title: Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay.
abstract: Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology.
authors.affiliation: Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, USA.
authors.forename: Kashonda
authors.initials: K
authors.lastname: Kelley
authors.forename: Angela
authors.initials: A
authors.lastname: Cosman
authors.forename: Phillip
authors.initials: P
authors.lastname: Belgrader
authors.forename: Brenda
authors.initials: B
authors.lastname: Chapman
authors.forename: Donna C
authors.initials: DC
authors.lastname: Sullivan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D002353:Carrier State / Q000175:diagnosis / Q000382:microbiology*; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009297:Nasal Mucosa / Q000382:microbiology; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins