Pathogen transcriptional profile in nasopharyngeal aspirates of children with acute respiratory tract infection

J Clin Virol. 2015 Aug;69:190-6. doi: 10.1016/j.jcv.2015.06.005. Epub 2015 Jun 10.

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.

Objectives: Detect viral and bacterial RNA in nasopharyngeal aspirates (NPA) from children aged 6-23 months with ARI using nCounter.

Study design: 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.

Conclusion: 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.

Keywords: ARI; Diagnostics; RSV; nCounter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / classification
  • Bacteria / genetics*
  • Bacteria / isolation & purification
  • Bacterial Infections / diagnosis
  • Gene Expression Profiling / methods*
  • Humans
  • Infant
  • Multiplex Polymerase Chain Reaction / methods
  • Nasopharynx / microbiology
  • Phylogeny
  • RNA, Bacterial / analysis*
  • RNA, Viral / analysis*
  • Respiratory Tract Infections / diagnosis
  • Respiratory Tract Infections / microbiology*
  • Sensitivity and Specificity
  • Virus Diseases / diagnosis
  • Viruses / classification
  • Viruses / genetics*
  • Viruses / isolation & purification

Substances

  • RNA, Bacterial
  • RNA, Viral