphylococcal species contained the mecA gene by PCR. Three samples containing non-MR staphylococcal species including Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis were negative for the mecA gene. The molecular assay results were consistent with culture in 59 of 60 (98%) of clinical samples tested. The one discrepant result was likely due to culture contamination.

Conclusions: This study demonstrates the feasibility of using PCR for the detection methicillin-resistant staphylococci in musculoskeletal infections. Patient samples can be directly analyzed for the presence of these organisms avoiding the unnecessary delays and shortcomings with culture techniques.

Significance: MecA PCR has the potential to become a beneficial adjunct or alternative to culture for identifying methicillin-resistant staphylococci in musculoskeletal infections. This assay may be particularly useful when delays in diagnosis would affect patient management. PCR could also be utilized in clinical settings where culture would be unreliable.