title: Quantitative multiplexed-tandem PCR for direct detection of bacteraemia in critically ill patients.
abstract: Culture remains the gold standard for diagnosis of blood stream infections (BSI), but its clinical utility is limited by slow turnaround times. Here we describe a method for rapid quantitative detection of bacterial DNA directly extracted from whole blood using a multiplexed tandem real-time PCR (MT-PCR) assay targeting Staphylococcus, Streptococcus, Pseudomonas, Enterococcus and Enterobacteriaceae 16S rDNA genes. Results were available less than 3.5 hours after blood collection with all five bacterial targets having limits of detection between 101and 103CFU/mL. A small-scale clinical evaluation of the assay using blood samples collected from 15 patients admitted to the Intensive Care Unit at our institution demonstrated 93.3% (14/15) concordance between MT-PCR and blood culture when detection of persistent bacterial DNAemia by MT- PCR was considered a true result. Further evaluation with clinical samples is needed; however, this method has potential as an effective rule-in diagnostic tool for bacteraemic sepsis and septic shock.
authors.affiliation: Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney and Westmead Hospital, Westmead, NSW, Australia; Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Ginn
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Briony
authors.initials: B
authors.lastname: Hazelton
authors.affiliation: Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney and Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Shereen
authors.initials: S
authors.lastname: Shoma
authors.affiliation: Intensive Care Unit, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Martin
authors.initials: M
authors.lastname: Cullen
authors.affiliation: Intensive Care Unit, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Solano
authors.affiliation: Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney and Westmead Hospital, Westmead, NSW, Australia; Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia. Electronic address: jonathan.iredell@sydney.edu.au.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Iredell
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D016470:Bacteremia / Q000175:diagnosis* / Q000235:genetics; D016638:Critical Illness / Q000453:epidemiology / Q000628:therapy; D004269:DNA, Bacterial / Q000032:analysis*; D016983:Enterococcus / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000175:diagnosis / Q000382:microbiology*; D013291:Streptococcus / Q000235:genetics
chemical_list: D004269:DNA, Bacterial
keywords: Bacteraemia; PCR; critical care; direct detection; sepsis