Specific detection of methicillin-resistant Staphylococcus aureus directly from clinical specimen by real-time polymerase chain reaction

Abstract number: 1134_03_160

Goffinet P., Louahabi A., Hougardy N.

Objectives:  

Although Staphylococcus aureus (S. aureus) is relatively easy to cultivate, culture methods require 24 h incubation and conventional identification methods may yield false-positive or false-negative results. Standard susceptibility and penicillin-binding protein latex agglutination tests are time-consuming because they require colonies to be isolated from 24 h culture or more. The correct identification of S. aureus and the detection of the mecA gene based on molecular methods is considered as a gold standard in the determination of methicillin-resistant S. aureus (MRSA). Many PCR for simultaneous detection of S. aureus specific gene target and mecA gene were developed, but cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without an isolation step. A more comprehensive real time PCR assay that targets both an S. aureus-specific gene and the mecA gene within a single PCR tube reaction using only one couple of primer and two adjacent fluorescent probes was established and evaluated.

Methods:  

The validation of the assay was performed using 12 MRSA strains and 31 other strains of Staphylococci (not MRSA). A positive result was obtained for all MRSA strains and not with the other strains.

437 screening swabs from anterior nares and throat were obtained from patients hospitalized in the intensive care unit. S. aureus strains were identified according to their characteristic growth morphologies, Gram stain characteristics, reaction to catalase, coagulase production, colour of colonies on SAID culture media (Biomerieux) and LightCycler staphylococcus kit (Roche Diagnostics). Resistance to oxacillin was measured with an AST-P536 card on the VITEK-2 instrument (Biomerieux) and/or LightCycler MRSA detection kit (Roche Diagnostics). In order to gain time, we included an automated DNA extraction protocol on a LC MagNA Pure instrument and we realised the real-time PCR on a LightCycler instrument.

Results:  

24 MRSA could distinctly be detected by this PCR assay, Vitek-2 and/or LightCycler Staphylococcus–MRSA detection kits. From these, 14 were detected both by PCR and Vitek-2, 4 only by Vitek-2 and 6 only by the new PCR assay. The per cent of agreement is 97.7%.

Conclusion:  

This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci. The turn-around-time for the whole process is only 4 hours.