Maxime Pichon1, Alexandre Gaymard1, Laurence Josset1, Martine Valette2, Gilles Millat3, Bruno Lina1, Vanessa Escuret4. 1. Hospices Civils de Lyon, Centre National de Référence des virus Influenzae France Sud, Laboratoire de Virologie, Institut des Agents Infectieux, Groupement Hospitalier Nord, F-69317, Lyon Cedex 04, France; Univ Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, CIRI, Inserm U1111, CNRS UMR5308, équipe Virpath, F-69372, Lyon Cedex 08, France. 2. Hospices Civils de Lyon, Centre National de Référence des virus Influenzae France Sud, Laboratoire de Virologie, Institut des Agents Infectieux, Groupement Hospitalier Nord, F-69317, Lyon Cedex 04, France. 3. Hospices Civils de Lyon, Plateforme de séquençage diagnostique, Centre de Biologie et de Pathologie Est, Groupement Hospitalier Est, F-69677, Bron, France. 4. Hospices Civils de Lyon, Centre National de Référence des virus Influenzae France Sud, Laboratoire de Virologie, Institut des Agents Infectieux, Groupement Hospitalier Nord, F-69317, Lyon Cedex 04, France; Univ Lyon, Université Lyon 1, Faculté de Médecine Lyon Est, CIRI, Inserm U1111, CNRS UMR5308, équipe Virpath, F-69372, Lyon Cedex 08, France. Electronic address: vanessa.escuret@univ-lyon1.fr.
Abstract
INTRODUCTION: The H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV. METHODS: RT-qPCR was performed using the ABI7500 platform. RT-ddPCR was carried out using the QX200 ddPCR platform. We strengthened our results by a NGS assay (Ion PGM™ sequencer). Discrimination performance and sensitivity of the RT-ddPCR assay were evaluated using mixes of wild type (WT) and mutated H275Y-NA-coding segments. RESULTS: The performance of RT-ddPCR was better than RT-qPCR, using NGS assay as a gold standard. RT-ddPCR was able to detect 0.28% oseltamivir-resistant IV in a WT IV population and 0.55% WT IV in an oseltamivir-resistant IV population. For the first patient, the H275Y-NA substitution was selected by oseltamivir treatment and reached about 50% of the IV population before dropping to less than 2% after treatment discontinuation which was under the lower limit of quantification by RT-qPCR and RT-ddPCR (<2%) after treatment stop. Then, five days after oseltamivir was re-introduced, the H275Y-NA substitution rose up to 100%. For the second patient, the H275Y-NA substitution reached about 30% two days after oseltamivir discontinuation. CONCLUSION: RT-ddPCR demonstrated better performances than classical RT-qPCR to estimate oseltamivir-resistant IV proportions. This technique could be used to detect earlier emergence of H275Y-NA substitution.
INTRODUCTION: The H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV. METHODS: RT-qPCR was performed using the ABI7500 platform. RT-ddPCR was carried out using the QX200 ddPCR platform. We strengthened our results by a NGS assay (Ion PGM™ sequencer). Discrimination performance and sensitivity of the RT-ddPCR assay were evaluated using mixes of wild type (WT) and mutated H275Y-NA-coding segments. RESULTS: The performance of RT-ddPCR was better than RT-qPCR, using NGS assay as a gold standard. RT-ddPCR was able to detect 0.28% oseltamivir-resistant IV in a WT IV population and 0.55% WT IV in an oseltamivir-resistant IV population. For the first patient, the H275Y-NA substitution was selected by oseltamivir treatment and reached about 50% of the IV population before dropping to less than 2% after treatment discontinuation which was under the lower limit of quantification by RT-qPCR and RT-ddPCR (<2%) after treatment stop. Then, five days after oseltamivir was re-introduced, the H275Y-NA substitution rose up to 100%. For the second patient, the H275Y-NA substitution reached about 30% two days after oseltamivir discontinuation. CONCLUSION: RT-ddPCR demonstrated better performances than classical RT-qPCR to estimate oseltamivir-resistant IV proportions. This technique could be used to detect earlier emergence of H275Y-NA substitution.
Authors: Cyril Barbezange; Louis Jones; Hervé Blanc; Ofer Isakov; Gershon Celniker; Vincent Enouf; Noam Shomron; Marco Vignuzzi; Sylvie van der Werf Journal: Front Microbiol Date: 2018-10-31 Impact factor: 5.640
Authors: A Bal; M Pichon; C Picard; J S Casalegno; M Valette; I Schuffenecker; L Billard; S Vallet; G Vilchez; V Cheynet; G Oriol; S Trouillet-Assant; Y Gillet; B Lina; K Brengel-Pesce; F Morfin; L Josset Journal: BMC Infect Dis Date: 2018-10-29 Impact factor: 3.090