"Real world" efficacy of bulevirtide in HBV/HDV-related cirrhosis including people living with HIV: Results from the compassionate use programme at INMI Spallanzani in Rome, Italy.

OBJECTIVES: We describe the preliminary results of bulevirtide compassionate use in patients with hepatitis B and delta virus (HBV/HDV)-related cirrhosis and clinically significant portal hypertension, including those living with HIV. METHODS: We conducted a prospective observational study of consecutive patients. Clinical evaluation, liver function tests, bile acid levels, HDV-RNA, HBV-DNA, hepatitis B surface antigen, and liver and spleen stiffness were assessed at baseline and after treatment months 1, 2, 3, 4, 6, 9, and 12. HIV-RNA and CD4+/CD8+ count were assessed in people living with HIV. The first drug injection was administered under nurse supervision, and counselling was provided and adherence reviewed at each visit. RESULTS: In total, 13 patients (61.5% migrants) were enrolled. The median treatment duration was 11 months. At month 6, mean alanine aminotransferase (ALT) levels fell by 64.5% and mean liver and spleen stiffness decreased by 8.6 and 0.9 kPa, respectively. The mean baseline HDV-RNA was 3.34 log IU/mL and 5.10 log IU/mL in people without and with HIV (n = 5) (p = 0.28), respectively. A similar mean decline was observed in both groups: -2.06 log IU/mL and -1.93 log IU/mL, respectively (p = 0.87). A combined response (undetectable HDV RNA or ≥ -2 log IU/mL decline vs. baseline, with ALT normalization) was achieved in 66% of subjects without and in 60% of patients with HIV. Patients with HIV showed persistently undetectable HIV-RNA and a progressive increase in CD4+/CD8+ cells during treatment. No patient discontinued bulevirtide because of adverse effects. CONCLUSIONS: Preliminary results suggest that bulevirtide is feasible and well-tolerated in populations with difficult-to-treat conditions, such as those with HIV/HBV/HDV co-infection and migrants, when special attention is given to patient education. HDV-RNA decline during treatment was similar in people living with and without HIV.

Third dose of SARS-CoV2 mRNA vaccination produces robust persistent cellular and humoral immune responses in liver transplant recipients.

Weaker responses have been described after two doses of anti-SARS-CoV2 vaccination in liver transplant recipients (LTRs). At the Italian National Institute for Infectious Diseases, 122 LTRs (84% males, median age 64 years) were tested for humoral and cell-mediated immune response after a third doses of anti-SARS-CoV2 mRNA vaccines. Humoral response was measured by quantifying anti-receptor binding domain and neutralizing antibodies; cell-mediated response was measured by quantifying IFN-γ after stimulation of T cells with SARS-CoV-2-specific peptides. Humoral and cellular responses improved significantly compared to the second vaccine dose; 86.4% of previous non-responders to the first 2 vaccine doses (N = 22) became responders. Mycophenolate mofetil-containing regimens were not associated with lower response rates to a third vaccine; shorter time since transplantation (<6 years) was associated with lower humoral and cellular responses to third vaccine. Protective antibodies against Omicron variant were detected in 60% of patients 12 weeks after third vaccine dose.

Coordinated cellular and humoral immune responses after two-dose SARS-CoV2 mRNA vaccination in liver transplant recipients.

Limited data are available on risks and benefits of anti-SARS-CoV2 vaccination in solid organ transplant recipients, and weaker responses have been described. At the Italian National Institute for Infectious Diseases, 61 liver transplant recipients underwent testing to describe the dynamics of humoral and cell-mediated immune response after two doses of anti-SARS-CoV2 mRNA vaccines and compared with 51 healthy controls. Humoral response was measured by quantifying both anti-spike and neutralizing antibodies; cell-mediated response was measured by PBMC proliferation assay with IFN-γ and IL-2 production. Liver transplant recipients showed lower response rates compared with controls in both humoral and cellular arms; shorter time since transplantation and multi-drug immunosuppressive regimen containing mycophenolate mofetil were predictive of reduced response to vaccination. Specific antibody and cytokine production, though reduced, were highly correlated in transplant recipients.

Daclatasvir, sofosbuvir with or without ribavirin for 24 weeks in hepatitis C genotype 3 cirrhosis: A real-life study.

INTRODUCTION AND AIM: Cirrhotic patients with hepatitis C virus genotype 3 infection show unsatisfactory outcomes after 12 weeks' treatment with direct antiviral agents. The National Italian Drug Agency allows 24 weeks of therapy in difficult-to-treat patients, including genotype 3 cirrhotics. Aim of this study was to evaluate efficacy and safety of a 24-week course of sofosbuvir plus daclatasvir±ribavirin in this population. MATERIALS AND METHODS: 106 consecutive cirrhotics (70.8% males, mean age 55.3±7.6 years) in 8 tertiary hepatology centers received sofosbuvir plus daclatasvir for 24 weeks. Ribavirin was administered in 85 (80.2%) based expected tolerability, at a mean dose of 964±202mg/day. Baseline Child-Pugh class was A 91.5%, B 6.6%, C 1.9%; mean baseline MELD was 8.5±2.7. RESULTS: All patients completed 12-week follow-up post-treatment, and 104 (98.1%) obtained sustained virological response (100% in ribavirin -treated patients vs. 90.4% without ribavirin; p=0.04). No worsening in renal and liver function was observed, no serious adverse events occurred. Two virological failures showed resistance associated variants (Y93H and S282T). CONCLUSION: An extended 24-week treatment with sofosbuvir plus daclatasvir+ribavirin obtained 100% efficacy in genotype 3 hepatitis C cirrhosis, with very limited side effects. The role of ribavirin seems crucial in this setting and should be administered if clinically feasible.

Lack of reduction in serum alpha-fetoprotein during treatment with direct antiviral agents predicts hepatocellular carcinoma development in a large cohort of patients with hepatitis C virus-related cirrhosis.

Risk of hepatocellular carcinoma (HCC) in hepatitis C virus cirrhotic patients treated with direct-acting antiviral agents (DAA) is still debating. We investigated it in a large cohort. The cohort comprised 1045 cirrhotic patients who completed treatment with DAA, with a median follow-up of 17.3 months after end of treatment (EOT), including 943 patients without history of HCC and 102 previously treated for HCC. The majority were men (59.9%), with compensated cirrhosis (88.8%), genotype 1b (44.7%). Univariate, multivariate analysis and Kaplan-Meier curves were performed to detect predictors of HCC in patients with and without reduction in alpha-fetoprotein (AFP) during treatment. SVR12 was 95.6%. HCC developed in 95 (9.9%), including 54 of 943 (5.7%) occurrent and 41 of 102 (39%) recurrent tumours. De novo were more often unifocal (P = 0.01) and curable (P = 0.03). AFP decreased from 16.1 ± 36.2 mg/dL (baseline) to 11.4 ± 55 mg/dL (EOT). At univariate analysis, predictors were a previous HCC, older age, higher model for end-stage liver disease, prolonged INR, lower platelets, baseline and EOT AFP, virological failure and no reduction in AFP during treatment. Kaplan-Meier curves showed lower incidence of HCC in patients showing any reduction in AFP (P = 0.001). Those with AFP <6 ng/mL had the lowest risk (P = 0.0002). At logistic regression, platelets (P = 0.009, OR 0.99 CI: 0.99-1.00), previous HCC (P < 0.000 01, OR: 10.76, 95% CI: 5.89-19.34) and no reduction in AFP during treatment (P = 0.0005, OR: 2.98, CI: 1.60-5.54) were independent predictors of HCC. In conclusion, risk of HCC after DAA treatment remains substantial. It is higher among patients with previous HCC, low platelets and without reduction in AFP during treatment.

Significance of detectable HCV RNA below the limit of quantification in patients treated with DAAs using standard and ultrasensitive protocols.

Predictive factors of HCV relapse after treatment with DAAs are poorly understood. In this study, we aimed to assess whether the residual viral load positivity observed during or at the end of treatment (EOT) has an impact on viral outcome. Blood samples were collected from 337 patients with genotypes (GT) 1a, 1b, 2, 3, and 4 HCV chronic infection, treated with DAAs to determine HCV RNA load by the Abbott RealTime HCV (ART) assay at treatment week (W) 4, at EOT, and 4, 12, 24 weeks after discontinuation. EOT and other samples with "detected <12/mL" (DNQ) were retested by an ultrasensitive protocol (USP) to confirm the result. Frequency of DNQ was analyzed in subgroups of patients and clinical conditions to assess potential correlations. At W4, 22% and 30.9% of the samples were undetectable and DNQ by ART assay, respectively, but no correlation for achieving SVR was found. In contrast, an HCV RNA cut-off of ≥50/mL at W4 was a significant predictor of therapy failure (P = 0.036, univariate analysis). At EOT, DNQ was associated to 12W treatment duration (P < 0.001) and GT1a infection (P = 0.036). Overall, 20/41 (48.8%) of DNQ samples at EOT or post-treatment W4, were confirmed by USP but only in a single case the patient experienced viral relapse. HCV RNA at W4 can predict SVR, irrespective to genotype or DAA regimen. HCV RNA DNQ at EOT is associated to shorter treatment duration and to GT1a, but is not a predictor of therapy failure.

Sofosbuvir plus daclatasvir with or without ribavirin is safe and effective for post-transplant hepatitis C recurrence and severe fibrosis and cirrhosis: A prospective study.

BACKGROUND: In 2012, an Italian Named Patient Program began for hepatitis C virus (HCV)-infected liver transplant (LT) recipients with advanced fibrosis, before approval of direct antiviral agents (DAA), to benefit severely ill patients. The aim of this "real-life" study was to assess treatment efficacy and safety with an extended course of daclatasvir (DCV) plus sofosbuvir (SOF) with or without ribavirin (RBV). METHODS: All HCV LT recipients with severe fibrosis in 15 Italian transplant centers were treated with DCV+SOF±RBV for 24 weeks; sustained virological response was assessed at 12 weeks post-treatment (SVR12). RESULTS: Eighty-seven patients were enrolled (75.9% males, mean age 58.4 ± 7.2 years, 83.9% genotype 1, 81.6% cirrhosis); 52 (59.8%) received RBV. Overall, 79 obtained SVR12 (90.8%): 100% in F3 and 88.7% in cirrhotics (91.5% in Child-Pugh A, 83.3% in Child-Pugh B and C). According to the treatment group, SVR was 80% in DCV + SOF group and 98.1% in SOF + DCV + RBV. Two virological relapses occurred during follow-up in cirrhotic patients who received DCV + SOF. Four cirrhotic patients in DCV + SOF group and 1 in DCV + SOF + RBV group died on treatment. CONCLUSION: An extended course of SOF plus DCV for 24 weeks, with or without RBV, is effective and well tolerated for the treatment of post-LT HCV recurrence with severe fibrosis.

Dendritic cells activation is associated with sustained virological response to telaprevir treatment of HCV-infected patients.

First anti-HCV treatments, that include protease inhibitors in conjunction with IFN-α and Ribavirin, increase the sustained virological response (SVR) up to 80% in patients infected with HCV genotype 1. The effects of triple therapies on dendritic cell (DC) compartment have not been investigated. In this study we evaluated the effect of telaprevir-based triple therapy on DC phenotype and function, and their possible association with treatment outcome. HCV+ patients eligible for telaprevir-based therapy were enrolled, and circulating DC frequency, phenotype, and function were evaluated by flow-cytometry. The antiviral activity of plasmacytoid DC was also tested. In SVR patients, myeloid DC frequency transiently decreased, and returned to baseline level when telaprevir was stopped. Moreover, an up-regulation of CD80 and CD86 on mDC was observed in SVR patients as well as an improvement of IFN-α production by plasmacytoid DC, able to inhibit in vitro HCV replication.

Prevention of hepatitis C recurrence by bridging sofosbuvir/ribavirin from pre- to post-liver transplant: a real-life strategy.

BACKGROUND & AIMS: Hepatitis C virus (HCV) re-infection following liver transplant (LT) is associated with reduced graft and patient survival. Before transplant, Sofosbuvir/Ribavirin (SOF/R) treatment prevents recurrent HCV in 96% of those patients achieving viral suppression for at least 4 weeks before transplant. We evaluated whether a bridging SOF-regimen from pre- to post-transplant is safe and effective to prevent HCV recurrence in those patients with less than 4 weeks of HCV-RNA undetectability at the time of transplant. METHODS: From July 2014 SOF/R was given in 233 waitlisted HCV cirrhotics with/without hepatocellular carcinoma (HCC) within an Italian Compassionate Program. One hundred patients were transplanted and 31 patients (31%) treated with SOF/R bridging therapy were studied. RESULTS: Liver transplant indication in bridge subgroup was HCC in 22 and decompensated cirrhosis in 9. HCV-genotype was 1/4 in 18 patients. SOF 400 mg/day and R (median dosage 800 mg/day) were given for a median of 35 days before LT. At transplant time, 19 patients were still HCV-RNA positive (median HCV-RNA 58 IU/mL). One recipient had a virological breakthrough at week 4 post-transplant; one died, on treatment, 1-month post-transplant for sepsis and 29/31 achieved a 12-week sustained virological response (94%). Acute cellular rejection occurred in three recipients. On September 2016, 30 recipients (97%) were alive with a median follow-up of 18 months (range 13-25). CONCLUSIONS: In patients with suboptimal virological response at LT, a bridging SOF/R regimen helps avoiding post-transplant graft reinfection.

Daclatasvir combined with sofosbuvir or simeprevir in liver transplant recipients with severe recurrent hepatitis C infection.

Daclatasvir (DCV) is a potent, pangenotypic nonstructural protein 5A inhibitor with demonstrated antiviral efficacy when combined with sofosbuvir (SOF) or simeprevir (SMV) with or without ribavirin (RBV) in patients with chronic hepatitis C virus (HCV) infection. Herein, we report efficacy and safety data for DCV-based all-oral antiviral therapy in liver transplantation (LT) recipients with severe recurrent HCV. DCV at 60 mg/day was administered for up to 24 weeks as part of a compassionate use protocol. The study included 97 LT recipients with a mean age of 59.3 ± 8.2 years; 93% had genotype 1 HCV and 31% had biopsy-proven cirrhosis between the time of LT and the initiation of DCV. The mean Model for End-Stage Liver Disease (MELD) score was 13.0 ± 6.0, and the proportion with Child-Turcotte-Pugh (CTP) A/B/C was 51%/31%/12%, respectively. Mean HCV RNA at DCV initiation was 14.3 × 6 log10 IU/mL, and 37% had severe cholestatic HCV infection. Antiviral regimens were selected by the local investigator and included DCV+SOF (n = 77), DCV+SMV (n = 18), and DCV+SMV+SOF (n = 2); 35% overall received RBV. At the end of treatment (EOT) and 12 weeks after EOT, 88 (91%) and 84 (87%) patients, respectively, were HCV RNA negative or had levels <43 IU/mL. CTP and MELD scores significantly improved between DCV-based treatment initiation and last contact. Three virological breakthroughs and 2 relapses occurred in patients treated with DCV+SMV with or without RBV. None of the 8 patient deaths (6 during and 2 after therapy) were attributed to therapy. In conclusion, DCV-based all-oral antiviral therapy was well tolerated and resulted in a high sustained virological response in LT recipients with severe recurrent HCV infection. Most treated patients experienced stabilization or improvement in their clinical status.

Rapid and preemptive evaluation of individual anti-hepatitis C virus treatment outcome capability by a short-term autologous liver tissue culture system.

Hepatitis C virus (HCV) standard of care (SOC) therapy is not effective in a large percentage of patients and its efficacy may be evaluated only after several weeks. The aim of this work was to set up an in vitro liver culture assay able to preemptively predict SOC outcome by using residual liver samples from HCV patients. The in vitro response to SOC was found associated with the in vivo treatment outcome with a concordance of 100%. A wider clinical trial on a larger patient group is necessary to fully evaluate the impact of this procedure on the clinical management of untreated HCV patients.

Determination of telaprevir in plasma of HCV-infected patients by HPLC-UV.

Telaprevir is a direct acting antiviral agent, used with pegylated interferon and ribavirin for the management of chronic hepatitis C virus (HCV) genotype 1 infection, in patients not responding to therapy with pegylated interferon and ribavirin only. Although 75% of patients achieve a sustained virological response after treatment with telaprevir, adverse drug-drug interactions and undesirable events often occur. Therefore, telaprevir monitoring is pivotal to improve the management of HCV infection. Here, the first High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method to quantify telaprevir in human plasma of HCV-genotype 1-infected patients is reported. The volume of the plasma sample was 700 µL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone). The extracted samples were reconstituted with 150 µL of 60/40 water/acetonitrile. Thirty microliters of these samples was injected into a HPLC-UV system, and the analytes were eluted on a X Terra(®) RP18 column (250 mm × 4.6 mm i.d.) with a particle size of 5 µm. The mobile phase (ammonium acetate buffer, 150 mM, pH 8.0, and methanol:acetonitrile 50:50) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 16 min; telaprevir was detected by UV at 276 and 286 nm. The calibration curve was linear from 312.5 to 20,000 ng/mL (r(2) > 0.996). The absolute recovery of telaprevir ranged between 89 and 93% at concentrations of quality control samples of 800, 4,000, 8,000, and 16,000 ng/mL. Both precision and accuracy were always <15%. The HPLC-UV method reported here: (i) has been validated, (ii) is currently applied to monitor telaprevir in plasma of HCV-infected patients, and (iii) appears useful in a routine laboratory. ,

Torquetenovirus Loads in Peripheral Blood Predict Both the Humoral and Cell-Mediated Responses to SARS-CoV-2 Elicited by the mRNA Vaccine in Liver Transplant Recipients.

Three years into the COVID-19 pandemic, mass vaccination campaigns have largely controlled the disease burden but have not prevented virus circulation. Unfortunately, many immunocompromised patients have failed to mount protective immune responses after repeated vaccinations, and liver transplant recipients are no exception. Across different solid organ transplant populations, the plasma levels of Torquetenovirus (TTV), an orphan and ubiquitous human virus under control of the immune system, have been shown to predict the antibody response after COVID-19 vaccinations. We show here a single-institution experience with TTV viremia in 134 liver transplant recipients at their first or third dose. We found that TTV viremia before the first and third vaccine doses predicts serum anti-SARS-CoV-2 Spike receptor-binding domain (RBD) IgG levels measured 2-4 weeks after the second or third dose. Pre-vaccine TTV loads were also associated with peripheral blood anti-SARS-CoV-2 cell-mediated immunity but not with serum SARS-CoV-2 neutralizing antibody titers.

High prevalence of asymptomatic SARS-CoV-2 infection in a cohort of liver transplant recipients in central Italy.

Asymptomatic subjects account for 25 to 45% of SARS-CoV-2 infections, and in particular, subjects on mild immunosuppressive therapy may have symptoms masked and could spread virus for an extended period of time. To determine the cumulative incidence of symptomatic and asymptomatic SARS-CoV-2 infections and associated risk factors, we conducted a prospective clinical and serological survey in a cohort of 278 liver transplant recipients (LTRs) from Central Italy. Three different serology tests were performed every 4 months in 259 LTRs between April 2020 and April 2021: one based on raw extract of whole SARS-CoV-2 virus and two on specific viral antigens (nucleoprotein and receptor binding domain) to detect specific IgG, IgM and IgA. Hundred fifteen LTRs who reported symptoms or close contact with a SARS-CoV-2-positive subject, or had a positive serological result underwent molecular testing by standard screening procedures (RT-PCR on naso-pharyngeal swab). Thirty-one past or active SARS-CoV-2 infections were identified: 14 had positive molecular test (64% symptomatic), and 17 had positive serology only (18% symptomatic). SARS-CoV-2 infection was not statistically related to gender, age, obesity, diabetes, renal impairment, type of anti-rejection therapy or time from transplant. Asymptomatic SARS-CoV-2 cases (61.3%) were more frequent in males and in those with glomerular filtrate rate >50 ml/min. Overall, the addition of repeated serology to standard diagnostic molecular protocols increased detection of SARS-CoV-2 infection from 5.1% to 10.9%. Anti-SARS-CoV-2 seroprevalence among our LTRs (11.2%) is comparable to the general population of Central Italy, considered a medium-impact area. Only one asymptomatic subject (6%) was found to carry SARS-CoV-2 in respiratory tract at the time of serological diagnosis.

Late Relapse and Reinfection in HCV Patients Treated with Direct-Acting Antiviral (DAA) Drugs.

The risk of hepatitis C virus (HCV) recurrence after direct-acting antiviral (DAA) treatment is <0.5%. However, the distinction between HCV RNA late relapse and reinfection still represents a challenge in virological diagnostics. The aim of this study was to employ next-generation sequencing (NGS) to investigate HCV RNA recurrence in patients achieving a sustained virologic response (SVR) at least six months post-treatment. NGS was performed on plasma samples from six HCV-positive patients (Pt1-6) treated with DAA. NGS of HCV NS5B was analyzed before treatment (T0), after HCV RNA rebound (T1), and, for Pt3, after a second rebound (T2). Reinfection was confirmed for Pt5, and for the first rebound observed in Pt3. Conversely, viral relapse was observed when comparing T0 and T1 for Pt6 and T1 and T2 for Pt3. Z-scores were calculated and used to predict whether HCV-positive patient samples at different time points belonged to the same quasispecies population. A low Z-score of <2.58 confirmed that viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2, while the Z-score for Pt4 was suggestive of possible reinfection. NGS data analyses indicate that the Z-score may be a useful parameter for distinguishing late relapse from reinfection.

The impact of the coronavirus disease 2019 pandemic on a central Italy transplant center.

Coronavirus disease 2019 (COVID-19) is challenging health care systems worldwide, raising the question of reducing the transplant program due to the shortage of intensive care unit beds and to the risk of infection in donors and recipients.We report the positive experience of a single Transplant Center in Rome, part of the National Institute for Infectious Diseases Lazzaro Spallanzani, one of the major national centers involved in the COVID-19 emergency.

Daclatasvir-based regimens in HCV cirrhosis: experience from the Italian early access program.

We reported the efficacy and safety data for daclatasvir (DCV)-based all-oral antiviral therapy in patients treated in the Italian compassionate-use program. 275 patients were included (202 male-73.5%, mean age: 57.4 years, 62 HIV-coinfected, 94 with recurrence of hepatitis C post-OLT). Forty-nine patients (17.8%) had Child-Pugh B, Genotype(G) distribution was: G1a:72 patients (26.2%), G1b:137 (49.8%); G3:40 (14.5%) and G4:26 (9.5%). Patients received DCV with sofosbuvir(SOF) (n = 221, 129 with ribavirin(RBV) or with simeprevir (SMV) or asunaprevir (ASU) (n = 54, 19 with RBV) for up to 24 weeks. Logistic regression was used to identify baseline characteristics associated with sustained virological response at week 12 post-treatment (SVR12). Liver function changes between baseline and follow up were assessed in 228 patients. 240 patients achieved SVR12 (87.3%), post transplant and HIV co-infected patients were equally distributed among SVR and no SVR (35% vs 34.3%; p = 0.56 and 24.2% vs 11.4%, p = 0.13, respectively). SVR rate was significantly higher with the combination DCV + SOF compared with DCV + SIM or ASU (93.2% vs 63.0%, p < 0.0001). Bilirubin value (OR: 0.69, CI95%: 0.54-0.87, p = 0.002) and regimen containing SOF (OR: 9.99, CI95%: 4.09-24.40; p < 0.001) were independently related with SVR. Mean albumin and bilirubin values significantly improved between baseline and follow-up week 12. DCV-based antiviral therapy was well tolerated and resulted in a high SVR when combined with SOF either in pre-transplant and in OLT patients and in "difficult to treat" HCV genotypes. Regimens containing DCV in combination with NS3 protease inhibitors obtained suboptimal results.

Characterization of Naturally Occurring NS5A and NS5B Polymorphisms in Patients Infected with HCV Genotype 3a Treated with Direct-Acting Antiviral Agents.

Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A (T79S, T107K, and T107S) and three NS5B (G166R, Q180K, and C274W) baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the novel L31F NS5A variant emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation.