title: Pathogen transcriptional profile in nasopharyngeal aspirates of children with acute respiratory tract infection.
abstract: BACKGROUND
  Acute respiratory tract infections (ARI) present a significant morbidity and pose a global health burden. Patients are frequently treated with antibiotics although ARI are most commonly caused by virus, strengthening the need for improved diagnostic methods.
  
  
  OBJECTIVE
  Detect viral and bacterial RNA in nasopharyngeal aspirates (NPA) from children aged 6-23 months with ARI using nCounter.
  
  
  METHODS
  A custom-designed nCounter probeset containing viral and bacterial targets was tested in NPA of ARI patients.
  
  
  RESULTS
  Initially, spiked control viral RNAs were detectable in ≥6.25 ng input RNA, indicating absence of inhibitors in NPA. nCounter applied to a larger NPA sample (n=61) enabled the multiplex detection of different pathogens: RNA viruses Parainfluenza virus (PIV 1-3) and RSV A-B in 21%, Human metapneumovirus (hMPV) in 5%, Bocavirus (BoV), CoV, Influenza virus (IV) A in 3% and, Rhinovirus (RV) in 2% of samples, respectively. RSV A-B was confirmed by Real Time PCR (86.2-96.9% agreement). DNA virus (AV) was detected at RNA level, reflecting viral replication, in 10% of samples. Bacterial transcripts from Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae and Chlamydophila pneumoniae were detected in 77, 69, 26, 8, 3 and 2% of samples, respectively.
  
  
  CONCLUSIONS
  nCounter is robust and sensitive for the simultaneous detection of viral (both RNA and DNA) and bacterial transcripts in NPA with low RNA input (<10 ng). This medium-throughput technique will increase our understanding of ARI pathogenesis and may provide an evidence-based approach for the targeted and rational use of antibiotics in pediatric ARI.
authors.affiliation: Centro de Pesquisas Gonçalo Moniz (CPqGM)-FIOCRUZ, Salvador, BA, Brazil; Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil.
authors.forename: Kiyoshi F
authors.initials: KF
authors.lastname: Fukutani
authors.affiliation: Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Department of Pediatrics, School of Medicine, Federal University of Bahia, Salvador, BA, Brazil.
authors.forename: Cristiana M
authors.initials: CM
authors.lastname: Nascimento-Carvalho
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Winke
authors.initials: W
authors.lastname: Van der Gucht
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Elke
authors.initials: E
authors.lastname: Wollants
authors.affiliation: Centro de Pesquisas Gonçalo Moniz (CPqGM)-FIOCRUZ, Salvador, BA, Brazil; Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Khouri
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: Dierckx
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Van Ranst
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Lieselot
authors.initials: L
authors.lastname: Houspie
authors.affiliation: Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil.
authors.forename: Maiara L
authors.initials: ML
authors.lastname: Bouzas
authors.affiliation: Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil.
authors.forename: Juliana R
authors.initials: JR
authors.lastname: Oliveira
authors.affiliation: Centro de Pesquisas Gonçalo Moniz (CPqGM)-FIOCRUZ, Salvador, BA, Brazil; Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Department of Pathology, School of Medicine, Federal University of Bahia, Salvador, BA, Brazil; Instituto de Investigação em Imunologia, São Paulo, SP, Brazil.
authors.forename: Aldina
authors.initials: A
authors.lastname: Barral
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Johan
authors.initials: J
authors.lastname: Van Weyenbergh
authors.affiliation: Centro de Pesquisas Gonçalo Moniz (CPqGM)-FIOCRUZ, Salvador, BA, Brazil; Post-Graduation Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Instituto de Investigação em Imunologia, São Paulo, SP, Brazil. Electronic address: camila@bahia.fiocruz.br.
authors.forename: Camila I
authors.initials: CI
authors.lastname: de Oliveira
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D001424:Bacterial Infections / Q000175:diagnosis; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007223:Infant; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009305:Nasopharynx / Q000382:microbiology; D010802:Phylogeny; D012329:RNA, Bacterial / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis; D014780:Viruses / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D012329:RNA, Bacterial; D012367:RNA, Viral
keywords: ARI; Diagnostics; RSV; nCounter