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

•H275Y-NA-induced oseltamivir resistance must be explored in patients with prolonged excretion of A(H1N1) influenza viruses.•ddPCR technologies could be an interesting alternative to sequencing technologies for viral resistance management.•Objective quantification of viral subpopulations may be optimized using open-source or manufacturers' software.

IntroductionThe 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.MethodsRT-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.ResultsThe 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.ConclusionRT-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.