title: Comparison of two commercial broad-range PCR and sequencing assays for identification of bacteria in culture-negative clinical samples.
abstract: Culturing has long been the gold standard for detecting aetiologic agents in bacterial infections. In some cases, however, culturing fails to detect the infection. To further investigate culture-negative samples, amplification and subsequent sequencing of the 16S rRNA gene is often applied. The aim of the present study was to compare the current method used at our Department of Clinical Microbiology, based on the MicroSeq ID system (Applied Biosystems, USA) with the Universal Microbe Detection (UMD) SelectNA kit (Molzym, Germany).
  
  
  
  76 culture-negative samples were first processed with the MicroSeq ID analysis, where total DNA was extracted and the 16S gene amplified and sequenced with the MicroSeq ID system. Samples were subsequently processed with the UMD SelectNA analysis, where human DNA was removed during the DNA extraction procedure and the 16S gene amplified in a real-time PCR and sequenced.
  
  
  
  22 of 76 samples (28.9%) were positive for bacteria with the UMD SelectNA, which was significantly more (p = 0.0055) than the MicroSeq ID where 11 of 76 samples (14.5%) were positive. The UMD SelectNA assay identified more relevant bacterial pathogens than the MicroSeq ID analysis (p = 0.0233), but also found a number of species that were considered contaminations.
  
  
  
  The UMD SelectNA assay was valuable for the identification of pathogens in culture-negative samples; however, due to the sensitive nature of the assay, extreme care is suggested in order to avoid false positives.
authors.affiliation: Department of Clinical Microbiology, Centre for Diagnostics, Rigshospitalet, Copenhagen, Denmark.|Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Camilla
authors.initials: C
authors.lastname: Stavnsbjerg
authors.affiliation: Department of Clinical Microbiology, Centre for Diagnostics, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Frimodt-Møller
authors.affiliation: Department of Clinical Microbiology, Centre for Diagnostics, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Moser
authors.affiliation: Department of Clinical Microbiology, Centre for Diagnostics, Rigshospitalet, Copenhagen, Denmark. tbjarnsholt@sund.ku.dk.|Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. tbjarnsholt@sund.ku.dk.
authors.forename: Thomas
authors.identifier: 0000-0002-8003-7414
authors.initials: T
authors.lastname: Bjarnsholt
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000254:growth & development; D001424:Bacterial Infections / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology*; D003132:Commerce; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005188:False Negative Reactions; D006801:Humans; D008828:Microbiological Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic
keywords: 16S PCR; Culture-negative samples; Molecular diagnostics; Universal Microbe Detection