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Antiviral Res. 2017 Aug 3;145:160-167. doi: 10.1016/j.antiviral.2017.07.021. [Epub ahead of print]

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

Author information

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.

KEYWORDS:

H275Y substitution; Influenza viruses; Next generation sequencing; Oseltamivir resistance; RT-droplet digital PCR; RT-qPCR

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