Relevant Resistance-associated Substitutions Research Articles

Emergence and Persistence of Resistance-Associated Substitutions in HCV GT3 Patients Failing Direct-Acting Antivirals.

Background: The hepatitis C virus has a high mutation rate, which results in the emergence of resistance-associated substitutions (RASs). Despite direct-acting antivirals (DAAs) efforts to treat chronically infected HCV genotype 3 (GT3) patients, there are concerns about the emergence and persistence of RASs in DAA failures. The objective of this study was to determine the prevalence of clinically relevant RASs in HCV NS5A and NS5B regions before and after treatment to better understand the role of RASs in treatment failures. Methods: Viral RNA was extracted before and after treatment from serum samples. NS5A and NS5B regions of HCV were amplified by nested PCR, followed by Sanger sequencing. The nucleotide sequences were aligned against HCV GT3 reference sequences, and amino acid substitutions were analyzed using the geno2pheno [hcv] webserver. Results: A total of 76 patients failing DAA therapy were stratified from the cohort of 1388. RASs were detected at the baseline in 15/76 patients and at relapse in 20/76 patients with cirrhosis and previously treated with interferons. The most prevalent NS5A RAS was Y93H found in all treatment-failing patients (14/54 in DCV vs. 6/22 in VEL), followed by A62S/T and A30K. No RASs were identified in NS5B. RASs that were present at the baseline persisted through the 24-week follow-up period and were enriched with emerging RASs during the treatment. The presence of RASs may be one of the causes of treatment failures in 26.3% of patients. Amino acid substitutions were present at the baseline in most of the patients with RASs against NS5A inhibitors. Patients with the baseline Y93H and/or A30K relapse more frequently than patients harboring A62S/T. Conclusion: Treatment-failing patients harbored NS5A RASs, and the most frequent were A30K (5/20), A62S/T (20/20), and Y93H (20/20). Direct resistance testing is recommended for optimizing re-treatment strategies in treatment-failing patients.

NS5A resistance – associated substitutions in chronic hepatitis C patients with direct acting antiviral treatment failure in Turkey

ObjectivesChronic hepatitis C (CHC) is now a more curable disease with new direct acting antivirals (DAA). Although high sustained virologic response rates, failures still occur in DAA regimens. Our objective in this study was to characterize the real-life presence of clinically relevant resistance – associated substitutions (RASs) in the HCV NS5A gene in CHC patients whose DAA regimen has failed. MethodsThe study enrolled 53 CHC patients who experienced failure with DAA regimen as the prospective longitudinal cohort between 2017–2019. Genotypic resistance testing was performed via the viral population sequencing method and The Geno2pheno HCV tool was used for RAS analysis. ResultsThe most frequent failure category was relapse (88%) followed by non-responder (12%). For a total of 36% of patients, RASs was detected in NS5A, Y93H was the most detected RAS in GT1b infected patients (89%). ConclusionsThis study establishes an HCV failure registry for Turkey in which samples were combined with clinical, virologic and molecular data of adult patients whose DAA therapy failed. RASs can occur in CHC patients with DAA treatment failures. Evaluation of RAS after DAA failure is very important before re-treatment is initiated to prevent virologic failure.

Impact of an Open Access Nationwide Treatment Model on Hepatitis C Virus Antiviral Drug Resistance.

Direct acting antivirals (DAAs) have revolutionized hepatitis C virus (HCV) treatment, but drug resistance could undermine proposed global elimination targets. Real‐world studies are needed to inform the impact of widespread DAA treatment on antiviral resistance in the community. The prevalence and range of posttreatment resistance‐associated substitutions (RASs) was determined in Australian patients with open access to DAAs through a wide range of prescribers. NS3, NS5A, and NS5B regions were amplified by polymerase chain reaction and analyzed by population sequencing. Clinically relevant RASs were identified using online databases (ReCALL and Geno2Pheno[hcv]). Of 572 samples, 60% were from genotype 3 and 27% from genotype 1a. Ninety‐two percent of people failed a DAA regimen containing an NS5A inhibitor, including 10% with a pangenotype regimen. NS5A RASs were detected in 72% of people with genotype 1 and 80% with genotype 3. For genotype 1, there was a range of RASs across the NS5A region, while for genotype 3, the Y93H RAS predominated (72%). The prevalence of NS3 RASs was higher in people exposed to an NS3 inhibitor (35% vs. 3.9%; P < 0.0001). NS5B resistance was rare, with a single case of sofosbuvir resistance. Multiclass drug resistance was found in 33% of people exposed to both NS3 and NS5A inhibitors. Conclusion: The high prevalence of NS5A RASs among people failing DAA therapy reinforces the importance of specific retreatment regimens, ideally guided by resistance testing. The impact of multiclass drug resistance on retreatment in people exposed to both NS3 and NS5A inhibitors needs to be assessed in real‐world studies. Surveillance for increasing antiviral resistance during treatment scale‐up is essential to maintain the efficacy of current DAA regimens.

Frequency of baseline NS5A resistance-associated substitutions in patients infected with genotype 1 of hepatitis C virus in Croatia

The backbone of current treatment for chronic Hepatitis C virus (HCV) infection are direct-acting antivirals targeting viral nonstructural proteins (NS3, NS4A, NS5A, NS5B). To date, there are six NS5A inhibitors approved for treatment of chronic HCV infection. The presence of drug-associated resistance substitutions is mainly due to fast error-prone replication, showing differential frequency between genotypes and subtypes. The aim of this study was to determine the frequency of baseline resistance to NS5A protein inhibitors in patients with genotype 1 HCV in Croatia. Resistance-associated substitutions (RAS) were detected by Sanger sequencing of HCV NS5A region amplified from 84 patients followed by phylogenetic analysis and analysis with Geno2Pheno algorithm. The frequency of NS5A RAS was 14.3% and highly dependent on viral subtype. The overall frequency of NS5A RAS was higher in patients infected with HCV subtype 1b (24.2%) than in those infected with HCV subtype 1a (7.8%). Overall, three resistance-conferring mutations were detected (Q30R, M28T and Y93H) along with two mutations (M28V and L31I) that cause reduced susceptibility to NS5A inhibitors. Analysis of the sequences showed two distinct subtype 1a clades with RAS detected in 4.3% (1/23) clade I and 10.7% (3/28) clade II sequences. Only a few distinct NS5A RAS were detected suggesting a high degree of homogeneity of the viral population. High frequency of clinically relevant NS5A RAS in Croatia suggest that the analysis of frequency and patterns of resistance mutations in local populations and evaluation of their possible clinical impact could be beneficial.

Baseline resistance-associated substitutions may impact DAA response among treatment failure chronic hepatitis C patients with pegylated interferon and ribavirin in real life.

To evaluate the impact of baseline resistance-associated substitutions (RASs) on direct-acting antiviral (DAA) treatment response among pegylated interferon in combination with ribavirin (PR) failing patients in a real-life setting. Blood samples and clinical data from 171 patients who failed PR treatment were collected. All of them received rescue DAA regimens. RAS identified in the NS3, NS5A and NS5B regions by Sanger sequencing method were compared by DAA regimen and HCV subtypes. We assessed sustained virological response at 12 weeks (SVR12) and evaluated the impact of baseline RASs on the effectiveness of DAA regimens in clinical practice. The overall SVR12 rates were: 89.47% (153/171), 92.1% (117/127) in patients without cirrhosis versus 81.8% (36/44) in those with cirrhosis, without significant difference (χ2=3.69, P=0.08); 87.9% in genotype (GT)1b patients (n=116) versus 93.8% in GT2a (n=32) versus 90.5% in GT3 (n=21) versus 100% in GT6 (n=2), without significant difference (χ2=1.02, P=0.84); 66.7% in asunaprevir (ASV) + daclatasvir (DCV) regimen (n=24) versus 94.0% in sofosbuvir (SOF)-based regimen (n=133), with significant difference (χ2=19.7, P=0.001). Our results showed that the prevalence of NS3, NS5A, NS5B RASs was 45.02%, 39.76% and 71.34%, respectively, and higher incidence of RASs in cirrhosis than without cirrhosis (81.8% versus 63.8%), with a statistically significant difference (χ2=4.92, P=0.03). In the ASV/DCV cohort (n=24), 4 of 11 patients (36.4%) with baseline NS3/NS5A RASs achieved SVR12, whereas 12 of 13 patients (92.3%) without RASs achieved SVR12, with significant difference (χ2=8.39, P=0.008). However, this relationship was not seen in the SOF-based subgroup (94.6% versus 92.7%; χ2=0.18, P=0.7). Treatment failure with DAAs occurred in 10.53% (n=18) of our study population, baseline NS5A substitution including L31M or Y93H (n=13) was the most frequently detected RAS, rescue regimen with velpatasvir (VEL)/SOF + ribavirin (RBV) for 12 weeks or 24 weeks was highly effective in patients who failed previous use of NS5A inhibitors, regardless of GT or cirrhosis. Natural RASs are common in Chinese patients failing with PR treatment. High prevalence of clinically relevant RASs (such as L31M, Y93H) supports the appropriateness of HCV resistance tests to properly guide DAA-based therapy. These findings might be used to select salvage therapies.

Naturally occurring NS5A and NS5B resistant associated substitutions in HCV and HCV/HIV patients in iranian population

The introduction of direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment promises shorter treatment duration, higher cure rates and fewer side effects. Naturally, occurring Resistance Associated Substitutions (RASs) are major challenge to the success of the HCV antiviral therapy. To determine the naturally occurring NS5A and NS5B RASs in Iranian HCV and HCV/human immunodeficiency virus (HIV) patients. A total of 209 DAA-naïve chronic HCV patients including 104 HCV mono-infected and 105 HCV/HIV co-infected cases were enrolled. Amplification and Sanger population sequencing of NS5A and NS5B regions of HCV genome were carried out. The amino acid sequence diversity of the NS5A and NS5B regions were analyzed using geno2pheno HCV. NS5A RASs were detected in 25.5% of HCV and 16.9% of HCV/HIV subjects. In HCV cases, clinically relevant RASs were L28M followed by M28Vand Q30H and Y93H/N. In HCV/HIV subjects, clinically relevant RASs were Y93H/N followed by L28M and P58T and M28V/T and Q30R. NS5B RASs were observed in 11.8% of HCV and 5.9% of HCV/HIV subjects. Clinically relevant substitutions were included V321A/I, C316Y, S282R and L159F. The major S282T mutation was not observed. The emergence of RASs is a growing issue in the setting of current treatment with DAAs. Although currently, screening of RASs is recommended before specific DAA regimens, it should be consider in patients with therapeutic failure and in the cases of retreatment.

Prevalence and Factors Related to Natural Resistance-Associated Substitutions to Direct-Acting Antivirals in Patients with Genotype 1 Hepatitis C Virus Infection.

This study aimed to assess the prevalence of natural resistance-associated substitutions (RASs) to NS3, NS5A and NS5B inhibitors in 86 genotype 1 Hepatitis C Virus (HCV)-infected patients from Buenos Aires, Argentina, and to determine their effect on therapy outcome. Additionally, virological, clinical and host genetic factors were explored as predictors of the presence of baseline RASs. NS3 RASs (39.2%) were more prevalent than NS5A RASs (25%) and NS5B RASs (8.9%). In the three regions, the frequencies of RASs were significantly higher in HCV-1b than in HCV-1a. The prevalence of Y93H, L159F and Q80K were 1.3%, 6.3% and 2.5%, respectively. IFNL3 CC genotype was identified as an independent predictor of the presence of baseline RASs in NS5A and NS3 genes (p = 0.0005 and p = 0.01, respectively). Sustained virologic response was achieved by 93.3% of the patients after receiving direct-acting antivirals (DAAs), although 48.7% of them showed baseline RASs related to the DAA-regimen. Notably, the prevalence of clinically relevant RASs in the three genes was lower than that observed around the world. The baseline presence of RASs in both subtypes did not appear to affect therapy outcome. These results support the need to evaluate resistance patterns in each particular country since RASs´ prevalence significantly vary worldwide.

Pre-treatment antiviral resistance in Australians with chronic hepatitis C: prevalence of NS3 and NS5A resistance data in the state of New South Wales.

Direct-acting antivirals (DAAs) have revolutionized HCV treatment, but the impact of antiviral resistance at a population level is still not clear. The majority of patients who fail DAA therapy develop resistance-associated substitutions (RASs), which can impact re-treatment. There is potential for resistance prevalence to rise in the community with treatment scale up, due to transmission of resistant virus. Monitoring for increasing antiviral resistance requires a reliable baseline, yet there are few published data on the prevalence of HCV resistance in Australia. The aim of this study was to determine the prevalence of RASs among untreated Australians with HCV genotype-1a infection, to inform ongoing surveillance. A cross-sectional study was performed at a single large university hospital pathology laboratory in Australia. Archived blood samples referred for HCV genotype testing were analysed. All patients were naive to DAAs. The prevalence of RASs in the HCV NS3 and NS5A regions was determined using Sanger based population sequencing. Of 379 samples tested, 34% contained DAA-resistant virus: 24% had resistance to NS3 protease inhibitors, 12% had NS5A inhibitor resistance and 4% of patients had resistance to both drug classes. Clinically relevant RASs conferring resistance against NS5A inhibitors ledipasvir, daclatasvir and elbasvir were detected in 5.8% of samples. This is the largest study of HCV antiviral drug resistance in Australia, which differs from resistance prevalence in the USA. The results provide valuable data on the baseline prevalence of HCV resistance, which can be used in the future to monitor for increasing antiviral resistance.

High frequency of multiclass HCV resistance-associated mutations in patients failing direct-acting antivirals: real-life data.

Direct-acting antiviral (DAA) therapy has dramatically increased sustained virological response rates in HCV-infected patients. However, resistance-associated substitutions (RAS) interfering with NS3- and NS5A-targeted therapy, still emerge. This real-life study analysed the type and frequency of RAS in rare cases of patients failing DAA regimens in 12 clinical centres in Israel. Blood samples and clinical data from 49 patients who failed various DAAs were collected. RAS identified in the NS3 and NS5A regions by population (Sanger) and next-generation sequencing (NGS) were compared by treatment regimen and HCV subtypes. The majority (71.4%, 35/49) of patients were infected with the genotype (GT)1b strain, while 12.2% (6/49) carried GT1a and 14.3% GT3a/b (7), GT4a (1) and GT1b/GT3a. RAS were identified in 85.7% (42/49) of failures, of which 90.5% (38/42) were clinically relevant RAS (known to be associated with a specific GT and DAA in patients failing therapy or those with more than twofold change in in vitro replicon assays). The most abundant RAS were 168A/E/Q/G/N/V (32.6%, 16/49) identified in NS3, and 93H/N (61.2%, 30/49), 31I/M/V (34.7%, 17/49) and 30R/H/K (12.2%, 6/49), identified in NS5A. Significantly more clinically relevant RAS were identified in NS5A (82.2%, 37/45) than in NS3 (35.7%, 10/28; P<0.01). While RAS were identified in all GT1a, GT3b and GT4a failures (100%, 10/10), only 71.8% (28/39) of GT1b or GT3a failures had RAS (P=0.09). In four cases, NGS identified additional clinically relevant RAS and in one patient, NGS deciphered coexistence of GT3a and GT1b infections. Our findings, together with additional real-life data, will contribute to the optimization of retreatment in DAA failure, when cost-related and suboptimal regimens must be employed.

Baseline dasabuvir resistance in Hepatitis C virus from the genotypes 1, 2 and 3 and modeling of the NS5B-dasabuvir complex by the in silico approach

ABSTRACTBackground: Current combination treatments with direct-acting antiviral agents (DAAs) can cure more than 95% of hepatitis C virus (HCV) infections. However, resistance-associated substitutions (RASs) may emerge and can also be present in treatment-naïve patients. Methods, results and discussion: In this study, a semi-pan-genotypic population sequencing method was developed and used to assess all NS5B amino acid variants between residue positions 310 and 564. Our method successfully sequenced more than 90% of genotype (GT) 1a, 1b, 2b and 3a samples. By using the population sequencing method with a cut-off of 20%, we found the dasabuvir RASs A553V and C445F to be a baseline polymorphism of GT 2b (8 out of 8) and GT 3a (18 out of 18) sequences, respectively. In GT 1a and 1b treatment-naïve subjects (n=25), no high-fold resistance polymorphism/RASs were identified. We further predicted dasabuvir’s binding pose with the NS5B polymerase using the in silico methods to elucidate the reasons associated with the resistance of clinically relevant RASs. Dasabuvir was docked at the palm-I site and was found to form hydrogen bonds with the residues S288, I447, Y448, N291 and D318. The RAS positions 316, 414, 448, 553 and 556 were found to constitute the dasabuvir binding pocket.

Pegylated-interferon plus ribavirin treatment does not alter the prevalence of resistance-associated substitutions to direct-acting antivirals in HCV genotype 1a patients.

BackgroundDirect-acting antiviral (DAA) resistance-associated substitutions (RASs) can jeopardize the effectiveness of DAAs in patients with hepatitis C virus (HCV). The selection pressure by pegylated-interferon (Peg-IFN) plus ribavirin (P/R) treatment may enhance HCV genome variation. However, whether P/R treatment alters the rate of change of RASs is still unclear.Materials and methodsWe retrieved the genomic sequences of HCV genotype (GT) 1a patients from GenBank, which included patients naïve to P/R (pre-IFN group) and those previously treated with P/R (post-IFN group). The sequences were aligned and analyzed by using MEGA 6.0 software. Clinically relevant RASs were summarized from the current medical literature.ResultsIn the cross-sectional study, the total prevalence of clinically relevant RASs was high, independent of the treatment group (pre-IFN: 219/403 [54.34%] vs post-IFN: 67/131 [51.15%]). The high prevalence was mainly detected in the NS3 region RAS at Q80 (40.69% vs 36.64%). The RASs in the NS5A region, such as M28, Q30, L31 and Y93, were uncommon (0%–5%). Similarly, all RASs showed no difference between the two groups. One exception was the RAS at I170 in the NS3 region, which was significantly higher in the post-IFN group than in the pre-IFN group. In the longitudinal study, similar results were observed. However, no difference in RAS at I170 was observed between the two groups. Finally, no clinically relevant RASs were detected in response to the DAA regimens approved for GT 1a patients treated with P/R.ConclusionOur results suggest that previous P/R treatment failure was not favorably associated with an increase in DAAs RASs present in GT1a patients. Our results support the American Association for the Study of Liver Diseases’ recommendations of DAA intervention in P/R-treated GT1a patients.

Low frequency of NS5A relevant resistance-associated substitutions to Elbasvir among hepatitis C virus genotype 1a in Spain: a cross-sectional study

Relevant resistance-associated substitutions (RASs) to elbasvir, the new HCV NS5A inhibitor, may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are few data outside clinical trials evaluating their prevalence and impact of elbasvir/grazoprevir. A multicenter cross-sectional study of 617 HCV-GT1a-infected individuals attended in 84 Spanish hospitals from the 17 Autonomous Communities and two Autonomous cities was performed. HCV population sequencing was used to identify RASs to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-GT1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%; respectively). Only 3.4% of patients had viruses with RASs that confer reduced susceptibility to elbasvir by geno2pheno[HCV] that identified exclusively the positions Q30H/R (n = 7) and Y93C/H/N (n = 8) as single mutations and Q30H + Y93H (n = 4) and Q30R + Y93H (n = 2) as double mutations considered as RASs to elbasvir. Lower prevalence of RASs to elbasvir in our HCV-GT1a-Spanish cohort was observed than reported previously in clinical trials. This information may be essential to guiding the implementation of elbasvir/grazoprevir in Spain, expected at the beginning of 2017 and the management of GT1a-infected patients.

Global epidemiology of HCV subtypes and resistance-associated substitutions evaluated by sequencing-based subtype analyses

HCV genotype, subtype, and presence of resistance-associated substitutions (RASs) are key determinants for the selection of direct-acting antiviral (DAA) treatment regimens. However, current HCV genotyping assays have limitations in differentiating between HCV subtypes, and RAS prevalence is largely undefined. The aim of this study was to investigate HCV epidemiology in 12,615 patient samples from 28 different countries across five geographic regions. We compared HCV genotype and subtypes using INNO-LiPA 2.0 vs. amplicon sequencing among 8,945 patients from phase II/III clinical trials of DAAs. Global HCV molecular epidemiology in 12,615 patients was investigated. Subtype RAS prevalence was determined by population or deep sequencing, and phylogenetic analyses investigating subtype diversity were performed. Although there was high concordance between INNO-LiPA and sequencing for genotype determination, INNO-LiPA was insufficient for subtype determination for genotype 2, 3, 4, and 6. Sequencing provided subtype refinement for 42%, 10%, 81%, and 78% of genotype 2, 3, 4, and six patients, respectively. Genotype discordance (genotype 2-genotype 1) was observed in 28 of 950 (3%) genotype 2 patients, consistent with inter-genotype recombinants. Sequencing-based analyses demonstrated variations in regional subtype prevalence, notably within genotype 2, 4 and 6. RAS prevalence varied by subtype, with the clinically relevant NS3 RAS Q80K found in genotype 1a, 5a and 6a and the NS5A RAS Y93H in genotype 1b, 3a, 4b, 4r and 7. Together, these analyses provide an understanding of subtyping accuracy and RAS distribution that are crucial for the implementation of global HCV treatment strategies. Hepatitis C virus (HCV) is highly variable, with seven genotypes and 67 subtypes characterized to date. The aim of this study was to i) compare two different methods of discriminating between genotypes; ii) investigate the prevalence of HCV subtypes for each genotype around the world; iii) find the prevalence of resistance-associated substitutions (RASs) in different subtypes. We found that both methods showed high concordance in genotype discrimination, but specific subtypes were not always identified accurately. Sequencing-based analyses demonstrated variations in regional subtype prevalence for some genotypes, notably within GT2, 4 and 6. RAS prevalence also varied by subtype. These variations could determine how successful different drugs are for treating HCV.

Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies.

Despite the excellent efficacy of direct-acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance-associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real-life DAA failures. Of the 200 virological failures that were analyzed in 197 DAA-treated patients, 89 with pegylated-interferon+ribavirin (PegIFN+RBV) and 111 without (HCV-1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS3/NS5A/NS5B was performed by home-made protocols, at failure (N=200) and whenever possible at baseline (N=70). The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non-responders. RAS prevalence varied according to IFN/RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS5A-RASs (96.1%), compared to NS3-RASs (75.9% with IFN, 70.5% without) and NS5B-RASs (66.6% with IFN, 20.4% without, in sofosbuvir failures). In the IFN-free group, RASs were higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, P<.001). Interestingly, 57.1% of DAA IFN-free non-responders had a misclassified genotype, and 3/4 sofosbuvir breakthroughs showed the major-RAS-S282T, while RAS-L159F was frequently found in sofosbuvir relapsers (18.2%). Notably, 9.0% of patients showed also extra target RASs, and 47.4% of patients treated with ≥2 DAA classes showed multiclass resistance, including 11/11 NS3+NS5A failures. Furthermore, 20.0% of patients had baseline-RASs, which were always confirmed at failure. In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second-line therapeutic tailoring.

Use of whole genome sequencing in the Dutch Acute HCV in HIV study: focus on transmitted antiviral resistance

ObjectiveWithin HIV-positive men having sex with men, the epidemic of hepatitis C virus (HCV) is ongoing. Transmission of resistant variants of HCV after failure of treatment with directly acting antivirals (DAA) could be a major threat to the effectivity of therapy. We determined whether HCV-resistant variants to DAAs were prevalent amongst patients with an acute HCV infection diagnosed in 2013 and 2014 in the Netherlands. MethodsTarget enrichment for viral nucleic acid separation and deep sequencing were used to recover whole HCV genomes of 50 patients with an acute HCV infection. The genomes were assembled by de novo assembly and analysed for known DAA resistance mutations. ResultsIn acute HCV infected treatment-naive patients, the relevant resistance-associated substitutions were Q80K (40%) in NS3/4a, M28V (24%) and Q30H combined with Y93H (2%) in NS5A and M414T (2%) or S556G (2%) in NS5b. Patients whose HCV infection failed to respond to boceprevir, peginterferon and ribavirin therapy developed mutations in NS3 at position T54A and R155K. ConclusionsTarget enrichment and whole genome sequencing were successfully applied directly on clinical samples from patients with an acute HCV infection.

HCV genotype-1 subtypes and resistance-associated substitutions in drug-naive and in direct-acting antiviral treatment failure patients.

Direct-acting antiviral (DAA) treatment regimens and response rates of patients with HCV genotype-1 (GT1) are currently considered subtype-dependent. Identification of clinically relevant resistance-associated substitutions (RASs) in the NS3 and NS5A proteins at baseline and in DAA failures, may also impact clinical decisions. In a multicentre cohort study (n=308), NS3 or NS5B sequencing (n=248) was used to discriminate between GT1 subtypes. The correlation between baseline NS3 and NS5A RASs on the 12-week sustained virological response (SVR12) rates of 160 of the patients treated with second-generation DAAs was also assessed. Post-treatment resistance analysis was performed on samples from 58 patients exhibiting DAA virological failure. GT1a, GT1b and GT1d subtypes were identified in 23.0%, 75.4% and 1.2% of tested samples. GT1b was most prevalent (97.7%, 128/131) among patients born in the former Soviet Union. The Q80K NS3 RAS was identified in 17.5% (10/57) of the GT1a carriers, most of whom were Israeli-born. NS3 and NS5A baseline RASs showed a negligible correlation with SVR12 rates. Treatment-emergent RASs were observed among 8.9% (4/45) and 76.9% (10/13) of first- and second-generation DAA failures, respectively, with D168V/E (NS3), Y93H and L31M (NS5A) being the most prevalent mutations. NS3 sequencing analysis can successfully discriminate between GT1 subtypes and identify NS3 amino acid substitutions. While pre-treatment NS3 and NS5A RASs marginally affect second-generation DAA SVR12 rates, post-treatment resistance analysis should be considered prior to re-therapy.