Ribavirin suppresses erythroid differentiation and proliferation in chronic hepatitis C patients.

Combination therapy with pegylated interferon (pegIFN) plus ribavirin (RBV) is the standard of care for chronic hepatitis C. One of the major treatment-related side effects is anaemia, attributed to RBV-induced haemolysis. However, haemolysis biomarkers are not present in all patients supporting the existence of other pathogenetic mechanisms. We studied the role of RBV in inducing haemolysis and its effects on erythropoiesis. In 18 hepatitis C virus (HCV) genotype 2 patients treated with pegIFN-alpha-2a (180 mcg/week) plus RBV (800 mg/day) for 24 weeks and in 10 hepatitis B virus (HBV) patients treated with pegIFN-alpha-2a (180 mcg/week) for 48 weeks, haemolysis was assessed by serum LDH, haptoglobin and reticulocyte count. Erythropoiesis was evaluated both ex vivo, analysing the clonogenic activity of patients' erythroid progenitors, as well as in vitro adding pegIFN and RBV to liquid cultures obtained from CD34+ cells of healthy volunteers. The majority of patients developed anaemia; the week 4 mean haemoglobin decrease was greater in HCV than in HBV patients (1.7 vs 0.47 g/dL, P = 0.01). Only three HCV patients (17%) and no HBV patients showed signs of haemolysis. The 15 nonhaemolytic HCV patients and all HBV patients showed a delay in erythroid differentiation, with a reduction in colony number and a relative increase in undifferentiated colony percentage. Haemolytic HCV patients had an increase in colony number at week 4 of therapy. In vitro, erythroid cell proliferation and differentiation were inhibited by both pegIFN and RBV. Both pegIFN and RBV have an inhibitory effect on erythroid proliferation and differentiation.

Feasibility and reproducibility of spleen transient elastography and its role in combination with liver transient elastography for predicting the severity of chronic viral hepatitis.

Liver transient elastography (L-TE) is a reliable, noninvasive predictor of disease severity in chronic liver disease of viral aetiology (CLD). Owing to the relationships among severity of CLD, portal hypertension and spleen involvement, the assessment of splenic stiffness (S-TE) may have an added value in staging CLD. Of 132 CLD patients of viral aetiology, 48 with myeloproliferative disorders (MD) and 64 healthy volunteers (HV), were concurrently investigated by both L-TE and S-TE. Liver disease severity was staged by liver biopsy (LB; Metavir) taken concurrently with TE examination and upper gastrointestinal tract endoscopy for gastro-oesophageal varices. The S-TE inter-observer agreement was analysed by an intra-class correlation coefficient (ICC); L-TE and S-TE accuracy was evaluated by receiver operating characteristic (ROC) curve analysis. Logistic regression analysis assessed the independent effect of L-TE and S-TE as predictors of hepatic fibrosis stage. S-TE failed in 22 CLD (16.6%), 12 (25%) MD and 12 (18%) HV. In the three groups, the ICC was 0.89 (0.84-0.92), 0.90 (0.85-0.94) and 0.86(0.80-0.91), respectively. In the CLD group, L-TE and S-TE independently predicted significant fibrosis (OR 5.2 and 4.6) and cirrhosis (OR 7.8 and 9.1), but at variance from L-TE, S-TE was independent from liver necroinflammation and steatosis. The NPV of S-TE for gastro-oesophageal varices was 100% using a 48 kPa cut-off. In CLD, spleen stiffness alone or in combination with hepatic stiffness can be reliably and reproducibly assessed by TE with the added value of improving the noninvasive diagnosis of severe liver disease and excluding the presence of oesophageal varices.

Inosine triphosphatase deficiency helps predict anaemia, anaemia management and response in chronic hepatitis C therapy.

Anaemia frequently complicates peginterferon/ribavirin therapy for chronic hepatitis C infection. Better prediction of anaemia, ribavirin dose reduction or erythropoietin (EPO) need, may enhance patient management. Inosine triphosphatase (ITPA) genetic variants are associated with ribavirin-induced anaemia and dose reduction; however, their impact in real-life clinic patient cohorts remains to be defined. We studied 193 clinic patients with chronic hepatitis C infection of mixed viral genotype (genotype 1/4 n = 123, genotype 2/3, n = 70) treated with peginterferon/ribavirin. Patients were genotyped for ITPA polymorphisms rs1127354 and rs7270101 using Taqman primers. Hardy-Weinberg equilibrium was present. Estimated ITPA deficiency was graded on severity (0-3, no deficiency/mild/moderate/severe, n = 126/40/24/3, respectively). Multivariable models tested the association with anaemia at 4 weeks of treatment [including decline in haemoglobin (g/dL); haemoglobin <10 g/dL and haemoglobin decline >3 g/dL]; ribavirin dose reduction and EPO use and explored sustained viral response (SVR) to peginterferon/ribavirin. More severe ITPA deficiency was associated with less reduction in haemoglobin level (P <0.001; R(2) = 0.34), less ribavirin dose reduction (OR 0.42; (95% CI = 0.23-0.77); P = 0.005) and less EPO use [OR 0.53; (0.30-0.94); P = 0.029]. ITPA deficiency was associated with SVR [OR: 1.70; (1.02-2.83); P = 0.041] independently of clinical covariates (adjusted R(2) = 0.31). In this clinical cohort, ITPA deficiency helped predict the risk of on-treatment anaemia, ribavirin dose reduction, need for EPO support and was associated with SVR. For patients on HCV regimens including peginterferon/ribavirin, testing for ITPA deficiency may have clinical utility.

Clinical drivers in naïve patient eligibility for treatment of chronic hepatitis C.

Hepatitis C virus (HCV) infection represents a major health problem, being a leading cause of cirrhosis and liver transplantation worldwide. Viral eradication achieved by Peginterferon and Ribavirin therapy is the only therapeutic option that can prevent fibrosis progression in chronic hepatitis and liver-related complications in cirrhotic patients. Unfortunately, the occurrence of potentially serious side effects argues against universal treatment of HCV-infected patients. Indeed most scientific societies suggest that eligibility for therapy be based on baseline factors, the so called clinical drivers for treatment eligibility. Current international guidelines recommend focusing on the severity of liver disease, likelihood of treatment response in terms of chances of sustained virological response (SVR) to antiviral therapy and risk of serious adverse events when making treatment decisions. However, evidence exists that treatment may benefit also patients with mild fibrosis and that baseline predictions of a SVR are inaccurate because of the key role of HCV kinetics while on-therapy. An extended treatment programme is further supported by the fact that an increase in the number of patients treated would ultimately result in a long-term reduction of liver-related deaths.

Comparative trials of peginterferon α2a and peginterferon α2b for chronic hepatitis C.

Standard of care for patients with chronic hepatitis C is pegylated interferon (pegIFN) combined with ribavirin (Rbv). It results in persistent viral eradication and prevents the progression of liver disease and the associated complications in about 50% of treated patients. Currently, two PegIFNs are available that differ significantly in terms of pharmacokinetic and pharmacodynamic profiles as a consequence of different pegylation chemistries. While the registration trials of the two therapeutic regimens demonstrated the superiority of each PegIFN vs the native IFN α2b, the superiority of one regimen over the other in terms of treatment efficacy remains unknown. Retrospective cohort studies and randomized prospective head-to-head trials have attempted to resolve the considerable controversy over this issue and support evidence-based treatment decisions.

High efficacy of direct-acting anti-viral agents in hepatitis C virus-infected cirrhotic patients with successfully treated hepatocellular carcinoma.

BACKGROUND: The efficacy of direct-acting anti-viral (DAA) therapy in patients with a history of hepatocellular carcinoma (HCC) is unknown. AIM: We prospectively evaluated whether previously treated HCC affects DAA efficacy in a large real-life cohort of cirrhotic patients. METHODS: From January to December 2015 all consecutive HCV mono-infected patients with cirrhosis and/or history of HCC attending 10 Italian tertiary liver centres were enrolled. Baseline characteristics and response to therapy were recorded. 1927 patients were enrolled (mean age: 62.1 ± 10.9 years; 1.205 males). Genotype 1 was the most frequent (67.9%) followed by genotypes 3 (12.4%), 2 (11.2%) and 4 (8.6%). 88.4% and 10.9% of cases were classified Child A and B, respectively, and 14 (<1%) cases were classified Child C. Ascites and hepatic encephalopathy occurred in 10.7% and 3.2% of patients, respectively. Varices were detected in 39.3% of patients. Suboptimal and optimal treatment was prescribed: 15.9% of patients received sofosbuvir/simeprevir, 33.4% sofosbuvir/ledipasvir, 20.2% a Viekirax + Exviera regimen, 15.7% sofosbuvir/ribavirin, 9.9% sofosbuvir/daclatasvir and 3.4% Viekirax; 1.3% of patients received an interferon-based regimen. RESULTS: The sustained virologic response (SVR) rate at intention-to-treat analysis was 95.1%. It differed significantly across Child classes, that is, 96.3%, 86.1% and 71.4% Child A, B and C, respectively (P < 0.0001) and across genotypes (P = 0.002). The SVR rate did not differ between patients with (95.0%) and those without previous HCC (95.1%). At multivariable analysis, SVR was significantly associated with HCV genotype, Child class. CONCLUSION: This large real-life study proves that the efficacy of DAA in cirrhotic patients is not impaired by successfully treated HCC.

Treatment of patients with chronic hepatitis C infection in Lombardia: a report by the Lombardia Hepatitis Network.

The arrival of potent directly acting antivirals (DAAs) for the treatment of chronic Hepatitis C virus (HCV) infection was a challenge for the regional health system of the Lombardia Region. Lombardia represents roughly 8% of the Italian territory but includes nearly 16% of the Italian population. In 2014, nearly 37,600 HCV patients were routinely followed-up in liver centers across the region; nearly 16,000 were classified as having advanced fibrosis or cirrhosis (Metavir F3-F4). The creation of a regional network was necessary to ensure uniformity in treatment access and treatment management. The first database analysis of the Lombardia Hepatitis Network was conducted in January 2016, and included data on 2432 patients who had received treatment from December 2014 to December 2015. The most prevalent HCV genotypes were HCV-1 found in 63% and HCV-3 found in 17%. Overall 90.4% patients achieved an SVR, SVR rates were 92.9% in HCV-1, 89.3% in HCV-2, 81.1% in HCV-3 and 88.9% in HCV-4.

Liver stiffness measurement reliability and main determinants of point shear-wave elastography in patients with chronic liver disease.

BACKGROUND: Liver stiffness (LS) measured by transient elastography (TE) accurately predicts the severity of chronic liver diseases (CLD). Point quantification shear-wave elastography (pSWE) is a new technique incorporated into a conventional ultrasound system for measuring LS. We evaluated pSWE feasibility, reproducibility and diagnostic accuracy in consecutively recruited CLD patients who concomitantly underwent TE and liver biopsy. AIM: To evaluate pSWE feasibility, reproducibility and diagnostic accuracy in consecutively recruited CLD patients who concomitantly underwent TE and liver biopsy. METHODS: Over 2 years 186 CLD patients (116 males, 132 viral hepatitis) consecutively underwent pSWE (10 valid measurements by ElastPQ) blindly performed by two raters. A further operator performed TE. Inter-observer agreement for pSWE was analysed by intraclass correlation coefficient (ICC) and correlated with histological liver fibrosis (METAVIR). Main determinants of pSWE were investigated by linear regression model. RESULTS: Three hundred and seventy-two (100%) reliable measurements were obtained by pSWE and 184 by TE (99%). LS was 8.1 ± 4.5 kPa for pSWE with the first rater and 8.0 ± 4.2 kPa with the second one vs. 8.8 ± 3.6 kPa for TE. pSWE ICC was 0.89 (95% CI 0.85-0.91), not influenced by age, sex, BMI, liver enzymes, liver aetiology. ICC increased over time with year 1 at 0.86 and 95% CI 0.81-0.90 vs. year 2 at 0.92 and 95% CI 0.87-0.95. Liver fibrosis was the only independent determinant of LS on pSWE. The AUROCs for diagnosing F ≥ 2, F ≥ 3 and F = 4 were 0.77, 0.85 and 0.88 for pSWE vs. 0.81, 0.88 and 0.94 for TE. After 1-year training they were 0.86, 0.94 and 0.91. CONCLUSION: Point quantification shear-wave elastography reliably and reproducibly evaluates liver stiffness, matching transient elastography for accuracy after a 1-year learning curve or 130 examinations.

Osmoregulation of vasopressin during acute lowering of arterial pressure by clonidine in moderate hypertension.

Hypertonic saline (100 mmol in 50 ml) was injected intravenously over 5 min in two groups of moderate essential hypertensive patients (group 1, n = 13; group 2, n = 6). In group 2, arterial pressure had been lowered by infusion of clonidine (0.3 mg in 100 ml saline), from 186 +/- 8/116 +/- 3 to 146 +/- 9/98 +/- 5 mmHg (mean +/- s.e.m.). The hypertonic stimulus increased the plasma osmolality of all subjects from 288 +/- 1 to 296 +/- 1 mosmol/kg (P less than 0.01). Plasma vasopressin increased from baseline values that were not significantly different (P less than 0.01) in each of the two groups. The increase in plasma vasopressin was significantly greater (P less than 0.05) in the group 2 hypertensives with a reduced arterial pressure (+7.81 +/- 1.79 pg/ml) than in the group 1 untreated hypertensives (+3.15 +/- 1.2 pg/ml). In our study, acute lowering of arterial pressure by clonidine did not significantly change baseline vasopressin, but facilitated osmotically induced vasopressin secretion.

High-resolution melting assay for genotyping of IFNL4-associated dinucleotide variant rs368234815.

Interferon-λ4-related dinucleotide variant rs368234815 TT/-G is strongly linked with rs12979860 polymorphism, the most important genetic marker connected to treatment-induced hepatitis C virus clearance. Due to additional baseline information that rs368234815 polymorphism could provide for the management of chronic hepatitis C, we developed and validated a high-resolution melting genotyping assay using 193 patients with chronic hepatitis C.

Allelic inhibition of displacement activity: a simplified one tube allele-specific PCR for evaluation of ITPA polymorphisms.

Recently, genome-wide association studies (GWAS) in patients with chronic hepatitis C virus (HCV) infection have identified two functional single nucleotide polymorphisms (SNPs) in the inosine triphosphatase (ITPA) gene, that are associated strongly and independently with hemolytic anemia in patients exposed to pegylated-interferon (Peg-IFN) plus ribavirin (RBV) combined therapy. Here has been developed a simplified allele discrimination polymerase chain reaction (PCR) assay named allelic inhibition of displacement activity (AIDA) for evaluation of ITPA polymorphisms. AIDA system relies on three unlabeled primers only, two outer common primers and one inner primer with allele-specific 3' terminus mismatch. DNA samples from 192 patients with chronic HCV infection were used to validate the AIDA system and results were compared with the gold standard TaqMan(®) SNP genotyping assay. Concordant data were obtained for all samples, granting for high specificity of the method. In conclusion, AIDA is a practical one-tube method to reproducibly and to assess accurately rs7270101 and rs1127354 ITPA SNPs.

Eukaryotic initiation factor 5B: a new player for the anti-hepatitis C virus effect of ribavirin?

The addition of the broad-spectrum antiviral agent ribavirin (RBV), a synthetic guanosine analog, to interferon-alpha (IFNα) monotherapy has been a major breakthrough in the treatment of patients with hepatitis C virus (HCV), as it greatly improved treatment response rates. Although several mechanisms of action have been proposed for RBV's antiviral activity, each with some experimental evidence, the precise mechanism by which it acts synergistically with IFNα has remained elusive. A cornerstone of the antiviral IFNα response is phosphorylation of the α subunit of eukaryotic initiation factor (eIF)2. This limits the availability of eIF2⋅GTP⋅Met-tRNA(i)(Met) ternary complexes, reduces formation of the 43S preinitiation complexes, ultimately blocking viral (and most cellular) mRNA translation. However recent studies indicated that translation driven by the HCV internal ribosome entry site (IRES) is insensitive to eIF2α phosphorylation. Particularly, in addition to the general eIF2-dependent pathway of translation, the HCV IRES makes use of a bacterial-like, eIF2-independent pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. Together, these observations support a model in which cellular stresses that induce eIF2α phosphorylation (e.g. treatment with IFNα) cause HCV IRES-directed translation to switch from an eIF2-dependent mode to an eIF5B-dependent mode, defining a tactic used by HCV to evade the INFα response. Eukaryotic eIF5B is a ribosome-dependent GTPase that is responsible for 80S complex formation in translation initiation but shows much lower affinities for GTP than to other GTPases, thus suggesting that it may mis-incorporate the RBV triphosphate (RTP) in place of GTP even at the RBV concentrations achieved in clinical use. Consequently, we theorize that RTP bound to eIF5B lowering its affinity for ribosome, blocks the 80S complex formation on HCV IRES inhibiting the eIF5B-dependent translation used by HCV to elude IFNα response. In conclusion, our hypothesis provides a mechanistic explanation for the phenomenon of RBV enhancement in INFα-based therapy.

Implications of PNPLA3 polymorphism in chronic hepatitis C patients receiving peginterferon plus ribavirin.

BACKGROUND: Homozygosity for the PNPLA3 p.I148M polymorphism influences steatosis and fibrogenesis in chronic hepatitis C (CHC). AIM: To evaluate the effect of p.148M/M on sustained virological response (SVR) and viral kinetics in patients who underwent antiviral therapy with peg-interferon and ribavirin, stratified according to viral genotype and fibrosis severity, and secondarily, the interaction with interleukin-28B ( IL28B ) genotype on liver damage. METHODS: In this observational study, we considered 602 treatment-naïve consecutive patients from tertiary referral centres in Milan and Vienna [61% genotype 1 (G1), 30% advanced fibrosis, 33% IL28B rs12979860 CC]. RESULTS: The p.148M/M genotype, detected in 8% of patients, did not influence SVR in the overall series (P = 0.29), but it was associated with SVR (3/17, 17% vs. 56/121, 46%; P = 0.034) and complete early viral response (4/17, 23% vs. 68/121, 56%; P = 0.018) in G1/4 patients with advanced fibrosis. After adjustment for age, viral load, IL28B CC genotype, treatment dose, and steatosis, p.148M/M remained a predictor of SVR in G1/4 patients with advanced fibrosis (OR 0.23, 95% CI 0.04-0.87). The p.148M/M genotype was associated with more advanced fibrosis in the overall series (P = 0.049), whereas the rs12979860 IL28B CC genotype only in patients negative for p.148M/M (P = 0.017), independently of age, BMI and alanine transaminase levels (OR 1.51, 95% CI 1.01-2.27). CONCLUSIONS: PNPLA3 p.148M/M genotype was negatively associated with SVR and early viral kinetics independently of steatosis, albeit only in difficult-to-cure G1/4 patients with advanced fibrosis, whereas stratification for the p.148M/M PNPLA3 genotype unmasked an association between IL28B CC genotype and more severe liver fibrosis.

Hepatitis C virus/human immunodeficiency virus coinfection in hemophiliacs: high rates of sustained virologic response to pegylated interferon and ribavirin therapy.

BACKGROUND: Progression of chronic hepatitis C virus (HCV) infection to end-stage liver disease is accelerated in patients coinfected with human immunodeficiency virus (HIV). HCV/HIV-coinfected hemophiliacs are no exception. Although eradication of HCV with pegylated interferon (Peg-IFN) plus ribavirin (Rbv) is the only approach to halt the progression of liver disease, the rates of sustained virologic response (SVR) in coinfected patients are attenuated as compared with those in HCV-monoinfected patients. Nonetheless, in HCV-infected hemophiliacs, who are considered to constitute a difficult-to-treat population, current treatment strategies yielded rates of SVR similar to those obtained in non-hemophiliacs. OBJECTIVES AND PATIENTS: In this open-label, prospective, multicenter study, the efficacy and safety of therapy with Peg-IFNalpha2a plus Rbv was evaluated in 34 HCV/HIV-coinfected adult hemophiliacs naive to previous antiviral therapy. METHODS: Peg-IFNalpha2a was administered at a dose of 180 mug subcutaneously once-weekly plus oral Rbv 1000-1200 mg day(-1) for 48 weeks, irrespective of HCV genotype. RESULTS: All but one patient (3%) completed the study, 15 (44%) achieved an SVR, and 13 (38%) required dose reduction of either drug. A rapid virologic response (HCV-RNA clearance at week 4; P = 0.01), a complete early virologic response (HCV RNA clearance at week 12; P = 0.005) and absence of cirrhosis (P = 0.04) were independent predictors of SVR. During a median post-treatment follow-up of 3 years, a steady increase in CD4+ cell count and CD4+/CD8+ cell ratio was observed in SVR patients. CONCLUSIONS: These results strongly support the use of anti-HCV therapy in HCV/HIV-coinfected hemophiliacs.

Recovery after L-DOPA treatment in peginterferon and ribavirin induced parkinsonism.

BACKGROUND: Hepatitis C virus (HCV) chronically infects approximately 2% of the European population. Antiviral therapy with pegInterferon-alpha (PegIFN) and ribavirin (Rbv) is the standard of care, leading to HCV eradication in roughly 50% of patients. IFN-based therapy has been associated with high rates (20%) of central nervous system side effects, but only a few case reports exist on extrapyramidal side effects. RESULTS: We report a 64-year-old man developing parkinsonism during PegIFN alfa-2a and ribavirin therapy for chronic hepatitis C. No improvement was observed after treatment discontinuation. Therefore, on the basis of previous clinical and experimental reports, levodopa-benserazide treatment was started. After substantial improvement, symptoms relapsed following drug tapering. CONCLUSIONS: This is the first case of parkinsonism in a Caucasian patient receiving PegIFN/Rbv therapy. The rapid and significant improvement of symptoms obtained in our patient with levodopa-benserazide, suggests that this therapy could be considered as first line symptomatic treatment.