Development and implementation of a 4plex real time-PCR assay for screening and detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus

Abstract number: P795

Kolman S., Ben-Nissan Y., Kilman A., Arieli C., Paitan Y.

Objectives: To develop a new real time-PCR (QR-PCR) assay for methicillin-resistant and methicillin-sensitive Staphylococcus aureus (MRSA, MSSA) detection with high and clinically satisfactory diagnostic values (Sensitivity, Specificity, PPV, NPV) and to implement it in a cost-effective, logistically feasible protocol in our infection control program.

Methods: We developed a 4-plex QR-PCR assay enabling detection of MRSA and MSSA overcoming most problems found in previously published assays and in commercial MRSA kits. The assay simultaneously detects in one PCR tube, a PCR internal control (inhibition and reagents integrity), the mecA gene, a S. aureus-specific gene and SCCmec:orfX region types 1 to 5. The assay was validated using 150 known staphylococcal strains. Analytical specificity was analyzed using 83 different bacterial and fungal species. Analytical sensitivity, direct swab sensitivity and detection of MRSA and MSSA in mixed population were evaluated. All samples were analyzed for mixed population and by conventional identification methods. The assay was implemented in our infection control program in a cost-effective, logistically feasible protocol.

Results: Validation with 150 known staphylococcal strains reviled 100% concordance with microbiological analysis. No cross reaction was observed to 83 different bacterial species (analytical specificity). The limit of detection was 20 CFU/PCR reaction for MRSA and 2 CFU/PCR for MSSA (analytical sensitivity). Direct swab sensitivity was 3000 CFU/ml for MRSA and 300 CFU/ml for MSSA corresponding to 150 CFU/swab or 20–25 CFU/PCR reaction for MRSA and to 15 CFU/swab or 2–4 CFU/PCR reaction for MSSA. No meaningful redaction was found in mixed population analysis.

During 2 years, out of 39,120 samples, we applied the assay on 4,482 samples suspected to contain staphylococci or S. aureus in 504 runs (2500 samples after exclusion of patient's duplicates). All calculated diagnostic values are >95–99% for MRSA detection and >91–99% for MSSA (Table 1). For logistical and economical reasons PCR is performed from colonies and results are available at the next day. Results are available in less then 3 hours if direct swab PCR is performed.

Conclusion: Our assay demonstrates very high diagnostics values, analytical sensitivity and specificity; suitable for direct swab analysis and can be incorporated into infection control programs in a cost-effective, logistically feasible protocol which will be presented.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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