title: Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.
abstract: BACKGROUND
  Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections.
  
  
  METHODS
  We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion.
  
  
  RESULTS
  87 samples [42 oropharyngeal (OP) and 45 expectorated sputum (ES)] were analyzed. 59 samples (68%) cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%), followed by FemA qPCR (52%) and 16SrRNA qPCR (34%). For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs.
  
  
  CONCLUSIONS
  Sequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.
authors.affiliation: University of Colorado, School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Johnson
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Edith T
authors.initials: ET
authors.lastname: Zemanick
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Accurso
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America.|Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Brandie D
authors.initials: BD
authors.lastname: Wagner
authors.affiliation: Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Robertson
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: J Kirk
authors.initials: JK
authors.lastname: Harris
mesh_terms: D000293:Adolescent; D001426:Bacterial Proteins / Q000235:genetics; D002648:Child; D003550:Cystic Fibrosis / Q000382:microbiology*; D005260:Female; D006801:Humans; D008297:Male; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012137:Respiratory System / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D012336:RNA, Ribosomal, 16S