- Email: [email protected]
High HCV subtype heterogeneity in a chronically infected general population revealed by high-resolution hepatitis C virus subtyping
Accepted Manuscript High Hcv subtype heterogeneity in a chronically infected general population Revealed by high-Resolution Hcv subtyping Dr Francisco Rodriguez-Frias, Leonardo Nieto, Josep Gregori, Damir Garcia-Cehic, Rosario Casillas, David Tabernero, Maria Homs, Maria Blasi, Marta Vila, Qian Chen, Victor Vargas, Lluís Castells, Lluís Viladomiu, Joan Genesca, Beatriz Minguez, Salvador Augustin, Judit Carbonell, Celia Perales, Maria Eugenia Soria, Miriam Asensio, Meritxell Llorens, Laura Ordeig, Cristina Godoy, Maria Buti, Rafael Esteban, Tomas Pumarola, Juan Ignacio Esteban, Josep Quer PII:
S1198-743X(17)30095-2
DOI:
10.1016/j.cmi.2017.02.007
Reference:
CMI 855
To appear in:
Clinical Microbiology and Infection
Received Date: 31 August 2016 Revised Date:
2 February 2017
Accepted Date: 4 February 2017
Please cite this article as: Rodriguez-Frias F, Nieto L, Gregori J, Garcia-Cehic D, Casillas R, Tabernero D, Homs M, Blasi M, Vila M, Chen Q, Vargas V, Castells L, Viladomiu L, Genesca J, Minguez B, Augustin S, Carbonell J, Perales C, Soria ME, Asensio M, Llorens M, Ordeig L, Godoy C, Buti M, Esteban R, Pumarola T, Esteban JI, Quer J, High Hcv subtype heterogeneity in a chronically infected general population Revealed by high-Resolution Hcv subtyping, Clinical Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT HIGH HCV SUBTYPE HETEROGENEITY IN A CHRONICALLY INFECTED GENERAL POPULATION
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REVEALED BY HIGH-RESOLUTION HCV SUBTYPING
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Francisco Rodriguez-Frias,a,b,c,*# Leonardo Nieto,a,d,* Josep Gregori,b, ,e,f Damir Garcia-Cehic,bf Rosario Casillas,a,b David Tabernero,a,b Maria Homs,a,b Maria Blasi,a Marta Vila,a Qian Chen,f Victor Vargas, b,c,f Lluís Castells, b,c,f Lluís Viladomiu, b,c,f Joan Genesca, b,c,f Beatriz Minguez, b,c,f Salvador Augustin, b,c,f Judit Carbonell,c,f Celia Perales,b,f Maria Eugenia Soria, f Miriam Asensio a,b , Meritxell Llorens f, Laura Ordeig f, Cristina Godoy a, Maria Buti,b,c,f Rafael Esteban,b,c,f Tomas Pumarolac,d, Juan Ignacio Esteban,b,c,f Josep Querb,c,f
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*Both authors have contributed equally to the work.
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Liver Pathology Unit, Department of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron (HUVH), 08035, Barcelona, Spain b Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, 28029, Madrid, Spain c Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain d Clinical Microbiology Deparment, HUVH, 08035, Barcelona, Spain e Roche Diagnostics SL, Sant Cugat del Vallès, 08174, Barcelona, Spain f Liver Unit, Liver Disease Laboratory, Internal Medicine Department, Vall d’Hebron Institut Recerca (VHIR)-HUVH), 08035, Barcelona, Spain
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Running Title: Prevalence HCV subtypes by UDPS in Barcelona
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Keywords: Hepatitis C virus, Subtypes, Mixed infections, High-resolution HCV subtyping
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Dr Francisco Rodriguez-Frias Liver Pathology Unit, Department of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, 08035, Barcelona, Spain Tel: 34 932746897 / 6896 E-mail addresses: [email protected], [email protected]
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Abstract: 249
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Main text (Abstract, References, Acknowledgment and Figure Legends not included): 2397
ACCEPTED MANUSCRIPT ABSTRACT
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Objectives: Accurately characterize the chronically-infected general HCV population in the
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Barcelona area by a highly sensitive subtyping method able to identify the 67 recognized
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hepatitis C virus (HCV) subtypes, and to discriminate mixed infections in a single individual.
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This information has strong implications on patient management, in deciding the optimal
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combination of direct-acting antivirals (DAAs) for each patient.
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Methods: HCV subtyping by UDPS. All HCV patients who attended Vall d'Hebron Hospital
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outpatient clinics from February 2015 to May 2016 (N=1473) were included.
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Results: The observed genotypes were: G1 (76.4%), G4 (9.8%), G3 (9.2%), G2 (3.5%) and G5
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(0.1%). Twenty-two subtypes were observed: 1b (53.6%), 1a (22.5%), 3a (9.0%), 4d (7.1%),
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4a (2.0%), and 2c (1.7%), with 16 low-prevalence subtypes accounting for the remaining
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3.2%. There was a worrisome 1% of mixed infections. Of note, G2 (3.5%) showed a high
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heterogeneity. An analysis stratified by age groups illustrated a clear predominance of G1b
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over G1a (84.5% vs 4.8%) in patients older than 63 years (N=516) and similar percentages of
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these subtypes in the 40 to 63 year-old group (N=824) (37.1% vs 32.0%) and the youngest
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group (<40) (N=133) (36.1% vs 32.3%).
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Conclusions: The observed subtype distribution shows a much higher heterogeneity than
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expected. Particularly G2 with few individuals, and mixed infections. The distribution by age
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groups suggests that all patients born before 1975 should be monitorized. The use of highly
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sensitive methods for HCV classification opens the door to acquiring a better acknowledge
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on the efficacy of each DAA-based treatment.
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ACCEPTED MANUSCRIPT INTRODUCTION
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The development of small-molecule compounds that directly inhibit the viral life cycle
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(DAA) has been a major milestone in the treatment of chronic hepatitis C virus (HCV)
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infection. These DAAs are inhibitors targeting non-structural proteins (NS3, NS5A and NS5B)
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that have essential functions in the viral life cycle (1). Sustained virological response (SVR)
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rates in HCV-infected patients may vary depending on the patient’s viral subtype (2-4).
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Infection with more than one subtype (mixed infection) may also affect the treatment
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outcome (5). Therefore, HCV subtyping and detection of mixed infections are emerging as
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key issues in deciding the most suitable combination therapy for affected patients (6-8).
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HCV is currently classified into 7 genotypes (numbered 1 to 7), and 67 subtypes within these
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genotypes (designated by lower case letters a to z) (9). In Spain, the reported genotype
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distribution is as follows: genotype (G) 1, 6%; G1b, 36%; G1a, 27%; G2, 2.70%; G3, 17%; and
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G4, 12% (10), which concurs with the worldwide genotype distribution (G1, 46.2%; G3,
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30.1%; G2, G4, and G6, 22.8%; and G5, <1%)(11;12). However, these data were obtained by
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classic low-resolution genotyping methods, known to have significant misclassification rates
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(13-17).
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Next-generation sequencing (NGS) enables thousands of clonal sequences to be obtained
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from a single sample and is currently the most powerful tool for analyzing viral quasispecies.
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Among the available NGS methods, the 454/GS-Junior technology has been successfully
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used to study of several aspects of the hepatitis virus quasispecies (17-23). Our laboratory
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recently developed a high-resolution method that can confidently identify the 67 HCV
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subtypes and is easily adaptable to detect new subtypes. Furthermore, the high coverage
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ACCEPTED MANUSCRIPT (2000-5000 sequences/patient) enables detection of mixed infections with high sensitivity
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(17).
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As effective DAA-based HCV therapy is subtype-dependent, it is important to know the
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distribution of subtypes in chronically infected patients and the percentage of patients with
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mixed infection in a particular geographic area. The aim of this study was to accurately
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characterize by subtypes the HCV chronically-infected population in the Barcelona area,
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using this high sensitive method.
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MATERIAL AND METHODS
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Blood samples were obtained from all consecutive patients diagnosed with active chronic
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HCV infection attended in the hepatology and infectious disease outpatient clinics of Vall
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d´Hebron University Hospital (Barcelona) between February 2015 and May 2016 regardless
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of their treatment status, as part of their routine workup. Samples were delivered to our
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laboratory within 8 hours after extraction and centrifuged; plasma was stored at -80ºC until
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analysis. Demographic data were obtained from the patients’ clinical records.
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HCV RNA was extracted using a total nucleic acid isolation (TNAI) kit in a COBAS/AmpliPrep
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system (Roche Diagnostic, West Sussex, UK). RNA amplification and deep sequencing were
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carried out according to a previously described method (17). Briefly, nested RT-PCR was
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performed using specific primers for the NS5B region. The PCR product form each plasma
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sample was quantified using the PicoGreen assay (Invitrogen, Carlsbad, CA, USA), and
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quality-analyzed using the BioAnalyzer DNA 1000 LabChip (Agilent, Santa Clara, CA, USA)
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prior to sequencing. PCR products from 20 patients individually identified by a genetic
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barcode were mixed in the same emulsion PCR reaction at equal molecular proportions, and
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ACCEPTED MANUSCRIPT then subjected to deep sequencing using the 454/GS-Junior platform (Roche, Branford, CA,
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USA). The 454/GS-Junior data then underwent a process of demultiplexing, quality filtering,
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haplotype clustering, and phylogenetic analysis using software developed by our laboratory
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and the Bioinformatics Clinical Services at Roche (Sant Cugat, Spain), as previously described
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(17). Phylogenetic analysis of the data was then performed using the DeepChek-HCV
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subtyping software by ABL, Luxemburg (https://www.ablsa.com), including a set of
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reference sequences for all subtypes (9).
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including equimolecular pooling, was automated using an Evo 75 robotic device (Tecan,
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Switzerland) to adapt it to routine laboratory practice and obtain results within 4 days.
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RESULTS
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Samples from 1473 consecutive patients were HCV-subtyped in routine practice to
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determine the most effective antiviral therapy for each patient. An average of 2500 reads
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(SD 500) for the NS5B region were obtained per patient. HCV genotype (G) 1 was found to
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be the most prevalent (76.4%), followed by G4 (9.8%) and G3 (9.2%). Genotype 2 had a low
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prevalence of 3.5% and only 1 G5 case was identified during the study period (0.1%). Mixed
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infections (Mx) with more than 1 HCV subtype were found in 1% of the samples (Figure 1a
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and Supplementary Table 1a).
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On subtype analysis, subtype 1b was the most prevalent (53.6%), followed by 1a (22.5%), 3a
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(9.0%), and 4d (7.1%). The remaining 7.8% included 18 minority subtypes (Figure 1b and
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Supplementary Table 1b), and 15 patients (1%) had mixed infections (Figure 1b and Table
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1). Most of the rare subtypes were seen in only a few individuals (1 to 8). G2, which was the
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less prevalent, showed the largest number of different subtypes (2a, 2b, 2c, 2i, 2j, 2m, 2q),
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ACCEPTED MANUSCRIPT followed by genotype 4 with 6 subtypes (4a, 4d, 4f, 4n, 4p, 4t) and genotype 1 with 5
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subtypes (1a, 1b, 1l, 1e, 1g, 1l). The mixed infections included subtypes 1a, 1b, 1l, 2c, 3a, 4a,
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4d, and 4f: 12 patients had 2 subtypes, 2 patients had 3 subtypes, and 1 patient had 4
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subtypes (Table 1).
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The number of cases was plotted against patient age, and a clearly bimodal distribution was
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observed, with modes at around 50 and 75 years of age (Figure 2). Three age groups were
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established based on this representation: <40 years, 40 to 63 years, and >63 years. The
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oldest group (>63) was mainly infected by subtype 1b (84.5%), followed by 1a (4.8%), and
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other subtypes, seen in very few individuals (Table 2). The middle-aged group (40-63)
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showed a large reduction in the prevalence of subtype 1b (37.1%) and a higher prevalence
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of 1a (32.0%), followed by 3a (12.5%), and 4d plus 4a (14.5%). The youngest group (<40)
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included a small percentage (9.03%) of HCV-infected patients (133/1473) and showed a
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subtype distribution profile similar to that of the middle-aged group: 1b (36.1%), 1a (32.3%),
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3a (16.5%) and 4d plus 4a (8.3%).
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DISCUSSION
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The novelty of this study is that HCV subtyping was carried out using a high-resolution NGS
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technique which enabled confident identification of the 67 recognized HCV subtypes and
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the detection of mixed infections. As the samples analyzed were from all consecutive HCV
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patients attended in our specialized outpatient clinics during the study period, these results
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are expected to be an accurate representation of HCV subtype distribution in the general
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population of the Barcelona metropolitan area (~1.5 million).
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ACCEPTED MANUSCRIPT Overall, HCV genotype distribution was similar to the results reported in other Spanish
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studies, in which G1 was the most prevalent (10;24). However, the prevalence of subtype 1b
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was higher in Barcelona (58%) than the values reported in both historical and recent studies
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performed in Spain (41.3% in 2006 (24) and 36% in 2015 (10). The reason for this difference
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may reside in the use of low-resolution techniques in these studies, which are unable to
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subtype all G1 viral strains (6% uncertainly for G1 in 2015 study), or because they combine
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results from different centers without a clear definition of the inclusion criteria. The
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percentage of G1a infections (22.5%) was similar to the values reported in the Spanish
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studies (24.1% in 2006 and 27% in 2015). Of note, 5 patients (all immigrants born in West
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Africa) were found to have infection of rare G1 subtypes (1e, 1g, 1l).
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G2 showed a very heterogeneous subtype distribution despite having a low prevalence
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(3.5%) in the population. Seven different G2 subtypes were identified, each found in few
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individuals: 2a (6 patients), 2b (1), 2c (25), 2i (4), 2j (8), 2m (1), and 2q (6). One patient with
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subtype 2j infection had a history of nonresponse to pegIFN+Rbv therapy. Genotype 2
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infections originated in Spain (46/51), Latin America (4/51), and (1/51) Russia.
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Genotype 3 was highly homogeneous. Among 134 patients with G3 infection, 133 had
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subtype 3a, whereas 1 immigrant from Pakistan had subtype 3b. Interestingly, the
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percentage of G3 cases has decreased considerably over the last 10 years, dropping from
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30.1% in 2006 (19) to 9.2% in the present study, and being surpassed by G4 (9.8%). This
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decrease may result from a combination of factors: genotype classification errors using low-
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resolution techniques, high SVR rates in G3 patients treated with pegIFN+Rbv, and deaths in
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HCV-infected intravenous drug abusers—for example, due to HIV coinfection.
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ACCEPTED MANUSCRIPT Six G4 subtypes were identified, with 4d being the most prevalent (105/145, 72.4%) and
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representing 7.1% of the total samples, followed by 4a (29/145, 20.03%), 4f (8/145, 5.5%),
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and 4n, 4p, and 4t with a single case each (1/145, 0.7%). Patients with subtypes 4a, 4f, 4n,
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4p, and 4t were immigrants born in Equatorial Guinea. Genotype 4 was the second most
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prevalent genotype in our chronically-infected HCV population, in contrast to the data
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reported in other Spanish studies, in which G3 was more prevalent than G4 (10;24). This
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difference may be related to the population cohort included: Aguilera et al enrolled patients
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genotyped in a number of different Spanish centers, whereas our study focused on patients
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followed in the specific setting of Barcelona. Subtype 4d was the most common, likely
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because in our area 4d is highly prevalent in the subgroup of men who have sex with men
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and are coinfected with HIV plus HCV (25). We identified one case of G5a, in an individual
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from Belgium, who may have been infected in South Africa.
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One finding of concern is the prevalence of mixed infections (1%), which could likely be
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higher in high-risk HCV-infected patients, such as intravenous drug addicts.
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On analysis of HCV subtype prevalence by age group, the oldest group (>63 years), which
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included individuals who lived through the Spanish Civil war and post-war period, were
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mainly infected by subtype 1b (84.5%). This group accounted for 35% of all HCV-infected
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patients seen in our outpatient facility. The middle-aged group (40-63 years), encompassed
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individuals born during the time when large hospitals were built within the publically-
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funded Spanish healthcare system and there was a peak in immigration to Barcelona from
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other Spanish regions. This group represented 55.9% of all HCV-infected patients
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(824/1473). Several international HCV clinical management guidelines (AASLD, EASL, and
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JSH) (26-28) recommend testing for the virus in all persons born between 1945 and 1965.
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ACCEPTED MANUSCRIPT Our data concur with this recommendation, but Spain did not participate in the Second
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World War (1939-1945), experiencing instead a civil war (1936-1939). Hence, our results
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suggest that these epidemiological studies should include patients born before 1975.
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A review of the recent history of waves of immigration to the Barcelona metropolitan area
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(since 1939) shows that there was no significant influx of refugees to Spain from any part of
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Europe (including France), despite Spain’s neutrality during the Second World War. This fact,
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together with the absence or minor incidence of immigration from the rest of Spain to
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Catalonia before the 1960s suggest that subtype 1b (86% in patients older than 63 years) is
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endemic in Catalonia, whereas genotypes 1a, 2, 3a, and 4 were imported in successive
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waves of immigration from other Spanish regions in the 1960s, and from Latin America and
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other HCV-endemic areas in the 1980s. Nonetheless, factors other than immigration should
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also be considered when discussing the introduction and transmission of particular HCV
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subtypes in a community. The changes in HCV subtype distribution may be the consequence
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of several factors that would differentially affect the specific regions of Europe: expansion of
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intravenous drug use, immigration to Europe from endemic areas (29), and high-risk
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behavior.
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The high-resolution NGS-based HCV subtyping used in this study can be widely applied to
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unequivocally determine the true prevalence of subtypes in a geographic area. HCV subtype
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distribution may have important implications in the design of healthcare management
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policies related to scheduling effective antiviral therapy. In this respect, G1 subtyping
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(1a/1b) provides relevant information concerning response rates, genetic barriers to
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resistance, and appropriate treatment. HCV treatment using DAA drug combinations is
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subtype-dependent in 1a and 1b patients, with subtype 1a being less susceptible than 1b to
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ACCEPTED MANUSCRIPT some of these therapies. International guidelines recommend genotyping/subtyping using
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an assay that provides accurate differentiation between these subtypes (27;28;30), although
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no recommendations have been provided for subtypes other than 1a and 1b. Nonetheless,
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the recommendations will likely be adapted based on the results of ongoing research in
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other subtypes, which is now possible with the use of high-resolution methods (17). For
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example, a recent study (31) showed that the Viekirax combination (paritaprevir/ritonavir+
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ombitasvir) with or without ribavirin is highly effective in G4 patients (100% SVRwith
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ribavirin and 91% without). However, in the ribavirin-free group, there were 3 failures (2
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relapse and 1 breakthrough) and all patients were found to have subtype 4d (3/16, 18.75%).
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All 3 developed resistance-associated variants (RAS) to NS3 and NS5A. Hence, treatment of
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subtype 4d patients should include ribavirin, and if this is not possible, a change in the DAA
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combination may be appropriate. In another study (32), 44 G4 patients were treated with
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Harvoni (ledipasvir+sofosbuvir) and reached an excellent SVR rate (93%); however, 2 of 3
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patients infected with subtype 4r failed. These results suggest that 4r patients should be
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treated using Harvoni combined with ribavirin and/or an NS3 protease inhibitor, or receive a
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different DAA combination. Although there is no available information on the efficacy of
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current DAA-based therapy in rare subtypes, it is not unreasonable to think that it will also
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be subtype-dependent. Thus, in the future, the use of high-resolution HCV subtyping may
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not be a simple recommendation, but instead, a necessary requirement before prescribing
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antiviral therapy in individual patients with HCV infection.
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CONCLUSIONS
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Our method of HCV subtyping based on deep sequencing of the NS5B amplicon, which has
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been routinely applied in Vall d'Hebron Hospital (Barcelona) for more than 18 months, has
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ACCEPTED MANUSCRIPT offered a highly accurate picture of the HCV population attending the publically-funded
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health system in the Barcelona area, including patients infected with more than one
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subtype. The study shows a richer map of HCV subtypes than was expected, and a highly
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dynamic contribution of these subtypes over the years, with somewhat different prevalence
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according to age.
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We believe that deep sequencing will soon become the gold standard technique for
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classification of HCV and possibly, any other viral infection. The coming generations of high-
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throughput sequencing are providing longer sequence capabilities and higher throughput at
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lower cost. There is now no reason to limit the use of NGS exclusively to research purposes.
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ACCEPTED MANUSCRIPT Acknowledgments
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This work was funded by Instituto de Salud Carlos III, PI12/01893, PI13/00456, PI15/00829
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and PI15/00856 cofinanced by the European Regional Development Fund (ERDF),
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GLD14/00296 from Gilead and CIBERehd (Centro de Investigación en Red de Enfermedades
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Hepáticas y Digestivas) which is funded by Instituto de Salud Carlos III, and by CDTI (Centro
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para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and
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Competitiveness (MINECO), IDI- 20151125. C.P. is supported by the Miguel Servet program
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of the Instituto de Salud Carlos III, grant CP14/00121) cofinanced by the European Regional
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Development Fund (ERDF). The authors thank Celine Cavallo (English native) for English
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language support and helpful editing suggestions. We are indebted to Roche Diagnostics
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Spain.
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ACCEPTED MANUSCRIPT Figure and table legends
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Figure 1. Prevalence of HCV genotypes (Fig. 1a) and subtypes (Fig. 1b) in the HCV
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chronically-infected general population in Barcelona. Mx stands for mixed infection
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(simultaneous infection by more than one HCV subtype).
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Figure 2. A) Histogram of the ages of HCV patients, which show a clear bimodal distribution.
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B) Histograms showing the distribution of HCV subtypes by age group.
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REFERENCES
264 (1) Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 2013 Jul;11(7):482-96.
267 268 269
(2) Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med 2011 Mar 31;364(13):1207-17.
270 271 272
(3) Poordad F, McCone J, Jr., Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med 2011 Mar 31;364(13):1195206.
273 274
(4) Zeuzem S, Soriano V, Asselah T, Bronowicki JP, Lohse AW, Mullhaupt B, et al. Faldaprevir and deleobuvir for HCV genotype 1 infection. N Engl J Med 2013 Aug 15;369(7):630-9.
275 276 277
(5) McNaughton AL, Thomson EC, Templeton K, Gunson RN, Leitch EC. Mixed genotype hepatitis C infections and implications for treatment. Hepatology 2013 Jun 6;doi:10.1002/hep.26554.
278 279 280
(6) Doyle JS, Hellard ME, Thompson AJ. The role of viral and host genetics in natural history and treatment of chronic HCV infection. Best Pract Res Clin Gastroenterol 2012 Aug;26(4):413-27.
281 282 283
(7) Lam AM, Espiritu C, Bansal S, Micolochick Steuer HM, Niu C, Zennou V, et al. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother 2012 Jun;56(6):3359-68.
284 285 286 287
(8) Mauss S, Berg T, Rockstroh J, Sarrazin C, Wedemeyer H. Short Guide to Hepatitis C. Mauss S, Berg T, Rockstroh J, Sarrazin C, Wedemeyer H, editors. 2012 edition. 2012. http://www.FlyingPublisher.com, The Flying Publisher Group. The Flying Publisher Guide. Ref Type: Serial (Book,Monograph)
288 289 290
(9) Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, et al. Expanded classification of hepatitis C Virus into 7 genotypes and 67 Subtypes: updated criteria and assignment web resource. Hepatology 2014;59(1):318-27.
291 292 293 294
(10) Aguilera A et al. Prevalencia Hepatitis C en España. http://www jano es/noticia-el-96-lospacientes-tratados-24892# 2015;I Congreso Nacional del Grupo de estudio de Hepatitis (Gehep) de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (Seimc)(25,Setimbre 2015. Vigo. Spain).
295 296
(11) Gower E, Estes CC, Hindman S, Razavi-Shearer H. Global epidemiology and genotype distribution od the hepatitis C virus. J Hepatol 2014;61(1):S45-S57.
297 298 299
(12) Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 2015 Jan;61(1):7787.
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ACCEPTED MANUSCRIPT (13) 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. J Clin Virol 2014 Jul;60(3):301-4.
303 304 305
(14) Hara K, Rivera MM, Koh C, Sakiani S, Hoofnagle JH, Heller T. Important factors in reliable determination of hepatitis C virus genotype by use of the 5' untranslated region. J Clin Microbiol 2013 May;51(5):1485-9.
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(15) Mallory MA, Lucic DX, Sears MT, Cloherty GA, Hillyard DR. Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing. J Clin Virol 2014 May;60(1):22-6.
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(16) Martro E, Gonzalez V, Buckton AJ, Saludes V, Fernandez G, Matas L, et al. Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions. J Clin Microbiol 2008 Jan;46(1):192-7.
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(17) Quer J, Gregori J, Rodriguez-Frias F, Buti M, Madejon A, Perez-Del-Pulgar S, et al. Highresolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. J Clin Microbiol 2015 Jan;53(1):219-26.
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(18) Rodriguez-Frias F, Tabernero D, Quer J, Esteban JI, Ortega I, Domingo E, et al. Ultra-deep pyrosequencing detects conserved genomic sites and quantifies linkage of drug-resistant amino acid changes in the hepatitis B virus genome. Plos One 2012;7(5):e37874.
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(19) Homs M, Buti M, Tabernero D, Quer J, Sanchez A, Corral N, et al. Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing. World J Gastroenterol 2012 Nov 14;18(42):6096-105.
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(20) Ramirez C, Gregori J, Buti M, Tabernero D, Camos S, Casillas R, et al. A comparative study of ultra-deep pyrosequencing and cloning to quantitatively analyze the viral quasispecies using hepatitis B virus infection as a model. Antiviral Res 2013 May;98(2):273-83.
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(21) Homs M, Caballero A, Gregori J, Tabernero D, Quer J, Nieto L, et al. Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution. Plos One 2014;9(11):e112306.
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(22) Gregori J, Esteban JI, Cubero M, Garcia-Cehic D, Perales C, Casillas R, et al. Ultra-deep pyrosequencing (UDPS) data treatment to study amplicon HCV minor variants. Plos One 2013;in press.
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(23) Gregori J, Salicru M, Domingo E, Sanchez A, Esteban J, Rodriguez-Frias F, et al. Inference with viral diversity indices: Clonal and NGS approaches. Bioinformatics 2013;in press.
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(24) Echevarria JM, Leon P, Pozo F, Avellon A. Follow-up of the prevalence of hepatitis C virus genotypes in Spain during a nine-year period (1996-2004). Enferm Infecc Microbiol Clin 2006 Jan;24(1):20-5.
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(25) Martinez-Rebollar M, Mallolas J, Perez I, Gonzalez-Cordon A, Lonca M, Torres B, et al. [Acute outbreak of hepatitis C in human immunodeficiency virus-infected patients]. Enferm Infecc Microbiol Clin 2015 Jan;33(1):3-8.
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ACCEPTED MANUSCRIPT (26) JSH. JSH Guidelines for the Management of Hepatitis C Virus Infection: A 2014 Update for Genotype 1. Hepatol Res 2014 Jan;44 Suppl S1:59-70.
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(27) EASL. Liver EAftSot. EASL Recommendations on the Treatment of hepatitis C. http://files easl eu/easl-recommendations-on-treatment-of-hepatitis-C/index html 2014.
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(28) American Association for the Study of Liver Diseases. Recommendations for Testing, Managing, and Treating Hepatitits C. http://hcvguidelines org/full-report/initialtreatment-hcv-infection-patients-starting-treatment 2014.
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(29) Esteban JI, Sauleda S, Quer J. The changing epidemiology of hepatitis C virus infection in Europe. J Hepatol 2008 Jan;48(1):148-62.
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(30) EASL. EASL Clinical Practice Guidelines: management of hepatitis C virus infection. J Hepatol 2014 Feb;60(2):392-420.
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(31) Hezode C, Asselah T, Reddy KR, Hassanein T, Berenguer M, Fleischer-Stepniewska K, et al. Ombitasvir plus paritaprevir plus ritonavir with or without ribavirin in treatment-naive and treatment-experienced patients with genotype 4 chronic hepatitis C virus infection (PEARL-I): a randomised, open-label trial. Lancet 2015 Jun 20;385(9986):2502-9.
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(32) Abergel A, Loustaud-Ratti V, Metivier S, Jiang D, Kersey K, Knox SJ, et al. Ledipasvir/Sofosbuvir treatment results in high SVR rates in patients with chronic genotype 4 and 5 HCV infection. Oral O056. Journal of Hepatology. 62[Supplement 2. 50th EASL meeting], S219. 2015.
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ACCEPTED MANUSCRIPT Table 1. Mixed infections, showing the percentage of each subtype, patient sex, and patient age at the time of sample collection (2015)
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Age, years 76 52 43 60 61 71 37 78 63 60 53 40 51 57 84
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Mixed Infection Subtypes 1a(51%)+1b(29%)+4d(20%) 1a(69.2%)+4d(30.7%) 1a+4a 1b(59%)+1a(17%)+3a(13%)+4d(10%) 1b(66%)+2c(34%) 1b(90%)+1a(10%) 1b(95%)+1a(5%) 1b(97%)+4d(1,3%) 1b(98%)+1a(2%) 1l(89%)+1b(6%)+1a(5%) 3a(66.5%)+1b(33.5%) 3a(84%)+4a(16%) 4d(78%),1a(21%) 4d(96%)+3a(4%) 4f(70%)+1b(30%)
ACCEPTED MANUSCRIPT Table 2. Prevalence of HCV subtypes by age group Number and percentage of patients infected by an HCV subtype related to the 3 age groups: younger than 40 years (<40), between 40 and 63 years (40-63), and older than 63 years (>63). Numbers inside parentheses are the percentage related to the total number of patients included in the study. The last column represents the total number
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of patients per subtype. Mx stands for mixed infection. Rows are sorted first by prevalence in decreasing order, and second by subtype lexicographic order.
>63
306 (37.1) 264 (32.0) 103 (12.5) 93 (11.3) 26 (3.2) 7 (0.8) 10 (1.2) 1 (0.1) 2 (0.2) 4 (0.5) 3 (0.4) 1 (0.1) 1 (0.1)
436 (84.5) 25 (4.8) 8 (1.6) 3 (0.6) 1 (0.2) 17 (3.3) 4 (0.8) 7 (1.4) 4 (0.8) 1 (0.2) 2 (0.4) 3 (0.6) 1 (0.2) 1 (0.2)
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2 (1.5) 1 (0.8) 1 (0.8)
40-63
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Age groups. Number of patients (percentage)
<40
1 (0.8)
1 (0.2) 1 (0.1) 1 (0.2) 1 (0.1) 1 (0.1) 1 (0.2)
1 (0.8) 1 (0.8)
All
790 (53.6) 332 (22.5) 133 (9.0) 105 (7.1) 29 (2.0) 25 (1.7) 15 (1.0) 8 (0.5) 8 (0.5) 6 (0.4) 6 (0.4) 4 (0.3) 2 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1)
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1a. HCV genotypes: chronically-infected general population
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1b. HCV subtypes: chronically-infected general population Other (5.7%)
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Figure 2
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S1198-743X(17)30095-2
DOI:
10.1016/j.cmi.2017.02.007
Reference:
CMI 855
To appear in:
Clinical Microbiology and Infection
Received Date: 31 August 2016 Revised Date:
2 February 2017
Accepted Date: 4 February 2017
Please cite this article as: Rodriguez-Frias F, Nieto L, Gregori J, Garcia-Cehic D, Casillas R, Tabernero D, Homs M, Blasi M, Vila M, Chen Q, Vargas V, Castells L, Viladomiu L, Genesca J, Minguez B, Augustin S, Carbonell J, Perales C, Soria ME, Asensio M, Llorens M, Ordeig L, Godoy C, Buti M, Esteban R, Pumarola T, Esteban JI, Quer J, High Hcv subtype heterogeneity in a chronically infected general population Revealed by high-Resolution Hcv subtyping, Clinical Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT HIGH HCV SUBTYPE HETEROGENEITY IN A CHRONICALLY INFECTED GENERAL POPULATION
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REVEALED BY HIGH-RESOLUTION HCV SUBTYPING
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Francisco Rodriguez-Frias,a,b,c,*# Leonardo Nieto,a,d,* Josep Gregori,b, ,e,f Damir Garcia-Cehic,bf Rosario Casillas,a,b David Tabernero,a,b Maria Homs,a,b Maria Blasi,a Marta Vila,a Qian Chen,f Victor Vargas, b,c,f Lluís Castells, b,c,f Lluís Viladomiu, b,c,f Joan Genesca, b,c,f Beatriz Minguez, b,c,f Salvador Augustin, b,c,f Judit Carbonell,c,f Celia Perales,b,f Maria Eugenia Soria, f Miriam Asensio a,b , Meritxell Llorens f, Laura Ordeig f, Cristina Godoy a, Maria Buti,b,c,f Rafael Esteban,b,c,f Tomas Pumarolac,d, Juan Ignacio Esteban,b,c,f Josep Querb,c,f
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*Both authors have contributed equally to the work.
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a
Liver Pathology Unit, Department of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron (HUVH), 08035, Barcelona, Spain b Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, 28029, Madrid, Spain c Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain d Clinical Microbiology Deparment, HUVH, 08035, Barcelona, Spain e Roche Diagnostics SL, Sant Cugat del Vallès, 08174, Barcelona, Spain f Liver Unit, Liver Disease Laboratory, Internal Medicine Department, Vall d’Hebron Institut Recerca (VHIR)-HUVH), 08035, Barcelona, Spain
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Running Title: Prevalence HCV subtypes by UDPS in Barcelona
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Keywords: Hepatitis C virus, Subtypes, Mixed infections, High-resolution HCV subtyping
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#
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Dr Francisco Rodriguez-Frias Liver Pathology Unit, Department of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, 08035, Barcelona, Spain Tel: 34 932746897 / 6896 E-mail addresses: [email protected], [email protected]
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Abstract: 249
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Main text (Abstract, References, Acknowledgment and Figure Legends not included): 2397
ACCEPTED MANUSCRIPT ABSTRACT
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Objectives: Accurately characterize the chronically-infected general HCV population in the
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Barcelona area by a highly sensitive subtyping method able to identify the 67 recognized
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hepatitis C virus (HCV) subtypes, and to discriminate mixed infections in a single individual.
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This information has strong implications on patient management, in deciding the optimal
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combination of direct-acting antivirals (DAAs) for each patient.
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Methods: HCV subtyping by UDPS. All HCV patients who attended Vall d'Hebron Hospital
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outpatient clinics from February 2015 to May 2016 (N=1473) were included.
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Results: The observed genotypes were: G1 (76.4%), G4 (9.8%), G3 (9.2%), G2 (3.5%) and G5
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(0.1%). Twenty-two subtypes were observed: 1b (53.6%), 1a (22.5%), 3a (9.0%), 4d (7.1%),
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4a (2.0%), and 2c (1.7%), with 16 low-prevalence subtypes accounting for the remaining
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3.2%. There was a worrisome 1% of mixed infections. Of note, G2 (3.5%) showed a high
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heterogeneity. An analysis stratified by age groups illustrated a clear predominance of G1b
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over G1a (84.5% vs 4.8%) in patients older than 63 years (N=516) and similar percentages of
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these subtypes in the 40 to 63 year-old group (N=824) (37.1% vs 32.0%) and the youngest
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group (<40) (N=133) (36.1% vs 32.3%).
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Conclusions: The observed subtype distribution shows a much higher heterogeneity than
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expected. Particularly G2 with few individuals, and mixed infections. The distribution by age
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groups suggests that all patients born before 1975 should be monitorized. The use of highly
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sensitive methods for HCV classification opens the door to acquiring a better acknowledge
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on the efficacy of each DAA-based treatment.
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ACCEPTED MANUSCRIPT INTRODUCTION
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The development of small-molecule compounds that directly inhibit the viral life cycle
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(DAA) has been a major milestone in the treatment of chronic hepatitis C virus (HCV)
59
infection. These DAAs are inhibitors targeting non-structural proteins (NS3, NS5A and NS5B)
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that have essential functions in the viral life cycle (1). Sustained virological response (SVR)
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rates in HCV-infected patients may vary depending on the patient’s viral subtype (2-4).
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Infection with more than one subtype (mixed infection) may also affect the treatment
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outcome (5). Therefore, HCV subtyping and detection of mixed infections are emerging as
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key issues in deciding the most suitable combination therapy for affected patients (6-8).
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HCV is currently classified into 7 genotypes (numbered 1 to 7), and 67 subtypes within these
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genotypes (designated by lower case letters a to z) (9). In Spain, the reported genotype
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distribution is as follows: genotype (G) 1, 6%; G1b, 36%; G1a, 27%; G2, 2.70%; G3, 17%; and
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G4, 12% (10), which concurs with the worldwide genotype distribution (G1, 46.2%; G3,
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30.1%; G2, G4, and G6, 22.8%; and G5, <1%)(11;12). However, these data were obtained by
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classic low-resolution genotyping methods, known to have significant misclassification rates
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(13-17).
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Next-generation sequencing (NGS) enables thousands of clonal sequences to be obtained
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from a single sample and is currently the most powerful tool for analyzing viral quasispecies.
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Among the available NGS methods, the 454/GS-Junior technology has been successfully
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used to study of several aspects of the hepatitis virus quasispecies (17-23). Our laboratory
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recently developed a high-resolution method that can confidently identify the 67 HCV
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subtypes and is easily adaptable to detect new subtypes. Furthermore, the high coverage
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ACCEPTED MANUSCRIPT (2000-5000 sequences/patient) enables detection of mixed infections with high sensitivity
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(17).
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As effective DAA-based HCV therapy is subtype-dependent, it is important to know the
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distribution of subtypes in chronically infected patients and the percentage of patients with
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mixed infection in a particular geographic area. The aim of this study was to accurately
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characterize by subtypes the HCV chronically-infected population in the Barcelona area,
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using this high sensitive method.
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MATERIAL AND METHODS
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Blood samples were obtained from all consecutive patients diagnosed with active chronic
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HCV infection attended in the hepatology and infectious disease outpatient clinics of Vall
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d´Hebron University Hospital (Barcelona) between February 2015 and May 2016 regardless
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of their treatment status, as part of their routine workup. Samples were delivered to our
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laboratory within 8 hours after extraction and centrifuged; plasma was stored at -80ºC until
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analysis. Demographic data were obtained from the patients’ clinical records.
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HCV RNA was extracted using a total nucleic acid isolation (TNAI) kit in a COBAS/AmpliPrep
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system (Roche Diagnostic, West Sussex, UK). RNA amplification and deep sequencing were
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carried out according to a previously described method (17). Briefly, nested RT-PCR was
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performed using specific primers for the NS5B region. The PCR product form each plasma
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sample was quantified using the PicoGreen assay (Invitrogen, Carlsbad, CA, USA), and
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quality-analyzed using the BioAnalyzer DNA 1000 LabChip (Agilent, Santa Clara, CA, USA)
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prior to sequencing. PCR products from 20 patients individually identified by a genetic
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barcode were mixed in the same emulsion PCR reaction at equal molecular proportions, and
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ACCEPTED MANUSCRIPT then subjected to deep sequencing using the 454/GS-Junior platform (Roche, Branford, CA,
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USA). The 454/GS-Junior data then underwent a process of demultiplexing, quality filtering,
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haplotype clustering, and phylogenetic analysis using software developed by our laboratory
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and the Bioinformatics Clinical Services at Roche (Sant Cugat, Spain), as previously described
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(17). Phylogenetic analysis of the data was then performed using the DeepChek-HCV
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subtyping software by ABL, Luxemburg (https://www.ablsa.com), including a set of
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reference sequences for all subtypes (9).
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including equimolecular pooling, was automated using an Evo 75 robotic device (Tecan,
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Switzerland) to adapt it to routine laboratory practice and obtain results within 4 days.
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RESULTS
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Samples from 1473 consecutive patients were HCV-subtyped in routine practice to
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determine the most effective antiviral therapy for each patient. An average of 2500 reads
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(SD 500) for the NS5B region were obtained per patient. HCV genotype (G) 1 was found to
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be the most prevalent (76.4%), followed by G4 (9.8%) and G3 (9.2%). Genotype 2 had a low
114
prevalence of 3.5% and only 1 G5 case was identified during the study period (0.1%). Mixed
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infections (Mx) with more than 1 HCV subtype were found in 1% of the samples (Figure 1a
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and Supplementary Table 1a).
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On subtype analysis, subtype 1b was the most prevalent (53.6%), followed by 1a (22.5%), 3a
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(9.0%), and 4d (7.1%). The remaining 7.8% included 18 minority subtypes (Figure 1b and
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Supplementary Table 1b), and 15 patients (1%) had mixed infections (Figure 1b and Table
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1). Most of the rare subtypes were seen in only a few individuals (1 to 8). G2, which was the
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less prevalent, showed the largest number of different subtypes (2a, 2b, 2c, 2i, 2j, 2m, 2q),
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ACCEPTED MANUSCRIPT followed by genotype 4 with 6 subtypes (4a, 4d, 4f, 4n, 4p, 4t) and genotype 1 with 5
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subtypes (1a, 1b, 1l, 1e, 1g, 1l). The mixed infections included subtypes 1a, 1b, 1l, 2c, 3a, 4a,
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4d, and 4f: 12 patients had 2 subtypes, 2 patients had 3 subtypes, and 1 patient had 4
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subtypes (Table 1).
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The number of cases was plotted against patient age, and a clearly bimodal distribution was
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observed, with modes at around 50 and 75 years of age (Figure 2). Three age groups were
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established based on this representation: <40 years, 40 to 63 years, and >63 years. The
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oldest group (>63) was mainly infected by subtype 1b (84.5%), followed by 1a (4.8%), and
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other subtypes, seen in very few individuals (Table 2). The middle-aged group (40-63)
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showed a large reduction in the prevalence of subtype 1b (37.1%) and a higher prevalence
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of 1a (32.0%), followed by 3a (12.5%), and 4d plus 4a (14.5%). The youngest group (<40)
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included a small percentage (9.03%) of HCV-infected patients (133/1473) and showed a
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subtype distribution profile similar to that of the middle-aged group: 1b (36.1%), 1a (32.3%),
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3a (16.5%) and 4d plus 4a (8.3%).
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DISCUSSION
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The novelty of this study is that HCV subtyping was carried out using a high-resolution NGS
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technique which enabled confident identification of the 67 recognized HCV subtypes and
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the detection of mixed infections. As the samples analyzed were from all consecutive HCV
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patients attended in our specialized outpatient clinics during the study period, these results
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are expected to be an accurate representation of HCV subtype distribution in the general
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population of the Barcelona metropolitan area (~1.5 million).
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ACCEPTED MANUSCRIPT Overall, HCV genotype distribution was similar to the results reported in other Spanish
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studies, in which G1 was the most prevalent (10;24). However, the prevalence of subtype 1b
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was higher in Barcelona (58%) than the values reported in both historical and recent studies
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performed in Spain (41.3% in 2006 (24) and 36% in 2015 (10). The reason for this difference
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may reside in the use of low-resolution techniques in these studies, which are unable to
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subtype all G1 viral strains (6% uncertainly for G1 in 2015 study), or because they combine
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results from different centers without a clear definition of the inclusion criteria. The
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percentage of G1a infections (22.5%) was similar to the values reported in the Spanish
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studies (24.1% in 2006 and 27% in 2015). Of note, 5 patients (all immigrants born in West
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Africa) were found to have infection of rare G1 subtypes (1e, 1g, 1l).
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G2 showed a very heterogeneous subtype distribution despite having a low prevalence
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(3.5%) in the population. Seven different G2 subtypes were identified, each found in few
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individuals: 2a (6 patients), 2b (1), 2c (25), 2i (4), 2j (8), 2m (1), and 2q (6). One patient with
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subtype 2j infection had a history of nonresponse to pegIFN+Rbv therapy. Genotype 2
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infections originated in Spain (46/51), Latin America (4/51), and (1/51) Russia.
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Genotype 3 was highly homogeneous. Among 134 patients with G3 infection, 133 had
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subtype 3a, whereas 1 immigrant from Pakistan had subtype 3b. Interestingly, the
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percentage of G3 cases has decreased considerably over the last 10 years, dropping from
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30.1% in 2006 (19) to 9.2% in the present study, and being surpassed by G4 (9.8%). This
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decrease may result from a combination of factors: genotype classification errors using low-
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resolution techniques, high SVR rates in G3 patients treated with pegIFN+Rbv, and deaths in
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HCV-infected intravenous drug abusers—for example, due to HIV coinfection.
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ACCEPTED MANUSCRIPT Six G4 subtypes were identified, with 4d being the most prevalent (105/145, 72.4%) and
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representing 7.1% of the total samples, followed by 4a (29/145, 20.03%), 4f (8/145, 5.5%),
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and 4n, 4p, and 4t with a single case each (1/145, 0.7%). Patients with subtypes 4a, 4f, 4n,
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4p, and 4t were immigrants born in Equatorial Guinea. Genotype 4 was the second most
169
prevalent genotype in our chronically-infected HCV population, in contrast to the data
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reported in other Spanish studies, in which G3 was more prevalent than G4 (10;24). This
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difference may be related to the population cohort included: Aguilera et al enrolled patients
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genotyped in a number of different Spanish centers, whereas our study focused on patients
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followed in the specific setting of Barcelona. Subtype 4d was the most common, likely
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because in our area 4d is highly prevalent in the subgroup of men who have sex with men
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and are coinfected with HIV plus HCV (25). We identified one case of G5a, in an individual
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from Belgium, who may have been infected in South Africa.
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One finding of concern is the prevalence of mixed infections (1%), which could likely be
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higher in high-risk HCV-infected patients, such as intravenous drug addicts.
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On analysis of HCV subtype prevalence by age group, the oldest group (>63 years), which
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included individuals who lived through the Spanish Civil war and post-war period, were
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mainly infected by subtype 1b (84.5%). This group accounted for 35% of all HCV-infected
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patients seen in our outpatient facility. The middle-aged group (40-63 years), encompassed
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individuals born during the time when large hospitals were built within the publically-
184
funded Spanish healthcare system and there was a peak in immigration to Barcelona from
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other Spanish regions. This group represented 55.9% of all HCV-infected patients
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(824/1473). Several international HCV clinical management guidelines (AASLD, EASL, and
187
JSH) (26-28) recommend testing for the virus in all persons born between 1945 and 1965.
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ACCEPTED MANUSCRIPT Our data concur with this recommendation, but Spain did not participate in the Second
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World War (1939-1945), experiencing instead a civil war (1936-1939). Hence, our results
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suggest that these epidemiological studies should include patients born before 1975.
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A review of the recent history of waves of immigration to the Barcelona metropolitan area
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(since 1939) shows that there was no significant influx of refugees to Spain from any part of
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Europe (including France), despite Spain’s neutrality during the Second World War. This fact,
194
together with the absence or minor incidence of immigration from the rest of Spain to
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Catalonia before the 1960s suggest that subtype 1b (86% in patients older than 63 years) is
196
endemic in Catalonia, whereas genotypes 1a, 2, 3a, and 4 were imported in successive
197
waves of immigration from other Spanish regions in the 1960s, and from Latin America and
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other HCV-endemic areas in the 1980s. Nonetheless, factors other than immigration should
199
also be considered when discussing the introduction and transmission of particular HCV
200
subtypes in a community. The changes in HCV subtype distribution may be the consequence
201
of several factors that would differentially affect the specific regions of Europe: expansion of
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intravenous drug use, immigration to Europe from endemic areas (29), and high-risk
203
behavior.
204
The high-resolution NGS-based HCV subtyping used in this study can be widely applied to
205
unequivocally determine the true prevalence of subtypes in a geographic area. HCV subtype
206
distribution may have important implications in the design of healthcare management
207
policies related to scheduling effective antiviral therapy. In this respect, G1 subtyping
208
(1a/1b) provides relevant information concerning response rates, genetic barriers to
209
resistance, and appropriate treatment. HCV treatment using DAA drug combinations is
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subtype-dependent in 1a and 1b patients, with subtype 1a being less susceptible than 1b to
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ACCEPTED MANUSCRIPT some of these therapies. International guidelines recommend genotyping/subtyping using
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an assay that provides accurate differentiation between these subtypes (27;28;30), although
213
no recommendations have been provided for subtypes other than 1a and 1b. Nonetheless,
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the recommendations will likely be adapted based on the results of ongoing research in
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other subtypes, which is now possible with the use of high-resolution methods (17). For
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example, a recent study (31) showed that the Viekirax combination (paritaprevir/ritonavir+
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ombitasvir) with or without ribavirin is highly effective in G4 patients (100% SVRwith
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ribavirin and 91% without). However, in the ribavirin-free group, there were 3 failures (2
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relapse and 1 breakthrough) and all patients were found to have subtype 4d (3/16, 18.75%).
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All 3 developed resistance-associated variants (RAS) to NS3 and NS5A. Hence, treatment of
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subtype 4d patients should include ribavirin, and if this is not possible, a change in the DAA
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combination may be appropriate. In another study (32), 44 G4 patients were treated with
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Harvoni (ledipasvir+sofosbuvir) and reached an excellent SVR rate (93%); however, 2 of 3
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patients infected with subtype 4r failed. These results suggest that 4r patients should be
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treated using Harvoni combined with ribavirin and/or an NS3 protease inhibitor, or receive a
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different DAA combination. Although there is no available information on the efficacy of
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current DAA-based therapy in rare subtypes, it is not unreasonable to think that it will also
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be subtype-dependent. Thus, in the future, the use of high-resolution HCV subtyping may
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not be a simple recommendation, but instead, a necessary requirement before prescribing
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antiviral therapy in individual patients with HCV infection.
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CONCLUSIONS
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Our method of HCV subtyping based on deep sequencing of the NS5B amplicon, which has
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been routinely applied in Vall d'Hebron Hospital (Barcelona) for more than 18 months, has
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ACCEPTED MANUSCRIPT offered a highly accurate picture of the HCV population attending the publically-funded
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health system in the Barcelona area, including patients infected with more than one
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subtype. The study shows a richer map of HCV subtypes than was expected, and a highly
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dynamic contribution of these subtypes over the years, with somewhat different prevalence
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according to age.
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We believe that deep sequencing will soon become the gold standard technique for
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classification of HCV and possibly, any other viral infection. The coming generations of high-
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throughput sequencing are providing longer sequence capabilities and higher throughput at
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lower cost. There is now no reason to limit the use of NGS exclusively to research purposes.
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ACCEPTED MANUSCRIPT Acknowledgments
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This work was funded by Instituto de Salud Carlos III, PI12/01893, PI13/00456, PI15/00829
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and PI15/00856 cofinanced by the European Regional Development Fund (ERDF),
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GLD14/00296 from Gilead and CIBERehd (Centro de Investigación en Red de Enfermedades
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Hepáticas y Digestivas) which is funded by Instituto de Salud Carlos III, and by CDTI (Centro
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para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and
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Competitiveness (MINECO), IDI- 20151125. C.P. is supported by the Miguel Servet program
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of the Instituto de Salud Carlos III, grant CP14/00121) cofinanced by the European Regional
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Development Fund (ERDF). The authors thank Celine Cavallo (English native) for English
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language support and helpful editing suggestions. We are indebted to Roche Diagnostics
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Spain.
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ACCEPTED MANUSCRIPT Figure and table legends
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Figure 1. Prevalence of HCV genotypes (Fig. 1a) and subtypes (Fig. 1b) in the HCV
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chronically-infected general population in Barcelona. Mx stands for mixed infection
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(simultaneous infection by more than one HCV subtype).
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Figure 2. A) Histogram of the ages of HCV patients, which show a clear bimodal distribution.
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B) Histograms showing the distribution of HCV subtypes by age group.
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REFERENCES
264 (1) Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 2013 Jul;11(7):482-96.
267 268 269
(2) Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med 2011 Mar 31;364(13):1207-17.
270 271 272
(3) Poordad F, McCone J, Jr., Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med 2011 Mar 31;364(13):1195206.
273 274
(4) Zeuzem S, Soriano V, Asselah T, Bronowicki JP, Lohse AW, Mullhaupt B, et al. Faldaprevir and deleobuvir for HCV genotype 1 infection. N Engl J Med 2013 Aug 15;369(7):630-9.
275 276 277
(5) McNaughton AL, Thomson EC, Templeton K, Gunson RN, Leitch EC. Mixed genotype hepatitis C infections and implications for treatment. Hepatology 2013 Jun 6;doi:10.1002/hep.26554.
278 279 280
(6) Doyle JS, Hellard ME, Thompson AJ. The role of viral and host genetics in natural history and treatment of chronic HCV infection. Best Pract Res Clin Gastroenterol 2012 Aug;26(4):413-27.
281 282 283
(7) Lam AM, Espiritu C, Bansal S, Micolochick Steuer HM, Niu C, Zennou V, et al. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother 2012 Jun;56(6):3359-68.
284 285 286 287
(8) Mauss S, Berg T, Rockstroh J, Sarrazin C, Wedemeyer H. Short Guide to Hepatitis C. Mauss S, Berg T, Rockstroh J, Sarrazin C, Wedemeyer H, editors. 2012 edition. 2012. http://www.FlyingPublisher.com, The Flying Publisher Group. The Flying Publisher Guide. Ref Type: Serial (Book,Monograph)
288 289 290
(9) Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, et al. Expanded classification of hepatitis C Virus into 7 genotypes and 67 Subtypes: updated criteria and assignment web resource. Hepatology 2014;59(1):318-27.
291 292 293 294
(10) Aguilera A et al. Prevalencia Hepatitis C en España. http://www jano es/noticia-el-96-lospacientes-tratados-24892# 2015;I Congreso Nacional del Grupo de estudio de Hepatitis (Gehep) de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (Seimc)(25,Setimbre 2015. Vigo. Spain).
295 296
(11) Gower E, Estes CC, Hindman S, Razavi-Shearer H. Global epidemiology and genotype distribution od the hepatitis C virus. J Hepatol 2014;61(1):S45-S57.
297 298 299
(12) Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 2015 Jan;61(1):7787.
AC C
EP
TE D
M AN U
SC
RI PT
265 266
ACCEPTED MANUSCRIPT (13) 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. J Clin Virol 2014 Jul;60(3):301-4.
303 304 305
(14) Hara K, Rivera MM, Koh C, Sakiani S, Hoofnagle JH, Heller T. Important factors in reliable determination of hepatitis C virus genotype by use of the 5' untranslated region. J Clin Microbiol 2013 May;51(5):1485-9.
306 307 308
(15) Mallory MA, Lucic DX, Sears MT, Cloherty GA, Hillyard DR. Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing. J Clin Virol 2014 May;60(1):22-6.
309 310 311 312
(16) Martro E, Gonzalez V, Buckton AJ, Saludes V, Fernandez G, Matas L, et al. Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions. J Clin Microbiol 2008 Jan;46(1):192-7.
313 314 315
(17) Quer J, Gregori J, Rodriguez-Frias F, Buti M, Madejon A, Perez-Del-Pulgar S, et al. Highresolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. J Clin Microbiol 2015 Jan;53(1):219-26.
316 317 318
(18) Rodriguez-Frias F, Tabernero D, Quer J, Esteban JI, Ortega I, Domingo E, et al. Ultra-deep pyrosequencing detects conserved genomic sites and quantifies linkage of drug-resistant amino acid changes in the hepatitis B virus genome. Plos One 2012;7(5):e37874.
319 320 321
(19) Homs M, Buti M, Tabernero D, Quer J, Sanchez A, Corral N, et al. Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing. World J Gastroenterol 2012 Nov 14;18(42):6096-105.
322 323 324
(20) Ramirez C, Gregori J, Buti M, Tabernero D, Camos S, Casillas R, et al. A comparative study of ultra-deep pyrosequencing and cloning to quantitatively analyze the viral quasispecies using hepatitis B virus infection as a model. Antiviral Res 2013 May;98(2):273-83.
325 326 327
(21) Homs M, Caballero A, Gregori J, Tabernero D, Quer J, Nieto L, et al. Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution. Plos One 2014;9(11):e112306.
328 329 330
(22) Gregori J, Esteban JI, Cubero M, Garcia-Cehic D, Perales C, Casillas R, et al. Ultra-deep pyrosequencing (UDPS) data treatment to study amplicon HCV minor variants. Plos One 2013;in press.
331 332
(23) Gregori J, Salicru M, Domingo E, Sanchez A, Esteban J, Rodriguez-Frias F, et al. Inference with viral diversity indices: Clonal and NGS approaches. Bioinformatics 2013;in press.
333 334 335
(24) Echevarria JM, Leon P, Pozo F, Avellon A. Follow-up of the prevalence of hepatitis C virus genotypes in Spain during a nine-year period (1996-2004). Enferm Infecc Microbiol Clin 2006 Jan;24(1):20-5.
336 337 338
(25) Martinez-Rebollar M, Mallolas J, Perez I, Gonzalez-Cordon A, Lonca M, Torres B, et al. [Acute outbreak of hepatitis C in human immunodeficiency virus-infected patients]. Enferm Infecc Microbiol Clin 2015 Jan;33(1):3-8.
AC C
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M AN U
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300 301 302
ACCEPTED MANUSCRIPT (26) JSH. JSH Guidelines for the Management of Hepatitis C Virus Infection: A 2014 Update for Genotype 1. Hepatol Res 2014 Jan;44 Suppl S1:59-70.
341 342
(27) EASL. Liver EAftSot. EASL Recommendations on the Treatment of hepatitis C. http://files easl eu/easl-recommendations-on-treatment-of-hepatitis-C/index html 2014.
343 344 345
(28) American Association for the Study of Liver Diseases. Recommendations for Testing, Managing, and Treating Hepatitits C. http://hcvguidelines org/full-report/initialtreatment-hcv-infection-patients-starting-treatment 2014.
346 347
(29) Esteban JI, Sauleda S, Quer J. The changing epidemiology of hepatitis C virus infection in Europe. J Hepatol 2008 Jan;48(1):148-62.
348 349
(30) EASL. EASL Clinical Practice Guidelines: management of hepatitis C virus infection. J Hepatol 2014 Feb;60(2):392-420.
350 351 352 353
(31) Hezode C, Asselah T, Reddy KR, Hassanein T, Berenguer M, Fleischer-Stepniewska K, et al. Ombitasvir plus paritaprevir plus ritonavir with or without ribavirin in treatment-naive and treatment-experienced patients with genotype 4 chronic hepatitis C virus infection (PEARL-I): a randomised, open-label trial. Lancet 2015 Jun 20;385(9986):2502-9.
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(32) Abergel A, Loustaud-Ratti V, Metivier S, Jiang D, Kersey K, Knox SJ, et al. Ledipasvir/Sofosbuvir treatment results in high SVR rates in patients with chronic genotype 4 and 5 HCV infection. Oral O056. Journal of Hepatology. 62[Supplement 2. 50th EASL meeting], S219. 2015.
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ACCEPTED MANUSCRIPT Table 1. Mixed infections, showing the percentage of each subtype, patient sex, and patient age at the time of sample collection (2015)
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Sex M M M F F F M F F M M M M F F
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Age, years 76 52 43 60 61 71 37 78 63 60 53 40 51 57 84
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Mixed Infection Subtypes 1a(51%)+1b(29%)+4d(20%) 1a(69.2%)+4d(30.7%) 1a+4a 1b(59%)+1a(17%)+3a(13%)+4d(10%) 1b(66%)+2c(34%) 1b(90%)+1a(10%) 1b(95%)+1a(5%) 1b(97%)+4d(1,3%) 1b(98%)+1a(2%) 1l(89%)+1b(6%)+1a(5%) 3a(66.5%)+1b(33.5%) 3a(84%)+4a(16%) 4d(78%),1a(21%) 4d(96%)+3a(4%) 4f(70%)+1b(30%)
ACCEPTED MANUSCRIPT Table 2. Prevalence of HCV subtypes by age group Number and percentage of patients infected by an HCV subtype related to the 3 age groups: younger than 40 years (<40), between 40 and 63 years (40-63), and older than 63 years (>63). Numbers inside parentheses are the percentage related to the total number of patients included in the study. The last column represents the total number
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of patients per subtype. Mx stands for mixed infection. Rows are sorted first by prevalence in decreasing order, and second by subtype lexicographic order.
>63
306 (37.1) 264 (32.0) 103 (12.5) 93 (11.3) 26 (3.2) 7 (0.8) 10 (1.2) 1 (0.1) 2 (0.2) 4 (0.5) 3 (0.4) 1 (0.1) 1 (0.1)
436 (84.5) 25 (4.8) 8 (1.6) 3 (0.6) 1 (0.2) 17 (3.3) 4 (0.8) 7 (1.4) 4 (0.8) 1 (0.2) 2 (0.4) 3 (0.6) 1 (0.2) 1 (0.2)
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2 (1.5) 1 (0.8) 1 (0.8)
40-63
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48 (36.1) 43 (32.3) 22 (16.5) 9 (6.8) 2 (1.5) 1 (0.8) 1 (0.8)
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Age groups. Number of patients (percentage)
<40
1 (0.8)
1 (0.2) 1 (0.1) 1 (0.2) 1 (0.1) 1 (0.1) 1 (0.2)
1 (0.8) 1 (0.8)
All
790 (53.6) 332 (22.5) 133 (9.0) 105 (7.1) 29 (2.0) 25 (1.7) 15 (1.0) 8 (0.5) 8 (0.5) 6 (0.4) 6 (0.4) 4 (0.3) 2 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1)
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1a. HCV genotypes: chronically-infected general population
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1b. HCV subtypes: chronically-infected general population Other (5.7%)
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A
Figure 2
B