Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis

Maxime Pichon (1, 2) , Alexandre Gaymard (1, 2) , Laurence Josset (1, 2) , Martine Valette (1) , Gilles Millat (3) , Bruno Lina (2, 1) , Vanessa Escuret (1, 2)

1 CNR des virus influenza - Laboratoire de Virologie [GH Nord HCL, Lyon]
2 Virpath - Virpath-Grippe, de l'émergence au contrôle -- Virpath-Influenza, from emergence to control
3 Hospices Civils de Lyon, Plateforme de séquençage diagnostique, Centre de Biologie et de Pathologie Est, Groupement Hospitalier Est, F-69677, Bron, France

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\texttrademark 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 (\textless2%) 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.

Mots clés: Antiviral Agents; Drug Resistance; Viral; H275Y substitution; High-Throughput Nucleotide Sequencing; Humans; Immunocompromised Host; Influenza A Virus; H1N1 Subtype; Influenza viruses; Influenza; Human; Molecular Diagnostic Techniques; Mutation; Missense; Neuraminidase; Next generation sequencing; Oseltamivir; Oseltamivir resistance; Real-Time Polymerase Chain Reaction; RT-droplet digital PCR; RT-qPCR; Viral Proteins

Domaines: Immunologie; Bactériologie; Virologie

Maxime Pichon, Alexandre Gaymard, Laurence Josset, Martine Valette, Gilles Millat, et al.. Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis. Antiviral Research, 2017, 145, pp.160-167. ⟨10.1016/j.antiviral.2017.07.021⟩. ⟨hal-01908998⟩