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Abstract

Background

To meet the target of Hepatitis C elimination in the UK, a rapid and high throughput method of genotyping is desirable. Current methods target the NS5B region with a nested PCR followed by Sanger sequencing. Drawbacks include insufficient information to guide second line treatment in the event of failure and poor sensitivity compared to assays targeting the 5’UTR.

Method

Previously tested HCV positive patient samples were selected and RNA extracted using an ultra-centrifugation manual extraction method. A two-step reverse transcription long range PCR generated amplicons which were quantified using the Qubit fluorometer and libraries prepared using the Nextera XT library preparation kit. Sequencing was performed using an Illumina Miseq and genomes were assembled using a Linex-based bioinformatics pipeline.

Results

A total of 150 samples were tested, with full HCV genomes obtained for genotypes 1–6 and the sensitivity of the assay corresponded to around 1000 IU  ml. Where previous sequence data for resistance associated variants (RAVs) was available, mutations were consistent. Furthermore, the assay identified a 2 k/1b recombinant and allowed the typing of previously unassigned genotypes, demonstrating its utility in the diverse local population.

Conclusion

This study displayed the utility of a whole genome sequencing approach to HCV. A major benefit of whole genome analysis was the inclusion of regions targeted by antiviral therapy such as NS5A and NS3. This allowed the identification of RAVs in patients experiencing treatment failure. Its application to dried blood spots is currently being investigated as part of a wider screening programme.

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/content/journal/acmi/10.1099/acmi.ac2019.po0534
2019-04-08
2019-12-11
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