Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay.
A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.
Francois
Patrice
P
Genomic Research Laboratory, Division of Infectious Diseases, University Hospitals of Geneva, CH-1211 Geneva 14, Switzerland. patrice.francois@hcuge.ch
Pittet
Didier
D
Bento
Manuela
M
Pepey
Béatrice
B
Vaudaux
Pierre
P
Lew
Daniel
D
Schrenzel
Jacques
J
D001426:Bacterial Proteins / Q000235:genetics; D006801:Humans; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D013212:Staphylococcus epidermidis / Q000302:isolation & purification
D001426:Bacterial Proteins; C096686:FemA protein, Bacteria; D011933:Reagent Kits, Diagnostic; C083616:mecA protein, Bacillus subtilis; D008712:Methicillin