Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, Panton–Valentine leucocidin (PVL), mecA and homologue mecALGA251

Background The recent discovery of a mecA homologue (mecALGA251) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecALGA251 could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing. Methods In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecALGA251, alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin]. Results and discussion The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecALGA251 from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecALGA251, including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecALGA251 in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.