**Author details**

Kok-Siong Poon1 , Julian Wei-Tze Tang2 and Evelyn Siew-Chuan Koay1,3\*

1 Department of Laboratory Medicine, National University Hospital, Singapore

2 University Hospitals of Leicester NHS, Leicester, United Kingdom

3 Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

\*Address all correspondence to: evelyn\_koay@nuhs.edu.sg

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**85**

*HCV Genotyping with Concurrent Profiling of Resistance-Associated Variants by NGS Analysis*

cobas® HCV GT is a new tool that accurately identifies Hepatitis C virus genotypes for clinical practice. PLoS

[9] Benedet M, Adachi D, Wong A, Wong S, Pabbaraju K, Tellier R, et al. The need for a sequencing-based assay to supplement the Abbott m2000 RealTime HCV Genotype II assay: A 1 year analysis. Journal of Clinical

[10] Larrat S, Poveda JD, Coudret C, Fusillier K, Magnat N, et al. Sequencing assays for failed genotyping with the versant hepatitis C virus genotype assay (LiPA), version 2.0. Journal of Clinical Microbiology. 2013;**51**:2815-2821

[11] Chueca N, Rivadulla I, Lovatti R, Reina G, Blanco A, et al. Using NS5B sequencing for hepatitis C virus genotyping reveals discordances with commercial platforms. PLoS One.

[12] Quer J, Gregori J, Rodríguez-Frias F, Buti M, Madejon A, et al. High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. Journal of Clinical Microbiology. 2015;**53**:219-226

[13] Kuiken C, Yusim K, Boykin L, Richardson R. The Los Alamos hepatitis C sequence database. Bioinformatics.

[14] Gonçalves Rossi LM, Escobar-Gutierrez A, Rahal P. Multiregion deep sequencing of hepatitis C virus: An improved approach for genetic relatedness studies. Infection, Genetics

and Evolution. 2016;**38**:138-145

[15] Escobar-Gutiérrez A, Vazquez-Pichardo M, Cruz-Rivera M, Rivera-Osorio P, Carpio-Pedroza JC, et al. Identification of hepatitis C virus

One. 2017;**12**:e0175564

Virology. 2014;**60**:301-304

2016;**11**:e0153754

2005;**1**:379-384

*DOI: http://dx.doi.org/10.5772/intechopen.84577*

[1] Murphy DG, Sablon E, Chamberland J,

[2] Ghany MG, Strader DB, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C: An update. Hepatology. 2009;**49**:1335-1374

[3] ASLD-IDSA HCV Guidance Panel. Hepatitis C guidance 2018 update: AASLD-IDSA recommendations for testing, managing, and treating hepatitis C virus infection. Clinical

[4] Germer JJ, Majewski DW, Rosser M, Thompson A, Mitchell PS, et al. Evaluation of the TRUGENE HCV 5'NC genotyping kit with the new GeneLibrarian module 312 for genotyping of hepatitis C virus from clinical specimens. Journal of Clinical Microbiology. 2003;**41**:4855-4857

[5] Verbeeck J, Stanley MJ, Shieh J, Celis L, Huyck E, et al. Evaluation of Versant hepatitis C virus genotype assay (LiPA) 2.0. Journal of Clinical Microbiology.

[6] González V, Gomes-Fernandes M, Bascuñana E, Casanovas S, Saludes V, et al. Accuracy of a commercially available assay for HCV genotyping and subtyping in the clinical

practice. Journal of Clinical Virology.

[7] Némoz B, Roger L, Leroy V, Poveda JD, Morand P, et al. Evaluation of the cobas® GT hepatitis C virus genotyping assay in G1-6 viruses including low viral loads and LiPA failures. PLoS One.

[8] Fernández-Caballero JA, Alvarez M, Chueca N, Pérez AB, García F. The

Infectious Diseases. 2018

2008;**46**:1901-1906

2013;**58**:249-253

2018;**13**:e0194396

Fournier E, Dandavino R, et al. Hepatitis C virus genotype 7, a new genotype originating from central Africa. Journal of Clinical Microbiology.

2015;**53**:967-972

**References**

*HCV Genotyping with Concurrent Profiling of Resistance-Associated Variants by NGS Analysis DOI: http://dx.doi.org/10.5772/intechopen.84577*

### **References**

*Bioinformatics Tools for Detection and Clinical Interpretation of Genomic Variations*

extended to cover GTs other than HCV 1a and 1b, namely GTs 2-6.

Diagnostics Singapore for funding the NGS reagents in this study.

The authors have no conflict of interest to declare.

, Julian Wei-Tze Tang2

\*Address all correspondence to: evelyn\_koay@nuhs.edu.sg

In conclusion, the genotyping results of the Vela NGS were found to be highly concordant with those of the LiPA method. Vela NGS refined the ST assignment in GT 6 and resolved previously indeterminate GTs reported by LiPA. Technically, the HCV Vela NGS was found to have consistent intra- and inter-run reproducibility in terms of GT and ST calling and RAVs identification. Detection of infections with multiple HCV GTs or STs is feasible by Vela NGS. Due to the assay design which relies on investigating the HCV sub-genomic regions, HCV recombinant strains may still be potentially missed. Deep sequencing allows sensitive identification of RAVs in the GT1a and 1b NS3, NS5A and NS5B regions, but the list of target RAVs is not exhaustive. We would also suggest the RAVs detection spectrum should be

We thank Cui-Wen Chua, Mui-Joo Khoo and Lily Chiu of the Department of Laboratory Medicine at the National University Hospital, Singapore, for their technical assistance in performing the NGS and LiPA analysis. We also thank Vela

**5. Conclusions**

**Acknowledgements**

**Conflict of interest**

**Author details**

Kok-Siong Poon1

of Singapore, Singapore

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Laboratory Medicine, National University Hospital, Singapore

3 Department of Pathology, Yong Loo Lin School of Medicine, National University

2 University Hospitals of Leicester NHS, Leicester, United Kingdom

and Evelyn Siew-Chuan Koay1,3\*

**84**

provided the original work is properly cited.

[1] Murphy DG, Sablon E, Chamberland J, Fournier E, Dandavino R, et al. Hepatitis C virus genotype 7, a new genotype originating from central Africa. Journal of Clinical Microbiology. 2015;**53**:967-972

[2] Ghany MG, Strader DB, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C: An update. Hepatology. 2009;**49**:1335-1374

[3] ASLD-IDSA HCV Guidance Panel. Hepatitis C guidance 2018 update: AASLD-IDSA recommendations for testing, managing, and treating hepatitis C virus infection. Clinical Infectious Diseases. 2018

[4] Germer JJ, Majewski DW, Rosser M, Thompson A, Mitchell PS, et al. Evaluation of the TRUGENE HCV 5'NC genotyping kit with the new GeneLibrarian module 312 for genotyping of hepatitis C virus from clinical specimens. Journal of Clinical Microbiology. 2003;**41**:4855-4857

[5] Verbeeck J, Stanley MJ, Shieh J, Celis L, Huyck E, et al. Evaluation of Versant hepatitis C virus genotype assay (LiPA) 2.0. Journal of Clinical Microbiology. 2008;**46**:1901-1906

[6] González V, Gomes-Fernandes M, Bascuñana E, Casanovas S, Saludes V, et al. Accuracy of a commercially available assay for HCV genotyping and subtyping in the clinical practice. Journal of Clinical Virology. 2013;**58**:249-253

[7] Némoz B, Roger L, Leroy V, Poveda JD, Morand P, et al. Evaluation of the cobas® GT hepatitis C virus genotyping assay in G1-6 viruses including low viral loads and LiPA failures. PLoS One. 2018;**13**:e0194396

[8] Fernández-Caballero JA, Alvarez M, Chueca N, Pérez AB, García F. The

cobas® HCV GT is a new tool that accurately identifies Hepatitis C virus genotypes for clinical practice. PLoS One. 2017;**12**:e0175564

[9] Benedet M, Adachi D, Wong A, Wong S, Pabbaraju K, Tellier R, et al. The need for a sequencing-based assay to supplement the Abbott m2000 RealTime HCV Genotype II assay: A 1 year analysis. Journal of Clinical Virology. 2014;**60**:301-304

[10] Larrat S, Poveda JD, Coudret C, Fusillier K, Magnat N, et al. Sequencing assays for failed genotyping with the versant hepatitis C virus genotype assay (LiPA), version 2.0. Journal of Clinical Microbiology. 2013;**51**:2815-2821

[11] Chueca N, Rivadulla I, Lovatti R, Reina G, Blanco A, et al. Using NS5B sequencing for hepatitis C virus genotyping reveals discordances with commercial platforms. PLoS One. 2016;**11**:e0153754

[12] Quer J, Gregori J, Rodríguez-Frias F, Buti M, Madejon A, et al. High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. Journal of Clinical Microbiology. 2015;**53**:219-226

[13] Kuiken C, Yusim K, Boykin L, Richardson R. The Los Alamos hepatitis C sequence database. Bioinformatics. 2005;**1**:379-384

[14] Gonçalves Rossi LM, Escobar-Gutierrez A, Rahal P. Multiregion deep sequencing of hepatitis C virus: An improved approach for genetic relatedness studies. Infection, Genetics and Evolution. 2016;**38**:138-145

[15] Escobar-Gutiérrez A, Vazquez-Pichardo M, Cruz-Rivera M, Rivera-Osorio P, Carpio-Pedroza JC, et al. Identification of hepatitis C virus

transmission using a next-generation sequencing approach. Journal of Clinical Microbiology. 2012;**50**:1461-1463

[16] Caraballo Cortes K, Rosińska M, Janiak M, Stępień M, Zagordi O, et al. Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center. PLoS One. 2018;**13**:e0194816

[17] Salmona M, Caporossi A, Simmonds P, Thélu MA, Fusillier K, et al. First next-generation sequencing fullgenome characterization of a hepatitis C virus genotype 7 divergent subtype. Clinical Microbiology and Infection. 2016;**22**:e1-e947

[18] Thomson E, Ip CL, Badhan A, Christiansen MT, Adamson W, et al. Comparison of next-generation sequencing technologies for comprehensive assessment of full-length hepatitis C viral genomes. Journal of Clinical Microbiology. 2016;**54**:2470-2484

[19] Pedersen MS, Fahnøe U, Hansen TA, Pedersen AG, Jenssen H, et al. A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus. Journal of Clinical Virology. 2018;**195**:49-56

[20] Del Campo JA, Parra-Sánchez M, Figueruela B, García-Rey S, Quer J, et al. Hepatitis C virus deep sequencing for sub-genotype identification in mixed infections: A real-life experience. International Journal of Infectious Diseases. 2018;**67**:114-117

[21] Manee N, Thongbaiphet N, Pasomsub E, Chantratita W. Clinical evaluation of a newly developed automated massively parallel sequencing assay for hepatitis C virus genotyping and detection of resistanceassociation variants Comparison with a line probe assay. Journal of Virological Methods. 2017;**249**:31-37

[22] Dirani G, Paesini E, Mascetra E, Farabegoli P, Dalmo B, et al. A novel next generation sequencing assay as an alternative to currently available methods for hepatitis C virus genotyping. Journal of Virological Methods. 2018;**251**:88-91

[23] Rodriguez C, Soulier A, Demontant V, Poiteau L, Mercier-Darty M, et al. A novel standardized deep sequencingbased assay for hepatitis C virus genotype determination. Scientific Reports. 2018;**8**:4180

[24] Bunchorntavakul C, Chavalitdhamrong D, Tanwandee T. Hepatitis C genotype 6: A concise review and response-guided therapy proposal. World Journal of Hepatology. 2013;**5**:496-504

[25] Schuermans W, Orlent H, Desombere I, Descheemaeker P, Van Vlierberghe H, et al. Heads or tails: Genotyping of hepatitis C virus concerning the 2k/1b circulating recombinant form. International Journal of Molecular Sciences. 2016;**17**

[26] Iles JC, Njouom R, Foupouapouognigni Y, Bonsall D, Bowden R, et al. Characterization of hepatitis c virus recombination in Cameroon by use of nonspecific next-generation sequencing. Journal of Clinical Microbiology. 2015;**53**:3155-3164

[27] Chui CK, Dong WW, Joy JB, Poon AF, Dong WY, et al. Development and validation of two screening assays for the hepatitis C virus NS3 Q80K polymorphism associated with reduced response to combination treatment regimens containing simeprevir. Journal of Clinical Microbiology. 2015;**53**:2942-2950

[28] Vicenti I, Falasca F, Sticchi L, Bruzzone B, Turriziani O, et al. Evaluation of a commercial real-time PCR kit for the detection of the Q80K

**87**

*HCV Genotyping with Concurrent Profiling of Resistance-Associated Variants by NGS Analysis*

*DOI: http://dx.doi.org/10.5772/intechopen.84577*

polymorphism in plasma from HCV genotype 1a infected patients. Journal of

[29] Pawlotsky JM. Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens. Gastroenterology. 2016;**151**:70-86

[30] Yoshida K, Hai TA, Teranishi Y, Kozuka R, et al. Long-term follow-up of resistance-associated substitutions in hepatitis C virus in patients in which direct acting antiviral-based therapy failed. International Journal of

Molecular Sciences. 2017;**18**

and asunaprevir. Journal of Gastroenterology. 2017;**52**:855-867

[31] Mawatari S, Oda K, Tabu K, Ijuin S, Kumagai K. New resistanceassociated substitutions and failure of dual oral therapy with daclatasvir

Clinical Virology. 2016;**76**:20-23

*HCV Genotyping with Concurrent Profiling of Resistance-Associated Variants by NGS Analysis DOI: http://dx.doi.org/10.5772/intechopen.84577*

polymorphism in plasma from HCV genotype 1a infected patients. Journal of Clinical Virology. 2016;**76**:20-23

*Bioinformatics Tools for Detection and Clinical Interpretation of Genomic Variations*

[22] Dirani G, Paesini E, Mascetra E, Farabegoli P, Dalmo B, et al. A novel next generation sequencing assay as an alternative to currently available methods for hepatitis C virus genotyping. Journal of Virological

[23] Rodriguez C, Soulier A, Demontant V, Poiteau L, Mercier-Darty M, et al. A novel standardized deep sequencingbased assay for hepatitis C virus genotype determination. Scientific

Methods. 2018;**251**:88-91

Reports. 2018;**8**:4180

2013;**5**:496-504

[24] Bunchorntavakul C,

Chavalitdhamrong D, Tanwandee T. Hepatitis C genotype 6: A concise review and response-guided therapy proposal. World Journal of Hepatology.

[25] Schuermans W, Orlent H, Desombere I, Descheemaeker P, Van Vlierberghe H, et al. Heads or tails: Genotyping of hepatitis C virus concerning the 2k/1b circulating

of Molecular Sciences. 2016;**17**

Foupouapouognigni Y, Bonsall D, Bowden R, et al. Characterization of hepatitis c virus recombination in Cameroon by use of nonspecific next-generation sequencing. Journal of Clinical Microbiology.

[27] Chui CK, Dong WW, Joy JB, Poon AF, Dong WY, et al. Development and validation of two screening assays for the hepatitis C virus NS3 Q80K polymorphism associated with reduced response to combination treatment regimens containing simeprevir. Journal of Clinical Microbiology.

[28] Vicenti I, Falasca F, Sticchi L, Bruzzone B, Turriziani O, et al. Evaluation of a commercial real-time PCR kit for the detection of the Q80K

[26] Iles JC, Njouom R,

2015;**53**:3155-3164

2015;**53**:2942-2950

recombinant form. International Journal

transmission using a next-generation sequencing approach. Journal of Clinical

[16] Caraballo Cortes K, Rosińska M, Janiak M, Stępień M, Zagordi O, et al. Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center.

[17] Salmona M, Caporossi A, Simmonds P, Thélu MA, Fusillier K, et al. First next-generation sequencing fullgenome characterization of a hepatitis C virus genotype 7 divergent subtype. Clinical Microbiology and Infection.

Microbiology. 2012;**50**:1461-1463

PLoS One. 2018;**13**:e0194816

[18] Thomson E, Ip CL, Badhan A, Christiansen MT, Adamson W, et al. Comparison of next-generation sequencing technologies for

comprehensive assessment of full-length hepatitis C viral genomes. Journal of Clinical Microbiology. 2016;**54**:2470-2484

[19] Pedersen MS, Fahnøe U, Hansen TA,

[20] Del Campo JA, Parra-Sánchez M, Figueruela B, García-Rey S, Quer J, et al. Hepatitis C virus deep sequencing for sub-genotype identification in mixed infections: A real-life experience. International Journal of Infectious

Pedersen AG, Jenssen H, et al. A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus. Journal of Clinical

Virology. 2018;**195**:49-56

Diseases. 2018;**67**:114-117

Methods. 2017;**249**:31-37

[21] Manee N, Thongbaiphet N, Pasomsub E, Chantratita W. Clinical evaluation of a newly developed automated massively parallel

sequencing assay for hepatitis C virus genotyping and detection of resistanceassociation variants Comparison with a line probe assay. Journal of Virological

2016;**22**:e1-e947

**86**

[29] Pawlotsky JM. Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens. Gastroenterology. 2016;**151**:70-86

[30] Yoshida K, Hai TA, Teranishi Y, Kozuka R, et al. Long-term follow-up of resistance-associated substitutions in hepatitis C virus in patients in which direct acting antiviral-based therapy failed. International Journal of Molecular Sciences. 2017;**18**

[31] Mawatari S, Oda K, Tabu K, Ijuin S, Kumagai K. New resistanceassociated substitutions and failure of dual oral therapy with daclatasvir and asunaprevir. Journal of Gastroenterology. 2017;**52**:855-867
