title: Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR: is viral quantification useful?
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.
authors.affiliation: Hospices Civils de Lyon, National Influenza Centre (South of France), Laboratory of Virology, Bron, France. maude.bouscambert-duchamp@chu-lyon.fr
authors.forename: M Bouscambert
authors.initials: MB
authors.lastname: Duchamp
authors.forename: J S
authors.initials: JS
authors.lastname: Casalegno
authors.forename: Y
authors.initials: Y
authors.lastname: Gillet
authors.forename: E
authors.initials: E
authors.lastname: Frobert
authors.forename: E
authors.initials: E
authors.lastname: Bernard
authors.forename: V
authors.initials: V
authors.lastname: Escuret
authors.forename: G
authors.initials: G
authors.lastname: Billaud
authors.forename: M
authors.initials: M
authors.lastname: Valette
authors.forename: E
authors.initials: E
authors.lastname: Javouhey
authors.forename: B
authors.initials: B
authors.lastname: Lina
authors.forename: D
authors.initials: D
authors.lastname: Floret
authors.forename: F
authors.initials: F
authors.lastname: Morfin
mesh_terms: D002648:Child; D002675:Child, Preschool; D004196:Disease Outbreaks; D005260:Female; D005814:Genes, Viral / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology; D008297:Male; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load / Q000379:methods