Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

Detection of MRSA on Infection Control Swabs Using an In-house Real-Time Multiplex PCR Screening Assay. (#341)

Luke Walters 1 , Gerhard Weldhagen , Mark Turra , Geoffrey Higgins
  1. Microbiology and Infectious Diseases, SA Pathology, ADELAIDE, SA, Australia

Methicillin-Resistant Staphylococcus aureus (MRSA) is a major concern in nosocomial environments throughout the world. Disease complications include wound infections, pneumonia, septicaemia, and endocarditis. The mechanism for resistance to -lactam antibiotics by S. aureus involves production of an altered penicillin binding protein “PBP2a”, coded for by the mecA gene which resides on a mobile gene cassette SCCmec.
To screen infection control swabs for direct detection of MRSA using molecular techniques, eliminating the need for screening cultures, reducing the time taken to identify colonised patients.
The assay designed to detect the SCCmec-orfX junction is a modified and expanded version of a previously published method (Huletsky et. al.), optimized to detect locally circulating strains. A new LNA (Locked Nucleic Acid) probe was incorporated into the assay based on NCBI database alignments and validation with typed strains. Four new primers to detect strains not covered by the original method were designed based on sequencing results. MecA and nuclease genes are also detected to complement the junction PCR with a computerised algorithm combining respective PCR values to produce a final result. PCR results were compared to MRSA chromogenic agar culture as a gold standard. In total the validation included 2056 Amies liquid media swabs, 230 MRSA isolates, 145 blood cultures, and 118 GeneXpert samples.
Parallel testing of infection control samples using the final computerised interpretation algorithm for MRSA detection yielded a sensitivity of 86.8%, specificity of 99.6%, positive predictive value of 76.5%, and most importantly a negative predictive value of 99.8%.
This cost effective, rapid assay is now in routine use and is performing well, with the ongoing combined efforts of the Microbiology and the Molecular Diagnostics laboratories.