Use of real-time polymerase chain reaction (rtPCR) as a diagnostic tool for influenza infection in a vaccine efficacy trial

Genetic and Antigenic Typing of Seasonal Influenza Virus Breakthrough Cases from a 2008-2009 Vaccine Efficacy Trial

“…In this study, and in agreement with a recent report, PCR represented a sensitive and accurate method for iden- tifying and typing influenza virus strains in samples from randomized prospective clinical trials (29). PCR also appeared to be more efficient at detecting A(H3N2) viruses than the culturebased methods, possibly related to the reduced sensitivity of culture method with A/Brisbane/10/07 lineage strains (29,39,40).…”

“…After thawing, these were cultured both on rhesus monkey kidney (RMK) cells and Madin-Darby canine kidney (MDCK) cells with incubation at 33 to 36°C for up to 2 weeks. Influenza virus A/B typing was performed on fixed cell cultures using standard immunofluorescence histology with influenza virus A-/B-specific antibodies (29).…”

“…Influenza virus detection and A/B typing were performed using quantitative real-time PCR (qPCR) targeting the matrix gene on RNA prepared from total nucleic acid extracted from frozen samples of nose/throat swabs, as described previously (29). Subtyping of influenza virus A-positive cases into seasonal A(H1N1) and A(H3N2) was performed in a separate reverse transcription-PCR (RT-PCR) assay using different sets of primers targeting the hemaggluti- Sequencing and sequence analysis.…”

“…In these trials, breakthrough cases are relatively infrequent, and the determination of antigenicity has been reliant on the conventional hemagglutination inhibition (HI) assay (27), which is limited by the availability of relevant reference strain ferret antisera (28) and the potential difficulties of cultivating sufficient virus from clinical samples. PCR has already been shown to be a more sensitive technique than culture at detecting influenza virus in nasal/throat swab samples from clinical breakthrough cases after vaccination (29). The aim of this study was to explore the relationship between HA1 domain sequences and antigenicities (determined by HI) of influenza virus strains isolated from clinical breakthrough cases.…”

Long-term immunogenicity of an AS03-adjuvanted influenza A(H1N1)pdm09 vaccine in young and elderly adults: An observer-blind, randomized trial

“…RT-qPCR was performed on viral RNA from the clinical samples as described previously [24]. Viral load values were quantified and the sample was considered positive when the measured viral load was equal to or above the assay cut-off [24].…”