title: Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.
abstract: The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands. Electronic address: marre.vandenbrand@vumc.nl.
authors.forename: Marre
authors.initials: M
authors.lastname: van den Brand
authors.affiliation: Department of Medical Microbiology, Maastricht University Medical Centre, P. Debyelaan 25, 6229HX Maastricht, The Netherlands. Electronic address: rph.peters@gmail.com.
authors.forename: Remco P H
authors.initials: RPH
authors.lastname: Peters
authors.affiliation: Microbiome, Troubadoursborgh 59, 3992BE Houten, The Netherlands. Electronic address: a.catsburg@vumc.nl.
authors.forename: Arnold
authors.initials: A
authors.lastname: Catsburg
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands. Electronic address: a.rubenjan@vumc.nl.
authors.forename: Anna
authors.initials: A
authors.lastname: Rubenjan
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands. Electronic address: ferdi_broeke@hotmail.com.
authors.forename: Ferdi J
authors.initials: FJ
authors.lastname: Broeke
authors.affiliation: Department of Pediatrics, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands. Electronic address: f.vandendungen@vumc.nl.
authors.forename: Frank A M
authors.initials: FAM
authors.lastname: van den Dungen
authors.affiliation: Department of Pediatrics, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands. Electronic address: m.vanweissenbruch@vumc.nl.
authors.forename: Mirjam M
authors.initials: MM
authors.lastname: van Weissenbruch
authors.affiliation: Department of Pediatrics, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands. Electronic address: am.vanfurth@vumc.nl.
authors.forename: A Marceline
authors.initials: AM
authors.lastname: van Furth
authors.affiliation: Department of Bioinformatics, University of Tartu, Ülikooli 18, 50090 Tartu, Estonia. Electronic address: triinu_k@ut.ee.
authors.forename: Triinu
authors.initials: T
authors.lastname: Kõressaar
authors.affiliation: Department of Bioinformatics, University of Tartu, Ülikooli 18, 50090 Tartu, Estonia. Electronic address: maido.remm@ut.ee.
authors.forename: Maido
authors.initials: M
authors.lastname: Remm
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, De Boelelaan 1118, 1081HZ Amsterdam, The Netherlands; Department of Medical Microbiology, Maastricht University Medical Centre, P. Debyelaan 25, 6229HX Maastricht, The Netherlands; Microbiome, Troubadoursborgh 59, 3992BE Houten, The Netherlands. Electronic address: p.savelkoul@vumc.nl.
authors.forename: Paul H M
authors.initials: PHM
authors.lastname: Savelkoul
authors.affiliation: Microbiome, Troubadoursborgh 59, 3992BE Houten, The Netherlands. Electronic address: mp.bos@vumc.nl.
authors.forename: Martine P
authors.initials: MP
authors.lastname: Bos
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001769:Blood / Q000382:microbiology*; D006801:Humans; D007231:Infant, Newborn; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000175:diagnosis* / Q000382:microbiology*; D013997:Time Factors
keywords: Late onset sepsis; Molecular diagnosis; Multiplex PCR; Neonatal sepsis; Rapid diagnosis; Real-time PCR