Chronic HCV-infected Individuals Research Articles

Role of Genetic and Epigenetic Modifications in the Progression of Hepatocellular Carcinoma in Chronic HCV Patients

Globally, hepatocellular carcinoma (HCC) is a significant cause of mortality and morbidity among chronically infected HCV patients. It is established that HCV is a primary risk factor for HCC progression. The treatment of HCV infection has been transformed by the introduction of DAAs with high rates of virological clearance. The reduction in cirrhosis-related consequences, particularly HCC, is the long-term objective of DAAs therapy for HCV. Although the risk of developing HCC is decreased in HCV patients who achieve a disease-sustaining virological response, these patients are nevertheless at risk, especially those with severe fibrosis and cirrhosis. Previous studies have shown that HCV induce several mechanisms of hepatocarcinogenesis in the host’s hepatic micro- and macro-environment, which leads to HCC progression. In an HCV-altered environment, compensatory liver regeneration favors chromosomal instability and irreversible alterations, which encourage hepatocyte neoplastic transformation and the development of malignant clones. These mechanisms involve a series of genetic and epigenetic modifications including host genetic factors, dysregulation of several signaling pathways, histone, and DNA modifications including methylation and acetylation. This review highlights the genetic and epigenetic factors that lead to the development of HCC in chronic HCV-infected individuals and can be targeted for earlier HCC diagnosis and prevention.

High-Resolution Genomic Profiling of Liver Cancer Links Etiology With Mutation and Epigenetic Signatures

Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization. To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection. Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway. We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

Oxidative Stress Induces Mitochondrial Compromise in CD4 T Cells From Chronically HCV-Infected Individuals.

We have previously shown that chronic Hepatitis C virus (HCV) infection can induce DNA damage and immune dysfunctions with excessive oxidative stress in T cells. Furthermore, evidence suggests that HCV contributes to increased susceptibility to metabolic disorders. However, the underlying mechanisms by which HCV infection impairs cellular metabolism in CD4 T cells remain unclear. In this study, we evaluated mitochondrial mass and intracellular and mitochondrial reactive oxygen species (ROS) production by flow cytometry, mitochondrial DNA (mtDNA) content by real-time qPCR, cellular respiration by seahorse analyzer, and dysregulated mitochondrial-localized proteins by Liquid Chromatography-Mass Spectrometry (LC-MS) in CD4 T cells from chronic HCV-infected individuals and health subjects. Mitochondrial mass was decreased while intracellular and mitochondrial ROS were increased, expressions of master mitochondrial regulators peroxisome proliferator-activated receptor 1 alpha (PGC-1α) and mitochondrial transcription factor A (mtTFA) were down-regulated, and oxidative stress was increased while mitochondrial DNA copy numbers were reduced. Importantly, CRISPR/Cas9-mediated knockdown of mtTFA impaired cellular respiration and reduced mtDNA copy number. Furthermore, proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance were significantly altered in HCV-CD4 T cells. These results indicate that mitochondrial functions are compromised in HCV-CD4 T cells, likely via the deregulation of several mitochondrial regulatory proteins.

Frequent occurrence of low-level positive autoantibodies in chronic hepatitis C

Evidence of autoimmune disease associated with hepatitis C virus (HCV)-infection has important clinical implications. A systematic profile of these autoantibodies in relevant clinical cohorts relative to healthy controls is needed to better inform current standard of care for chronic hepatitis C. Samples from an Australian cohort of chronic HCV-infected subjects (n=127) were tested for the presence of 19 diagnostic autoantibodies and compared with data available from a control cohort representing a general Caucasian population (n=198). Chronic HCV-infected individuals had a greater number of autoantibodies than controls (p<0.0001). Anti-nuclear antibodies (ANA) followed by anti-smooth muscle antibodies (SMA) were the most frequently detected autoantibodies within the HCV cohort and significantly more than in the control cohort (p<0.0001 and p=0.006, respectively). However, for most autoantibody assays the 95th percentile approximated the reference value for positivity. None of the autoantibodies were significantly associated with age or sex for the HCV cohort, except SMA positivity that was significantly higher in chronic HCV-infected male subjects (p<0.0001). Autoantibodies found in chronic HCV-infected subjects were commonly low positive and not disease-specific. Accordingly, general screening for autoimmunity in HCV-infected subjects should not be performed unless there is high clinical suspicion of an underlying autoimmune disease.

Untreated alcohol use disorder in people who inject drugs: a major barrier to HCV treatment uptake

Abstract Aims Although people who inject drugs (PWID) are the core at-risk population in the hepatitis C (HCV) epidemic in industrialized countries, few initiate treatment. Alcohol use disorder (AUD), common within this population, has been identified as a barrier to HCV treatment uptake in the general population. We investigated whether the arrival of new and well-tolerated HCV treatments (direct-acting antivirals: DAA) has improved HCV treatment uptake in French PWID compared with former treatments (pegylated interferon-based treatments: Peg-IFN). Methods Analyses were based on exhaustive care delivery data collected between 2012 and 2016 (study period) in the French national healthcare reimbursement database. Chronic HCV-infected individuals who received opioid agonist therapy (OAT) at least once during the study period were included. Separate analyses were performed for 2012-2013 (Peg-IFN era, 19,700 patients) and 2014-2016 (DAA era, 22,545 patients). Using discrete-time Cox proportional hazards models, we tested for associations between AUD (i.e. AUD-related long-term illness status, diagnosis coding during hospitalization or AUD pharmacological treatment) and first HCV treatment delivery, after adjusting for gender, age, complementary universal health cover, liver disease severity and type of OAT received. Results Incidence rate of HCV treatment uptake per 100 person-years [95% confidence interval] was 6.56 [6.30-6.84] and 5.70 [5.51-5.89], for Peg-IFN-based treatment (2012-2013) and DAA (2014-2016), respectively. After multiple adjustment, people with AUD not receiving related medication had 30% and 14% lower Peg-IFN-based treatment and DAA uptake, respectively, than those without AUD (hazard ratio [95%CI]: 0.70 [0.62-0.80] and 0.86 [0.78-0.94]). No difference was observed between those treated for AUD and those without AUD. Conclusions Despite the benefits of DAA, untreated AUD is still a major barrier to HCV treatment access for PWID in France. Key messages AUD is still a barrier to HCV treatment for French PWID. DAA availability improved HCV treatment uptake for PWID with AUD.

Retrieval and re-evaluation of previously diagnosed chronic hepatitis C infections lost to medical follow-up in the Netherlands.

Many individuals previously diagnosed with chronic hepatitis C virus (HCV) infection are likely to be lost to medical follow-up and, therefore, remain untreated despite new highly effective drug treatment, direct acting antivirals. We aim to identify and retrieve these chronic HCV-infected individuals to re-evaluate them and offer treatment. Possible chronic HCV infections were identified from test results of the medical microbiological laboratory, notifications to the public health service, and the hospital registries over the past 15 years were checked in South Limburg, the Netherlands. Individuals were contacted based on the physician-patient relationship of the gastroenterologist or microbiologist (retrieval). Individuals were informed about the new treatment options, offered an HCV-RNA test, and if still positive, referred to the gastroenterologist for treatment (re-evaluation). In total, 689 individuals with a positive anti-HCV test in the past were identified, 308 (45%) were eligible for retrieval, 90 (29%) of them were retrieved, 34 (38%) of those retrieved were re-evaluated, 19 (56%) of those tested were HCV-RNA positive, and 12 (63%) of these individuals were offered treatment. During every step of the retrieval chain, many patients were lost. Nevertheless, with substantial effort, we were able to identify, retrieve, and positively re-evaluate a limited number of individuals with a possible chronic HCV infection who were lost to medical follow-up (19 patients). With this case-finding approach, we were able to prevent potential severe complications in these patients and contribute to a small step in the eradication of HCV in the Netherlands.

Rheumatoid factor and immunoglobulin M mark hepatitis C-associated mixed cryoglobulinaemia: an 8-year prospective study

ObjectivesThe prevalence and factors of hepatitis C virus (HCV) -associated mixed cryoglobulinaemia in Asia remain elusive, and we aimed to investigate these topics. MethodsAn 8-year prospective cohort study was conducted in 678 consecutive Taiwanese individuals with chronic HCV infection (438 completed an anti-HCV therapy course). ResultsOf 678 individuals, 437 (64.5%) had mixed cryoglobulinaemia and 20 (2.9%) had mixed cryoglobulinaemic syndrome. At baseline, IgM (cut-off >122 mg/dL), triglycerides and IgG levels, and HCV genotype 3 were independently associated with mixed cryoglobulinaemia. Rheumatoid factor (RF) levels were associated with mixed cryoglobulinaemic syndrome (cut-off >12.2 IU/mL). At 24 weeks post-therapy, the 362 individuals with a sustained virological response (SVR) had higher cured (106/362 (29.3%) versus 10/76 (13.2%), p = 0.003) and lower persistent (100/362 (27.6%) versus 33/76 (43.4%), p = 0.003) mixed cryoglobulinaemia rates than non-SVR patients. Among SVR patients, compared with baseline levels, RF, IgG and IgM levels decreased, except in individuals with new mixed cryoglobulinaemia. Pre-therapy IgM levels were associated with 24-week post-therapy new (95% CI of OR 1.002–1.023) and persistent (95% CI of OR 1.004–1.015) mixed cryoglobulinaemia in SVR patients. After up to 8 years, 24-week post-therapy IgM levels were associated with mixed cryoglobulinaemia in SVR patients (9/51; 17.64%; 95% CI of HR 1.004–1.011). Among 17 SVR patients with pre-therapy mixed cryoglobulinaemic syndrome, 5 (29.4%) had long-term mixed cryoglobulinaemia and 4 (23.5%) had mixed cryoglobulinaemic syndrome. ConclusionsOver 60% of chronic HCV-infected individuals had mixed cryoglobulinaemia, and 17.64% of SVR patients had mixed cryoglobulinaemia 8 years post-therapy. Pre-therapy RF and IgM levels marked HCV-associated mixed cryoglobulinaemic syndrome and mixed cryoglobulinaemia, respectively.

The Role of Hepatitis C Virus Core Antigen Testing in the Era of Direct Acting Antiviral Therapies: What We Can Learn from the Protease Inhibitors.

Direct-acting antiviral (DAA) therapies have revolutionised the treatment of hepatitis C virus (HCV). The financial cost of DAAs however is significant, and first generation protease inhibitors (PIs) also require frequent monitoring of viral RNA levels to guide treatment. In this context, we examined the relevance of HCV antigen testing to evaluate the potential role in monitoring virological response to HCV antiviral treatment with the PI-based triple therapies, telaprevir (TVR) and boceprevir (BOC). Chronic HCV-infected individuals (n = 152) enrolled in the Irish Hepatitis C Outcomes Research Network (ICORN) study were prospectively analysed for baseline markers and the early viral kinetics associated with SVR. The sustained virological response (SVR) rates in the cohort receiving TVR and BOC were 87.3% and 73.8%, respectively. Baseline factors associated with successful outcome in TVR therapy were age (P = 0.0098), IFNL3 genotype (P = 0.0330) and viral load (P = 0.0456). RNA level at week 4 (P = 0.0068) and viral antigen negativity at week 2 (P = 0.0359) were predictive of SVR for TVR-based therapy. In BOC therapy, prior interferon treatment (P = 0.0209) and IFNL3 genotype (P = 0.0410) were baseline predictors of SVR. Evidence of viraemia based either on viral RNA or antigen at week 4 predicted SVR in these patients. Our data showed that rapid decline of HCV antigen to negative level at week 2 in TVR treatment and <0.96 log fmol/l in BOC treatment after commencement of PI triple therapy were associated with SVR. HCV antigen measurement should be considered as a potential alternative for monitoring treatment response during DAA-based regimens.

Matched population-based study examining the risk of type 2 diabetes in people with and without diagnosed hepatitis C virus infection.

Meta-analyses have found hepatitis C virus (HCV) infection to be associated with an increased risk of type 2 diabetes mellitus (T2DM). Here, we examine this association within a large population-based study, according to HCV RNA status. A data-linkage approach was used to examine the excess risk of diagnosed T2DM in people diagnosed with antibodies to HCV (anti-HCV) in Scotland (21929 anti-HCV(+ves) ; involving 15827 HCV RNA(+ves) , 3927 HCV RNA(-ves) and 2175 with unknown RNA-status) compared to that of a threefold larger general population sample matched for gender, age and postcode (65074 anti-HCV(-ves) ). To investigate effects of ascertainment bias the following periods were studied: up to 1year before (pre-HCV)/within 1year of (peri-HCV)/more than 1year post (post-HCV) the date of HCV-diagnosis. T2DM had been diagnosed in 2.9% of anti-HCV(+ves) (including 3.2% of HCV RNA(+ves) and 2.3% of HCV RNA(-ves) ) and 2.7% of anti-HCV(-ves) . A higher proportion of T2DM was diagnosed in the peri-HCV period (i.e. around the time of HCV-diagnosis) for the anti-HCV(+ves) (22%) compared to anti-HCV(-ves) (10%). In both the pre-HCV and post-HCV periods, only those anti-HCV(+ves) living in less deprived areas (13% of the cohort) were found to have a significant excess risk of T2DM compared to anti-HCV(-ves) (adjusted odds ratio in the pre-HCV period: 4.0 for females and 2.3 for males; adjusted hazard ratio in the post-HCV period: 1.5). These findings were similarly observed for both HCV RNA(+ves) (chronic) and HCV RNA(-ves) (resolved). In the largest study of T2DM among chronic HCV-infected individuals to date, there was no evidence to indicate that infection conveyed an appreciable excess risk of T2DM at the population level.

Genetic variants in the apoptosis gene BCL2L1 improve response to interferon-based treatment of hepatitis C virus genotype 3 infection.

Genetic variation upstream of the apoptosis pathway has been associated with outcome of hepatitis C virus (HCV) infection. We investigated genetic polymorphisms in the intrinsic apoptosis pathway to assess their influence on sustained virological response (SVR) to pegylated interferon-α and ribavirin (pegIFN/RBV) treatment of HCV genotypes 1 and 3 infections. We conducted a candidate gene association study in a prospective cohort of 201 chronic HCV-infected individuals undergoing treatment with pegIFN/RBV. Differences between groups were compared in logistic regression adjusted for age, HCV viral load and interleukin 28B genotypes. Four single nucleotide polymorphisms (SNPs) located in the B-cell lymphoma 2-like 1 (BCL2L1) gene were significantly associated with SVR. SVR rates were significantly higher for carriers of the beneficial rs1484994 CC genotypes. In multivariate logistic regression, the rs1484994 SNP combined CC + TC genotypes were associated with a 3.4 higher odds ratio (OR) in SVR for the HCV genotype 3 (p = 0.02). The effect estimate was similar for genotype 1, but the association did not reach statistical significance. In conclusion, anti-apoptotic SNPs in the BCL2L1 gene were predictive of SVR to pegIFN/RBV treatment in HCV genotypes 1 and 3 infected individuals. These SNPs may be used in prediction of SVR, but further studies are needed.

Progression to advanced liver fibrosis in HIV-HCV-coinfected patients and prioritization of new hepatitis C therapies.

Because of their high cost, the use of direct-acting antivirals (DAAs) is being restricted by many governments to chronic HCV-infected individuals with advanced liver fibrosis. However, response rates are lower and toxicities more frequent in this subset of patients. All HIV-HCV-coinfected patients followed for at least 3 years at one reference clinic were identified. Liver fibrosis progression (LFP) was defined as a shift from Metavir F0-F2 to F3-F4 estimates (>9.5 KPa) using elastometry. A total of 527 HIV-HCV-coinfected patients were identified, of whom 344 had F0-F2 at baseline. Pegylated interferon/ribavirin therapy was given to 205 patients with null/mild fibrosis, of whom 92 (44.9%) achieved sustained virological response (SVR). After a mean follow-up of 53 months, LFP occurred in 5.4% SVR, 25.7% non-SVR and 18% untreated patients (P=0.005). In multivariate analysis, only achievement of SVR prevented LFP (adjusted hazard ratio 2.1; 95% CI 1.1, 4.1; P=0.01). In 139 untreated patients, only greater baseline elastometry values predicted LFP in multivariate analysis (adjusted hazard ratio 1.84; 95% CI 1.03, 3.3; P=0.03). The area under the receiver operating characteristic (AUROC) curve was 79%. A discriminant threshold of 7.1 kPa gave 68% sensitivity and 82% specificity. In the absence of successful treatment, more than 20% of HIV-HCV-coinfected patients with null/mild liver fibrosis progress to advanced fibrosis within 5 years. Patients with >7.1 kPa (Metavir F2) display the highest risk. Therefore, all coinfected patients with any significant liver fibrosis should be considered as candidates for new DAA-based therapies.

Positive ratio of specific antibodies to F protein in serum samples from chronic HCV-infected patients using an enzyme-linked immunosorbent assay

Although some studies have reported a positive ratio of specific antibodies to the alternative reading frame protein in an enzyme-linked immunosorbent assay test, our data from meta-analysis provide evidence supporting the presence of circulating anti-F protein antibodies. We collected studies focused on hepatitis C virus (HCV) and F protein. From an initial identification of 460 articles, we selected 16 studies that were randomized-controlled trials (RCTs). The results of the Mantel-Haenszel test showed that a statistically significant number of studies reported an effective value in chronic HCV-infected individuals (P<0.00001). We concluded that compared with healthy individuals, the positive ratio of F protein detection was higher in chronic HCV-infected individuals; the odds ratio was 63.61 [95% confidence interval (CI)=28.69, 141.06]. The values for chronic HCV-infected individuals were significantly different from those for non-HCV-infected individuals; the odds ratio was 53.43 (95% CI=23.33, 122.35). The positive ratio of the core protein was higher than that of F protein (rate difference=-38%, 95% CI=-42, -35%). We concluded that F protein elicits specific antibodies in most chronic HCV-infected individuals. Further, we confirmed the results of previous reports. The relationship between anti-F protein antibody and HCV coinfection still needs to be confirmed with further studies. Considering the high polymorphism rate of HCV, further studies are still needed for the selection of synthetic peptides from F protein that can coat the wells on microplates and serve as a commercial reagent.

Raltegravir switch improves hepatitis C transaminitis in HIV‐1 and hepatitis C (HCV) co‐infected individuals

Purpose of the studyHCV is one of the most relevant co‐morbidities seen in HIV‐infected individuals as evidenced by the negative impact that HIV exerts on the course of HCV infection. Despite remarkable results on HIV infection alone, the impact of highly active antiretroviral therapy (HAART) on liver disease in co‐infection remains unknown. We sought to explore the impact of raltegravir (RAL) on amino transferase (ALT) in HIV/HCV co‐infected individuals.MethodsHIV‐infected individuals co‐infected with HCV within the last 5 years receiving non‐integrase inhibitor containing HAART with a subsequent switch to RAL‐containing HAART were identified from a retrospectively maintained outpatient database. Patient demographics were extracted. Biochemical, virological and immunological parameters were collated and individuals received pegylated interferon with ribavirin were excluded. ALT levels at switch and post switch were compared using Kruskal‐Wallis test. Spearman's Rank correlation was used to assess the relationship between ALT and HCV‐RNA.Summary of resultsTwenty‐seven HIV‐HCV co‐infected individuals were identified between January 2007 and January 2012 and seven individuals were excluded. Median age was 44 years (range: 31–68). Five had acute and fifteen had chronic HCV infection during the switch. Twenty (100%) had HIV‐RNA‐1&lt;40 copies/mL at time of RAL switch. In chronic HCV infected individuals, median ALT levels at the time of switch were 465 IU/L, decreased significantly to 179 IU/L 1 month following switch (p=0.0261) and to 140 IU/L 6 months later (p=0.0225). On the other hand, in acute HCV infected individuals median ALT levels were 1005 IU/L at time of switch but decreased significantly to 220 IU/L 1 month later (p=0.0034) and to 35 IU/L 6 month later (p=0.0026). Sustained improvement in ALT levels from baseline to 1 month and up to 24 weeks after switch to RAL was observed in both groups but the reduction in ALT levels was statistically more significant in acutely infected individuals. ALT and HCV‐RNA levels showed a positive correlation at 6 months pre, post and at time of switch both in acute and chronic HCV‐infected individuals (Spearman's Rank correlation).ConclusionIn our study, RAL had a favourable effect on the liver up to 24 weeks after switch in HIV/HCV infected individuals.image

Distributional characteristics of CD25 and CD127 on CD4+ T cell subsets in chronic HCV infection

Attenuated CD4+ T-cell-mediated immune responses are involved in persistence of HCV infection, but the mechanism remains undefined. In this study, the proportions of CD4+ T cell subsets, naïve, central memory, effector memory and effector cells, along with CD25 (IL-2R alpha) and CD127 (IL-7R alpha) expression on different CD4+ T cell subsets, were measured by polychromatic flow cytometry in 24 chronic HCV-infected individuals and 21 healthy controls. A significant decrease in naïve CD4+ T cells and an increase of central memory and effector memory CD4+ T cells were found in HCV-infected patients compared with healthy controls. HCV-infected patients showed a lower level of CD127 expression in all CD4+ T cells subsets, especially in central memory and effector CD4+ T cells. In terms of total CD4+ T cells, an increase in CD25+ regular T cells (CD4+ CD25+ CD127lo) was found in HCV-infected patients. Interestingly, naïve CD4+ T cells showed increased CD25 expression, while effector memory and effector CD4+ T cells had lower CD25 expression. These data indicated that variations in different fractions of CD4+ T cells, including the phenotypic profile and expression level of CD25 and CD127, may be associated with low efficiency of immune response in chronic HCV infection. These results will strengthen the understanding of pathogenesis and dysfunction of CD4+ T cell immunity during long-term HCV persistence.

Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis.

Natural killer (NK) cells constitute the first line of host defense against invading pathogens. They usually become activated in an early phase of a viral infection. Liver is particularly enriched in NK cells, which are activated by hepatotropic viruses such as hepatitis C virus (HCV). The activated NK cells play an essential role in recruiting virus-specific T cells and in inducing antiviral immunity in liver. They also eliminate virus-infected hepatocytes directly by cytolytic mechanisms and indirectly by secreting cytokines, which induce an antiviral state in host cells. Therefore, optimally activated NK cells are important in limiting viral replication in this organ. This notion is supported by the observations that interferon treatment is effective in HCV-infected persons in whom it increases NK cell activity. Not surprisingly, HCV has evolved multiple strategies to counter host's NK cell response. Compromised NK cell functions have been reported in chronic HCV-infected individuals. It is ironic that activated NK cells may also contribute toward liver injury. Further studies are needed to understand the role of these cells in host defense and in liver pathology in HCV infections. Recent advances in understanding NK cell biology have opened new avenues for boosting innate and adaptive antiviral immune responses in HCV-infected individuals.