Comparative efficacy and safety of Sofosbuvir/Velpatasvir and Danoprevir for the treatment of chronic hepatitis C: the real-world data in China.

BACKGROUND: Sofosbuvir/Velpatasvir (Epclusa, ECS) is the first pan-genotype direct-acting antiviral agent (DAA) for hepatitis C virus (HCV) infection, and Danoprevir (DNV) is the first DAA developed by a Chinese local enterprise, which is suitable for combined use with other drugs to treat genotype 1b chronic hepatitis C. However, previous reports have never compared the real-world data of ECS and DNV. PATIENTS AND METHODS: 178 chronic hepatitis C patients were retrospectively recruited, and 94cases were accepted with Sofosbuvir/Velpatasvir ± Ribavirin (ECS group), and others (n = 84 treated with DNV combination therapy (DNV group). The HCV genotype, virological response, adverse effects and some laboratory biochemical indexes were contrasted between above two groups in the real world study. RESULTS: DNV group had significantly lower level of alpha-fetoprotein (AFP), lower rates of decompensated cirrhosis ( P < 0.05). ECS group possessed more 6a (31.91% vs.13.10%) while DNV group was provided with more 1b (48.81% vs. 22.34%) patients. Significantly poor liver function was detected in ECS group at 4-week treatment (ALT and AST) and 12-week follow-up (AST) (all P < 0.05). The SVR12 undetectable rates of both groups were 100%, and no serious event was observed during the treatment and follow-up in both groups. CONCLUSION: In this retrospective real-world study, the efficacy of DNV combined therapy is similar to Sofosbuvir/Velpatasvir ± Ribavirin for chronic HCV infection, and the safety is comparable. DNV based therapy is a promising regimen for chronic hepatitis C.

Catalytic hairpin assembly triggering amplified DNAzyme-feedback for sensitive detection of hepatitis C virus genotype 1b.

Developing a simple, reliable, and sensitive hepatitis C virus (HCV) genetic sensing platform is of great significance for diagnosing diseases and selecting appropriate antiviral treatments. Herein, a tandem nucleic acid amplification strategy for sensitive detection of HCV genotype 1b (HCV-1b) was developed by stringing the catalytic hairpin assembly (CHA) and the triggered DNAzyme amplifier. The hairpin reactants were initiated by the target to produce lots of triggering double-stranded DNA sequences which can efficiently activate the subsequent blocked DNAzyme. Thereby, the continuous cleavage of substrate was realized, resulting in the fluorescence signal amplification. The DNA-based isothermal CHA-DNAzyme (CDz) sensing platform was successfully applied for sensitive detection of HCV-1b with the limit of detection (84 pM) and showed good selectivity. Moreover, the practical detection of target DNA in the complex biologic matrix indicated that the developing strategy had good potential for early HCV infection diagnosis.

Hepatitis C virus alters the morphology and function of peroxisomes.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

Determination of hepatitis C virus viremia and genotype distribution in Turkish citizens and immigrants from 2018 to 2022.

Hepatitis C virus (HCV) infection is an important public health problem with potential risk for Turkey. In order to contribute to the epidemiological data, we aimed to investigate the changes in seroprevalence, viremia rates, and genotypes in the last five years in HCV patients in the southern region of Turkey, which has received heavy migration in recent years, according to demographic criteria. In our study, we analyzed the results retrospectively with demographic data. Conducted at a single center, the study involved 259,875 anti-HCV antibody tests administered between January 2018 and July 2022. The study revealed a prevalence of 0.5% for HCV antibody positivity and a viremia prevalence of 0.1%. Among Turkish nationals, the most common genotypes were GT1 (65.1%), while foreign nationals, mainly of Syrian and Ukrainian origin, showed GT4 (52.3%) as the predominant genotype (p<0.001 for both). Although GT2 (7.4% vs. 4.5%) and GT3 (23.3% vs. 13.6%) were relatively higher in Turkish nationals compared to foreign nationals, the difference was not statistically significant (p=0.750 and p=0.154, respectively). This highlights the importance of continuous monitoring and public health efforts to address the potential impact of these demographic shifts on HCV epidemiology in the region.

Treatment Outcomes of Sofosbuvir/Velpatasvir/Voxilaprevir in Direct-Acting Antiviral-Experienced Hepatitis C Virus Patients: A Systematic Review and Meta-Analysis.

About 5% of chronic hepatitis C (CHC) patients experienced treatment failure with direct-acting antiviral (DAA) treatment. The global data on the practice and treatment outcomes of Sofosbuvir/Velpatasvir/Voxilaprevir (SOF/VEL/VOX) in DAA-experienced CHC patients remains sparse. We performed a systematic review and meta-analysis to evaluate the efficacy and safety of SOF/VEL/VOX as a salvage treatment in DAA-experienced CHC patients. We searched five electronic databases from inception to 31 January 2023. The study outcomes were SVR12 and treatment-related adverse effects, with subgroup analysis performed based on genotype, cirrhosis, HCC, prior SOF/VEL exposure, and region. We identified and analyzed data from 24 studies (2877 DAA-experienced CHC patients); 17.2% had prior SOF/VEL exposure, 25% received ribavirin with SOF/VEL/VOX, and 42% had pre-treatment resistance-associated substitution (RAS) testing performed. Eastern Mediterranean had a higher pooled SVR12 than the America and Europe regions (p < 0.05). Predictors of SOF/VEL/VOX failure were genotype 3, active HCC, baseline cirrhosis, and prior SOF/VEL. Baseline RAS mutation and ribavirin supplementation were not associated with higher SVR12. Treatment discontinuation because of drug-related adverse events was uncommon (10 studies, 0.2%). In summary, SOF/VEL/VOX is efficacious and safe for retreatment in DAA-experienced CHC patients, even with RAS mutation. Our findings support SOF/VEL/VOX as a first-line rescue treatment for DAA-experienced CHC patients.

Circulating miRNA-192 and miR-29a as Disease Progression Biomarkers in Hepatitis C Patients with a Prevalence of HCV Genotype 3.

MicroRNAs miR-29a and miR-192 are involved in inflammatory and fibrotic processes of chronic liver disease, and circulating miR-29a is suggested to diagnose fibrosis progression due to hepatitis C virus (HCV) infection. This study aimed to evaluate the expression profile of circulating miR-192 and 29a in a patient cohort with a high frequency of HCV genotype-3. A total of 222 HCV blood samples were collected and serum were separated. Patients were classified into mild, moderate, and severe liver injury based on their Child-Turcotte-Pugh CTP score. RNA was isolated from the serum and used for quantitative real-time PCR. The HCV genotype-3 (62%) was the predominant HCV genotype. In HCV patients, the serum miR-192 and miR-29a levels were significantly upregulated in comparison to healthy controls (p = 0.0017 and p = 0.0001, respectively). The progression rate of miR-192 and 29a in the patient group with mild was highly upregulated compared to patients with moderate and severe hepatitis infection. The ROC curve of miR-192 and miR-29a of moderate liver disease had a significant diagnostic performance compared to the other HCV-infected groups. The increase in miR-29a and miR-192 serum levels was even slightly higher in patients with HCV genotype-3 than in non-genotype-3 patients. In conclusion, serum miR-192 and miR-29a levels significantly increased during the progression of chronic HCV infection. The marked upregulation in patients with HCV genotype-3 suggests them as potential biomarkers for hepatic disease, independently of the HCV genotype.

Gender-Specific Differences in Serum Sphingomyelin Species in Patients with Hepatitis C Virus Infection-Sphingomyelin Species Are Related to the Model of End-Stage Liver Disease (MELD) Score in Male Patients.

Hepatitis C virus (HCV) replication depends on cellular sphingomyelin (SM), but serum SM composition in chronic HCV infection has been hardly analyzed. In this work, 18 SM species could be quantified in the serum of 178 patients with chronic HCV infection before therapy with direct-acting antivirals (DAAs) and 12 weeks later, when therapy was completed. Six SM species were higher in the serum of females than males before therapy and nine at the end of therapy; thus, sex-specific analysis was performed. Type 2 diabetes was associated with lower serum levels of SM 36:2;O2 and 38:2;O2 in men. Serum SM species did not correlate with the viral load in both sexes. Of note, three SM species were lower in males infected with HCV genotype 3 in comparison to genotype 1 infection. These SM species normalized after viral cure. SM 38:1;O2, 40:1;O2, 41:1;O2, and 42:1;O2 (and, thus, total SM levels) were higher in the serum of both sexes at the end of therapy. In males, SM 39:1;O2 was induced in addition, and higher levels of all of these SM species were already detected at 4 weeks after therapy has been started. Serum lipids are related to liver disease severity, and in females 15 serum SM species were low in patients with liver cirrhosis before initiation of and after treatment with DAAs. The serum SM species did not correlate with the model of end-stage liver disease (MELD) score in the cirrhosis and the non-cirrhosis subgroups in females. In HCV-infected male patients, nine SM species were lower in the serum of patients with cirrhosis before DAA treatment and eleven at the end of the study. Most of the SM species showed strong negative correlations with the MELD score in the male cirrhosis patients before DAA treatment and at the end of therapy. Associations of SM species with the MELD score were not detected in the non-cirrhosis male subgroup. In summary, the current analysis identified sex-specific differences in the serum levels of SM species in HCV infection, in liver cirrhosis, and during DAA therapy. Correlations of SM species with the MELD score in male but not in female patients indicate a much closer association between SM metabolism and liver function in male patients.

Genotypes of Hepatitis C Virus and Efficacy of Direct-Acting Antiviral Drugs among Chronic Hepatitis C Patients in a Tertiary Care Hospital.

Hepatitis C virus (HCV) chronic infection is a major causative factor for several chronic liver diseases, including liver cirrhosis, liver cell failure, and hepatocellular carcinoma. The HCV has seven major genotypes. Genotype 4 is the most prevalent genotype in the Middle East, including Saudi Arabia, followed by genotype 1. The HCV genotype affects the response to different HCV treatments and the progression of liver disease. Currently, combinations of direct-acting antiviral drugs (DAAs) approved for the treatment of HCV achieve high cure rates with minimal adverse effects. Because real-world data from Saudi Arabia about the efficacy of DAAs are still limited, this study was conducted to assess the effectiveness of DAAs in treating patients with chronic hepatitis C and to identify the variables related to a sustained virologic response (SVR) in a real-world setting in Saudi Arabia. This prospective cohort study included 200 Saudi patients with chronic HCV who were 18 years of age or older and had been treated with DAAs at King Abdul-Aziz Specialized Hospital in Taif, Saudi Arabia, between September 2018 and March 2021. The response to treatment was assessed by whether or not an SVR had been achieved at week 12 post treatment (SVR12). An SVR12 was reached in 97.5% of patients. SVR12 rates were comparable for patients of different ages, between men and women, and between patients with and without cirrhosis. In addition, the SVR12 rates did not differ according to the infecting HCV genotype. In this study, the presence of cirrhosis and the patient's gender were independent predictors of who would not reach an SVR12 (known here as the non-SVR12 group) according to the results of univariate and multivariate binary logistic regression analyses based on the determinants of SVR12. In this population of patients with chronic HCV infection, all DAA regimens achieved very high SVR12 rates. The patients' gender and the presence of cirrhosis were independent factors of a poor response.

High real-world effectiveness of 12-week elbasvir/grazoprevir without resistance testing in the treatment of patients with HCV genotype 1a infection in Norway.

BACKGROUND AND AIMS: The recommended treatment duration of hepatitis C virus (HCV) genotype 1a (GT1a) infection with elbasvir/grazoprevir (EBR/GZR) in the presence of a high baseline viral load and resistance associated substitutions (RAS) is 16 weeks with ribavirin added. The objective of this study was to evaluate the real-world effectiveness of 12 weeks of EBR/GZR without ribavirin and regardless of baseline viral load and RAS testing. METHOD: This retrospective, observational cohort study was performed at five Norwegian hospitals that did not systematically utilize RAS testing. All adult patients with chronic HCV GT1a and compensated liver disease who had received 12 weeks of EBR/GZR without ribavirin and baseline RAS testing, were included. The primary endpoint was sustained virologic response at week 12 (SVR12), or if not available, at week 4 (SVR4). RESULTS: We included 433 patients and attained SVR data on 388. The mean age was 45.7 years (22-73 years). 67.2% were male. HIV co-infection was present in 3.8% (16/424) and cirrhosis in 4% (17/424). The viral load was >800 000 IU/mL in 55.0% (235/427) of patients. Overall SVR was achieved in 97.2% (377/388). SVR was achieved in 98.3% (169/172) of those with viral load ≤800 000 IU/mL and in 96.2% (202/210) of those with viral load >800 000 IU/mL. CONCLUSION: We observed high SVR rates among patients with HCV GT1a infection treated with EBR/GZR for 12 weeks without ribavirin, with no regard to baseline viral load and no RAS testing.

Circulating microRNAs as predictors of response to sofosbuvir + daclatasvir + ribavirin in in HCV genotype-4 Egyptian patients.

BACKGROUND: MicroRNAs (miRNAs) play an important role in various diseases, including HCV infection, the aim of the current study was to evaluate the potential use of serum miRNAs as biomarkers for diagnosis, prognosis, and prediction of responses to direct acting antivirals (sofosbuvir + daclatasvir + ribavirin) in HCV-4 patients. METHODS: The serum expression profiles of four liver-associated miRNAs (miRNA-122, 155, 196 and 29) were assessed in 160 HCV-4 patients and 50 healthy controls using real-time PCR prior to therapy. RESULTS: miR-122 and miR-155 showed upregulation in HCV-4 patients compared to healthy controls while miR-196 and miR-29 showed downregulation in HCV-4 patients. ROC curve analyses revealed that the four-studied miRNAs could be valuable biomarkers for predicting response to DAAs with AUC 0.973 for miR-122, 0.878 for miR-155, 0.808 for miR-29 and 0.874 for miR-196 respectively. Univariate logistic regression analysis revealed that miR-196 level is positive predictor for SVR, whereas miR-122,155 levels are negative predictors of response. Multivariate logistic regression analysis revealed that miR-196 is the most significant in predicting response to treatment (p value = 0.011). CONCLUSION: To the best of our knowledge, the current study provided the first clinical evidence of the potential use of circulating miRNAs (miR; 122, 155, 196 and 29) as biomarkers of CHC in HCV-4 patients receiving the new DAA regimen (SOF/DAV + RIB), which is a strong motivator for further studies.

From Bench to Bedside: Clinical and Biomedical Investigations on Hepatitis C Virus (HCV) Genotypes and Risk Factors for Albuminuria.

An extrahepatic manifestation of nephropathies can be a feature of the chronic hepatitis C virus (HCV) infection. Albuminuria is a major risk factor for nephropathies and chronic kidney disease (CKD). The correlation between HCV genotypes and albuminuria is still unclear. In this study, investigations have been done for the biomedical tools and methodologies used in the National Health and Nutrition Examination Survey (NHANES) public database. We searched the 2007−2016 NHANES public database to retrieve data regarding the different HCV genotypes and clinical scenarios. This study attempted to investigate the impacts of HCV genetic diversity, associated comorbidities, and racial differences on albuminuria. The urine albumin/creatinine ratio (ACR) was the primary endpoint. Among 40,856 participants, 336 participants with positive and 237 with negative HCV RNA tests were analyzed, excluding 14,454 participants with negative HCV antibodies and 25,828 which were missed. After controlling for sex, race, education level, smoking, diabetes mellitus, hepatitis B, alcohol use, and body mass index (BMI) with a generalized linear equation, HCV genotype 2 was more likely than any other genotype to cause albuminuria based on the urine ACR (p < 0.001). The generalized linear equation also demonstrated a significantly higher urine ACR, including hepatitis B (p < 0.001), diabetes mellitus (p < 0.001), and smoking (p = 0.026). In summary, the patients with HCV genotype 2 presented with increased albuminuria in comparison with other HCV genotypes in this 10-year retrospective analysis. HCV infection could be a risk factor of CKD; early diagnosis and appropriate treatment may improve clinical outcomes.

Hepatitis C Infection as a Risk Factor for Hypertension and Cardiovascular Diseases: An EpiTer Multicenter Study.

Hepatitis C infection is one of the main reasons for liver cirrhosis and hepatocellular carcinoma. In recent years, more and more is being heard about extrahepatic manifestations of the hepatitis C infection including its possible influence on the development of hypertension and cardiovascular diseases. In the given work, the frequency analysis of the incidence of hypertension and cardiovascular diseases among 2898 HCV-infected patients treated in Poland and the assessment of their relevance to the HCV genotype and the progression of liver fibrosis can be found. The prevalence of hypertension in the group of analyzed patients was 39% and was significantly associated with old age (OR = 1.08 (1.07-1.08)) and female sex, as well as the progression of liver fibrosis (OR = 1.54 (1.29-1.85)). Hypertension was found in 47.6% of patients with F4 fibrosis, 42.1% of patients with F3 fibrosis, and 25% of patients with F1 fibrosis. The incidence of cardiovascular disease in the studied group of patients was as follows: all incidents, 131 (4.52%); including ischemic heart disease 104, (3.95%); stroke, 2 (0.07%); atherosclerosis, 21 (0.72%); and aneurysms, 4 (0.14%). The obtained results prove that the prevalence of cardiovascular diseases is significantly associated with the advanced age of patients and the progression of liver fibrosis. The relevance of sex and the HCV genotype to the prevalence frequency of cardiovascular diseases in the study group has not been proven. This being the case, no differences in the frequency of their incidence depending on the HCV genotype, including genotype 3, was found. Hepatitis C infection as a non-classical risk factor for cardiovascular disease and hypertension does require further studying.

Serum Ceramide Species Are Associated with Liver Cirrhosis and Viral Genotype in Patients with Hepatitis C Infection.

Hepatitis C virus (HCV) infection affects ceramide metabolism, and, here, we have evaluated associations of eight serum ceramide species with viral load, viral genotype, and disease markers in 178 patients with chronic HCV. In this cohort, ceramide d18:1;O2/16:0 was higher in the serum of the 20 diabetic patients compared to the patients without this complication. Moreover, ceramide d18:1;O2/24:0 was negatively correlated with age. Of note, all but ceramide d18:1;O2/16:0 and 26:0 were diminished in the serum of patients with liver cirrhosis and, with the exception of ceramide d18:1;O2/16:0, were negatively correlated with the model for end-stage liver disease (MELD) score. Most of the serum ceramides are carried in low-density lipoprotein (LDL), which rises following effective direct-acting antiviral (DAA) therapy. Ceramide d18:1;O2/24:0 recovered in parallel with LDL, whereas ceramide d18:1;O2/18:0 declined. Genotype-3-infected patients had the lowest ceramide levels, which were comparable to other genotypes after DAA treatment. Notably, ceramide d18:1;O2/23:0 and 24:0 were negatively correlated with the MELD score in patients with liver cirrhosis at the end of DAA therapy. Long-chain (LC) ceramides show adverse effects, whereas very-long-chain (VL) species have protective functions in the liver. The ratio of VL/LC ceramides was higher in non-cirrhosis patients than cirrhosis patients and further increased at the end of therapy in this subgroup. In summary, our study shows that serum ceramide levels are related to liver cirrhosis and viral genotype. Whether the more favorable serum ceramide profile in non-cirrhosis patients, before and after DAA therapy, is of pathophysiological importance needs further investigation.

Prevalence of Hepatitis C Virus Infection, Genotypes and Subtypes in Migrants from Pakistan in Barcelona, Spain.

Background: Hepatitis C virus (HCV) is a major cause of chronic liver infection with 71 million people infected worldwide. Pakistan has the second highest prevalence of HCV infection and more than half (52%) of Pakistani living in Spain reside in Barcelona. The aim of this study was to analyse the seroprevalence and viraemic rate and determine the genotypes and subtypes of HCV among Pakistanis living in the southern metropolitan area of Barcelona. Methods: We included all Pakistani patients seeking primary healthcare in the southern metropolitan area of Barcelona from August 2011 to July 2014. Serum samples were screened for HCV antibodies. HCV viral load was determined by reverse transcription polymerase chain reaction and genotypes and subtypes were performed using Versant HCV Genotype and/or deep-sequencing. Screening for hepatitis B virus (HBV) was also carried out. Results: Among 5877 Pakistani patients, 565 (9.61%) were screened for anti-HCV antibodies, with 68 (12.04%) being positive. The viral load was determined in 65, with 31 presenting active infection and the viraemic rate was 47.69% (95% confidence interval 36.02-59.62). HCV genotyping and subtyping were performed in 24 individuals. Most infections corresponded to HCV genotype 3 (91.67%), and high resolution HCV subtyping was performed in 18 samples, 16 of which presented subtype 3a. One subject presented HBV coinfection with undetectable HBV DNA. During the study period, we identified a possible case of HCV vertical transmission followed by spontaneous viraemia clearance in a chronically infected mother with a C/T IL28B genetic polymorphism. Conclusion: These results suggest that general HCV screening protocols in patients from high prevalence countries, such as Pakistan, would be helpful to identify and treat active HCV infections. This could avoid further transmission and contribute to building targeted health policies for micro-elimination of HCV infection in specific communities.

Hepatitis C virus genotypes and viremia in a tertiary hospital in Istanbul, Turkey.

INTRODUCTION: The World Health Organization estimates that 71 million people with chronic HCV infection lived worldwide in 2015. HCV is a globally prevalent pathogen, that genotype1 is the most common. In this study, the prevalence of anti-HCV, distributions of HCV genotype, and viremia rates in patients with chronic hepatitis C were evaluated. METHODOLOGY: In this retrospective single-center study, anti-HCV results of 197,081 patients were evaluated between 2017 and 2020. Quantitative HCV-RNA PCR tests were performed on the Rotor-Gene Q real-time PCR instrument. HCV genotypes determination of 546 samples was carried out with the Gen-C 2.0 Reverse Hybridization strip and HCV Genotype Plus Real-TM kit. RESULTS: The prevalence of anti-HCV was 0.95% and viremic HCV infection was 0.3% (610/197,081). HCV viremia rate was 33.17%. HCV viremia rate was highest in 2017 (52.36%) and the lowest in 2020 (18.3%) (p < 0.001). Genotype1 (72%) was the most common genotype, followed by genotype3 (14.1%), and genotype4 (8.8%). The most common subtypes were determined as genotype1b (56.2%) and genotype1a (13.2%). The viral load was higher in patients infected with genotype5. CONCLUSIONS: In this study, the rate of viremic HCV infection was found to be 0.3%. This rate was lower than the worldwide rate of HCV viremia. The distribution of HCV genotypes was like the global data. The identification of circulating genotypes and subtypes is essential for epidemiological purposes and remains important in the choice of treatment in patients with chronic HCV.

Hesperidin identified from Citrus extracts potently inhibits HCV genotype 3a NS3 protease.

BACKGROUND: Hepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries. METHODS: The full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI-MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease. RESULTS: Fusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC50 values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MSn analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC50 value of 11.34 ± 3.83 µg/mL. CONCLUSIONS: A FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a.

Sero-reactivity to three distinct regions within the hepatitis C virus alternative reading frame protein (ARFP/core+1) in patients with chronic HCV genotype-3 infection.

Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5 % of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three -20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47 % for ARFP-P2, 5.9 % for ARFP-P3 and 100 % for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.

Gender-Specific Frequency Distribution of Hepatitis C Virus Genotypes in Punjab province, Pakistan: A Clinically Significant Descriptive Cross-Sectional Study.

BACKGROUND: Hepatitis C virus (HCV) is the major cause of liver cirrhosis, chronic liver disease, and hepatocellular carcinoma. More than 10 million individuals are living with HCV infection in Pakistan. Due to unawareness, very little information is known about HCV genotype occurrence in Punjab, the largest province of Pakistan. Identification of HCV genotype is very important for HCV treatment because different genotypes of HCV respond differently to antiviral therapy. OBJECTIVE: The purpose of this research was to determine the distribution frequency of different HCV genotypes in the Punjab province and to demonstrate the distribution pattern of HCV genotypes in different age groups and sexes. MATERIALS AND METHODS: In this study, we performed HCV genotyping of 3692 samples collected from different sites of the Punjab province, Pakistan. Among 3692 samples, 1755 (47.5%) were males and 1937 (52.4%) were females. RESULTS: A total of 3692 samples were subjected to HCV genotyping and 2977 (81%) patients were genotyped successfully, whereas 715 (19%) patients were found to be HCV not detected. Our study demonstrated that among typeable genotypes, 3a constituted 2582 (69.9%) patients followed by 1a (n = 280) 7.5%, 1b (n = 64) 1.7%, 2a (n = 6) 0.16%, genotype 4 (n = 10) 0.27%, 3+4 (n = 2) 0.56%, 1a+2a (n = 11) 0.29%, 1b+2a (n = 1) 0.02%, 1a+1b (n = 1) 0.02%, and 1a+1b+3 (n = 1) 0.02% patients. HCV genotype distribution was evaluated gender wise and in different age groups like 0-12, 13-18, 19-59, and >60 years. All the HCV genotypes were equally distributed among men and women. The most affected age group was 19-59 years as compared to other age groups. CONCLUSION: The most frequently distributed HCV genotype in Punjab was found to be genotype 3a followed by genotype 1a, and only 0.94% of infected patients had a mixed genotype infection. Genotype 1a was found to be increasing significantly in the studied population. With these results, it can be assumed that genotype 3a may be replaced by genotype 1a with the passage of time. If this happens, this situation will be challenging in terms of antiviral therapy.

Impact of HCV Eradication on Lipid Metabolism in HIV/HCV Coinfected Patients: Data from ICONA and HepaICONA Foundation Cohort Study.

OBJECTIVES: HCV shows complex interactions with lipid metabolism. Our aim was to examine total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) changes in HIV/HCV coinfected patients, after achieving sustained virological response (SVR), according to different HCV genotypes and specific antiretroviral use. METHODS: HIV/HCV coinfected patients, enrolled in the ICONA and HepaICONA cohorts, who achieved DAA-driven SVR were included. Paired t-tests were used to examine whether the pre- and post-SVR laboratory value variations were significantly different from zero. ANCOVA regression models were employed to estimate the causal effect of SVR and of PI/r use on lipid changes. The interaction between the effect of eradication and HCV genotype was formally tested. RESULTS: six hundred and ninety-nine HIV/HCV coinfected patients were enrolled. After HCV eradication, a significant improvement in liver function occurred, with a significant decrease in AST, ALT, GGT, and total plasmatic bilirubin. TC and LDL-C significantly increased by 21.4 mg/dL and 22.4 mg/dL, respectively (p < 0.001), after SVR, whereas there was no evidence for a change in HDL-C (p = 0.45) and triglycerides (p = 0.49). Notably, the TC and LDL-C increase was higher for participants who were receiving darunavir/ritonavir, and the TC showed a more pronounced increase among HCV genotype 3 patients (interaction-p value = 0.002). CONCLUSIONS: complex and rapid changes in TC and LDL-C levels, modulated by HCV genotype and PI/r-based ART combinations, occurred in HIV/HCV coinfected patients after SVR. Further studies are needed to evaluate the clinical impact of these changes on the long-term risk of cardiovascular disease.

HCV Genotype Distribution of Patients with Chronic Hepatitis C in Istanbul.

OBJECTIVES: Hepatitis C virus (HCV), which has no protective vaccine, is a common cause of chronic hepatitis, which is a severe public health threat. There are differences in nucleotide and amino acid sequences in different regions of the HCV genome. As a result of these differences, HCV has been shown to have at least seven major genotypes and many subtypes. In Turkey, the prevalence of genotype 1 is between 51.7% and 97.1%, the highest rate among all genotypes, while subtype 1b is the genotype with the highest rate. It is important to detect mixed genotype infection reliably as it causes treatment failure. This study aims to reveal the distribution of the HCV genotypes in our hospital in Istanbul over the years and to contribute to the epidemiological data of Turkey. METHODS: For this purpose, 385 patient samples sent to Sisli Hamidiye Etfal Training and Research Hospital, Clinical Microbiology Laboratory for HCV genotype determination between January 2016 and June 2019 were evaluated retrospectively. Anti-HCV was screened by enzyme immunoassay and confirmation was performed by Line immunoassay. HCV genotyping assays targeting highly conserved 5'UTR and most variable region NS5B regions were used. RESULTS: The most common genotype was genotype 1 (81.3%) with 313 cases and subtypes 1a and 1b were detected at the rates of 10.9% and 67.8%, respectively. In addition, genotype 3, 2, 4, 5 were detected at the rates of 8.8%, 3.4%, 2.9%, 0.8%, respectively and mixed genotype was found in 2.9% of cases. Although genotype 5 is seen in South Africa, it is found in the Middle East region, albeit at a low rate. In our study, it was observed that genotype 5 was detected in different years from patients of Syrian origin. CONCLUSION: In this study, genotype 1 was the most common genotype with a rate of 81.3% and subtype 1b was 67.8%, in accordance with the literature. However, genotypes 3, 2, 4 and 5 were also present at low rates. It is important to monitor these rare genotypes since some of them are dominant in surrounding countries. In addition, 2.9% of HCV mixed genotype was detected and this should be considered concerning management of HCV infection.