Glecaprevir

HCV resistance-associated substitutions following direct-acting antiviral therapy failure – Real-life data from Poland

Miłosz Parczewski, Ewa Janczewska, Arkadiusz Pisula, Dorota Dybowska, Władysław Łojewski, Adam Witor, Marta Wawrzynowicz-Syczewska, Łukasz Socha, Rafał Krygier, Brygida Knysz, Joanna Musialik, Anna Urban´ska, Kaja Scheibe, Jerzy Jaroszewicz
a Department of Infectious, Tropical Diseases and Immune Deficiency, Pomeranian Medical University in Szczecin, Szczecin, Poland
b Department of Basic Medical Sciences, The School of Health Sciences in Bytom, Medical University of Silesia, Bytom, Poland
c ID Clinic, Mysłowice, Poland
d Department of Infectious Diseases and Hepatology, Ludwik Rydygier Collegium Medicum, Bydgoszcz Faculty of Medicine, Nicolaus Copernicus University, Torun´,Poland
e Department of Infectious Diseases, Regional Hospital in Zielona G´ora, Zielona G´ora, Poland
f Hospital for Infectious Diseases, Chorz´ow, Poland
g Department of Infectious Diseases, Hepatology and Liver Transplantation, Pomeranian Medical University in Szczecin, Szczecin, Poland
h Outpatients Hepatology Department, State University of Applied Sciences, Konin, Poland
i Department of Infectious Diseases, Liver Diseases and Acquired Immune Deficiencies, Wrocław Medical University, Wrocław, Poland
j Department of Nephrology, Transplantation and Internal Medicine, Medical University of Silesia in Katowice, Katowice, Poland
k Department of Infectious Diseases and Hepatology, Medical University of Silesia, Katowice, Poland

A B S T R A C T
Background: This study analysed the NS3 and NS5A mutation frequencies, persistence and drug susceptibility in a cohort of real-life patients, with failed hepatitis C virus (HCV) therapy following directly acting antiviral (DAA) treatment.
Methods: NS3/NS5A Sanger sequences from 105 patients infected with HCV genotype (G) 1a (6,5.7%), G1b (94,89.5%), G3a (4,3.8%), and G4 (1,1.0%) post DAA treatment failure were analysed. NS3 and NS5A resistance- associated substitutions (RASs) were identified using the geno2pheno algorithm and associated with clinical variables. Time trends were examined using logistic regression.
Results: NS5A RAS were found in 87.9% of sequences derived from patients exposed to this class of agents, whereas NS3 RAS was found in 59.1% of HCV protease-exposed subjects. The frequency of the NS3 RAS increased with fibrosis stage, from 40.0% among F0/F1 individuals to 81.8% among patients with liver cirrhosis (F4, p = 0.094). NS5A mutation frequencies were 7.6% for 28A/V/M, 10.6% for 30 K/Q/R, 42.4% for 31I/F/M/ V, and 75.8% for 93H. For NS3, the most common RASs were 56F–23.7%, 168A/E/I/Y/T/V–14.0%, and 117H–5.4%. Susceptibility to glecaprevir/pibrentasvir, velpatasvir/voXlaprevir, and elbasvir/grazoprevir was retained in 92.9%, 43.4%, and, 25.3% of patients, respectively. The frequency of NS3 RAS decreased with time elapsed from failure to sampling (p = 0.034 for trend). NS5A RAS frequency remained stable over the 24-months.
Conclusions: Following DAA treatment failure, NS5A and NS3 RASs were common with increasing frequency among patients with advanced liver disease. In most cases, despite the presence of RASs, susceptibility to DAA combinations with higher genetic barrier was retained.

1. Introduction
Infection with hepatitis C virus (HCV) remains a significant cause of liver disease, which may lead to liver cirrhosis and the development of hepatocellular carcinoma. According to current World Health Organi-zation estimates, approXimately 71 million people worldwide are NS5A mutation frequencies in a cohort of real-life patients, with failed chronically infected with HCV (Blach et al., 2017; European Union, 2017). From 1990 to 2013, the number of deaths associated with viral hepatitis increased from 0.89 million (95% CI: 0.86–0.94 million) to 1.4 million (95% CI: 1.38–1.54 million) (Stanaway et al., 2016). The inci- dence of infection varies significantly depending on the region, ranging from 1% in Central and Western Europe, North America, Australia, Latin America, the Caribbean Islands, Southeast Africa, and highly developed Asian countries such as Japan and Singapore to 1–2% in East and South East Asia, East Europe, and West Sub-Saharan Africa. Coun- tries with an endemically high (up to 7% of the population) incidence of HCV infections include those in the Central Asian region, including Egypt, Syria, Pakistan, Uzbekistan, Georgia, and Mongolia (Collabora- tors, 2017). Historically, the predominant route of infection has been associated with medical procedures, blood product transfusions, and the use of intravenous psychoactive substances. Recently, an increase in the frequency of new infections through sexual contact has been observed, especially in men who have sex with men (Boesecke et al., 2015). The revolution related to the use of directly acting antivirals (DAAs) in HCV treatment allows the elimination of infection in 90–95% of patients. The available drugs include inhibitors of 2–3 viral proteins that replaced the treatment of pegylated interferon with ribavirin (RBV). However, DAA treatment failure remains an issue even with widespread access to these therapies because unsuccessfully treated infection may progress to more advanced liver disease and potentially limit the efficacy of HCV elimi- nation efforts.
Drug resistance-associated substitutions (RASs) associated with decreased sensitivity to DAA might lead to a reduction in therapeutic efficacy, resulting in the need for retreatment. Substitutions that decrease DAA sensitivity have been reported for all drug classes used, e.g. protease (NS3), NS5A, and NS5B inhibitors. The development of drug resistance to antiviral agents depends on an array of factors, including drug efficacy, genetic barrier to resistance, therapy adherence, viral replication activity (“fitness”), and the host genetic variability (Silva et al., 2015; Wang et al., 2017; Wargo and Kurath, 2012). Both natural subtype-related HCV variability and errors introduced by the viral po- lymerase, combined with a replication capacity of 1010–1013 virions/ day, result in the rapid selection of drug-resistant variants under therapy pressure (Bartolini et al., 2015; Neumann et al., 1998; Ribeiro et al., 2012). Even after 2 weeks from treatment initiation, resistance- associated variants have been observed in cases of treatment failure (Dietz et al., 2015; Ehrenberg et al., 2014; Guan et al., 2014; Guan et al., 2015; Halfon and Locarnini, 2011). Despite infrequent therapy failure with currently used pangenotypic agents, identification of resistance in failing cases remains a viable strategy guiding virologic response prior to retreatment (European Association for the Study of the Liver et al., 2020).
In Poland estimated frequency of HCV infection in the population is 0.4–0.5% of the population, which translates into 150 thousand of active cases. It is estimated, that the majority of cases remain undiagnosed, with approXimately 31% of total number of patients diagnosed so far (Halota et al., 2020). Majority of observed infections are related to the iatrogenic transmission route, however historically in the end of the last century injection drug use related infections were also common. Recently, increase in HCV infections among men-who-have-sex-with- men was observed, which was confirmed in the phylogenetic analyses (Parczewski et al., 2019). Genotype 1b remains dominant (82%), fol- lowed by genotype 3 (11.3%), genotype 4 (3.5%) and genotype 1a (3.2%).Directly acting antivirals have become available in the country since 2015, with evolving access, currently glecaprevir, voXilaprevir, sofosbuvir, ledipasvir, pibrentasvir and velpatasvir are included in the HCV therapy following DAA treatment. We analysed the susceptibility to NS3 and NS5A inhibitors in the cohort to reflect possible salvage options and select optimal second-line therapy following treatment failure. Lastly, we investigated the persistence of mutations over 24 months to reflect the mutation decay.

2. Material and methods
2.1. Study population
The present study focused on patients presenting with NS3 and NS5A DAA regimen failure using multicentre samples collected between 2015 and 2019. For the study samples patients with HCV-RNA > 1000 IU/ml after at least 3 months of DAA therapy termination were sequenced. The samples were collected at various time points after virologic failure at the discretion of the treating physician. Patients were treated in the following Polish centres: Bydgoszcz, Bytom, Chorzo´w, Konin, Mysło- wice, Szczecin, Katowice, Wrocław, and Zielona Go´ra. In total, 105 post DAA treatment failure NS3/NS5A sequences from patients infected with genotype (G) 1a (6, 5.7%), G1b (94, 89.5%), G3a (4, 3.8%), and G4 (1,1.0%) were analysed. The study protocol was approved by the Bioethical Committee of Pomeranian Medical University, approval number KB- 0012/26/17. The patient data were coded and anonymous, patients provided informed consent for sequencing.
For all patients, the following clinical data were collected: age, sex, HCV transmission route, hepatitis B (HB) virus coinfection status (defined as HBs antigen positive or anti-HBcore antibody status), alanine and aspartate aminotransferase activity, HCV-RNA level at the date of sampling, and liver fibrosis stage, assessed based on histopathology or transient elastography (FibroScan™, Echosens, Paris, France) or real- time shear wave elastography (AiXplorer™, Super-Sonic Imagine S.A., AiX-en-Provence, France). Liver fibrosis assessment expressed on the METAVIR scoring system was available for 88 patients. All patients were of European-descent and Polish ethnicity, HCV monoinfected, and no history of injection drug use or HIV coinfection. For all patients, a his- tory of DAA-based treatment was collected, with exact timepoints for the therapy initiation and termination available for 86 (81.9%) individuals.

2.2. Genotypic drug resistance assessment
NS3 and NS5A sequencing was performed using previously pub- lished Sanger methodology with sequence assembly performed using the Recall online tool with 15% threshold miXed base calls (Woods et al., 2012). NS3 and NS5A sequence pair was available for 99 samples, NS5A only for 4 samples and NS3 only for one sample.
For all patients, the HCV genotype was verified using the OXford HCV automated subtyping and COMET subtyping tools. NS3 and NS5A RASs were identified using the geno2pheno algorithm HCV v.0.92. For interpretation of drug resistance, any variant scored as “reduced sus- ceptibility” or “resistant” to NS3 or NS5A inhibitors was included. If the antiviral agent was unlicensed for the genotype, susceptibility was not analysed. Only mutations associated with either reduced susceptibility or resistance in the geno2pheno algorithm were further analysed. Therefore For NS5A, the following positions were included: 28, 30, 31, and 93, and for NS3: 36, 41, 43, 54, 55, 56, 80, 117, 122, 155, 156, 158,166,168, 170, 174, and 175. RAS frequencies were calculated for the entire dataset and separately for NS3, NS5A, and NS3 NS5A exposures.

2.3. Statistical analyses
Statistical comparisons were performed using chi-square tests for categorical variables. All continuous variables were distributed in a non- linear manner; therefore, the U-Mann Whitney test was used for ana- lyses. Computations were performed using Statistica 13.0 PL software (Statasoft, Poland). P values 0.05 were considered statistically signif- icant. Time trends were examined using logistic regression (R statistical platform, version 3.1.0) for binary (resistant reduced susceptibility vs susceptible) variables.

3. Results
3.1. Population characteristics and overall DAA resistance
The majority of patients in the analysed cohort were male (n = 61, 58.1%), with a median age of 54 years (Interquartile range – IQR: 42–61 years). Of these, 49 (55.7%) had either advanced liver fibrosis or cirrhosis. The median time from the last day of exposure to the sequencing sample was 11 (5.1–15) months, with a median HCV viral load of 6.21 (5.89–6.53) log IU/mL at this time point. The majority (89.5%) of the patients presenting with antiviral treatment failure were infected with G1b (Table 1). Pegylated interferon/RBV boceprevir, simeprevir, or telaprevir treatment failures were observed in 38 (36.2%) cases and in 27 (25.7%) individuals showing failed NS3 NS5A in- hibitor therapy, with the most common being asunaprevir/dalclatasvir (19, 18.2%) or less frequently, grazoprevir/elbasvir combinations (6, 5.7%). NS3NS5ANS5B inhibitor (ombitasvir/pibrentasvir/rito- navir dasabuvir) combinations were used in 28 (26.6%) cases, whereas 11 (10.5%) participants were exposed to NS5A inhibitor NS5B RBV, most commonly, ledipasvir/sofosbuvir (9, 8.6%) (Sup- plemental Table 1).
For the entire dataset (n 105), NS5A variants associated with reduced susceptibility or resistance to NS5A and NS3 RAS were found in 60% and 61.0% of sequences, respectively (Figs. 1 and 2, blue bars). However, NS5A RAS were found in 87.9% of sequences derived from patients exposed to this class of agents (58/66 cases), whereas NS3 RAS was found in 59.1% of HCV protease-exposed subjects (55/93 cases).
If analysed by codon positions, NS5A mutation frequencies among NS5A exposed cases were 7.6% for 28A/V/M, 10.6% for 30 K/Q/R, 42.4% for 31I/F/M/V, and 75.8% for 93H. NS5A RAS at multiple po- sitions was observed in 29 (50%) of the exposed cases (Fig. 1). In the NS3 exposed group, the most common RASs to this drug class were observed in the following codon positions: 56F–23.7%, 168A/E/I/Y/T/V–14.0%, and 117H–5.4% (Fig. 2). When mutation frequency was analysed based on failure of the antiviral regimen, NS3 168G, NS5A 31F/I/M/V, and 93H RASs were less frequent among patients showing failure of pegy- lated interferon NS3 RBV therapy than of the NS5A inhibitor- containing regimens. It should be also noted, that NS3 NS5A NS5B inhibitor RBV DAA combinations are associated with more common selection of 168G variant (Fig. 3).

3.2. Clinical characteristics and RAS
Among patients exposed to NS3 inhibitors, alanine aminotransferase activity was higher among patients with observed NS3 RAS [median:
53.0 (IQR: 42.0–70.5) IU/mL vs 35.9 (IQR: 28.0–56.0) IU/mL, p =
0.014] or NS3 and NS5A RAS [median: 57.0 (IQR: 51.0–100.0) IU/mL vs
28.0 (IQR: 22.0–36.0) IU/mL, p 0.0004]. In the latter group, signifi- cantly higher aspartate aminotransferase activity was observed [me- dian: 70.0 (IQR: 54.0–122.2) IU/mL vs 44.0 (IQR: 29.0–52.0) IU/mL, p 0.003] (Table 2). The frequency of the NS3 drug resistance variants increased with fibrosis stage, from 40.0% among F0/F1 individuals to 81.8% among patients with liver cirrhosis (F4, p 0.094). NS3 RASs were more frequent among patients with advanced liver fibrosis (F3) or
cirrhosis (n = 29, 72.5%) compared to F0–F2 cases (n = 18, 48.7%, p = 0.03), and for patients with cirrhosis (F4, n = 18, 81.8%) compared to F0–F3 cases (n 29, 52.7%, p 0.023).
Similarly, in the group exposed to both HCV protease and NS5A in- hibitors, there was an increase in the frequency of observed NS3 NS5A RAS frequency with liver fibrosis progression from 33% in F0/F1 to 80% in F4, with resistance-associated variants more common among F3 and F4 patients (n 17, 70.8%) than lower fibrotic stages (F0–F2, n 8, 40%, p 0.025), and cases with cirrhosis (n 12, 80%) compared to non-cirrhotic ones (n 12, 44.8%, p 0.039). Among NS5A exposed cases, no association of analysed variables with RAS presence was found.

3.3. Antiviral drug susceptibility in the analysed population
For the entire analysed dataset and dataset of only NS3 exposed cases, individual NS3 inhibitor susceptibilities ranged from 48.0%/ 49.5% (grazoprevir) to 99%/98.9% (glecaprevir). For NS5A inhibitors, the highest number of susceptible sequences was observed for pibren- tasvir (92.2% for the entire dataset and 87.5% for those exposed only to NS5A) with the lowest susceptibility for daclatasvir (40.4% and 12.3%, respectively). EXcept for pibrentasvir, < 20% of sequences obtained from individuals exposed to NS5A inhibitors showed susceptibility to other class agents (Supplemental table 2). Among the currently recommended DAA combinations (EASL guidelines 2020), full susceptibility of the NS3/NS5A combination gle- caprevir/pibrentasvir was observed in 92.9% of patients, 43.4% for velpatasvir/voXlaprevir, with 25.3% susceptibility for elbasvir/grazo- previr (Supplemental table 3). 3.4. Mutation decay after DAA exposure Time elapsed from drug exposure to sample collection, median (IQR) months 11 (5.1–15) The median collection time after the last day of exposure was 11 (IQR: 5.1–15) months. To calculate the trend associated with RASs decay Alanine aminotransferase activity (ALT), median (IQR) IU/ml 60 (43–86) Aspartate aminotransferase activity (AST), median (IQR) IU/ ml 51 (33–73) over time we calculated NS3 and NS5A mutation frequency following the last day of DAA exposure at monthly intervals, for a maximum of 24 HCV viral load at sequencing, median (IQR) log IU/ml 6.21 months. The frequency of NS3 variants decreased significantly over Liver fibrosis stage, n (%) (5.89–6.53) time, from a mean of 75% for the samples collected within 3 months Fig. 1. NS5A resistance in the entire cohort exposed either to NS3 or NS5A, NS5A only exposed individuals and following a combination therapy with NS3 plus NS5A. Fig. 2. NS3 resistance in the entire cohort exposed either to NS3 or NS5A, NS3 only exposed individuals and following a combination therapy with NS3 plus NS5A. 4. Discussion In the current study, we presented data on NS3 and NS5A RAS fre- quencies in a cohort of patients with DAA treatment failure from Poland, with the majority of treatment failures observed for G1b, which remains the most common genotype observed in the country (Panasiuk et al., 2013). Samples were collected randomly at different time points following HCV-RNA detection after treatment termination. A significant proportion of patients had either advanced liver fibrosis or cirrhosis (>55%), which are known factors for preexisting RAS (Parczewski et al., 2019) and reduced DAA treatment response (Degasperi et al., 2019; European Association for the Study of the Liver et al., 2020). In patients with decompensated cirrhosis of the liver, the frequency of the Y93H mutation can be as high as 50% (Curry et al., 2015).
The majority of treatment failures were observed for the par- itaprewir/ritonavir + ombitasvir ± dasabuvir ± RBV combination, followed by asunaprevir/dalclatasvir. These combinations contain low genetic barrier agents, known to select for Y56H, R155K, and D168V/E in NS3, and M28T and Q30R in NS5A (Itakura et al., 2020; Krishnan et al., 2015a; Krishnan et al., 2015b; McPhee et al., 2012). All mentioned variants were common in our dataset. The presence of Y93H mutation is strongly associated with asunaprevir/dalclatasvir or elbasvir/grazo- previr treatment failure (Huang et al., 2019; Iio et al., 2017). Moreover, in treatment-experienced patients, the combination of grazoprevir and elbasvir despite good overall efficacy (96.2%) was previously associated with lower efficacy in patients with RAS in the NS3 region (Forns et al., 2015). The 93H/N/R mutation alone and in combination with 31I/F/M/ V was the most common NS5A RAS observed in our cohort, with a strong association with failure of NS5A inhibitor-containing combination. The presence of L31M/V/I and Y93H mutations has been previously asso- ciated with reduced efficacy of therapy with ombitasvir/paritaprevir/ritonavir and dasabuvir with sofosbuvir + RBV (Fedorchenko et al.,NS3 mutation patterns by failing combination 2020).
The frequency of NS3 alone and NS5 NS5A RAS combination following treatment failure increased with fibrosis stage, a phenomenon we have observed previously for the treatment of patients from Poland (Parczewski et al., 2019), which is in agreeance with the non-response to DAA observed in patients with advanced fibrosis in real-world data (Zarebska-Michaluk et al., 2019). In the HCT-target cohort, highrhq prevalence of NS3 and 2 classes of RAS have been observed in patients experiencing liver cirrhosis (Wang et al., 2018). Potential explanations include suboptimal exposure of NS3-inhibitors due to sinusoidal capil- larisation or the presence of portal hypertension. However, it could also be caused by longer disease duration and faster progression in subjects with RAS.
Drug susceptibility analysis performed both separately and for combinations included in the current EASL guidelines showed that the highest number of susceptible patients was observed for gelcaprevir (99%) and paritaprevir (81.2%) for the NS3 class and 92.2% for pibrentasvir (supplementary table 3). Patients showed notably higher susceptibility to glecaprevir/pibrentasvir (92.95%) than to velpatasvir and voXilaprevir (43%) recommended in combination with sofosbuvir as the fiXed-dose therapy for the retreatment of patients with or without compensated cirrhosis presenting post DAA treatment failure. This data, however should be interpreted with consideration of data from the Magellan-1 study, which indicated low drug resistance barrier of gle- caprevir/pibrentasvir when used for re-treatment of DAA failing cases, with necessity to extend the therapy to 16 weeks (Poordad et al., 2018). Previous data indicate that all the above-mentioned agents with higher genetic barriers to resistance demonstrate efficacy in treating in- dividuals with previous DAA failures. In a Phase 3 Polaris-4 study, among regimen failures (97% of whom were resistant to NS3 or NS5A), the combination of velpatasvir/sofosbuvir and voXilaprevir was associ- ated with >90% treatment efficacy irrespective of HCV genotype or the presence of cirrhosis (Zeuzem et al., 2020). Similar data were obtained from the POLARIS-1 trial, which was also performed in the NS5A failure group with sustained virologic response observed in 98% of velpatasvir/ sofosbuvir/voXilaprevir-treated cases (Bourliere et al., 2017). In addi-tion, the combination of glecaprevir/pibrentasvir used in subjects with NS3 or NS5A resistance resulted in >90% virologic responses, however, this combination proved to be less efficacious among patients infected with G1a (Poordad et al., 2018), and was infrequent among patients analysed for the present study.
We confirmed the trend for NS3, but not for NS5A mutation decay. The frequency of NS3 RASs decreased significantly among samples collected at more distant time points from the DAA treatment termina- tion, reflecting the increasing susceptibility to these agents over time. This is consistent with the sequencing data indicating that mutations in the NS3 region were eliminated 6–14 months after exposure (Howe et al., 2015; Sullivan et al., 2013). It was also observed in the present study that NS5A variants remained common and stably frequent over time, which reflects the high replicative fitness associated with these RASs. A lack of elimination of NS5A has been observed previously (Krishnan et al., 2020; Wyles and Gutierrez, 2014; Yoshimi et al., 2015). In a study by Dvory-Sobol et al. [42], the presence of RAS was detected in 82% of cases, even 92 weeks following treatment failure. This phe- nomenon is important for DAA retreatment because circulating resis- tance variants might limit salvage treatment options.
Limitations of the present study are associated with the non- systematic sampling of patients for resistance analyses. However, due to differences in the follow-up strategies among HCV treatment centres, other sample collection strategies proved impossible.
In conclusion, as suggested by the current EASL guidelines, resis- tance analyses provide valuable data to guide treatment decisions following HCV treatment failure (European Association for the Study of the Liver et al., 2020). Both NS5A and NS3 RASs are common and pre- sent in the majority of DAA unsuccessfully treated individuals. Sur- veillance of the emerging mutation pattern allows us to preselect the second-line treatment with optimal efficacy, considering the high rates of sustained virologic response in the treatment-experienced group and contributing to HCV elimination in the near future.

References
Bartolini, B., Selleri, M., Garbuglia, A.R., Giombini, E., Taibi, C., Lionetti, R., D’Offizi, G., Capobianchi, M.R., 2015. HCV NS3 quasispecies in liver and plasma and dynamics of telaprevir-resistant variants in breakthrough patients assessed by UDPS: A case study. J. Clin. Virol. 72, 60–65.
Blach, S., Zeuzem, S., Manns, M., Altraif, I., Duberg, A.-S., Muljono, D.H., Waked, I.,Alavian, S.M., Lee, M.-H., Negro, F., Abaalkhail, F., Abdou, A., Abdulla, M., Rached, A.A., Aho, I., Akarca, U., Al Ghazzawi, I., Al Kaabi, S., Al Lawati, F., Al Namaani, K., Al Serkal, Y., Al-Busafi, S.A., Al-Dabal, L., Aleman, S., Alghamdi, A.S., Aljumah, A.A., Al-Romaihi, H.E., Andersson, M.I., Arendt, V., Arkkila, P., Assiri, A. M., Baatarkhuu, O., Bane, A., Ben-Ari, Z., Bergin, C., Bessone, F., Bihl, F., Bizri, A.R., Blachier, M., Blasco, A.J., Mello, C.E.B., Bruggmann, P., Brunton, C.R., Calinas, F., Chan, H.L.Y., Chaudhry, A., Cheinquer, H., Chen, C.-J., Chien, R.-N., Choi, M.S., Christensen, P.B., Chuang, W.-L., Chulanov, V., Cisneros, L., Clausen, M.R.,
Cramp, M.E., Craxi, A., Croes, E.A., Dalgard, O., Daruich, J.R., de Ledinghen, V., Dore, G.J., El-Sayed, M.H., Ergo¨r, G., Esmat, G., Estes, C., Falconer, K., Farag, E., Ferraz, M.L.G., Ferreira, P.R., Flisiak, R., Frankova, S., Gamkrelidze, I., Gane, E., García-Samaniego, J., Khan, A.G., Gountas, I., Goldis, A., Gottfredsson, M., Grebely, J., Gschwantler, M., Pessoˆa, M.G., Gunter, J., Hajarizadeh, B., Hajelssedig, O., Hamid, S., Hamoudi, W., Hatzakis, A., Himatt, S.M., Hofer, H., Hrstic, I., Hui, Y.-T., Hunyady, B., Idilman, R., Jafri, W., Jahis, R., Janjua, N.Z., Jarˇcuˇska, P., Jeruma, A., Jonasson, J.G., Kamel, Y., Kao, J.-H., Kaymakoglu, S., Kershenobich, D., Khamis, J., Kim, Y.S., Kondili, L., Koutoubi, Z., Krajden, M., Krarup, H., Lai, M.-S., Laleman, W., Lao, W.-C., Lavanchy, D., La´zaro, P., Leleu, H.,Lesi, O., Lesmana, L.A., Li, M., Liakina, V., Lim, Y.-S., Luksic, B., Mahomed, A.,Maimets, M., Makara, M., Malu, A.O., Marinho, R.T., Marotta, P., Mauss, S.,Memon, M.S., Correa, M.C.M., Mendez-Sanchez, N., Merat, S., Metwally, A.M.,Mohamed, R., Moreno, C., Mourad, F.H., Müllhaupt, B., Murphy, K., Nde, H., Njouom, R., Nonkovic, D., Norris, S., Obekpa, S., Oguche, S., Olafsson, S., Oltman, M., Omede, O., Omuemu, C., Opare-Sem, O., Øvrehus, A.L.H., Owusu- Ofori, S., Oyunsuren, T.S., Papatheodoridis, G., Pasini, K., Peltekian, K.M.,Phillips, R.O., Pimenov, N., Poustchi, H., Prabdial-Sing, N., Qureshi, H., Ramji, A., Razavi-Shearer, D., Razavi-Shearer, K., Redae, B., Reesink, H.W., Ridruejo, E., Robbins, S., Roberts, L.R., Roberts, S.K., Rosenberg, W.M., Roudot-Thoraval, F., Ryder, S.D., Safadi, R., Sagalova, O., Salupere, R., Sanai, F.M., Avila, J.F.S., Saraswat, V., Sarmento-Castro, R., Sarrazin, C., Schmelzer, J.D., Schr´eter, I., Seguin- DevauX, C., Shah, S.R., Sharara, A.I., Sharma, M., Shevaldin, A., Shiha, G.E., Sievert, W., Sonderup, M., Souliotis, K., Speiciene, D., Sperl, J., St¨arkel, P., Stauber, R.E., Stedman, C., Struck, D., Su, T.-H., Sypsa, V., Tan, S.-S., Tanaka, J., Thompson, A.J., Tolmane, I., Tomasiewicz, K., Valantinas, J., Van Damme, P., van der Meer, A.J., van Thiel, I., Van Vlierberghe, H., Vince, A., Vogel, W., Wedemeyer, H., Weis, N., Wong, V.W.S., Yaghi, C., Yosry, A., Yuen, M.-F.,Yunihastuti, E., Yusuf, A., Zuckerman, E., Razavi, H., 2017. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study.
Lancet Gastroenterol. Hepatol. 2, 161–176.Boesecke, C., Grint, D., Soriano, V., Lundgren, J.D., d’Arminio Monforte, A., Mitsura, V. M., Chentsova, N., Hadziosmanovic, V., Kirk, O., Mocroft, A., Peters, L., Rockstroh, J. K., 2015. Hepatitis C seroconversions in HIV infection across Europe: which regions and patient groups are affected? Liver Int. 35, 2384–2391.
Bourliere, M., Gordon, S.C., Flamm, S.L., Cooper, C.L., Ramji, A., Tong, M., Ravendhran, N., Vierling, J.M., Tran, T.T., Pianko, S., Bansal, M.B., de Ledinghen, V., Hyland, R.H., Stamm, L.M., Dvory-Sobol, H., Svarovskaia, E., Zhang, J., Huang, K.C., Subramanian, G.M., Brainard, D.M., McHutchison, J.G., Verna, E.C., Buggisch, P., Landis, C.S., Younes, Z.H., Curry, M.P., Strasser, S.I., Schiff, E.R., Reddy, K.R., Manns, M.P., Kowdley, K.V., Zeuzem, S., Polaris, Investigators, P.-., 2017.
Sofosbuvir, Velpatasvir, and VoXilaprevir for previously treated HCV infection. N. Engl. J. Med. 376, 2134–2146.
Collaborators, T.P.O.H.C.V, 2017. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study. Lancet Gastroenterol. Hepatol. 2, 161–176.
Curry, M.P., O’Leary, J.G., Bzowej, N., Muir, A.J., Korenblat, K.M., Fenkel, J.M., Reddy, K.R., Lawitz, E., Flamm, S.L., Schiano, T., 2015. Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. N. Engl. J. Med. 373, 2618–2628.
Degasperi, E., Spinetti, A., Lombardi, A., Landonio, S., Rossi, M.C., Pasulo, L., Pozzoni, P., Giorgini, A., Fabris, P., Romano, A., Lomonaco, L., Puoti, M., Vinci, M., Gatti, F., Carolo, G., Zoncada, A., Bonfanti, P., Russo, F.P., Aghemo, A., Soria, A., Centenaro, R., Maggiolo, F., Rovere, P., Pasin, F., Paon, V., Faggiano, G., Vario, A., Grossi, G., Soffredini, R., Carriero, C., Paolucci, S., Noventa, F., Alberti, A., Lampertico, P., Fagiuoli, S., Lombardia, N., Veneto Study, G., 2019. Real-life effectiveness and safety of sofosbuvir/velpatasvir/voXilaprevir in hepatitis C patientswith previous DAA failure. J. Hepatol. 71, 1106–1115.
Dietz, J., Susser, S., Berkowski, C., Perner, D., Zeuzem, S., Sarrazin, C., 2015.
Consideration of viral resistance for optimization of direct antiviral therapy of hepatitis C virus genotype 1-infected patients. PLoS One 10, e0134395.
Ehrenberg, A.E., Schmuck, B., Anwar, M.I., Gustafsson, S.S., Stenberg, G., Danielson, U. H., 2014. Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors. J. Enzyme Inhib. Med. Chem. 29, 868–876.
European Association for the Study of the Liver, Electronic address, E.E.E, Clinical Practice Guidelines Panel, C, Representative, E.G.B, Panel, M, 2020. EASL recommendations on treatment of hepatitis C: final update of the series. J. Hepatol. 73, 1170–1218.
European Union, H.C.V.C, 2017. Hepatitis C virus prevalence and level of intervention required to achieve the WHO targets for elimination in the European Union by 2030: a modelling study. Lancet Gastroenterol. Hepatol. 2, 325–336.
Fedorchenko, S.V., Martynovych, T., Klimenko, Z., Yanchenko, V., Solianyk, I., 2020. Retreatment of patients with chronic hepatitis C, subtype 1b and cirrhosis, who failed previous direct-acting antiviral therapy including first- and second-generation NS5A inhibitors with ombitasvir/paritaprevir/ritonavir, dasabuvir sofosbuvir ribavirin. J. Viral Hepat. 27, 548–551.
Flisiak, R., Zarebska-Michaluk, D., Jaroszewicz, J., Lorenc, B., Klapaczynski, J., Tudrujek-Zdunek, M., Sitko, M., Mazur, W., Janczewska, E., Pabjan, P., Dybowska, D., Buczynska, I., Czauz-Andrzejuk, A., Belica-Wdowik, T., Berak, H.,
Krygier, R., Piasecki, M., Dobracka, B., Citko, J., Piekarska, A., Socha, L., Deron, Z., Tronina, O., Laurans, L., Bialkowska, J., Tomasiewicz, K., Halota, W., Simon, K., Pawlowska, M., 2020. Changes in patient profile, treatment effectiveness, and safety during 4 years of access to interferon-free therapy for hepatitis C virus infection. Pol. Arch Intern. Med. 130, 163–172.
Forns, X., Gordon, S.C., Zuckerman, E., Lawitz, E., Calleja, J.L., Hofer, H., Gilbert, C., Palcza, J., Howe, A.Y.M., DiNubile, M.J., 2015. Grazoprevir and elbasvir plus ribavirin for chronic HCV genotype-1 infection after failure of combination therapy containing a direct-acting antiviral agent. J. Hepatol. 63, 564–572.
Guan, Y., Sun, H., Li, Y., Pan, P., Li, D., Hou, T., 2014. The competitive binding between inhibitors and substrates of HCV NS3/4A protease: a general mechanism of drug resistance. Antivir. Res. 103, 60–70.
Guan, Y., Sun, H., Pan, P., Li, Y., Li, D., Hou, T., 2015. EXploring resistance mechanisms of HCV NS3/4A protease mutations to MK5172: insight from molecular dynamics simulations and free energy calculations. Mol. BioSyst. 11, 2568–2578.
Halfon, P., Locarnini, S., 2011. Hepatitis C virus resistance to protease inhibitors.
J. Hepatol. 55, 192–206.
Halota, W., Flisiak, R., Juszczyk, J., Malkowski, P., Pawlowska, M., Simon, K., Tomasiewicz, K., 2020. Recommendations of the polish group of experts for HCV for the treatment of hepatitis C in 2020. Clin. EXp. Hepatol. 6, 163–169.
Howe, A.Y.M., Long, J., Nickle, D., Barnard, R., Thompson, S., Howe, J., Alves, K., Wahl, J., 2015. Long-term follow-up of patients receiving boceprevir for treatment of chronic hepatitis C. Antivir. Res. 113, 71–78.
Huang, C.F., Hung, C.H., Cheng, P.N., Bair, M.J., Huang, Y.H., Kao, J.H., Hsu, S.J., Lee, P.
L., Chen, J.J., Chien, R.N., Peng, C.Y., Lin, C.Y., Hsieh, T.Y., Cheng, C.H., Dai, C.Y.,
Huang, J.F., Chuang, W.L., Yu, M.L., 2019. An open-label, randomized, active- controlled trial of 8 versus 12 weeks of Elbasvir/Grazoprevir for treatment-naive patients with chronic hepatitis C genotype 1b infection and mild fibrosis (EGALITE Study): impact of baseline viral loads and NS5A resistance-associated substitutions. J. Infect. Dis. 220, 557–566.
Iio, E., Shimada, N., Abe, H., Atsukawa, M., Yoshizawa, K., Takaguchi, K., Eguchi, Y., Nomura, H., Kuramitsu, T., Kang, J.H., Matsui, T., Hirashima, N., Tsubota, A., Kusakabe, A., Hasegawa, I., Miyaki, T., Shinkai, N., Fujiwara, K., Nojiri, S., Tanaka, Y., 2017. Efficacy of daclatasvir/asunaprevir according to resistance- associated variants in chronic hepatitis C with genotype 1. J. Gastroenterol. 52,94–103.
Itakura, J., Kurosaki, M., Kakizaki, S., Amano, K., Nakayama, N., Inoue, J., Endo, T., Marusawa, H., Hasebe, C., Joko, K., Wada, S., Akahane, T., Koushima, Y., Ogawa, C.,
Kanto, T., Mizokami, M., Izumi, N., 2020. Features of resistance-associated substitutions after failure of multiple direct-acting antiviral regimens for hepatitis C. JHEP Rep. 2, 100138.
Krishnan, P., Tripathi, R., Schnell, G., Reisch, T., Beyer, J., Dekhtyar, T., Irvin, M.,
Xie, W., Larsen, L., Podsadecki, T., Pilot-Matias, T., Collins, C., 2015a. O057: Long- term follow-up of treatment-emergent resistance-associated variants in NS3, NS5A and NS5B with paritaprevir/r-, ombitasvir- and dasabuvir-based regimens.
J. Hepatol. 62, S220.
Krishnan, P., Tripathi, R., Schnell, G., Reisch, T., Beyer, J., Irvin, M., Xie, W., Larsen, L., Cohen, D., Podsadecki, T., Pilot-Matias, T., Collins, C., 2015b. Resistance analysis of baseline and treatment-emergent variants in hepatitis C virus genotype 1 in the AVIATOR study with paritaprevir-ritonavir, ombitasvir, and dasabuvir. Antimicrob. Agents Chemother. 59, 5445–5454.
Krishnan, P., Tripathi, R., Schnell, G., Reisch, T., Beyer, J., Dekhtyar, T., Irvin, M., Xie, W., Larsen, L., Podsadecki, T., Pilot-Matias, T., Collins, C., 2020. O057: Long- term follow-up of treatment-emergent resistance-associated variants in NS3, NS5A and NS5B with paritaprevir/r-, ombitasvir- and dasabuvir-based regimens.
J. Hepatol. 62, S220.
McPhee, F., Friborg, J., Levine, S., Chen, C., Falk, P., Yu, F., Hernandez, D., Lee, M.S., Chaniewski, S., Sheaffer, A.K., Pasquinelli, C., 2012. Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir. Antimicrob. Agents Chemother. 56, 3670–3681.
Neumann, A.U., Lam, N.P., Dahari, H., Gretch, D.R., Wiley, T.E., Layden, T.J., Perelson, A.S., 1998. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-α therapy. Science 282, 103–107.
Panasiuk, A., Flisiak, R., Mozer-Lisewska, I., Adamek, A., Tyczyno, M., Halota, W., Pawlowska, M., Stanczak, J., Berak, H., Wawrzynowicz-Syczewska, M., Boron- Kaczmarska, A., Lapinski, T.W., Grzeszczuk, A., Piekarska, A., Tomasiewicz, K., Jablkowski, M., Kryczka, W., Zarebska-Michaluk, D., Stepien, P., Garlicki, A.M., Kozlowska, J., Wiercinska-Drapalo, A., Zasik, E., Mazur, W., Dobracka, B., Dobracki, W., Simon, K., Ryzko, J., Pawlowska, J., Dzierzanowska-Fangrat, K., Januszkiewicz-Lewandowska, D., Szenborn, L., Zaleska, I., Rokitka, M., Strawinska, E., Balinowska, K., Smiatacz, T., Stalke, P., Sikorska, K., Lakomy, A., Zdrojewski, M., Lachowicz, A., 2013. Distribution of HCV genotypes in Poland. Przegl. Epidemiol. 67 (11–16), 99–103.
Parczewski, M., Kordek, J., Janczewska, E., Pisula, A., Lojewski, W., Socha, L., Wawrzynowicz-Syczewska, M., Bociaga-Jasik, M., Szymczak, A., Cielniak, I., Siwak, E., Mularska, E., Aksak-Was, B., Urbanska, A., Lubke, N., 2019. Hepatitis C virus (HCV) genotype 1 NS5A resistance-associated variants are associated with advanced liver fibrosis independently of HCV-transmission clusters. Clin. Microbiol. Infect. 25, 513 e511-513 e516.
Poordad, F., Pol, S., Asatryan, A., Buti, M., Shaw, D., Hezode, C., Felizarta, F., Reindollar, R.W., Gordon, S.C., Pianko, S., Fried, M.W., Bernstein, D.E., Gallant, J., Lin, C.W., Lei, Y., Ng, T.I., Krishnan, P., Kopecky-Bromberg, S., Kort, J., Mensa, F.J., 2018. Glecaprevir/Pibrentasvir in patients with hepatitis C virus genotype 1 or 4 and past direct-acting antiviral treatment failure. Hepatology 67, 1253–1260.
Ribeiro, R.M., Li, H., Wang, S., Stoddard, M.B., Learn, G.H., Korber, B.T., Bhattacharya, T., Guedj, J., Parrish, E.H., Hahn, B.H., 2012. Quantifying the diversification of hepatitis C virus (HCV) during primary infection: estimates of the in vivo mutation rate. PLoS Pathog. 8, e1002881.
Silva, T., Cortes Martins, H., Coutinho, R., Leitao, E., Silva, R., Padua, E., 2015.
Molecular characterization of hepatitis C virus for determination of subtypes and detection of resistance mutations to protease inhibitors in a group of intravenous drug users co-infected with HIV. J. Med. Virol. 87, 1549–1557.
Stanaway, J.D., Flaxman, A.D., Naghavi, M., Fitzmaurice, C., Vos, T., Abubakar, I., Abu- Raddad, L.J., Assadi, R., Bhala, N., Cowie, B., 2016. The global burden of viral hepatitis from 1990 to 2013: findings from the global burden of disease study 2013. Lancet 388, 1081–1088.
Sullivan, J.C., De Meyer, S., Bartels, D.J., Dierynck, I., Zhang, E.Z., Spanks, J., Tigges, A. M., Ghys, A., Dorrian, J., Adda, N., 2013. Evolution of treatment-emergent resistant variants in telaprevir phase 3 clinical trials. Clin. Infect. Dis. 57 (2), 221–229. https://doi.org/10.1093/cid/cit226.
Wang, G.P., Terrault, N., Reeves, J.D., Liu, L., Li, E., Zhao, L., Lim, J.K., Morelli, G.,
Kuo, A., Levitsky, J., Sherman, K.E., Frazier, L.M., Ramani, A., Peter, J., Akuskevich, L., Fried, M.W., Nelson, D.R., 2018. Prevalence and impact of baseline resistance-associated substitutions on the efficacy of ledipasvir/sofosbuvir or simeprevir/sofosbuvir against GT1 HCV infection. Sci. Rep. 8, 3199.
Wang, H., Guo, C., Chen, B.Z., Ji, M., 2017. Computational study on the drug resistance mechanism of HCV NS3 protease to BMS-605339. Biotechnol. Appl. Biochem. 64, 153–164.
Wargo, A.R., Kurath, G., 2012. Viral fitness: definitions, measurement, and current insights. Curr. Opin. Virol. 2, 538–545.
Woods, C.K., Brumme, C.J., Liu, T.F., Chui, C.K., Chu, A.L., Wynhoven, B., Hall, T.A., Trevino, C., Shafer, R.W., Harrigan, P.R., 2012. Automating HIV drug resistance genotyping with RECall, a freely accessible sequence analysis tool. J. Clin. Microbiol. 50, 1936–1942.
Wyles, D.L., Gutierrez, J.A., 2014. Importance of HCV genotype 1 subtypes for drug resistance and response to therapy. J. Viral Hepat. 21, 229–240.
Yoshimi, S., Imamura, M., Murakami, E., Hiraga, N., Tsuge, M., Kawakami, Y.,Aikata, H., Abe, H., Hayes, C.N., Sasaki, T., Ochi, H., Chayama, K., 2015. Long term persistence of NS5A inhibitor-resistant hepatitis C virus in patients who failed daclatasvir and asunaprevir therapy. J. Med. Virol. 87, 1913–1920.
Zarebska-Michaluk, D., Buczynska, I., Simon, K., Tudrujek-Zdunek, M., Janczewska, E., Dybowska, D., Sitko, M., Dobracka, B., Jaroszewicz, J., Pabjan, P., Klapaczynski, J., Laurans, L., Mazur, W., Socha, L., Tronina, O., Parczewski, M., Flisiak, R., 2019. Real world experience of Glecaprevir in chronic hepatitis C retreatment with genotype specific regimensin nonresponders to previous interferon-free therapy. Can. J. Gastroenterol. Hepatol. 2019, 4029541.
Zeuzem, S., Flamm, S.L., Tong, M.J., Vierling, J.M., Pianko, S., Buggisch, P., de Ledinghen, V., Hyland, R.H., Wu, X., Svarovskaia, E.S., 2020. A Randomized, Controlled, Phase 3 Trial of Sofosbuvir/Velpatasvir/VoXilaprevir or Sofosbuvir/ Velpatasvir for 12 Weeks in Direct Acting Antiviral-EXperienced Patients with Genotype 1–6 HCV Infection: The POLARIS-4 Study, 63, 59A-59A.