tetano
Editor, Senior Moderator
Antiviral Res. 2017 Aug 2. pii: S0166-3542(17)30431-X. doi: 10.1016/j.antiviral.2017.07.021. [Epub ahead of print]
[h=1]Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis.[/h] Pichon M[SUP]1[/SUP], Gaymard A[SUP]1[/SUP], Josset L[SUP]1[/SUP], Valette M[SUP]2[/SUP], Millat G[SUP]3[/SUP], Lina B[SUP]1[/SUP], Escuret V[SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]INTRODUCTION:[/h] 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.
[h=4]METHODS:[/h] 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.
[h=4]RESULTS:[/h] 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.
[h=4]CONCLUSION:[/h] 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.
Copyright ? 2017. Published by Elsevier B.V.
[h=4]KEYWORDS:[/h] H275Y substitution; Influenza viruses; Next generation sequencing; Oseltamivir resistance; RT-droplet digital PCR; RT-qPCR
PMID: 28780426 DOI: 10.1016/j.antiviral.2017.07.021
[h=1]Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis.[/h] Pichon M[SUP]1[/SUP], Gaymard A[SUP]1[/SUP], Josset L[SUP]1[/SUP], Valette M[SUP]2[/SUP], Millat G[SUP]3[/SUP], Lina B[SUP]1[/SUP], Escuret V[SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]INTRODUCTION:[/h] 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.
[h=4]METHODS:[/h] 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.
[h=4]RESULTS:[/h] 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.
[h=4]CONCLUSION:[/h] 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.
Copyright ? 2017. Published by Elsevier B.V.
[h=4]KEYWORDS:[/h] H275Y substitution; Influenza viruses; Next generation sequencing; Oseltamivir resistance; RT-droplet digital PCR; RT-qPCR
PMID: 28780426 DOI: 10.1016/j.antiviral.2017.07.021