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Misidentification of a Hepatitis C Virus (HCV) recombinant form leading to treatment failure with new direct acting antivirals

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HAL Id: hal-02265854

https://hal-normandie-univ.archives-ouvertes.fr/hal-02265854

Submitted on 12 Aug 2019

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Misidentification of a Hepatitis C Virus (HCV)

recombinant form leading to treatment failure with new direct acting antivirals

Amandine Decroos, Fabienne de Oliveira, Marie Leoz, Sylvie Larrat, Ghassan Riachi, Thomas Mourez

To cite this version:

Amandine Decroos, Fabienne de Oliveira, Marie Leoz, Sylvie Larrat, Ghassan Riachi, et al.. Misiden- tification of a Hepatitis C Virus (HCV) recombinant form leading to treatment failure with new direct acting antivirals. Journée Normande de Recherche Biomédicale, Sep 2016, Rouen, France.

�hal-02265854�

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Misidentification of a Hepatitis C Virus (HCV) recombinant form leading to treatment failure with new direct acting antivirals

Amandine DECROOS1, Fabienne DE OLIVEIRA1, Marie LEOZ1, Sylvie LARRAT2, Ghassan RIACHI3, Thomas MOUREZ1

1- Groupe de Recherche sur l’Adaptation Microbienne, Normandie Univ, UNIROUEN, UNICAEN, GRAM, F-76000 Rouen, France 2- Univ. Grenoble Alpes, UVHCI, F-38000 Grenoble, France

3- Service d’Hépato-gastro-entérologie, CHU de Rouen, F-76000 Rouen, France

CONCLUSION

We confirm that analysing a single region of the genome to extrapolate the HCV genotype leads to misidentification of HCV recombinant forms

The misidentification of these forms leads to treatment failure. In the case presented here the additional cost of the treatment, follow-up and laboratory analysis was estimated above 70 000 euros

We urgently need rapid and easy methods for the whole genome sequencing of HCV to avoid such costly consequences

All suspicions of recombinant strains can be investigated by the method described herein

The use of new Direct Acting Antivirals (DAA) specific of Hepatitis C Virus (HCV), has greatly improved the virological suppression, and for the most recent ones, the biological and clinical tolerance of HCV treatment. Most of the DAA do not haveyet a pangenotypic action, which means that most of them have a predominant action on specific HCV genotype. This observation led to different recommendations in the treatment of HCV genotype 1, 2, 3, and 4 infections.

Genotyping methods may target different regions of the HCV genome, although only the whole genome sequencing could confirm the correct genotype. We report the case of a patient, infected with an unusual recombinant 2k/1b recombinant HCV form which was falsely diagnosed with an HCV genotype 2 infection, and treated with an unadapted treatment, leading to a treatment failure.

INTRODUCTION PATIENT

In 2005, a 42-year-old Ukrainian male, was diagnosed with an HCV infection, which was genotyped as a genotype 2 using the Genekit HCV 5’NC (Visible). In May 2014, he started a 12 week treatment with sofosbuvir (400 mg daily) and ribavirin (600 mg twice daily), following our national guidelines for genotype 2 treatment. The pre-treatment HCV RNA plasma viral load (pVL) was 5.7 log UI/mL. Four weeks after, the pVL was <12 UI/mL and remained undetectable until the end of treatment in August 2014. Two weeks after, the pVL was detectable, and rose to 6.1 log UI/mL eight weeks after the end of the treatment.

After this virological failure, a retreatment was decided in July 2015 with the same drug association but for 24 weeks. In the meantime, we decided to check the genotype using a previously described method targeting the NS5B region.

Both 5’ Core and 3’ NS5B region of the genome were sequenced to confirm the presence of a putative recombinant virus

A cDNA covering the region from E1 to NS3 was synthesised, and sequenced by the Sanger method with a set of 15 primers adapted from previously published methods

Bioinformatic analysis was performed by NCBI Genotyping, BLAST, Neighbour Joining phylogenetic analysis and SimPlot sequences

METHODS

Regions targeted for genotyping assay

Proteins targeted by HCV direct acting antivirals Region amplified to determine

the recombination breakpoint

RESULTS

Sequencing of the Core region at the beginning of the genome confirmed the phylogenetic proximity to genotype 2k HCV

Sequencing of the NS5B region at the end of the genome confirmed the phylogenetic proximity to genotype 1b HCV

Comparison of the NS5B gene to HCV 1a and 1b reference strains, and recombinant 2k/1b described in Russia and Cyprus made us focus the region from E1 to NS3 to search for the recombination breakpoint, as the breakpoint is usually situated in this part of the genome in these circulating strains

Similarity of the complete sequence of 1699 nucleotides, from E1 to NS3, was compared to different genotype query sequences to identify the recombination breakpoint using SimPlot software

SimPlot analysis showed a recombination breakpoint at the final part of the NS2 gene as described below

Similarity plot of the 2k/1b E1 to NS3 HCV region

Plot was generated by the SimPlot software with 200 nucleotides (nt) windows, 20 nt increments, and the Kimura 2-parameter method with a transition-transversion (Ts/Tv) ratio of 2.0.

The sample was queried against 3 different 2k/1b HCV recombinants in red, green and yellow, a genotype 1b reference strain in blue, and a genotype 2k reference strain in grey [HQ537005 (2k1b), AY587845 (2k1b), HQ537006 (2k1b), AB031663 (2k), AB016785 (1b)].

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