Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR : is viral quantification useful?

Authors

  • M. Bouscambert Duchamp,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author
  • J. S. Casalegno,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author
  • Y. Gillet,

    1.  Hospices Civils de Lyon, Groupement Hospitalier Est, Hôpital femme-mère-enfant, Paediatric units, Bron, France
    Search for more papers by this author
  • E. Frobert,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author
  • E. Bernard,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    Search for more papers by this author
  • V. Escuret,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author
  • G. Billaud,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    Search for more papers by this author
  • M. Valette,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author
  • E. Javouhey,

    1.  Hospices Civils de Lyon, Groupement Hospitalier Est, Hôpital femme-mère-enfant, Paediatric units, Bron, France
    Search for more papers by this author
  • B. Lina,

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author
  • D. Floret,

    1.  Hospices Civils de Lyon, Groupement Hospitalier Est, Hôpital femme-mère-enfant, Paediatric units, Bron, France
    Search for more papers by this author
  • F. Morfin

    1.  Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron
    2.  Université de Lyon, Université Lyon 1 Virologie et Pathologie Humaine, CNRS FRE 3011, Lyon Cedex
    Search for more papers by this author

Corresponding author and reprint requests: M. Bouscambert Duchamp, Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology – Bât A3, 59 Boulevard Pinel, F-69677 Bron Cedex, France
E-mail: maude.bouscambert-duchamp@chu-lyon.fr

Abstract

Clin Microbiol Infect 2010; 16: 317–321

Abstract

The emergence of the influenza A(H1N1) 2009 virus prompted the development of sensitive RT-PCR detection methods. Most are real time RT-PCRs which can provide viral quantification. In this manuscript, we describe a universal influenza A RT-PCR targeting the matrix (M) gene, combined with an RNaseP RT-PCR. These PCRs allow the detection of all influenza A virus subtypes, including A(H1N1)2009, together with a real-time assessment of the quality of the specimens tested. These PCR procedures were evaluated on 209 samples collected from paediatric patients. Viral loads determined through Ct values were corrected according to the RNaseP Ct value. The mean viral load in the collected samples was estimated to be 6.84 log RNA copies/mL. For poor quality samples (RNaseP Ct > 27), corrections resulted in +3 to +8 Ct values for the M gene RT-PCR. Corrected influenza Ct values were lower in late samples. No correlation was established between viral loads and clinical severity or duration of disease.This study shows that real time RT-PCR targeting the matrix gene is a reliable tool for quantification of type A influenza virus but emphasises the need for sample quality control assessment through cellular gene quantification for reliable estimation of the viral load. This method would be useful for disease management when repeated specimens are collected from an infected individual.

Ancillary