Hepatitis C Virus RNA Titers Research Articles

Retrospective study evaluating association of colorectal tumors and hepatitis C virus.

Chronic hepatitis C virus (HCV) has been associated with hepatic and extrahepatic malignancies. Limited studies have shown an association between colorectal adenomas and HCV populations. To study the prevalence of colorectal adenomas in patients with HCV compared to the general population and to evaluate if it is an independent risk factor for colorectal adenomas. Patients were divided into HCV and non-HCV based on their HCV RNA titers. Patients with alcoholic liver disease, hepatitis B infection, and inflammatory bowel disease were excluded. Continuous variables were analyzed using the Mann-Whitney U test, and categorical variables using χ 2 with P < 0.05 were considered statistically significant. The significant covariates (independent variables) were matched in both groups by propensity score matching, followed by multivariate regression analysis. Of the 415 patients screened, 109 HCV patients and 97 non-HCV patients with colonoscopy results were included in the study. HCV patients were older, had a smoking history, had less frequent aspirin use, and had a lower body mass index (BMI) (P < 0.05). The HCV cohort had a significantly increased number of patients with adenomas (adenoma detection rate of 53.2% vs 34%. P = 0.006). We performed a propensity-matched multivariate analysis where HCV infection was significantly associated with colorectal adenoma (OR: 2.070, P = 0.019). Our study shows a significantly higher rate of adenomas in HCV patients compared to the general population. Prospective studies would help determine if the increase in adenoma detection lowers the risk for colorectal cancer.

A Composite Score for Predicting Vertical Transmission of Hepatitis C: A Multicenter Study.

Prevention of the vertical transmission of the hepatitis C virus (HCV) presents an obstetric challenge. There are no approved antiviral medications for the treatment or prevention of HCV for pregnant patients. We aimed to create a composite score to accurately identify a population of pregnant patients with HCV who have high potential for vertical transmission. In a retrospective, multicenter cohort study, we identified pregnant patients with hepatitis C with linked data to their infants who have had HCV RNA or HCV antibody testing. Demographic data, including age and race/ethnicity, as well as clinical and laboratory data, including tobacco/alcohol use, infections, liver function tests, the HCV RNA titer, HCV antibody, HCV genotype, absolute lymphocyte count, and platelet count, were collected. Data were analyzed using logistic regression and receiver operating characteristics (ROCs) and internally validated using the forward selection bootstrap method. We identified 157 pregnant patients and 163 corresponding infants. The median maternal delivery age was 29 (IQR: 25-33) years, and the majority (141, or 89.8%) were White. A high HCV RNA titer, high absolute lymphocyte count, and high platelet count were associated with vertical transmission. A high HCV RNA titer had an AUROC of 0.815 with sensitivity, specificity, a positive predictive value, and a negative predictive value of 100.0%, 59.1%, 17.6%, and 100.0%, respectively. A composite score combining the three risk factors had an AUROC of 0.902 (95% CI = 0.840-0.964) but with a risk of overfitting. An HCV RNA titer alone or a composite score combining the risk factors for HCV vertical transmission can potentially identify a population of pregnant patients where the rate of vertical transmission is high, allowing for potential interventions during antepartum care.

CLEC18A Impairs Phagocytosis by Reducing FcγRIIA Expression and Arresting Autophagosome-Lysosome Fusion.

Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P < 0.005). We demonstrated that CLEC18A could inhibit FcγRIIA expression in a dose-dependent manner through the production of NOX-2-dependent reactive oxygen species to impair the uptake of ICs. Additionally, CLEC18A suppresses the Rab7 expression that is induced by starvation. Overexpressed CLEC18A does not affect autophagosome formation but does reduce the recruitment of Rab7 to autophagosomes, thereby retarding the maturation of autophagosomes and affecting autophagosome-lysosome fusion. We offer a novel molecular machinery with which to understand the association of HCV infection with autoimmunity and propose that CLEC18A may act as a candidate biomarker for HCV-associated MC. IMPORTANCE During infection, the host immune system produces cellular factors to protect against pathogen invasion. However, when the immune response overreacts and there is dysregulated cytokine homeostasis, autoimmunity occurs following an infection. We identified a cellular factor that is involved in HCV-related extrahepatic manifestation, namely, CLEC18A, which is expressed abundantly in hepatocytes and phagocytes. It inhibits HCV replication in hepatocytes by interacting with Rab5/7 and enhancing type I/III IFN expression. However, overexpressed CLEC18A inhibited FcγRIIA expression in phagocytes to impair phagocytosis. Furthermore, the interaction between CLEC18A and Rab5/7 may reduce the recruitment of Rab7 to autophagosomes and thereby retard autophagosome maturation and cause immune complex accumulation. A decreasing trend in CLEC18A levels that was accompanied by reduced HCV RNA titers and diminished cryoglobulin was observed in the sera of HCV-MC patients after direct-acting antiviral therapy. CLEC18A may be used for the evaluation of anti-HCV therapeutic drug effects and could be a potential predisposing factor for the development of MC syndrome.

A composite score for predicting vertical transmission of hepatitis C: a multicenter study

Prevention of vertical transmission of hepatitis C virus (HCV) presents an obstetric challenge. We aimed to create a composite score to accurately isolate a population of pregnant women with HCV who will have a high rate of vertical transmission. In a retrospective, multicenter, cohort study, we identified pregnant women with hepatitis C with linked data to their infants who have had HCV RNA or HCV antibody testing. Demographic data including age and race/ethnicity as well as clinical and laboratory data including tobacco/alcohol history, infectious history, liver function tests, HCV RNA titer, HCV antibody, HCV genotype, absolute lymphocyte count, and platelet count were collected. Data were analyzed by logistic regression and receiver operating characteristic (ROC). We identified 157 pregnant women and 163 corresponding infants. The median maternal delivery age was 29 (IQR: 25-33) years, and the majority (141 or 89.8%) were white, high HCV RNA titer, high absolute lymphocyte count and high platelet count were associated with vertical transmission. A composite score combining the three risk factors ha an AUROC of 0.934 (95% CI= 0.883-0.985), superior to any of the three risk factors individually. The sensitivity, specificity and positive predictive value were 100.0%, 86.4%, and 42.9%, respectively. A composite score combining risk factors HCV vertical transmission can isolate a population of pregnant women where the rate of vertical transmission is high, allowing for potential interventions during antepartum or intrapartum care.

Second hepatitis C virus transmission by blood components since introduction of mandatory NAT screening in Germany.

Viral safety of blood products in Germany has improved significantly over the last two decades. We describe the second documented transfusion-transmitted (TT) episode for the hepatitis C virus (HCV) in Germany since mandatory nucleic acid amplification techniques (NAT) screening was introduced in 1999. When a repeat donor who had tested negative for anti-HCV tested positive for HCV RNA by NAT in a minipool (MP) of eight, a look-back procedure was initiated. Qualitative, quantitative and genotyping assays were used to investigate the titers of the quarantined fresh frozen plasma (FFP) from the donor and a serum sample from the recipient of the pooled platelet concentrate (PPC). Amplified products of 5'UTR and HVR1 were used for sequence comparison to characterize the HCV genomic identity of donor and recipient samples. All NAT tests utilized in this procedure were able to detect a low HCV RNA titer (~15 IU/ml) in the FFP from the donation. Dilution of FFP by factor 8 was performed to mimic an MP, and the detection rate correlated well with the claimed sensitivity of the tests. Analysis of donor and recipient samples revealed genotype 3a viral transmission confirmed by sequence analysis. This TT HCV case could have been prevented by individual donation (ID) NAT. However, a low titer blood donation in the window period (WP) is very rare. Residual risk calculation for TT HCV in the WP revealed that, compared to MP-NAT testing, ID-NAT would improve blood safety only marginally.

Safety and efficacy of sofosbuvir-velpatasvir to treat chronic hepatitis C virus infection in treatment-naive patients in Rwanda (SHARED-3): a single-arm trial

Hepatitis C virus (HCV) genotype 4 is the predominant type of HCV found in sub-Saharan Africa. Various genotype 4 subtypes, such as 4r, frequently have resistance-associated substitutions that can increase rates of treatment failure with common direct-acting antiviral regimens. In-vitro studies suggest that the NS5A inhibitor velpatasvir is effective against viral isolates containing such resistance-associated substitutions, but its clinical efficacy against genotype 4 non-a/d subtypes in sub-Saharan Africa remains to be confirmed. We aimed to evaluate the safety and efficacy of sofosbuvir-velpatasvir among adults chronically infected with HCV and naive to direct-acting antiviral treatment in Rwanda, where genotype 4 non-a/d subtypes predominate. In this single-arm prospective trial, we enrolled adults (age ≥18 years) in Rwanda who had chronic HCV infection and a plasma HCV RNA titre of at least 1000 IU/mL. Patients were referred from hospitals with HCV treatment programmes throughout Rwanda and were sequentially enrolled and assessed for eligibility at a single study site. Individuals with decompensated liver disease or hepatitis B virus co-infection were excluded. Participants were given an oral fixed-dose combination tablet of sofosbuvir (400 mg) and velpatasvir (100 mg) once-daily for 12 weeks. The primary endpoint was the proportion of participants with a sustained virological response 12 weeks after completion of treatment (SVR12) in the intention-to-treat population. Viral sequencing of the NS5A and NS5B genes was done at baseline for all participants and end of follow-up (week 24) for participants who did not have SVR12. This study is registered with ClinicalTrials.gov (NCT03888729) and is completed. Between Sept 23, 2019, and Jan 10, 2020, 73 individuals were screened for eligibility, of whom 12 (16%) were excluded and 61 (84%) were enrolled. 40 (66%) participants were female, 21 (34%) were male, median age was 64 years (IQR 51-74), and median baseline HCV viral load was 5·7 log10 IU/mL (5·2-6·2). The genotypes identified among the participants were 4k (28 [46%] participants), 4r (11 [18%]), 4v (eight [13%]), 4q (five [8%]), 4l (three [5%]), 4b (one [2%]), 4c (one [2%]), and one undetermined genotype 4 subtype. Three isolates could not be sequenced and were of indeterminate genotype. Of the 55 HCV isolates that were successfully sequenced, all had at least two NS5A resistance-associated substitutions. 59 (97% [95% CI 89-99]) participants had SVR12. 18 (30%) participants had grade 3 adverse events (including 12 [20%] with hypertension), and none had grade 4 adverse events. Four (7%) participants had serious adverse events, including one asthma exacerbation, one abscess, one uterine myoma, and one pelvic fracture related to a motor vehicle accident. No serious adverse events were attributed to the study drug and no adverse event resulted in discontinuation of the study drug. A 12-week regimen of sofosbuvir-velpatasvir is safe and efficacious in treating chronic HCV genotype 4 infection in patients in Rwanda. This regimen could be an effective treatment option in regions known to have a high prevalence of HCV genotype 4 of diverse non-a/d subtypes. Gilead Sciences.

Safety and efficacy of sofosbuvir–velpatasvir–voxilaprevir for re-treatment of chronic hepatitis C virus infection in patients with previous direct-acting antiviral treatment failure in Rwanda (SHARED-3): a single-arm trial

Hepatitis C virus (HCV) genotype 4 non-a/d subtypes, which frequently have NS5A resistance-associated substitutions, are highly prevalent in sub-Saharan Africa. These subtypes, particularly genotype 4r, have been associated with higher rates of failure of treatment regimens containing the NS5A inhibitors ledipasvir or daclatasvir, which are the most accessible direct-acting antivirals in low-income countries. Clinical evidence regarding the efficacy of re-treatment options for these subtypes is limited. We aimed to evaluate the safety and efficacy of sofosbuvir-velpatasvir-voxilaprevir for the treatment of adults in Rwanda with chronic HCV infection, predominantly of genotype 4, and a history of direct-acting antiviral treatment failure. In this single-arm prospective trial, we enrolled adults (aged ≥18 years) with a HCV RNA titre of at least 1000 IU/mL, and a documented history of direct-acting antiviral failure. Patients were assessed for eligibility at a single study site after referral from hospitals with HCV treatment programmes throughout Rwanda, and participants for whom sofosbuvir-ledipasvir treatment had failed in the previous SHARED trial were also included. Participants with decompensated liver disease or hepatitis B virus co-infection were excluded. Participants were treated once daily with an oral fixed-dose combination tablet containing sofosbuvir (400 mg), velpatasvir (100 mg), and voxilaprevir (100 mg) for 12 weeks. The primary endpoint was the proportion of participants with a sustained virological response 12 weeks after completion of treatment (SVR12) in the intention-to-treat population. Viral sequencing of NS3, NS5A, and NS5B genes was done at baseline in all participants and at end of follow-up (week 24) in participants with treatment failure. The study is registered with ClinicalTrials.gov (NCT03888729) and is completed. Between Sept 23, 2019, and Jan 10, 2020, 49 individuals were screened and 40 participants were enrolled. 20 (50%) were female, 20 (50%) were male, median age was 63 years (IQR 56-68), and median HCV viral load was 6·2 log10 IU/mL (5·8-6·5) at baseline. The genotype subtypes identified were 4r (18 [45%] participants), 4k (six [15%]), 4b (five [13%]), 4q (four [10%]), 4l (two [5%]), 4a (one [3%]), 4m (one [3%]), and 3h (one [3%]). One (3%) genotype 4 isolate could not be subtyped, and one (3%) isolate was of unknown genotype. All successfully sequenced isolates (33 [83%]) had at least two NS5A resistance-associated substitutions and 25 (63%) had three or more. 39 (98% [95% CI 87-100]) participants had SVR12. Seven (18%) participants had a total of ten grade 3, 4, or 5 adverse events, including three (8%) cases of hypertension, and one (3%) case each of cataract, diabetes, gastrointestinal bleeding, joint pain, low back pain, vaginal cancer, and sudden death. Four of these events were categorised as serious adverse events resulting in hospitalisation. The one sudden death occurred at home from an unknown cause 4 weeks after the completion of treatment. No serious adverse event was determined to be related to the study drug or resulted in treatment discontinuation. A 12 week course of sofosbuvir-velpatasvir-voxilaprevir is safe and efficacious for the re-treatment of individuals infected with HCV genotype 4 non-a/d subtypes with frequent baseline NS5A resistance-associated substitutions, following failure of previous direct-acting antiviral treatment. Improved affordability and access to sofosbuvir-velpatasvir-voxilaprevir in regions with these subtypes is crucial. Gilead Sciences.

Combination treatment with sofosbuvir and ribavirin for patients diagnosed with hepatitis C genotype 2: A real-world, single-center study

Background and study aimsThe demand for treatments for viral hepatitis using direct antiviral agents (DAAs) has increased; however, few real-world clinical studies are available. The objective of this study was to evaluate the efficacy and safety of sofosbuvir combined with ribavirin for patients with chronic hepatitis C (CHC) genotype 2 (GT2). Patients and methodsA total of 106 consecutive CHC GT2 patients treated with sofosbuvir plus ribavirin between May 2016 and August 2018 (median age: 52.5 years, male: 51 [48.1%], treatment-naïve patients: 98 [92.5%]) were analyzed. The primary endpoint was sustained virologic response at 12 weeks (SVR12). The secondary endpoint was the occurrence of side effects during treatment. ResultsOf a total of 106 patients with CHC GT2, 103 were genotype 2a (97.2%), and 3 were 2b (2.8%). SVR12 was confirmed in 105 of 106 patients (99.1%). The one patient with treatment failure had combined liver cirrhosis and hepatocellular carcinoma. Twenty-five patients had liver cirrhosis in addition to hepatitis C virus (HCV) (Child-Turcotte-Pugh (CTP)-A, n = 24; C, n = 1), and SVR12 was confirmed in 24 of these patients (96.0%). The mean HCV RNA titer was 2,629,159 IU/ml. Reductions in haemoglobin levels occurred in 23 patients during treatment (3.0 mg/dL, mean), and consequently, ribavirin dose reduction was required (365.2 mg, mean). ConclusionSofosbuvir plus ribavirin was highly effective for the treatment of patients with CHC GT2 and had no serious, treatment-related adverse effects.

Relationship between Duffy Antigen Receptor for Chemokines and Clinical Characteristics in Han People with Chronic Hepatitis C Infection in Dalian, China

Background and Objectives: Most patients with untreated chronic hepatitis C virus (HCV) infection develop hepatic fibrosis. Hepatic disease progression is monitored with hematological markers (alanine aminotransferase [ALT], aspartate aminotransferase [AST], albumin and platelet [PLT] count, AST/ALT ratio, AST/PLT ratio index [APRI], and fibrosis 4 score [FIB-4]) and FibroScan. The present study aimed to investigate the association between Duffy antigen/chemokines receptor (DARC) polymorphisms and clinical parameters in the Han people with chronic hepatitis C infection in Dalian, China. Materials and Methods: This cohort study was performed on 245 Han people with chronic HCV at Dalian infectious hospital during April-December 2015. The participants of the research were selected using the consecutive sampling method. The DARC genotyping was performed using the TaqMan probe method and transient elastography was measured by FibroScan. Results: Based on the findings, DARC polymorphisms correlated with ALT concentrations (FY*A/FY*A vs. FY*A/FY*B, P=0.025). However, the DARC polymorphism did not have an association with HCV RNA titers (FY*A/FY*A vs. FY*A/FY*B, P=0.241) or hepatic fibrosis (FY*A/FY*A vs. FY*A/FY*B, P=0.325). Moreover, correlation analyses showed that APRI (P&lt;0.001, rho=0.603) and FIB-4 (P&lt;0.001, rho=0.698) were useful predictors of hepatic fibrosis in chronic HCV infection. Besides, HCV RNA titers (P=0.327) and hepatic injury markers (P=0.814, 0.198, 0.767, and 0.171 for ALT, AST, ALB, and AST/ALT, respectively) were not useful for the estimation of the fibrosis stage in patients with chronic hepatitis C. Conclusion: The FY*A allele is a potentially valuable protective factor against hepatocyte damage in chronic HCV-infected patients.

971 Eradication of Hepatitis C by Increasing Identification Rates of the Hepatitis C Virus (HCV) in Baby Boomers Using the Health Maintenance Tab

INTRODUCTION: Chronic HCV is one of the leading causes of cirrhosis and hepatocellular carcinoma in the United States. Current national data shows 75% of HCV infected adults are born between 1945–1965 (baby boomers). The USPSTF recommends one-time screening of this population for HCV. The aim of our study is to assess the impact on the rate of detection of HCV by implementing a screening tool in the health maintenance tab for this subset of high-risk individuals and improving treatment rates METHODS: In March 2017, our health center established a Health Maintenance tab for Screening HCV for all baby boomers.Yearly data regarding the number of patients screened, patients who had positive screening, data on confirmatory testing and the number that finally received treatment was collected between March 2012-February 2018. The percentage of patients screened and later confirmed positive was calculated. RESULTS: We compared the screening of baby boomers 5 years prior to the health maintenance tab and 1 year after it was introduced. In 2012, a total of 30915 patients were seen in the health facility with 1.83% screened, followed by 2.57% of 30679 patients in 2013, 2.4% from 29614 patients in 2014, 1.8% from 29081 patients in 2015, 1.7% from 28887 patients in 2016 and 7.8% from 29896 in 2017. There was 4.5 times increase in the screening of patients after the intervention. The number of patients who were screened positive with HCV Antibody from March 2012 to Feb 2018 were 15, 15, 55, 73, 64 and 182 in the consecutive years. The number of patients that had confirmatory testing with HCV RNA titers from 2012 to 2018 was 80%, 60%, 83%, 80%, 71.8% and 77.4%. Of those who had HCV RNA titers ordered between 2012 to 2018, the number of patients who were confirmed positive were 9 in 2012, 4 in 2013, 30 in 2014, 35 in 2015, 18 in 2016. The year after the intervention identified 73 patients who had confirmed Hepatitis RNA titers. Of the patients identified as having the infection, 4 were treated in 2012, 2 in 2013, 25 in 2014, 22 in 2015, 18 in 2016 and 52 after the intervention. CONCLUSION: Screening baby boomers using the health maintenance tab in the Electronic health record identified more patients who may in fact have Subclinical HCV. Identifying these individuals earlier would help initiate necessary treatment promptly and help prevent chronic complications. However, treatment of identified patients remains a challenge as other socioeconomic problems prevents individuals getting complete eradication of the infection

2382 Successful Hepatitis C Virus Antibody-Positive Donor Liver Transplantation in Acute Sickle Cell Intrahepatic Cholestasis: A Case Report

INTRODUCTION: Sickle cell hepatopathy (SCH) is the umbrella term referring to liver dysfunction in sickle cell disease (SCD) patients. We report a case of acute intrahepatic cholestasis (ASCIH) with underlying cirrhosis managed with hepatitis C virus (HCV) antibody-positive donor liver transplantation (LT). CASE DESCRIPTION/METHODS: A 29-year-old African American male with known medical history of SCD (Hb SS) and cirrhosis presented to the medical intensive liver unit with vaso-occlusive pain crises and jaundice. Patient was afebrile with benign exam. Labs were consistent with obstructive jaundice (total serum bilirubin of 57 mg/dL, direct serum bilirubin of 30 mg/dL), and elevated liver enzymes (alkaline phosphatase 306 U/L, aspartate transaminase 227 U/L and alanine transaminase 54 U/L). He had significant leukocytosis 38.6 k/uL, low hemoglobin 6.3 g/dL and thrombocytopenia 39 k/uL. Sodium level was 149 mg/dl and INR was 2.3. Computed tomography scan showed changes consistent with cirrhosis but no evidence of ductal dilatation. He developed encephalopathy after 24 hours of presentation, acute kidney injury (serum creatinine 3.57 mg/dl) and respiratory failure requiring endotracheal intubation. He was diagnosed with acute liver failure secondary to ASCIH after a non-revealing extensive workup for acute liver failure. Exchange transfusion was started without improvement in hepatic function or mental status. His MELD-Na (Model for End-Stage Liver Disease-Sodium) score was 43 and thus patient was listed for urgent LT. He received an HCV positive donor liver after 9 days of hospitalization. Explanted liver biopsy showed widespread sinusoidal dilatation with innumerable clusters of sickled red blood cells, scattered pigmented histiocytes, and cholestasis. Following transplant, exchange transfusion was resumed and immunosuppression treatment started with mycophenolate, tacrolimus and steroid taper. Glecaprevir/Pibrentasvir started for high HCV RNA titer. He was discharged after 23 days with total bilirubin of 4.2 mg/dL and normalization of his liver enzymes. He was doing well during his 4 months follow up. DISCUSSION: We present a rare case of acute sickle cell intrahepatic cholestasis treated with successful LT. This represents the 1st case of a positive HCV donor LT and the 20th LT case in SCD. ASCIH is the most severe, often fatal, form of SCH. Our goal with this case is to increase the awareness of this important condition and emphasize the role of liver transplantation as a potential treatment.

Evaluation of Dried Blood Spots as a Feasible Alternative to Plasma for the Detection and Quantification of Hepatitis C Virus in a Tropical Setting: A Pilot Study

AbstractIntroduction: Confirmatory diagnosis of hepatitis C virus (HCV) infection (HCV RNA detection) is essential before start of the therapy. HCV RNA detection is not available in many parts of India. Shipment of plasma from distant places to referral laboratories may affect HCV RNA titres. Dried blood spots (DBS) provide an easy alternative for transporting samples to centres where HCV RNA testing is done. Aim: Evaluation of DBS as a feasible alternative to plasma for HCV diagnosis. Methods: In this cross-sectional study, 40 consecutive patients’ blood samples were collected from patients referred from the Liver Clinic. Whole blood was spotted onto two Whatman 903TM cards. One card was incubated at ≥37°C and other at 4°C for 15 days, after drying. DBS was eluted and run in Abbott RealTime HCV assay. HCV was also quantified using the Abbott ARCHITECT HCV core antigen assay for 29 of the study patients. Results were compared with normal plasma values. Results: The median log HCV RNA value (in log10 IU/mL) of plasma was 5.74, with normalised DBS it was 4.92 (≥37°C) and 4.66 (4°C); difference in plasma and DBS median log values was 0.82 (≥37°C) and 1.08 (4°C) logs, respectively. Interclass correlation values were 0.943, P < 0.0001 (≥37°C) and 0.950, P < 0.0001 (4°C), showing high agreement. The median HCV core antigen value (in fmol/L) for plasma was 325.35, whereas it was 4.77 (≥37°C) and 4.64 (4°C) for DBS samples. Conclusions: DBS can be used for sampling patients from distant resource-limited settings as an alternative to plasma for HCV RNA estimation. Larger studies are required to evaluate the feasibility of DBS in the Indian subcontinent, especially for HCV core antigen estimation.

Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5'UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication.

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5′UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5′UTR nucleotides (nt) 95–110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528–8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95–110 or within the genomic RNA location comprising nt 8528–8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95–110 and NS5B nt 8528–8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528–8543 is essential for optimal virus replication.

High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log10 FFU/mL and 10.4 log10 IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.

Evaluation of in vitro screening and diagnostic kits for hepatitis C virus infection

BackgroundTo detect infection by hepatitis C virus (HCV), a reliable kit with high sensitivity and specificity is indispensable. Detection kits for anti-HCV antibodies (anti-HCV) are used for screening, and quantification kits for HCV RNA and core antigen are used for definite diagnosis of HCV infection. ObjectivesWe evaluated the performance of these kits using International Standards and a regional reference panel with HCV negative and positive specimens. Study designIn vitro diagnostic kits (10 anti-HCV, two HCV RNA, and three HCV core antigen) were included. ResultsNearly all specimens in the regional reference panel were correctly identified by all anti-HCV detection kits (one false-positive was observed in one kit). Both HCV RNA quantification kits also correctly identified and quantified HCV RNA titers, without genotype-specific differences. Among the HCV core antigen kits, International Standard values were inconsistent. The sensitivities of these kits were insufficient to detect HCV in positive specimens in the regional reference panel. ConclusionsIn vitro diagnostic kits assessing anti-HCV and HCV RNA have sufficient sensitivities and specificities to screen and detect HCV infection. However, HCV core antigen quantification kits have some limitations in their sensitivities and consistencies for diagnosis of HCV infection. Quality control with International Standards and a regional reference panel is important to maintain the performances of diagnostic kits for HCV infection and to verify the clinical reliability of these kits.

Incidentally detected asymptomatic hepatitis C virus infection with significant fibrosis: Possible impacts on management.

Liver biopsy may be considered in patients with hepatitis C virus (HCV) infection to assess the severity of liver injury and stage of fibrosis, thereby guiding therapeutic decisions. In addition, advanced stage also necessitates surveillance for hepatocellular carcinoma. The aim of this study was to assess whether transaminase (alanine transaminase [ALT]) levels and RNA titers correlate with the histological activity index (HAI) and fibrosis (F) stage in asymptomatic patients with incidentally detected HCV (IDHCV). Retrospective evaluation of liver biopsies was done in 113 patients with IDHCV, diagnosed during routine screening. Decision of liver biopsy was made on the basis of age, genotype, acceptable clinical, hematological, and biochemical profiles, and willingness of the patients to undergo treatment. Serum ALT levels, HCV RNA titers, and genotypes were correlated with HAI and F stage. Genotyping was done in 77 of the 113 patients, of which genotype 3 was seen in 43 and genotype 1 in 25 patients. A higher fibrosis stage (Ishak's >F2) was noted in 23.8% of the biopsies. Serum ALT showed a significant correlation with the HAI score on liver biopsy (P = 0.01) but not with the stage of fibrosis (P = 0.52). HCV RNA titers did not reveal any correlation with HAI score or fibrosis stage. Serum transaminases and HCV RNA titers are poor predictors of disease severity and fibrosis. Since HCV shows a slow disease progression, higher stage may predict a worse prognosis irrespective of the low viral RNA load. Liver biopsy may help guide therapeutic decisions in IDHCV infection.

Distribution of Hepatitis C Virus Genotypes in Patients with Major β-Thalassemia in Mashhad, Northeast Iran

BACKGROUND Hepatitis C virus (HCV) is considered to be the major cause of post-transfusion hepatitis in patients with thalassemia. We aimed to determine the HCV prevalence, genotypes, and viral load among patients with major β-thalassemia in Mashhad, Iran. METHODS Medical records of all 550 patients with major β-thalassemia who referred to ThalassemiaHemophilia Center of Mashhad (Sarvar Clinic) were reviewed from October to November 2011. Plasma samples of the patients were tested for the presence of anti-HCV antibodies by enzyme linked immunosorbent assay. Real-time polymerase chain reaction (PCR) was used to determine viral genotype and HCV RNA titer. RESULTS HCV antibodies were detected in 37 individuals (6.73%) including 17 men and 20 women with mean age of 25.2 ± 8.4 years. The PCR analysis was performed for 27 patients, of whom HCV RNA was detected in 17 patients (63.0%). Viral titers were investigated in 14 subjects and a high viral load more than 600000 copies/mL was observed in 6 patients (42.9%). The most prevalent genotypes were 3a (50.0%) followed by 1a (37.5%). No significant correlation was found between genotype and age, sex, serum ferritin, liver tests, and HCV RNA titer. CONCLUSION HCV infection among patients with thalassemia is more common than general population in Mashhad, northeast Iran. The dominant HCV subtype is 3a followed by 1a. These findings could help health authorities to provide preventive measures, and practitioners to choose the right protocol of treatment for the patients.

Hepatic tropism of hepatitis C virus infection

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a major cause of life-threatening liver diseases such as liver cirrhosis and hepatocellular carcinoma. In current research, we aimed to clarify the mechanism of hepatic tropism of HCV infection. Although non-hepatic cells could not permit replication of HCV RNA, exogenous expression of liver specific miRNA, miR-122 facilitated efficient replication of viral RNA through direct interaction with 5'UTR of viral genome, indicating that miR-122 is one of the key determinants for hepatic tropism of HCV infection. In spite of efficient replication of viral RNA, formation of infectious particles was not observed in non-hepatic cells exogenously expressing miR-122. We found that expression of apolipoprotein E (ApoE) facilitated the formation of infectious HCV particles in non-hepatic cells, indicating that not only miR-122 but also ApoE participate in tissue tropism of HCV infection. To understand the exact roles of miR-122 and apolipoproteins in hepatic tropism of HCV, we established miR-122 and ApoB/ApoE knockout (KO) Huh7 cells, respectively. Although slight increase of intracellular HCV RNA and infectious titers in the culture supernatants was observed, propagation of HCV was impaired in miR-122 KO Huh7 cells. After serial passages of HCV in miR-122 KO cells, we obtained an adaptive mutant that possessed G28A substitutions in the 5'UTR of the HCV genome and exhibited efficient translation and replication in both miR-122 KO Huh7 and non-hepatic cells without exogenous expression of miR-122. These results suggest that HCV mutants replicating in non-hepatic cells in an miR-122-independent manner participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. Deficiency of both ApoB and ApoE strongly inhibited the formation of infectious HCV particles. Interestingly, expression not only of ApoE but also of ApoA or ApoC could rescue the production of infectious HCV particles in ApoB/ApoE KO cells, suggesting that exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles.

Add-on effects of fluvastatin in simeprevir/pegylated-interferon/ribavirin combination therapy for patients with genotype 1 hepatitis C virus infection: A randomized controlled study.

The Japan Society of Hepatology guidelines indicate that hepatitis C virus (HCV) protease inhibitor combination therapy with simeprevir (SMV), pegylated-interferon (Peg-IFN), and ribavirin (RBV) is a therapeutic option for patients who fail to respond to a direct direct-acting antiviral-containing regimen. However, treatment outcomes have room for improvement. Fluvastatin (FLV) add-on treatment in Peg-IFN and RBV combination therapy for HCV-infected patients significantly improved the sustained virologic response (SVR), but the add-on effect of FLV on SMV combination therapy is not well understood. This was a prospective, randomized, multicenter study in which a total of 61 HCV genotype 1b-infected patients were recruited and 60 eligible patients were randomly allocated to two groups that received 12weeks of SMV/Peg-IFN/RBV followed by 12weeks of Peg-IFN/RBV with or without 24weeks of FLV. The SVR rate and adverse events were compared between the two groups. Thirty-one patients were allocated to the FLV add-on group and 29 patients were allocated to the control group. Baseline clinical factors, including median age, baseline platelet count, alanine aminotransferase level, HCV RNA titer, Fibrosis-4 index, and rate of IL28B minor genotype, were all similar between the two groups. The rapid virologic response, end-of-treatment response rates, SVR rates at 24weeks after treatment, and safety profiles were also similar between the two groups. This prospective, randomized, multicenter study indicated that FLV had no add-on effect when given with SMV/Peg-IFN/RBV combination therapy for genotype 1b HCV-infected patients.

Interferon alpha treatment stimulates interferon gamma expression in type I NKT cells and enhances their antiviral effect against hepatitis C virus.

Interferon (IFN) inhibits hepatitis C virus (HCV) replication through up-regulation of intrahepatic IFN-stimulated gene expression but also through activation of host immune cells. In the present study, we analyzed the immune cell-mediated antiviral effects of IFN-α using HCV-infected mice. Urokinase-type plasminogen activator (uPA)-severe combined immunodeficiency (SCID) mice with transplanted human hepatocytes were infected with genotype 1b HCV and injected with human peripheral blood mononuclear cells (PBMCs). IFN-α treatment following human PBMC transplantation resulted in a significant reduction in serum HCV RNA titers and a higher human CD45-positive mononuclear cell chimerism compared to mice without human PBMC transplantation. In mice with human PBMCs treated with IFN-α, serum concentrations of IFN-γ increased, and natural killer T (NKT) cells, especially type I NKT cells, produced IFN-γ. Mice in which IFN-γ signaling was blocked using antibody or in which transplanted PBMCs were depleted for type I NKT cells showed similar levels of anti-HCV effect compared with mice treated only with IFN-α. These results show that IFN-α stimulates IFN-γ expression in type 1 NKT cells and enhances the inhibition of HCV replication. We propose that type 1 NKT cells might represent a new therapeutic target for chronic hepatitis C patients.