title: Rapid identification of Gram-positive pathogens and their resistance genes from positive blood culture broth using a multiplex tandem RT-PCR assay.
abstract: The early initiation of targeted antibiotic therapy in patients with bacteraemia and septic shock impacts favourably on outcomes. Rapid methods are therefore increasingly employed for bacterial identification directly from positive blood culture bottles, but with variable success. We evaluated the performance of the Gram Positive 12 multiplex tandem PCR (MT-PCR) assay (AusDiagnostics; catalogue no. 6202, version 07) containing targets for the identification of staphylococci including Staphylococcus aureus, streptococci including Streptococcus pneumoniae, enterococci including Enterococcus faecalis and Enterococcus faecium and their common antibiotic resistance genes (mecA, vanA, vanB). A total of 673 aerobic and anaerobic blood culture broths demonstrating Gram-positive cocci on microscopy were analysed in parallel with traditional phenotypic methods. Amplification of the internal control was inhibited in 79/673 (11.7 %) samples; however, MT-PCR identification was in concordance with phenotypic identification to the genus level in 96.6 % (537/556) of the remaining monomicrobial specimens and to the species level, where applicable, in 100 % (172/172) of samples. MT-PCR identification for 94.7 % (36/38) of polymicrobial samples matched traditional phenotypic identification. Meticillin and vancomycin susceptibility results determined by MT-PCR in blood culture broths demonstrated complete agreement with those determined by phenotypic methods in all 143 Staphylococcus aureus isolates and eight E. faecium isolates, respectively. Gram-positive pathogens and their key antibiotic resistance markers were reliably identified with the MT-PCR assay within 3 h of a positive blood culture result.
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia.
authors.forename: Briony J
authors.initials: BJ
authors.lastname: Hazelton
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia.
authors.forename: Lee C
authors.initials: LC
authors.lastname: Thomas
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia.
authors.forename: Tuba
authors.initials: T
authors.lastname: Unver
authors.affiliation: Sydney Emerging Infectious Diseases Institute, University of Sydney, Westmead Hospital, Westmead, New South Wales, Australia.|Centre for Research Excellence in Critical Infection, Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales, Australia.|Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Iredell
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D016470:Bacteremia / Q000175:diagnosis / Q000382:microbiology*; D001426:Bacterial Proteins / Q000235:genetics; D019729:Carbon-Oxygen Ligases / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D016983:Enterococcus / Q000145:classification / Q000187:drug effects / Q000235:genetics; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006801:Humans; D008712:Methicillin / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction; D046915:Penicillin-Binding Proteins; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012772:Shock, Septic / Q000175:diagnosis / Q000382:microbiology; D013210:Staphylococcus / Q000145:classification / Q000187:drug effects / Q000235:genetics; D013291:Streptococcus / Q000145:classification / Q000187:drug effects / Q000235:genetics; D014640:Vancomycin / Q000494:pharmacology; D020713:Vancomycin Resistance / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D012336:RNA, Ribosomal, 16S; C071437:VanA ligase, Bacteria; C079913:VanB protein, Enterococcus; C487789:mecA protein, Staphylococcus aureus; D014640:Vancomycin; D019729:Carbon-Oxygen Ligases; D008712:Methicillin