title: High frequency of occupied attB regions in Norwegian Staphylococcus aureus isolates supports a two-step MRSA screening algorithm.
abstract: Rapid nucleic acid amplification tests for methicillin-resistant Staphylococcus aureus (MRSA) diagnostics commonly target the mec resistance gene, genes specific for S. aureus, and the integration site for the SCCmec resistance cassette, orfX. Due to poor specificity when these target genes are used individually, additional culture is required to verify positive results. The combination of these targets is useful, but the optimal algorithm may depend on the presence of the genetic markers in S. aureus isolates, as well as the prevalence of MRSA in a population. The aim of the present study was to identify a rapid, low-cost, and functional screening algorithm in order to reduce the response time for MRSA diagnostics. An in-house orfX-SCCmec polymerase chain reaction (PCR) assay was established and evaluated. The results were compared with an existing mec/nuc PCR assay and traditional culture. Methicillin-sensitive S. aureus (MSSA) that tested false-positive in the orfX-SCCmec PCR assay were further investigated with full genome sequencing using the Ion PGM™ System to verify results and causality. Based on these data, a two-step screening algorithm with initial mec/nuc PCR followed by orfX-SCCmec PCR on positive samples was suggested and tested on 1443 patient samples. 22.5 % of MSSA isolates tested false-positive with the orfX-SCCmec PCR. Full genome sequencing of these isolates identified genetic variation in the attB region of S. aureus, including empty cassette variants and non-mec SCC. The suggested two-step MRSA screening algorithm allowed us to report MRSA results for 95.6 % of all samples and 99 % of MRSA-negative samples after one day.
authors.affiliation: Department of Health Sciences, Oslo and Akershus University College, Oslo, Norway. hege.tunsjo@hioa.no.|Department of Microbiology and Infection Control, Akershus University Hospital, Lørenskog, Norway. hege.tunsjo@hioa.no.
authors.forename: H S
authors.initials: HS
authors.lastname: Tunsjø
authors.affiliation: Bioinformatics Core Facility, Department of Core Facilities, Institute of Cancer Research, Radium Hospital, part of Oslo University Hospital, Oslo, Norway.
authors.forename: S
authors.initials: S
authors.lastname: Kalyanasundaram
authors.affiliation: Institute for Cancer Genetics and Informatics, Radium Hospital, part of Oslo University Hospital, Oslo, Norway.
authors.forename: M M
authors.initials: MM
authors.lastname: Worren
authors.affiliation: Department of Microbiology and Infection Control, Akershus University Hospital, Lørenskog, Norway.|Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
authors.forename: T M
authors.initials: TM
authors.lastname: Leegaard
authors.affiliation: Institute of Clinical Medicine, University of Oslo, Oslo, Norway.|Department of Clinical Molecular Biology and Laboratory Sciences (EpiGen), Akershus University Hospital, Lørenskog, Norway.
authors.forename: A E F
authors.initials: AEF
authors.lastname: Moen
mesh_terms: D000465:Algorithms; D001287:Attachment Sites, Microbiological; D002353:Carrier State / Q000175:diagnosis / Q000382:microbiology; D003365:Costs and Cost Analysis; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009664:Norway; D016133:Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013997:Time Factors