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Evaluation of an Isothermal Signal Amplification Method for Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Patient-Screening Swabs

Molecular Diagnostics and Typing Unit, Public Health Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH,¹ Cytocell Ltd., Adderbury, Oxford OX17 3SN, United Kingdom²

Received 21 January 2003/ Returned for modification 4 March 2003/ Accepted 1 April 2003

A new molecular assay (CytAMP) utilizing isothermal signal-mediated amplification of RNA was evaluated for rapid detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). The assay targeted the coa (coagulase) and mecA genes, thereby simultaneously identifying S. aureus and methicillin (oxacillin) resistance. Results were obtained in approximately 3.5 h as a color signal in 96-well microtiter plates. The detection limit was between 2 x 10⁵ and 10⁶ CFU/assay, equivalent to 4 x 10⁶ to 2 x 10⁷ CFU/ml in an overnight broth. This level of growth was obtained with an initial inoculum of 10 to 50 CFU. The CytAMP assay and a mecA-femB PCR assay both detected 113 MRSA strains among 396 clinical isolates of bacteria (CytAMP sensitivity and specificity were both 100% relative to those of PCR). Conventional culture detected 109 MRSA strains, but with 19 false-positive and 23 false-negative results relative to both molecular methods. Discrepancies were also observed for 100 enrichment broths containing MRSA screening swabs, with 11 broths culture negative but PCR positive. CytAMP and PCR were more in agreement, but six broths were CytAMP negative and PCR positive. Five of these contained 10² to 10⁵ CFU/assay (below the CytAMP detection limit of 2 x 10⁵ CFU/assay), and the sixth contained 10⁶ CFU/assay. Overall, culture and CytAMP had similar sensitivities and specificities relative to those of PCR, but the CytAMP assay enabled swabs to be analyzed as a batch following overnight incubation in enrichment broth, with results reported before 12 noon the next day.

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