Resistance-associated Substitutions Research Articles

Resistance-associated substitutions and response to treatment in a chronic hepatitis C virus infected-patient: an unusual virological response case report

BackgroundDirect-Acting agents (DAAs) target and inhibit essential viral replication proteins. They have revolutionized the treatment of Hepatitis C virus (HCV) infection reaching high levels of sustained virologic response. However, the detection of basal resistance-associated substitutions (RASs) to DAAs in naïve patients could be important in predicting the treatment outcome in some patients exhibiting failures to DAA-based therapies. Therefore, the aim of this work was to evaluate the presence of RASs as minority variants within intra-host viral populations, and assess their relationship to response to therapy on a multiple times relapser patient infected chronically with HCV.Case presentationA male HCV infected-patient with a genotype 1a strain was evaluated. He had previously not responded to dual therapy (pegylated interferon-α plus ribavirin) and was going to start a direct-acting agent-based therapy (DAAs). He showed no significant liver fibrosis (F0). Viral RNA was extracted from serum samples taken prior and after therapy with DAAs (sofosbubir/ledipasvir/ribavirin). NS5A and NS5B genomic regions were PCR-amplified and the amplicons were sequenced using Sanger and next-generation sequencing (NGS) approaches. RASs were searched in in-silico translated sequences for all DAAs available and their frequencies were determined for those detected by NGS technology. Sanger sequencing did not reveal the presence of RASs in the consensus sequence neither before nor after the DAA treatment. However, several RASs were found at low frequencies, both before as well as after DAA treatment. RASs found as minority variants (particularly substitutions in position 93 within NS5A region) seem to have increased their frequency after DAA pressure. Nevertheless, these RASs did not become dominant and the patient still relapsed, despite perfect adherence to treatment and having no other complications beyond the infection (no significant fibrosis, no drug abuse).ConclusionsThis report shows that some patients might relapse after a DAA-based therapy even when RASs (pre- and post-treatment) are detected in very low frequencies (< 1%) within intra-host viral populations. Increased awareness of this association may improve detection and guide towards a personalized HCV treatment, directly improving the outcome in hard-to-treat patients.

Distribution of naturally -occurring NS5B resistance-associated substitutions in Egyptian patients with chronic Hepatitis C

NS5B polymerase inhibitors represent the cornerstone of the present treatment of Hepatitis C virus infection (HCV). Naturally occurring substitution mutations to NS5B inhibitors have been recorded. The current study intended to demonstrate possible natural direct acting antiviral (DAA)-mutations of the HCV NS5B region in HCV patients in Minia governorate, Egypt. Samples were collected from 27 treatment-naïve HCV patients and 8 non-responders. Out of 27 treatment-naïve patients, 17 NS5B sequences (amino acids 221-345) from treatment-naïve patients and one sample of non-responders were successfully amplified. Nucleotide sequences have been aligned, translated into amino acids, and compared to drug resistance mutations reported in the literature. NS5B amino acid sequence analysis ensures several novel NS5B mutations existence (more than 40 substitution mutations) that have not been previously documented to be correlated with a resistant phenotype. It was found that K304R (82.4%), E327D and P300T (76.5% each) substitutions were the most distributed in the tested samples, respectively. S282T, the major resistance mutation that induces high sofosbuvir-resistance level in addition to other reported mutations (L320F/C) and (C316Y/N) were not recognized. Q309R mutation is a ribavirin-associated resistance, which was recognized in one strain (5.9%) of genotype 1g sequences. Besides, one substitution mutation (E237G) was identified in the successfully amplified non-responder sample. Our study showed various combinations of mutations in the analyzed NS5B genes which could enhance the possibility of therapy failure in patients administered regimens including multiple DAA.

Phylogenetic and Molecular Analyses of More Prevalent HCV1b Subtype in the Calabria Region, Southern Italy.

Hepatitis C virus subtype 1b (HCV1b) is still the most prevalent subtype worldwide, with massive expansion due to poor health care standards, such as blood transfusion and iatrogenic procedures. Despite safe and effective new direct antiviral agents (DAA), treatment success can depend on resistance-associated substitutions (RASs) carried in target genomic regions. Herein we investigated transmission clusters and RASs among isolates from HCV1b positive subjects in the Calabria Region. Forty-one NS5B and twenty-two NS5A sequences were obtained by Sanger sequencing. Phylogenetic analysis was performed using the maximum likelihood method and resistance substitutions were analyzed with the Geno2pheno tool. Phylogenetic analysis showed sixteen statistically supported clusters, with twelve containing Italian sequences mixed with foreign HCV1b isolates and four monophyletic clusters including only sequences from Calabria. Interestingly, HCV1b spread has been maintained by sporadic infections in geographically limited areas and by dental treatment or surgical intervention in the metropolitan area. The L159F NS5B RAS was found in 15 isolates and in particular 8/15 also showed the C316N substitution. The Y93H and L31M NS5A RASs were detected in three and one isolates, respectively. The A92T NS5A RAS was found in one isolate. Overall, frequencies of detected NS5B and NS5A RASs were 36.6% and 22.7%, respectively. For the eradication of infection, improved screening policies should be considered and the prevalence of natural RASs carried on viral strains.

Real-world re-treatment outcomes of direct-acting antiviral therapy failure in patients with chronic hepatitis C.

Direct-acting antiviral (DAA) drugs are associated with high (>95%) sustained virological response at 12 weeks (SVR12) in chronic hepatitis C (CHC) patients. There is a paucity of data regarding the characteristics and re-treatment outcomes of DAA treatment failure patients. In a retrospective analysis of the prospectively collected database, we assessed the outcomes of re-treatment among patients with previous DAA failure. Patients' characteristics, viral characteristics, including resistance-associated substitutions (RAS) in a subgroup of patients, SVR12, and clinical outcomes were studied. Of 40 patients with DAA failure, among whom 36 were retreated, mean age was 45.7 years, 63.9% (n = 23) were male, 63.9% (n = 23) had agenotype-3 infection and 63.9% (n = 23) were cirrhotic. The re-treatment regimens included a combination of pan-genotypic DAA, mainly sofosbuvir and velpatasvir with or without ribavirin. Three patients who declined retreatment and one who was still on treatment was excluded. For patients who completed re-treatment, SVR12 was 100% irrespective of genotypes. SVR12 among genotype 3 was 75% (15 of 20) when lost to follow-up was considered a treatment failure. Six patients died due to liver-related causes, including five (83.3%) with hepatocellular carcinoma. RAS analysis in 17 randomly selected patients did not reveal any dominant substitutions in NS5A or NS5B region affecting SVR12, though several novel mutations were observed. In conclusion, re-treatment of CHC patients with prior DAA failure using pan-genotypic DAA is associated with high SVR12 rates irrespective of genotype or the presence of RAS.

Risk Assessment of Hepatitis C Virus Resistance to Ns3/4a Protease Inhibitors (Pis)

Hepatitis C virus (HCV) persistently infects more than 175-185 million people globally. Drug resistance to anti-HCV DAAs therapeutics particularly NS3/4A has been a major concern for efficacy of HCV treatment due to generation of HCV quasispecies. Risk determination of the resistance of HCV to anti-NS3/4A inhibitors was accomplished using ViPR algorithm that computes and analyzes resistance-associated amino acid substitutions in HCV NS3 protein target site and reveal altered response to NS3/4A PIs. Spot comparison and data visualization was carried out using ncbi.nlm.nih.gov protein sequence graphics and R-package for Ubuntu version 12.04. Resistance to multiple NS3/4A PIs (telaprevir, simeprevir, faldaprevir and asunaprevir) was exhibited by HCV genotype 1 variant D1194A/E/T/G-NS3 (a substitution hot spot) while the risk of resistance to telaprevir (V1062 L NS3 variant) and simeprevir (S1148R variant) was demonstrated by HCV genotype 2. Genotype 3 & 4 revealed resistance against only telaprevir. Another almost pan-genotype (genotype 2,3,4,5 and unclassified) substitution hot spot was identified as anti-telaprevir variant V1062 L. Single substitution conferring multiple anti-NS3/4A PIs resistance was observed in D1194A NS3 variant (anti-simeprevir, anti-faldaprevir, anti-asunaprevir in genotype 1), Q1106K NS3 variant (anti-simeprevir, anti-faldaprevir in genotype 5 and 6) and T1080S NS3 variant (anti-faldaprevir, anti-telaprevir in genotype 5 and unclassified NS3 sequence). In addition to that, variant S1148G exhibited increased sensitivity to simeprevir in case of genotype 1. Because of a higher degree of HCV genomic variability, NS3 variants with decreased susceptibility to NS3/4A PIs exists, and therefore the resistance profile along with potency, adverse effect of PIs is noteworthy and should be well-considered while developing different protease inhibitors for effective treatment of HCV patients.

HCV genome-wide analysis for development of efficient culture systems and unravelling of antiviral resistance in genotype 4

ObjectiveHCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at...

Naturally Occurring Resistance Associated Substitutions in Non-Cirrhotic, Treatment Naive HCV-HIV Co-Infected Patients Does Not Affect the Treatment Response for Anti-HCV Antiviral Therapy.

PurposeLimited literature on the prevalence of baseline resistance associated substitutions (BL-RAS) among HCV–HIV co-infected patients and their association with treatment outcomes is available especially from India. Hence, the present study aimed to study naturally occurring RAS among non-cirrhotic HCV–HIV co-infected patients and their impact on the response to anti-HCV therapy.Patients and MethodsIn this retrospective study, archived blood samples of 80 HCV–HIV co-infected patients, before anti-HCV therapy initiation, were tested for substitutions at the drug acting sites (NS5a and NS5b) in the HCV genome by direct PCR sequencing.ResultsBL-RAS were seen in 19 (23.7%) patients. As well as BL-RAS, all patients were given sofosbuvir (SOF) 400 mg+ daclatasvir (DCV) 60 mg for 12 weeks. Overall, sustained virological response (SVR) was achieved in 63 (78.8%) patients, in 13 with BL-RAS and in 50 without BL-RAS. All the SVR failure cases (n=17) were retreated with SOF (400 mg) +DCV (60 mg)+ ribavirin (RBV) for 24 weeks. SVR was eventually attained in 14 (82.3%) patients, in 4/6 (66.6%) with BL-RAS and in 10/11 (91%) without BL-RAS. On univariate analysis, age more than 30 years (OR: 11.6; 95% CI: 3.0–45.5, p-value<0.001) and female gender (OR: 8.6; 95% CI: 1.1−69, p-value <0.009) were found to be significant factors associated with the attainment of SVR.ConclusionBL-RAS are common in HCV–HIV co-infected patients. The existence of BL-RAS, however, did not affect the attainment of SVR among non-cirrhotic, treatment naive HCV–HIV co-infected patients.

Baseline Hepatitis C Virus NS5A Resistance-Associated Polymorphisms in Patients With and Without Human Immunodeficiency Virus Coinfection in Mexico.

Objective: We aimed to evaluate the frequency and associated factors of baseline NS5A resistance-associated substitutions (RASs) in patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) monoinfection with genotype 1b (GT1b) or genotype 1a (GT1a). Moreover, we performed a phylogenetic analysis to evaluate the pattern of clustering among samples of patients with RASs. Results: Fifty-five patients were infected with GT1a, of whom 44 (80%) were HIV-infected patients. RAS prevalence in GT1a was 14% (6/44) and distributed as follows: 5 (11%) harbored M28V and 1 (2%) A92T. Twenty-four patients were infected with HCV GT1b, of whom only 5 (21%) were HIV coinfected; RASs were found in 17/24 (71%) patients, as follows: Y93H+F37L+Q54H (1/24), Y93H+F37L (1/24), P58S (1/24), L31F+F37L (1/24), F37L+H/Q54H (3/24), and F37L (10/24). Only GT1b was significantly associated with RASs (adjusted odds ratio 16.37; 95% confidence interval 2.74-97.48; p = 0.002) in the multivariate analysis. A cluster of sequences from HIV/HCV GT1a patients was found; however, we did not find phylogenetic relationships among sequences with NS5A RASs. Conclusions: In our population of HCV-infected patients, the frequency of NS5A RASs at baseline was somewhat similar to the previously reported worldwide rate. HCV GT1b showed the most significant association with harboring of NS5A RASs. Of note, despite there being clusters among sequences of HIV-coinfected patients, NS5A RASs were not transmitted.

Resistance-associated substitutions after sofosbuvir/velpatasvir/voxilaprevir triple therapy failure.

Direct-acting antivirals (DAAs) resolve chronic HCV infection in >95% of patients, but a small percentage do not respond to DAA-based therapy. These may be difficult to treat because of resistance-associated substitutions (RAS) emerging after treatment failure. Triple therapy with sofosbuvir (SOF)/velpatasvir (VEL)/voxilaprevir (VOX) is the recommended retreatment after DAA-based failure. However, in rare cases, failure to triple therapy occurs, and there is little information characterizing the viruses that relapse. To determine the RAS profile after failing SOF/VEL/VOX, and seek suitable alternatives for retreatment, samples from 5 patients were analysed using MiSeq Illumina deep sequencing before and after triple therapy. All patients were men, aged 59-78years, 2 HCV genotype (G) 1b and 3 G3a. The most prevalent NS3 substitutions after SOF/VEL/VOX failure were Y56F and A166T. Four patients had the NS5A RAS, Y93H, after triple failure, and Y93H was observed in both G1b patients before retreatment and after SOF/ledipasvir failure. In 2 G3a patients, Y93H appeared at triple failure, and on the other G3a, A30K persisted in 100% of viral genomes. Finally, G1b patients showed C316N in NS5B, associated with SOF failure, but G3a patients had no known NS5B substitutions. HCV RAS analysis identified the following substitutions present at higher rates after triple failure: Y56F in NS3 (G1b), A166T in NS3 (G3a), A30K or Y93H in NS5A, and C316N in NS5B (G1b). A RAS-based salvage treatment (SOF+glecaprevir/pibrentasvir+RBV) was successfully used in one G3a patient.

From the Editor’s Desk...

From the Editor’s Desk...

HCV Diagnosis and Sequencing Using Dried Blood Spots from Patients in Kinshasa (DRC): A Tool to Achieve WHO 2030 Targets.

The World Health Organization has established an elimination plan for hepatitis C virus (HCV) by 2030. In Sub-Saharan Africa (SSA) access to diagnostic tools is limited, and a number of genotype 4 subtypes have been shown to be resistant to some direct-acting antivirals (DAAs). This study aims to analyze diagnostic assays for HCV based on dried blood spots (DBS) specimens collected in Kinshasa and to characterize genetic diversity of the virus within a group of mainly HIV positive patients. HCV antibody detection was performed on 107 DBS samples with Vidas® anti-HCV and Elecsys anti-HCV II, and on 31 samples with INNO-LIA HCV. Twenty-six samples were subjected to molecular detection. NS3, NS5A, and NS5B regions from 11 HCV viremic patients were sequenced. HCV seroprevalence was 12.2% (72% with detectable HCV RNA). Both Elecsys Anti-HCV and INNO-LIA HCV were highly sensitive and specific, whereas Vidas® anti-HCV lacked full sensitivity and specificity when DBS sample was used. NS5B/NS5A/NS3 sequencing revealed exclusively GT4 isolates (50% subtype 4r, 30% 4c and 20% 4k). All 4r strains harbored NS5A resistance-associated substitutions (RAS) at positions 28, 30, and 31, but no NS3 RAS was detected. Elecsys Anti-HCV and INNO-LIA HCV are reliable methods to detect HCV antibodies using DBS. HCV subtype 4r was the most prevalent among our patients. RASs found in subtype 4r in NS5A region confer unknown susceptibility to DAA.

Secondary substitutions in the hemagglutinin and neuraminidase genes associated with neuraminidase inhibitor resistance are rare in the Influenza Resistance Information Study (IRIS)

Amino acid substitutions in influenza virus neuraminidase (NA) that cause resistance to neuraminidase inhibitors (NAI) generally result in virus attenuation. However, influenza viruses may acquire secondary substitutions in the NA and hemagglutinin (HA) proteins that can restore viral fitness. To assess to which extent this happens, the emergence of NAI resistance substitutions and secondary – potentially compensatory – substitutions was quantified in influenza viruses of immunocompetent individuals included in the Influenza Resistance Information Study (IRIS; NCT00884117). Known resistance substitutions were detected by mutation specific RT-PCR in viruses of 57 of 1803 (3.2%) oseltamivir-treated individuals, including 39 individuals infected with A/H1N1pdm09 [H275Y] virus and 18 with A/H3N2 [R292K] virus. A total of fifteen and ten other amino acid substitutions were acquired in HA and NA respectively, of A/H1N1pdm09, A/H3N2 and influenza B viruses upon treatment with oseltamivir but none of these was associated with resistance to oseltamivir. All cultured viruses with the known resistance substitutions H275Y or R292K showed reduced susceptibility to oseltamivir in the NA-star assay. Upon next-generation sequencing, the vast majority of NAI resistant A/H1N1pdm09 and A/H3N2 viruses had no resistance-associated secondary substitutions at high frequency. Only in two A/H1N1pdm09 [H275Y] viruses, the potentially compensatory secondary substitutions HA-D52N and NA-R152K were detected. We conclude that the emergence of secondary substitutions that may restore viral fitness upon the emergence of known influenza virus NAI resistance substitutions was a rare event in this immunocompetent population.

Analysis of the effect of HCV resistance-associated substitutions on the short-term efficacy of DAA after single administration in three phase Ib clinical trials

Analysis of the effect of HCV resistance-associated substitutions on the short-term efficacy of DAA after single administration in three phase Ib clinical trials

Resistance analysis and treatment outcomes in hepatitis C virus genotype 3-infected patients within the Italian network VIRONET-C.

This study aimed to investigate the role of resistance-associated substitutions (RASs) to direct-acting-antivirals (DAAs) in HCV genotype 3 (GT3). Within the Italian VIRONET-C network, a total of 539 GT3-infected patients (417 DAA-naïve and 135 DAA-failures, of them, 13 at both baseline and failure) were analysed. Sanger sequencing of NS3/NS5A/NS5B was performed following home-made protocols. The majority of patients were male (79.4%), 91.4% were injection drug users, 49.3% were cirrhotic and 13.9% were HIV co-infected. Phylogenetic analysis classified sequences as GT3a-b-g-h (98%-0.4%-0.2%-1.2%) respectively. Overall, 135 patients failed a DAA regimen: sofosbuvir (SOF)/daclatasvir (DCV) or velpatasvir (VEL)±ribavirin (RBV) (N=91/15) and glecaprevir (G)/pibrentasvir (P) (N=9). Moreover, 14.8% of patients were treated with suboptimal regimens for GT3: 3D±RBV (Paritaprevir/r+Ombitasvir+Dasabuvir, N=15), SOF+Simeprevir (SIM) (N=1) or SOF/Ledipasvir (LDV)±RBV (N=4). RAS prevalence was 15.8% in DAA-naïve patients. At failure, 81.5% patients showed at least one RAS: 11/25 (44.0%) in NS3, 109/135 (80.7%) in NS5A, 7/111 (6.3%) in NS5B SOF-failures. In NS5A-failures, Y93H RAS was the most prevalent (68.5% vs 5.1% DAA-naïve, P<.001) followed by A30K (12.7% vs 2.8% in DAA-naïve, P<.001). Analysing baseline samples, a higher prevalence of NS5A-RASs was observed before treatment in DAA-failures (5/13, 38.5%) vs DAA-naïves (61/393, 15.5%, P=.04). Regarding 228 DAA-naïve patients with an available outcome, 93.9% achieved a SVR. Interestingly, patients with baseline Y93H and/or A30K had SVR rate of 72.2% vs 95.7% for patients without NS5A-RASs (P=.002). In this real-life GT3 cohort, the majority of failures harboured resistant variants carrying NS5A-RASs, the most frequent being Y93H. The presence of natural NS5A-RASs before treatment was associated with failure. Further analyses are needed to confirm this observation, particularly for the new current regimens.

Low level of baseline resistance in recently HCV-infected men who have sex with men with high-risk behaviours

ObjectivesPresence of baseline hepatitis C virus (HCV) resistance-associated substitutions (RASs) can impair treatment outcome of direct-acting antivirals. We investigated the prevalence of pre-treatment HCV resistance among recently HCV-infected men who have sex with men (MSM) with high risk behaviours, either human immunodeficiency virus (HIV) co-infected or at high risk of HIV acquisition and under pre-exposure prophylaxis (PrEP). MethodsNS5A and NS3 fragments were deep sequenced on pre-treatment samples of 72 subjects using Illumina MiSeq paired-end sequencing technology. Ultra-deep sequencing data were analysed by SmartGene® platform. RASs mentioned in the literature were analysed and interpreted depending on genotype (GT) at 10% cut-off. ResultsHCV genotyping showed 36 (50.0%) GT1a, 31 (43.1%) GT4d and 5 (6.9%) GT3a infections. Fifty-five patients (76.4%) were co-infected with HIV and 15 (20.8%) received PrEP. In GT1a viruses, NS3 RASs were found in 4/30 viruses (13.3%; S122 G/N, R155 K and I170 V) and Q80 K polymorphism was present in 14/30 viruses (46.7%). No NS3 RASs were detected in GT4d and GT3a viruses. NS5A RASs were detected in 3/36 GT1a viruses (8.3%; Q30E/R, L31 M and H58 L). NS5A subtype-specific polymorphisms L30R and T58 P were found at high frequencies in 31/31 (100%) and 16/31 (51.6%) GT4d viruses, respectively. One RAS M31 L was also observed along with the polymorphisms L30R and T58 P. No NS5A RASs were detected in GT3a viruses. ConclusionA low level of RASs to NS3 and NS5A inhibitors in pre-treatment samples was detected in the study population. Our findings reassure the clinical management of HCV infection in this high-risk population.

Risk factors for difficult-to-treat hepatitis C virus genotype 4r in Rwanda and implications for elimination in sub-Saharan Africa.

In sub-Saharan Africa, there exist distinct HCV genotype (GT) subtypes harbouring resistance-associated substitutions to commonly used non-structural protein 5A (NS5A) inhibitor-based direct-acting antiviral (DAA) regimens. In particular, GT4r subtype has demonstrated high rates of treatment failure. In the absence of routine viral sequencing in sub-Saharan Africa, it is important to identify sociodemographic, epidemiologic, and clinical characteristics that may be associated with GT4r infection. Methods: A secondary analysis was performed on data from 300 adults with HCV GT4 enrolled in a prospective trial assessing the safety and efficacy of sofosbuvir-ledipasvir in Rwanda in 2017. The association between characteristics at enrolment and GT subtype was assessed by chi-square analysis and logistic regression. In multivariate analysis, there were a higher proportion of participants with GT4r subtype with age <40years (OR: 3.6, 95% CI: 1.3-10.5, p=0.02), previous hospitalization (OR: 2.5, 95% CI: 1.3-5.0, p=0.006), previous surgery (OR: 2.2, 95% CI: 1.1-4.2, p=0.03), cirrhosis (OR: 3.2, 95% CI: 1.3-7.5, p=0.008) and baseline HCV RNA >1 million IU/ml (OR: 3.4, 95% CI: 1.6-6.9, p=0.001). Rwandan adults with GT4r are more likely to be younger, have a history of hospital admissions and surgeries and have more active or advanced liver disease compared to those with other GT4 subtypes. In the absence of advanced diagnostics to assess GT subtype, patients with these characteristics may warrant closer monitoring for treatment failure or alternative DAA regimens. More treatment experience with diverse DAA regimens is urgently needed for GT subtypes particular to this region.

NS34A resistance-associated substitutions in chronic hepatitis C in Upper Egypt and regression of liver fibrosis after direct-acting antiviral therapy

BackgroundViral resistance-associated substitutions (RASs) can develop in the setting of DAAs therapy (i.e., emerging RASs). Long-term monitoring of fibrosis regression after achieving SVR to simiprevir (SMV)/sofosbuvir (SOF) is essential. The aim of this study was to determine the prevalence of baseline and emerging NS34A RASs in chronic HCV patients in Upper Egypt and to assess the impact of SMV/SOF therapy on liver stiffness.ResultsThe enrolled 59 patients had HCV genotype 4a without any baseline RASs in the NS34A region. 96.6% (57/59) of patients achieved sustained virological response (SVR12). Of the two patients who failed to achieve SVR12, one of them developed emerging RASs Q80K in the NS34A region. Seventy-two weeks after SMV/SOF therapy, the percentage of patients with liver fibrosis stage (F2, F3, and F4) decreased from 75.4% before treatment to 42.1% after treatment. The combination of SOF and SMV appeared to be well tolerated.ConclusionsAll patients had HCV genotype 4a without any baseline RASs in the NS34A region. In addition, there was improvement of non-invasive measures of liver fibrosis in patients who achieved SVR, 72 weeks after SMV/SOF therapy.

Prevalence and Pattern of Resistance in NS5A/NS5B in Hepatitis C Chronic Patients Genotype 3 Examined at a Public Health Laboratory in the State of São Paulo, Brazil.

PurposeGlobally, it is estimated that 71 million people are chronically infected with hepatitis C, and 10–20% of these will develop cirrhosis and hepatocellular carcinoma. The development of new direct-acting antiviral (DAA) drugs has contributed to sustained virological response (SVR), eliminating the infection and achieving cure of chronic hepatitis C. However, treated patients can develop HCV resistance to DAAs, which can contribute to the failure of treatment. Here, we aimed to evaluate the prevalence and specific pattern of NS5A and NS5B resistance-associated substitutions (RAS) in samples from patients chronically infected with HCV genotype 3a at a public health laboratory, Instituto Adolfo Lutz, São Paulo, Brazil.Patients and MethodsSerum samples from the enrolled individuals were submitted to “in-house” polymerase chain reaction amplification of NS5A and NS5B non-structural protein genes, which were then sequenced by Sanger method.ResultsA total of 170 and 190 samples were amplified and analyzed for NS5A and NS5B, respectively. For NS5A, 20 (12.0%) samples showed some important RAS; 16 (9.0%) showed some type of substitution and 134 (79.0%) showed no polymorphism. No sample showed any RAS for NS5B.ConclusionThis study found important RAS in samples from naïve chronic HCV patients in some areas from São Paulo. The most prevalent were A62S, A30K, and Y93H, which could indicate an increase in resistance to some DAAs used in HCV treatment.

Prevalence of Naturally-Occurring NS5A and NS5B Resistance-Associated Substitutions in Iranian Patients With Chronic Hepatitis C Infection.

BackgroundHepatitis C virus (HCV), non-structural 5A (NS5A), and non-structural 5B (NS5B) resistance-associated substitutions (RASs) are the main causes of failure to direct-acting antiviral agents (DAAs). NS5A and NS5B RASs can occur in patients with HCV infection naturally and before exposure to DAAs.ObjectivesThis study aimed to evaluate naturally-occurring NS5A and NS5B RASs in Iranian patients with HCV genotype 1a (HCV-1a) and -3a infections.MethodsIn this cross-sectional study, viral RNA was extracted from serum specimens. NS5A and NS5B regions were amplified using RT-PCR followed by DNA sequencing. The results of nucleotide sequences were aligned against reference sequences of HCV-1a and -3a and the amino acid substitutions were analyzed using geno2pheno [hcv] web application.ResultsAmong 135 patients with hepatitis C, NS5A amino acid substitutions/RASs were identified in 26.4% and 15.9% of patients with HCV-1a and -3a infections, respectively. The identified amino acid substitutions/RASs in the NS5A region of patients with HCV-1a infection were M28T/V/I 11.1%, Q30R/H 4.2%, L31M 1.4%, and H58Y/P/C/D/Q/S/T 16.7%. Y93H substitution was not found in HCV-1a sequences. In patients with HCV-3a infection, NS5A amino acid substitutions/RASs were A30T/K 9.5%, L31F 1.6%, P58S/T/C 3.2%, Y93H 3.2%, and Y93N 3.2%. No resistance substitutions were identified in NS5B sequences from patients with HCV-1a and -3a infections.ConclusionIn this study, baseline amino acid substitutions/RASs were only identified in the NS5A region in Iranian patients with HCV-1a and -3a infections, and the prevalence of these amino acid substitutions/RASs were in accordance with similar studies. There were no RASs in the HCV-1a and -3a NS5B region.

Elbasvir and grazoprevir for the treatment of hepatitis C

ABSTRACT Introduction: Hepatitis C is one of the leading causes of chronic liver disease. The direct-acting-antivirals has revolutionized the chronic hepatitis C treatment. DAAs can achieve a sustained virological response rate >95% in different populations. Area covered: This review summarizes the pharmacokinetics, pharmacodynamics, efficacy, and safety of Elbasvir/Grazoprevir (EBR/GZR). Expert opinion: EBR/GZR is a combination of NS5A and NS3/4A inhibitors. The performance in the EBR/GZR combination’s safety and tolerability is appreciated in clinical treatment. EBR/GZR also has a higher barrier to resistance-associated substitutions. Based on clinical trials and real-world experience, elbasvir/grazoprevir is effective in the HCV GT1, 4 infections.