Hepatitis C Virus RNA Concentration Research Articles

DNA-directed formation of plasmonic core-satellite nanostructures for quantification of hepatitis C viral RNA.

Hepatitis C virus (HCV) continues to be a significant public health challenge, affecting an estimated 71 million people globally and posing risks of severe liver diseases. Despite advancements in treatments, diagnostic limitations hinder the global elimination efforts targeted by 2030. This study introduces an innovative diagnostic approach, integrating catalytic hairpin assembly (CHA) with plasmonic core-satellite gold nanoparticle (AuNP) assemblies, to enable sensitive and specific detection of HCV RNA. We optimized the stoichiometry of DNA hairpins to form highly stable three-way junctions (3WJs), minimizing non-specific reactions in an enzyme-free, isothermal amplification process. The resulting dual-transduction biosensor combines colorimetric and surface-enhanced Raman spectroscopy (SERS) techniques, utilizing the Raman reporter malachite green isothiocyanate (MGITC) for signal generation. Our system targets a conserved 23-nucleotide sequence within the HCV 5'-UTR, essential for RNA replication, facilitating pan-genotypic HCV detection that complements direct-acting antiviral strategies. We evaluated the biosensor's efficacy using fluorescence spectroscopy, native PAGE, AFM, and TEM. Findings indicate that the 60 nm core AuNPs surrounded by 20 nm satellite AuNPs achieved a ten-fold increase in sensitivity over the 10 nm satellites, detecting HCV RNA concentrations as low as 1.706 fM. This sensitivity is crucial, given the extremely low viral loads present during early infection stages. Our research demonstrates the promise of enzyme-free molecular biosensors for HCV, with the potential to provide cost-efficient, rapid, point-of-care testing, although further sensitivity enhancements are needed to address the challenges of early-stage detection.

Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay

Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 12a (CRISPR–Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the RT-LAMP-coupled CRISPR–Cas12 assay yielded 96% sensitivity, 100% specificity, and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentrations as low as 10 ng/µL (an estimated 2.38 Log10 IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings.

CORRELATION BETWEEN THE ANTIBODY RESPONSE TOWARD SPECIFIC HCV PROTEINS AND HCV VIRAL LOAD

Background: Hepatitis C virus (HCV) is an RNA virus causing acute or chronic infection and affecting more than 2% of population worldwide. The firstline tests for diagnosis of HCV infection are 3rd or 4th generation enzyme immunoassays - ELISA and CIA. They indicate the presence of antibodies against HCV in serum. These tests are characterized by high sensitivity and specificity, but they cannot distinguish past, acute or chronic infection, and sometimes produce false positive results. Confirmatory tests, such as recombinant immunoblot-line immune assay (LIA), and quantitative PCR, are used to validate the positive antibody response. The recombinant immunoblot assay can be used to determine the specificity of antibody to HCV. The aim of the present study is to determine the correlation between the anti-HCV response in confirmatory immunoblot assay and the HCV viral load, measured by PCR. Materials and methods: Twenty-nine anti-HCV positive sera were included in the study. Third generation ELISA assay was used for anti-HCV screening of the samples and for detection of anti-HCV antibodies against specific HCV proteins. Third generation line immunoassay INNO-LIA HCV Score, based on the principle of an enzyme immunoassay, was used as a confirmatory test. The HCV viral load was measured by quantitative PCR method – Abbott Real Time HCV (Abbott Molecular Inc., USA) with linear sensitivity range from 1.08 Log 10 IU/ml (12 [IU/ml]) to 8.00 Log 10 IU/ml (100 000 000 [IU/ml]). Results: HCV RNA was quantified in all studied samples. Ten of 29 serum samples (34%, Group I) were HCV RNA negative. The rest of the samples were HCV RNA positive as follows: 3 serums were with minimal viral load from < 12 to 10 000 IU/ml (10%, Group II); 3 serum samples –between 10 000 and 100 000 IU/ml (10%, Group III); 10 serum samples – between 100 000 and 1 000 000 IU/ml (34%, Group IV) and in 3 serum samples HCV RNA concentration was over 1 000 000 IU/ml (10%, Group V). Conclusion: HCV screening strategies involving anti-HCV detection by ELISA combined with recombinant immunoblot assay can be the method of choice in laboratories with limited equipment and finances.

Mathematical modeling of hepatitis C RNA replication, exosome secretion and virus release.

Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response.

Contribution of Hepatitis C Infection to a Large Cohort of Cryoglobulin-Positive Patients: Detection and Characteristics.

Cryoglobulins (CGs) are cold precipitating immunoglobulins, and hepatitis C virus (HCV) infection is its most common cause. The purpose of the study was to determine the contribution of HCV in a large cohort of CG. Biological characteristics and specificity of CGs in HCV patients were compared to non-HCV subjects. Cryoglobulin analysis included isotype, clonality, concentration, and rheumatoid factor (RF) in cryoprecipitate and serum complement and RF. This study is an extension of the study carried out on a cohort of 13,439 patients tested for CGs from all medical units, in which 1,675/13,439 (12.5%) patients had a CG, and 680/1,675 (40.6%) had HCV serology or viral load determination (HCV RNA). Among these 680 CG patients tested for HCV, 325 of 680 (47.8%) HCV patients (272 HCV RNA+ and 45 HCV RNA− patients) were compared to 355/680 (52.2%) non-HCV subjects. After a positive detection of CG, HCV status was determined only for 37.7% (256/680) of patients, allowing the diagnosis of a previously unknown HCV infection for 39.8% (102/256). Concentration of HCV RNA+ CGs (median = 80.5 mg/L) was significantly higher than that of HCV RNA− CG (median = 50.5 mg/L, p = 0.001) and HCV− CG (median = 32 mg/L, p < 0.0001). There was no difference of median CG concentration between HCV RNA− patients and non-HCV subjects. Rheumatoid factor titer was significantly higher in type II CG compared to type III CG in HCV RNA+ patients (254 ± 720 vs. 15 ± 21 IU/mL, p < 0.0001) and non-HCV subjects (333 ± 968 vs. 16.8 ± 26 IU/mL, p = 0.0004). Complement functional activity CH50 was lower in HCV RNA+ patients (36 ± 24 U/mL) and in HCV RNA− patients (32 ± 21 U/mL) than in non-HCV subjects (50 ± 25 U/mL, p = 0.001 and p = 0.004). In conclusion, HCV infection and treatment influence CG characteristics. It is essential, and far from always tested, to determine the HCV status of patients with mixed CG, and conversely to search for CG in patients with HCV infection.

Prevention of viral transmission during lung transplantation with hepatitis C-viraemic donors: an open-label, single-centre, pilot trial

A substantial proportion of organ donors test positive for hepatitis C virus (HCV) infection. To date, only a few studies have evaluated the safety of using lungs from these donors for transplantation, and no direct interventions to donor organs have been done with the aim of preventing HCV transmission via organ transplantation. We aimed to assess the safety and efficacy of lung transplantation in humans from HCV-positive donors to HCV-negative recipients after application of ex-vivo lung perfusion (EVLP) plus ultraviolet C (UVC) perfusate irradiation. We did a single centre, prospective, open-label, non-randomised trial in which donor lungs from HCV-viraemic donors (HCV-positive) were transplanted into HCV-negative recipients at Toronto General Hospital, University Health Network (Toronto, ON, Canada). Donors were younger than 65 years old and tested positive for HCV by nucleic acid testing. Donors who tested positive for hepatitis B virus, HIV, human T-lymphotropic virus 1 or 2 were excluded. Recipients were on the lung transplant waiting list without significant liver disease (stage 2 fibrosis or higher were excluded) or active HCV infection. Before implantation, all HCV-positive donor lungs were treated with EVLP with or without UVC perfusate irradiation to reduce the concentration of HCV RNA and infectivity. For the first week after transplantation, patients' HCV RNA blood concentrations were measured once daily, then once per week for 12 weeks. All patients received 12 weeks of oral sofosbuvir 400 mg plus velpatasvir 100 mg, starting at least 2 weeks after transplantation. The primary endpoint was a composite of survival and HCV-free status at 6 months after transplantation in all patients who received HCV-positive lungs. Patient outcomes such as survival, time in hospital, and incidence of acute rejection were compared between those receiving HCV-positive lungs and all patients who received HCV-negative lung transplants during the study period. The study is registered with ClinicalTrials.gov, NCT03112044. From Oct 1, 2017, to Nov 1, 2018, 209 patients had a transplantation; of 27 donors who were HCV-positive and initially considered, 22 were suitable for transplantation. The remaining 187 donors were HCV-negative. Before implantation, 11 of the HCV-positive donor lungs were treated with EVLP alone and the other 11 were treated with EVLP plus UVC. Lung disease, urgency status, and positive donor-recipient HLA crossmatch were similar between the patients who received HCV-positive and HCV-negative lungs. 20 (91%) patients in the HCV-positive group developed HCV viraemia within the first week after transplantation and had sofosbuvir plus velpatasvir treatment, starting at a median of 21 days after transplantation (IQR 16·76-24·75). Donor organ treatment with EVLP plus UVC was associated with significantly lower recipient viral loads in blood within the first week after transplantation than with EVLP alone (median of 167 IU/mL [IQR 20-12 000] vs 4390 IU/mL [1170-112 000] at day 7; p=0·048) and prevented transmission in two (18%) of 11 patients. All 20 infected patients achieved negative HCV PCR within 6 weeks of treatment initiation. The primary endpoint of survival and HCV-free status at 6 months after transplantation was achieved in 19 (86%) of 22 patients in the HCV-positive group. 6-month survival was 95% in recipients receiving lungs from HCV-viraemic donors versus 94% in recipients receiving lungs from HCV-negative donors. The most common grade 3-4 adverse events in the HCV-positive group were respiratory complications (five [23%]) and infections (four [18%]). Serious adverse events requiring admission to hospital occurred in ten (45%) patients. One (5%) patient who did not develop HCV infection died at day 31 from multiorgan failure related to pseudomonas sepsis. Two patients presented with HCV relapse within 3 months after sofosbuvir plus velpatasvir completion and required retreatment. Early and intermediate clinical outcomes were not significantly different between patients receiving viraemic HCV donor lungs and HCV-negative donor lungs. Donor organ treatment with UVC perfusate irradiation during EVLP significantly decreased HCV viral loads within the first 7 days after transplantation and shows the proof-of-concept for a novel approach of minimising viral load ex vivo before transplantation, with intent of preventing donor-recipient transmission. Canadian Institutes of Health Research.

Treatment of chronic hepatitis C virus infection in Rwanda with ledipasvir–sofosbuvir (SHARED): a single-arm trial

Limited treatment data are available for hepatitis C virus (HCV) in sub-Saharan Africa, especially for genotype 4. Our objective was to establish the safety and efficacy of ledipasvir-sofosbuvir for chronic HCV genotype 1 or 4 infection in adults in Rwanda. We did a single-arm trial to evaluate the safety and efficacy of ledipasvir-sofosbuvir in Rwandan adults with chronic HCV infection at a single study site (Rwanda Military Hospital, Kigali, Rwanda). We enrolled individuals aged 18 years or older with HCV genotype 1 or 4 infection and a plasma HCV RNA concentration of more than 1000 IU/mL at screening. All participants were given ledipasvir (90 mg) and sofosbuvir (400 mg) in a single combination tablet once daily for 12 weeks. We established HCV genotype using an Abbott platform, and HCV subtype with PCR amplification. The primary endpoint was the proportion of participants with a sustained virological response 12 weeks after therapy (SVR12). All patients enrolled in the study were included in the primary endpoint analyses. This study is registered with ClinicalTrials.gov, number NCT02964091. 300 participants were enrolled between Feb 6, 2017, and Sept 18, 2017, and the follow-up period was completed on March 1, 2018. On genotyping, 248 (83%) participants were reported as having genotype 4, four (1%) genotype 1, and 48 (16%) both genotype 1 and genotype 4. Subsequent viral sequencing showed all participants actually had genotype 4 infection with subtype 4k (134 [45%]), subtype 4r (48 [16%]), subtype 4q (42 [14%]), and subtype 4v (24 [8%]) predominating. Overall, 261 (87%, 95% CI 83-91) participants achieved SVR12. In participants with genotype 4r, SVR12 was observed in 27 (56%, 95% CI 41-71) participants versus 234 (93%, 90-96) individuals with other subtypes. There were no drug-related treatment discontinuations due to ledipasvir-sofosbuvir. The most common adverse events were hypertension (97 [32%]), headache (78 [26%]), dizziness (61 [20%]), and fatigue (56 [19%]). There were six serious adverse events; none were assessed to be due to the study drug. 296 participants had data for pill counts at week 4 and 8; 271 (92%) had 100% adherence and only one (<1%) had an adherence of less than 90%. This is the first large-scale prospective study reporting direct-acting antiviral outcomes in sub-Saharan Africa. The high adherence and treatment success without intensive support measures or highly specialised clinical providers, and lack of treatment discontinuations due to adverse events support the feasibility of HCV treatment decentralisation and scale-up in sub-Saharan Africa. Genotype 4r is uniquely expressed in this region and associated with high rates of treatment failure, suggesting a need for rigorous test-of-cure in clinical practice and consideration of the use of newer pangenotypic direct-acting antiviral regimens in this region. Gilead Sciences.

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay.

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R 2 = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.

Deferred treatment with sofosbuvir–velpatasvir–voxilaprevir for patients with chronic hepatitis C virus who were previously treated with an NS5A inhibitor: an open-label substudy of POLARIS-1

Direct-acting antiviral regimens containing NS5A inhibitors are highly effective treatments for chronic hepatitis C virus (HCV) infection, but are not always successful. In the POLARIS-1 phase 3 study, sofosbuvir-velpatasvir-voxilaprevir for 12 weeks was highly effective in the treatment of chronic HCV infection in patients previously treated with a direct-acting antiviral regimen containing an NS5A inhibitor. We aimed to assess the efficacy and safety of sofosbuvir-velpatasvir-voxilaprevir in patients from the deferred treatment group of POLARIS-1, who were initially assigned to masked placebo treatment. This open-label, deferred treatment substudy was done at 73 clinical sites (hospitals and clinics) in the USA, France, Canada, the UK, Germany, Australia, and New Zealand. Patients who received placebo in the primary study and who did not have a new clinically significant illness at the post-treatment week 4 assessment were eligible to enter this substudy. Participants received a combination tablet of sofosbuvir (400 mg), velpatasvir (100 mg), and voxilaprevir (100 mg) once daily for 12 weeks. The primary efficacy outcome was achievement of sustained virological response (defined as HCV RNA concentration below the lower limit of quantification) 12 weeks after the end of treatment (SVR12). The primary safety outcome was the proportion of patients who discontinued treatment due to adverse events. This study is registered with ClinicalTrials.gov, number NCT02607735, and the EU Clinical Trials Register, number 2015-003455-21. 152 patients received placebo in the primary study and were potentially eligible for participation in the open-label substudy, of whom 147 were enrolled from March 30, 2016, to Oct 12, 2016. All 147 patients completed treatment, and 143 (97%; 95% CI 93-99) achieved SVR12. Four (3%) patients had virological relapse; all had HCV genotype 1a infection and one also had compensated cirrhosis. The most common adverse events were fatigue (31 [21%]), headache (29 [20%]), diarrhoea (28 [19%]), and nausea (21 [14%]). No deaths, treatment discontinuations, or treatment-related serious adverse events occurred. Supporting the results from the blinded portion of the phase 3 primary study, the single-tablet regimen of sofosbuvir-velpatasvir-voxilaprevir for 12 weeks was safe, well tolerated, and highly effective in patients with chronic HCV infection who had previous treatment failure with NS5A inhibitor-containing regimens. A salvage regimen for this population represents an important advance for patients with limited retreatment options. Gilead Sciences.

Multicenter Comparison Study of both Analytical and Clinical Performance across Four Roche Hepatitis C Virus RNA Assays Utilizing Different Platforms

ABSTRACTThe efficacy of antiviral treatment for chronic hepatitis C virus (HCV) infection is determined by measuring HCV RNA at specific time points throughout therapy using highly sensitive and accurate HCV RNA assays. This study compared the performances of two recently developed real-time PCR HCV RNA assays, cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and cobas HCV for use on the cobas 4800 system (cobas 4800 HCV), with those of two established assays, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2 (CAP/CTM v2) and the Cobas TaqMan HCV test, version 2 for use with the High Pure system (HPS/CTM v2). The limits of detection (LODs) and linearity at lower concentrations (5 to 1000 IU/ml) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1 to 4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1 to GT 4. Results from cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (<0.3 log10 IU/ml difference between expected and observed results) with LODs of 8.2 IU/ml and 11.7 IU/ml, respectively, for GT 1. The new assays showed excellent agreement with results from CAP/CTM v2 and HPS/CTM v2 in samples with quantifiable viral loads. The concordances using the 6 million IU/ml cutoff were high among all four assays (90 to 94%). In conclusion, the cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear and correlate well with the established Roche assays used in clinical practice.

INVESTIGATION OF THE SIGNIFICANCE OF DETECTION OF THE NUCLEOCAPSID ANTIGEN OF THE HEPATITIS C VIRUS IN CHILDREN WITH CHRONIC HEPATITIS C

The aim of this study was to investigate the clinical and diagnostic significance of the detection of core-antigen of hepatitis C virus (HCV) in children with chronic hepatitis C (CHC). Sera of 101 patients and 24 non-infected participants were analyzed. Core-antigen was determined by chemiluminescent method using «ARCHITECT HCV Ag» (Abbott, USA). HCV RNA was detected by highly sensitive OT-PCA. The presence of liver fibrosis was established by non-invasive methods. Antibody to core-antigen was detected by ELISA using commercial test-systems. A correlation was found between concentration of core-antigen and HCV RNA, viral subtypes, ALT level, and presence of liver fibrosis. High sensitivity and specificity of the chemiluminescent method for the detection of core antigen in children with CHC was shown. For the first time three ranges of core-antigen concentrations were proposed.

Ombitasvir, paritaprevir, and ritonavir plus ribavirin for chronic hepatitis C virus genotype 4 infection in Egyptian patients with or without compensated cirrhosis (AGATE-II): a multicentre, phase 3, partly randomised open-label trial

In Egypt, chronic hepatitis C virus (HCV) infection occurs in around 10% of the population (about 8 million individuals), and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and mortality. Although HCV genotype 4 constitutes about 20% of HCV infections worldwide, the prevalence in Egypt is more than 90%. We assessed the efficacy and safety of the two direct-acting antiviral drugs ombitasvir, an NS5A inhibitor, and paritaprevir, an NS3/4A protease inhibitor dosed with ritonavir, plus ribavirin in treatment of chronic HCV infection in Egypt. AGATE-II was a phase 3, open-label, partly randomised trial in patients with chronic HCV genotype 4 infection recruited from five academic and hepatology centres in Egypt. Patients were HCV treatment-naive or treatment-experienced with interferon-based regimens. Eligible patients were aged 18 years or older, and had been chronically infected with HCV genotype 4 for at least 6 months with a plasma HCV RNA concentration of more than 1000 IU/mL at screening. Patients without cirrhosis were assigned to receive 12 weeks of 25 mg ombitasvir, 150 mg paritaprevir, and 100 mg ritonavir orally once daily plus weight-based ribavirin. Patients with compensated cirrhosis were randomly assigned (1:1) to receive the same treatment for either 12 weeks or 24 weeks. Randomisation was stratified by previous pegylated interferon and ribavirin treatment experience using a web-based interactive response technology system and computer-generated schedules prepared by personnel from the funder's statistics department. Investigators were masked to randomisation schedules and were informed of each patient's assigned treatment by the interactive response technology system immediately after allocation. The primary endpoint was the proportion of patients with a sustained virological response (HCV RNA <15 IU/mL) 12 weeks after the last dose of study drug (SVR12). All patients who received at least one dose of study drugs were included in the primary and safety analysis. This study is registered with ClinicalTrials.gov, number NCT02247401. Between Nov 4, 2014, and March 16, 2015, we screened 182 patients with HCV infection, of whom 160 were eligible for inclusion; 100 patients were assessed as not having cirrhosis and were given 12 weeks of treatment, and 60 patients assessed as having cirrhosis were randomly assigned to the 12-week treatment group (n=31) or the 24-week treatment group (n=29). 94 (94%; 95% CI 88-97) of 100 patients in the without cirrhosis group, 30 (97%; 84-99) of 31 patients in the cirrhosis 12-week treatment group, and 27 (93%; 78-98) of 29 patients in the cirrhosis 24-week treatment group achieved SVR12. The most common adverse events in patients without cirrhosis were headache (41 [41%]) and fatigue (35 [35%]). Fatigue occurred in nine (29%) patients in the cirrhosis 12-week treatment group and 11 (38%) patients in the cirrhosis 24-week treatment group, and headache occurred in nine (29%) patients in the cirrhosis 12-week treatment group and in 10 (35%) patients in the cirrhosis 24-week treatment group. Adverse events were predominantly mild or moderate in severity, and laboratory abnormalities were not clinically meaningful. No patients discontinued treatment because of an adverse event. One serious adverse event in the group without cirrhosis was attributed to study drugs by the investigators; the patient had deep venous thrombosis. Ombitasvir, paritaprevir, and ritonavir plus ribavirin for 12 weeks achieved SVR12 in a high proportion of patients and was well tolerated in Egyptian patients with HCV genotype 4 infection with or without compensated cirrhosis. Extension of treatment to 24 weeks in patients with cirrhosis did not improve the proportion of patients achieving SVR12. A shorter duration regimen could be useful to address the significant burden of HCV genotype 4 infection in patients with compensated cirrhosis. AbbVie.

Maximum levels of hepatitis C virus lipoviral particles are associated with early and persistent infection.

Hepatitis C virus (HCV) is bound to plasma lipoproteins and circulates as an infectious lipoviral particle (LVP). Experimental evidence indicates that LVPs have decreased susceptibility to antibody-mediated neutralisation and higher infectivity. This study tested the hypothesis that LVPs are required to establish persistent infection, and conversely, low levels of LVP in recent HCV infection increase the probability of spontaneous HCV clearance. LVP in non-fasting plasma was measured using the concentration of HCV RNA bound to large >100 nm sized lipoproteins after ex vivo addition of a lipid emulsion, that represented the maximum concentration of LVP (maxi-LVP). This method correlated with LVP in fasting plasma measured using iodixanol density gradient ultracentrifugation. Maxi-LVP was measured in a cohort of 180 HCV participants with recent HCV infection and detectable HCV RNA from the Australian Trial in Acute Hepatitis C (ATAHC) and Hepatitis C Incidence and Transmission Study in prison (HITS-p) cohorts. Spontaneous clearance occurred in 15% (27 of 180) of individuals. In adjusted analyses, low plasma maxi-LVP level was independently associated with spontaneous HCV clearance (≤827 IU/ml; adjusted odds ratio 3.98, 95% CI: 1.02, 15.51, P = 0.047), after adjusting for interferon lambda-3 rs8099917 genotype, estimated duration of HCV infection and total HCV RNA level. Maxi-LVP is a biomarker for the maximum concentration of LVP in non-fasting samples. Low maxi-LVP level is an independent predictor of spontaneous clearance of acute HCV.

Ledipasvir-sofosbuvir in patients with hepatitis C virus genotype 5 infection: an open-label, multicentre, single-arm, phase 2 study

Data about the response of hepatitis C virus (HCV) genotype 5 to approved and experimental treatment regimens are scarce. We assessed the efficacy and safety of combination therapy with the NS5A inhibitor ledipasvir and the NS5B polymerase inhibitor sofosbuvir in patients with HCV genotype 5. We did this open-label, multicentre, single-arm, phase 2 trial at five hospitals in France. Eligible patients were at least 18 years old and had chronic infection with HCV genotype 5, with plasma HCV RNA of at least 10,000 IU/mL. We used BLAST analyses of NS5B partial sequences to establish the genotype and subtype at screening. Patients were given a fixed-dose combination tablet of 90 mg ledipasvir and 400 mg sofosbuvir orally once per day for 12 weeks. The primary endpoint was the proportion of patients with a sustained viral response, defined as HCV RNA concentration less than 15 IU/mL at 12 weeks after the end of treatment (SVR12). We analysed efficacy and safety in all patients who received at least one dose of ledipasvir-sofosbuvir. This trial is registered with EudraCT, number 2013-003978-27, and with ClinicalTrials.gov, number NCT02081079. From March 7 to June 10, 2014, we recruited 41 patients, including 21 who were treatment naive and 20 who were treatment experienced. All patients were of white ethnic origins. All 41 patients who started treatment completed the full 12 weeks of treatment and had undetectable HCV RNA at their final treatment visit. In the overall study population, 39 (95%, 95% CI 83-99) of 41 patients achieved SVR12. SVR12 was achieved by 20 (95%, 76-100) of the 21 patients who were treatment naive and 19 (95%, 75-100) of the 20 patients who were treatment experienced. Eight (89%) of nine patients with cirrhosis achieved SVR12, whereas 31 (97%) of the 32 patients without cirrhosis achieved SVR12. The two patients who did not reach SVR12 both had IL28B TT genotype and had viral relapse within 4 weeks of the end of treatment. The most common adverse events were asthenia (16 [39%] patients), headache (11 [27%] patients), and fatigue (four [10%] patients). One patient had a serious adverse event, worsening depression, which we judged to be unrelated to study treatment. The oral regimen of ledipasvir-sofosbuvir is an effective and well-tolerated treatment for patients with HCV genotype 5 infection who are treatment naive or treatment experienced. Gilead Sciences.

New Therapies, Evidence, and Guidance in Hepatitis C Management: Expert Practices and Insights from an Educational Symposium at the AMCP 27th Annual Meeting Expo.

The 2013-2014 approvals of new direct-acting antiviral (DAA) therapies for hepatitis C virus (HCV) infection have engendered a paradigm shift in HCV treatment and management, offering the potential for a cure at a population level. The availability of the highly effective and relatively safe DAAs prompted revisions to guidance recommendations based on new clinical trial evidence. In the context of this paradigm shift and considerations of the costs associated with the new DAAs, managed care professionals face new questions and challenges regarding HCV treatment and management approaches. To address the continuing education needs of this group, PRIME Education, Inc. (PRIME) conducted a symposium on HCV at the 27th Annual Meeting Expo of the Academy of Managed Care Pharmacy. Moderated by Michael R. Stinchon, Jr., RPh, the program panel featured 2 internationally recognized leaders in hepatitis C treatment and research: Norah Terrault, MD, MPH, and Alex Monto, MD. To summarize the educational symposium presentations and discussions. This article is organized by key questions that the panelists and attendees raised for discussion during the 2-hour symposium. The questions addressed methods for assessing liver fibrosis; comprehensive patient assessment to inform treatment decisions; the influence of viral load on decisions about treatment duration; the role of ribavirin in optimizing treatment efficacy; unmet treatment needs for patients with HCV genotype 3 or advanced liver disease; and managed care strategies for patient education, adherence promotion, and care coordination. In answering attendee questions on these issues, the expert panelists presented established evidence, and recognizing limitations to current evidence and guidance recommendations, they discussed applications of clinical judgment and offered their views and practices regarding individualized care for patients with HCV. In response to questions about the utility of noninvasive methods for assessing liver fibrosis, the expert panel presented a comparative overview of the methodology, accuracy, risks, limitations, and costs of noninvasive tests and liver biopsy. Discussion highlighted the strengths of noninvasive methods for diagnosing advanced disease and cirrhosis and the methods' limitations that pose barriers to ensuring that patients receive necessary antiviral therapy. Based on guidance recommendations, treatment should be prioritized in patients with advanced fibrosis or cirrhosis (Metavir score F3 to F4). While acknowledging the importance of this recommendation, the symposium panelists also argued that making effective decisions about whom, and when, to treat requires a more comprehensive clinical approach to patient assessment and adjusting recommended priorities according to individual patient considerations. This approach involves evaluating outcomes such as extrahepatic complications, including those affecting quality of life, functional status, and work productivity. In response to questions regarding decisions about DAA therapy duration based on viral load, the panel engaged the audience in thinking critically about evidence-based cutoff values and natural fluctuations of HCV RNA concentrations. Discussions centered on the importance of clinical judgment to ensure that the treatment duration promotes the highest efficacy and avoids risks of relapse. The panel responded to several audience questions about the role of ribavirin in new DAA regimens. Evidence-based presentations and discussions focused on patient-specific factors that must be considered to inform effective decisions about adding ribavirin. The panel took a similar approach to answering questions about emerging challenges and the difficult-to-treat populations of patients with HCV genotype 3 or advanced liver disease. The symposium concluded with presentation of, and discussion on, managed care strategies for educating patients about appropriate HCV medication use, improving adherence, and coordinating care provided by the interprofessional team. The availability of new DAAs for HCV raises new questions and challenges for managed care professionals, especially regarding prioritizing patients for immediate therapy as well as treatment and management approaches that account for the needs of individual patients and subpopulations. The educational symposium summarized in this article directly addressed key questions and challenges through presentations of evidence, guidance recommendations, and interactive discussions on the views and practices of international leaders in HCV treatment and research.

Ledipasvir and sofosbuvir for hepatitis C genotype 4: a proof-of-concept, single-centre, open-label phase 2a cohort study

Worldwide, although predominantly in low-income countries in the Middle East and Africa, up to 13% of hepatitis C virus (HCV) infections are caused by HCV genotype 4. For patients with HCV genotype 1, the combination of ledipasvir and sofosbuvir has been shown to cure high proportions of patients with excellent tolerability, but this regimen has not been assessed for the treatment of HCV genotype 4. We assessed the efficacy, safety, and tolerability of 12 weeks of combination therapy with ledipasvir and sofosbuvir for patients with chronic HCV genotype 4 infections. In this single-centre, open-label cohort, phase 2a trial, patients with HCV genotype 4 who were treatment naive or interferon treatment experienced (HIV-negative) were sequentially enrolled at the Clinical Center of the National Institutes of Health, Bethesda, MD, USA. We gave patients 12 weeks of ledipasvir (90 mg) and sofosbuvir (400 mg) as a single combination tablet once per day. The primary efficacy endpoint was sustained viral response at 12 weeks (SVR12), as measured by the proportion of patients with HCV RNA concentrations less than the lower limit of quantification (COBAS TaqMan HCV test, version 1.0, 43 IU/mL). The primary safety endpoint was the frequency and severity of adverse events. We did our analyses on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT01805882. Between Sept 16, 2013, and Nov 2, 2014, we recruited 21 patients. 20 (95%) of 21 patients completed 12 weeks of treatment and achieved SVR12 (95% CI 76-100), including seven patients with cirrhosis. One patient was non-adherent to study drugs and withdrew from the study, but was included in the intention-to-treat analysis. No patients discontinued treatment because of adverse events and no grade 3 or 4 adverse events occurred that were related to study medications. The most common adverse events were diarrhoea (two patients), fatigue (three patients), nausea (two patients), and upper respiratory infections (two patients). Ledipasvir and sofosbuvir treatment for 12 weeks was well tolerated by patients with HCV genotype 4 and resulted in 100% SVR for all patients who received all 12 weeks of study drugs, irrespective of previous treatment status and underlying liver fibrosis. This is the first report of a single-pill, all-oral, interferon-free, ribavirin-free treatment for patients with HCV genotype 4. NIAID, National Cancer Institute and Clinical Center Intramural Program. The study was also supported in part by a Cooperative Research and Development Agreement between NIH and Gilead Sciences.

Virological response after 6 week triple-drug regimens for hepatitis C: a proof-of-concept phase 2A cohort study

Direct-acting antiviral drugs have a high cure rate and favourable tolerability for patients with hepatitis C virus (HCV). Shorter courses could improve affordability and adherence. Sofosbuvir and ledipasvir with ribavirin have high efficacy when taken for 8 weeks but not for 6 weeks. We assessed whether the addition of a third direct-acting antiviral drug to sofosbuvir and ledipasvir would allow a shorter treatment duration. In this single-centre, open-label, phase 2A trial, we sequentially enrolled treatment-naive patients with HCV genotype 1 infection into three treatment groups: 12 weeks of sofosbuvir and ledipasvir; 6 weeks of sofosbuvir, ledipasvir, and GS-9669; or 6 weeks of sofosbuvir, ledipasvir, and GS-9451. Patients and investigators were not masked to treatment assignment. The primary endpoint was the propotion of patients with sustained viral response at 12 weeks after treatment completion (SVR12), assessed by serum HCV RNA concentrations lower than 43 IU/mL (the lower limit of quantification). We did an intention-to-treat analysis for the primary endpoint and adverse events. This study is registered with ClinicalTrials.gov, number NCT01805882. Between Jan 11, 2013, and Dec 17, 2013, we enrolled 60 patients, and sequentially assigned them into three groups of 20. We noted an SVR12 in all 20 patients (100%, 95% CI 83-100) allocated to sofosbuvir and ledipasvir for 12 weeks; in 19 (95%, 75-100) of the 20 patients allocated to sofosbuvir, ledipasvir, and GS-9669 for 6 weeks (one patient relapsed 2 weeks after completion of treatment); and in 19 (95%, 75-100%) of the 20 patients allocated to sofosbuvir, ledipasvir, and GS-9451 for 6 weeks (one patient was lost to follow-up after reaching sustained viral response at 4 weeks). Most adverse events were mild and no patients discontinued treatment. Two serious adverse events occurred (pain after a post-treatment liver biopsy and vertigo), both unrelated to study drugs. In this small proof-of-concept study, two different three-drug regimens that were given for 6 weeks resulted in high cure rates for HCV infection with excellent tolerability. Addition of a third potent direct-acting antiviral drug can reduce the duration of treatment required to achieve sustained viral response in patients with chronic HCV genotype 1 infection without cirrhosis. National Institute of Allergy and Infectious Diseases (NIAID), National Cancer Institute and Clinical Center Intramural Program, German Research Foundation, National Institutes of Health, Gilead Sciences.

Comparison of three methods for extraction of HCV RNA in sera collected from individuals with hyperlipidemia, hyperbilirubinemia and hyperglobulinemia

Efficient detection of HCV RNA in serum is critical for the diagnosis of hepatitis C virus (HCV) infection. Various nucleic acid extraction methods have been used to extract viral RNA for real-time reverse transcription PCR (RT-PCR). However, the efficiencies of extraction methods for HCV RNA in sera collected from individuals with hyperlipidemia, hyperbilirubinemia and hyperglobulinemia have not been investigated. In the present study, the efficiencies of three extraction methods, i.e., Trizol, guanidine isothiocyanate and silica nanoparticles, were evaluated and compared. All serum samples were collected from HCV-infected patients. For serum samples in which bilirubin, lipids and globulins were all within the normal range, the medians of HCV RNA concentration with Trizol, guanidine isothiocyanate and silica method were 2.25×104, 2.80×104 and 3.26×105IU/ml HCV RNA respectively (n=180). For hyperlipidemia serum samples, the medians were 6.70×103, 8.79×103 and 1.10×106 respectively (n=158). For hyperbilirubinemia serum samples, the medians were 5.71×104, 1.59×105 and 1.09×106 respectively (n=107). For hyperglobulinemia serum samples, the medians were 3.44×104, 3.10×104 and 3.06×105 respectively (n=71). The medians were highest with silica method from all these types of serum samples. The silica method is, therefore, efficient for HCV RNA extraction even for sera from hyperlipidemia, hyperbilirubinemia and hyperglobulinemia patients.

Efficacy and safety of 12 weeks versus 18 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and patients with previous null response with or without cirrhosis (C-WORTHY): a randomised, open-label phase 2 trial

There is a high medical need for an interferon-free, all-oral, short-duration therapy for hepatitis C virus (HCV) that is highly effective across diverse patient populations, including patients with cirrhosis or previous null response to pegylated interferon (peginterferon) plus ribavirin (PR-null responders). We aimed to assess the efficacy, safety, and effective treatment duration of grazoprevir (an HCV NS3/4A protease inhibitor) combined with elbasvir (an HCV NS5A inhibitor) with or without ribavirin in patients with HCV genotype 1 infection with baseline characteristics of poor response. The C-WORTHY trial is a randomised, open-label phase 2 trial of grazoprevir plus elbasvir with or without ribavirin; here we report findings for two cohorts of previously untreated patients with cirrhosis (cohort 1) and those with previous PR-null response with or without cirrhosis (cohort 2) enrolled in part B of the study. Eligible patients were adults aged 18 years or older with chronic HCV genotype 1 infection and HCV RNA concentrations of 10 000 IU/mL or higher in peripheral blood. We randomly assigned patients to receive grazoprevir (100 mg daily) and elbasvir (50 mg daily) with or without ribavirin for 12 or 18 weeks. Randomisation was done centrally with an interactive voice response system; patients and study investigators were masked to treatment duration up to week 12 but not to treatment allocation. The primary endpoint was the proportion of patients achieving HCV RNA less than 25 IU/mL at 12 weeks after end of treatment (SVR12), assessed by COBAS TaqMan version 2.0. This study is registered with ClinicalTrials.gov, number NCT01717326. We describe findings for 253 patients enrolled in cohort 1 (n=123) or cohort 2 (n=130). In cohort 1, we randomly assigned 60 patients to the 12-week regimen (31 with ribavirin and 29 with no ribavirin) and 63 to the 18-week regimen (32 with ribavirin and 31 with no ribavirin); in cohort 2, we randomly assigned 65 patients to the 12-week regimen (32 with ribavirin and 33 with no ribavirin) and 65 to the 18-week regimen (33 with ribavirin and 32 with no ribavirin. High SVR12 rates were achieved irrespective of the use of ribavirin or extension of the treatment duration from 12 to 18 weeks; SVR12 rates ranged from 90% (95% CI 74-98; 28/31; cohort 1, 12 weeks, ribavirin-containing) to 100% (95% CI 89-100; 33/33; cohort 2, 18 weeks, ribavirin-containing). Among patients treated for 12 weeks with grazoprevir plus elbasvir without ribavirin, 97% (95% CI 82-100, 28/29) of patients in cohort 1 and 91% (76-98, 30/33) of patients in cohort 2 achieved SVR12. Adverse events reported in more than 10% of patients were fatigue (66 patients, 26% [95% CI 21-32]), headache (58 patients, 23% [95% CI 18-29]), and asthenia (35 patients, 14% [95% CI 10-19]). Treatment with grazoprevir plus elbasvir, both with and without ribavirin and for both 12 and 18 weeks' treatment duration, showed high rates of efficacy in previously untreated patients with cirrhosis and previous PR-null responders with and without cirrhosis. These results support the phase 3 development of grazoprevir plus elbasvir. Merck & Co, Inc.

Pharmacokinetics and Pharmacodynamics of Setrobuvir, an Orally Administered Hepatitis C Virus Non-Nucleoside Analogue Inhibitor

PurposeNew antiviral agents with activity against hepatitis C virus (HCV) are needed to optimize treatment for chronic hepatitis C (CHC). We evaluated the pharmacokinetics of setrobuvir (a non-nucleoside HCV polymerase inhibitor) in healthy volunteers (study 1 & 2) and its antiviral efficacy in patients with genotype 1, noncirrhotic treatment-naive CHC (study 3). MethodsThree studies investigated the pharmacokinetics and pharmacodynamics of setrobuvir. First, sequential cohorts of volunteers were randomly assigned to receive single oral doses of setrobuvir 400 to 3000 mg or placebo in a double-blind, ascending dose study. In the second study, volunteers were randomly assigned to receive multiple doses of setrobuvir (400 or 800 mg once daily [QD] or 600 mg twice a day [BID]). In the third study, patients with genotype 1 CHC received setrobuvir (200, 400, or 800 mg) or placebo BID for 3 days. FindingsAfter single doses of setrobuvir (400–3000 mg) to volunteers in a fasted state, peak Cmax and AUC0–∞ increased in a less than dose-proportional manner. The mean apparent t½z ranged from 22.0 to 31.3 hours and was not dose related. Cmax and AUC increased significantly (4.3- and 6.3-fold, respectively) in volunteers who received 2000 mg with a high-fat meal versus fasting. After multiple oral doses, steady state was achieved after 7 days of dosing (400 or 800 mg QD and 600 mg BID) and accumulation was dose-independent. Mean day 14 plasma exposure increased in a less than dose-proportional manner in volunteers who received 400 and 800 mg QD, but it was more than dose-proportional in volunteers receiving 600 mg BID. Dose did not affect the mean t½z (range, 24.1–26.6 hours), apparent oral clearance (0.254–0.516 L/h), or apparent volume of distribution (9.60–18.1 L). In patients with CHC, dose-related reductions in HCV RNA concentration were apparent within 24 hours of the start of treatment. Reductions from baseline to the end of treatment (day 3) in patients treated with setrobuvir 200, 400, and 800 mg BID were –2.1, –2.2, and –2.9 log10 IU/mL, respectively (vs ≤0.1 log10 IU/mL with placebo). Reductions in HCV RNA were greater in patients with genotype 1b (range, –2.7 to –3.1 log10 IU/mL) than in patients with genotype 1a (range, –1.3 to –2.7 log10 IU/mL). Setrobuvir was well tolerated, with no serious adverse events. ImplicationsThe steady state pharmacokinetics of setrobuvir appear to be dose proportional, and setrobuvir produces a mean reduction of 2.9 log10 IU/mL in HCV RNA over 3 days in patients with genotype 1 (a and b) treated with 800 mg BID. ClinicalTrials.gov identifier: NCT00782353