TaqMan Hepatitis C Virus Research Articles

Prevalence of Hepatitis C Viral Infection among Diabetes Mellitus Patients in Qassim Region, Saudi Arabia

The worldwide prevalence of Diabetes Mellitus (DM) associated with Hepatitis C Virus (HCV) infection are reported with higher rates of morbidity and mortality. The frequency of HCV is approximately 3-4 million cases each year and in parallel the incidence of DM is increasing alarmingly. World Health Organization (WHO) has specified that DM will be the 7th leading cause of mortality by 2030. The increasing association between HCV and DM has been indicated by some significant reports recently. HCV infection leads to hepatic steatosis and rapid insulin resistance, which in turn upsurges the risk factors for hepatic fibrosis and hepatocellular carcinoma. This study is designed to examine the association between HCV and DM, and different risk factors associated with HCV infection in Qassim region, Kingdom of Saudi Arabia (KSA). A total of 634 blood samples were obtained from diabetic and non-diabetic patients. These blood samples were first screened for HCV infection by enzyme-linked immunosorbent assay (ELISA) and positive samples were again confirmed by TaqMan HCV quantitative test and the viral load in different samples was estimated. The HCV prevalence was identified as 2.5% in diabetic patients with a positive association between HCV and DM (RR= 1.24, OR= 1.77) which is not significant statistically. However, the HCV prevalence among diabetic females was significantly different from males (p<0.05). The behavioural factors had no significant impact to acquire HCV infection. This study indicated a positive association between HCV and DM. Gender was an association factor in the HCV and DM status. Further studies with larger sample size is significant to properly assess the temporal relationship between HCV and DM.

Rescue use of Pegylated Interferon in Dialysis Patient who Failed to Respond Sofosbuvir.

Rescue use of Pegylated Interferon in Dialysis Patient who Failed to Respond Sofosbuvir.

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.

Molecular epidemiology and viral load analysis of hepatitis C virus genotypes from Sindh, Pakistan

The present study was aimed to assess the molecular epidemiology of Hepatitis C Virus (HCV) genotypes and viral load (VL) of HCV infected patients. A total of 261 anti-HCV positive patients were enrolled for VL analysis using COBAS AmpliPrep/COBAS TaqMan HCV and Abbott Real Time HCV Genotype II Assay Kit to determine the genotypes. The data demonstrated that 60.15% (n=157) samples were from females and 39.85% (n=104) from male patients. Serum markers such as Bilirubin, SGPT, PT, APTT, and α-fetoproteins were analyzed by commercial kits and Cobas-C 501, Sysmex Ca500 and Cobas-C 601 automatic analyzers as per manufacturer’s guidelines. All patients were above 15 years age and their mean age was 39 years. Male to female ratio was 0.66% (104/157). Age-wise ddistribution revealed that 31-45 years age group comprised majority of the patients, whereas lowest number belonged to age group 61 and above. HCV genotyping data revealed five different genotypes/subtypes. Genotype 3a was most frequent accounting 76.24% (n=199) followed by Genotype 1a (2.29%), 1b (2.68%), 2a (4.98%). Moreover, 13.79% (n=36) samples revealed untypable genotype. Subtype 1b had highest mean VL of 6.84 log10 IU/ml among all other genotypes. The data of serum markers analysis revealed a slight fluctuation but appeared under the normal range. However, Bilirubin was slightly lower in Genotype 1a as compared with other genotypes while elevated α-fetoprotein was found in patients infected with untypable genotypes. In summary, Genotype 3a was predominant genotype throughout Sindh and subtype 1b had highest viral load compared with subtypes 3a and 2a. Keywords: Genotyping; Hepatitis C; HCV; Prevalence; Serum markers http://dx.doi.org/10.19045/bspab.2021.100037

Fully automated rapid quantification of Hepatitis C Virus RNA in human plasma and serum by integrated on-chip RT-qPCR and capillary electrophoresis

The quantification of hepatitis C virus (HCV) is essential for the management of chronic hepatitis C therapy. We have developed a fully automated microfluidic RT-qPCR system for rapid quantitative detection of HCV RNA in human EDTA-plasma and serum, and the performance of the method was assessed. The platform for the assay, µTASWako g1 Fully Automated Genetic Analyzer, performs automated sample preparation and RNA extraction, followed by amplification and detection on an integrated RT-qPCR-CE (capillary electrophoresis (CE)) microfluidic chip. The total assay time from sample input to data output is less than 120 minutes. The HCV assay has a linear quantitative range of 15 to 107 IU/mL, with a limit of detection (LOD) of 10.65 IU/mL in EDTA-plasma and 12.43 IU/mL in serum. The assay has a reproducibility of SD ≤ 0.16 log10 IU/mL and an accuracy of ≤ 0.22 log10 IU/mL difference when compared to the assigned values. The main HCV genotypes 1 to 6 are detected with an accuracy of ± 0.3 log10 IU/mL. The assay is specific for HCV RNA and is free of interference from non-HCV pathogens, elevated levels of anti-viral and anti-bacterial drugs, and common endogenous interferents. In the linear quantitative range, the assay is highly correlated with the Roche cobas AmpliPrep/cobas TaqMan HCV Test, version 2.0 (r2 = 0.949). As the assay is highly sensitive, accurate and specific, and provides reliable quantification of HCV in plasma and serum, it can potentially be applicable for monitoring the therapy and management of HCV infection.

The new Xpert HCV viral load real-time PCR assay accurately quantifies hepatitis C virus RNA in serum and whole-blood specimens

Background: Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification are essential to diagnose and monitor the virological response to antiviral treatment and the emergence of resistance. Objective and study design: The aim of this study was to assess the ability of the new Xpert HCV Viral Load assay to accurately detect and quantify HCV RNA in serum and in whole blood collected on dried blood spot (DBS). Serum and whole blood from a large series of patients chronically infected with different HCV genotypes were tested in parallel for HCV RNA detection and quantification. Results: A significant relationship between HCV RNA levels measured with the Xpert HCV Viral Load assay and the two commercial real-time PCR comparators (Abbott RealTime HCV test and Cobas AmpliPrep/Cobas Taqman HCV 2.0 test) was found in serum as well as in whole blood specimens. Conclusions: The Xpert HCV Viral Load assay accurately quantifies HCV RNA regardless of the HCV genotype and can thus confidently be used to detect active HCV infection in serum and in whole blood specimens.

36 Institutional Adherence to CDC Guidelines for Hepatitis C Virus Screening Reveals Added Utility of Confirmatory Molecular Testing

Current Centers for Disease Control and Prevention (CDC) recommendations for hepatitis C virus (HCV) screening include confirmation of reactive HCV antibody (HCVAB) results for all patients and confirmation of nonreactive results for patients who may have been exposed within the past six months. Our aim was to assess adherence to these recommendations at a 1,250-bed tertiary care hospital in the Midwestern United States. In cases for which confirmatory testing was performed, our secondary aim was to determine the accuracy of the HCVAB screen for the assessment of active HCV infection. We retrospectively identified consecutive HCVAB samples tested by the ADVIA Centaur Immunoassay System over a period of seven years (January 2010 to December 2016) and by the COBAS AmpliPrep/COBAS TaqMan HCV RNA viral load assay (HCVRNA) over the same time period. The HCVAB test results were recorded as well as the percentage of reactive and nonreactive results that received confirmatory testing within 30 days. When both tests were performed, we calculated the concordance rates. Using the HCVRNA as the gold standard, we calculated the positive predictive values (PPVs) and negative predictive values (NPVs) of the HCVAB for the detection of active HCV disease within the patient population receiving both tests within 30 days of each other and within the patient population receiving both tests within one day of each other. Over the seven-year period, 66,066 samples were tested for HCVAB; 61,976 (93.8%) were negative, and 4,090 (6.2%) were positive. Of the 4090 HCVAB samples with positive results, 3,607 HCVAB samples underwent HCVRNA testing within 30 days, suggesting 88.2% adherence to CDC guidance for confirmation of positive HCVAB results. Of the 61,976 negative HCVAB results, 1,888 (3.0%) received confirmatory HCVRNA testing within 30 days. Among the 5,495 HCVAB samples followed up by HCVRNA testing within 30 days, there were 892 false-positive (HCVAB-positive, HCVRNA-negative) and 171 false-negative (HCVAB-negative, HCVRNA-positive) results, leading to a HCVAB PPV/NPV of 75.3%/90.6%. When the analysis was limited to HCVAB/HCVRNA test pairs that were taken within 1 day of each other (n = 1,790), the PPV/NPV improved to 78.6%/96.0%. The adherence to CDC confirmatory recommendations for patients with positive HCVAB results is high (88.2%), and a large proportion of patients with negative HCVAB results do not receive additional testing (97.0%). A small but significant proportion of patients with negative HCVAB tests will have a positive viral load within 30 days and even within one day. Although repeat testing in our study was likely to occur primarily among high-risk patients, creating the potential for bias, these findings suggest that patients with ongoing risk factors for HCV should receive follow-up testing with HCVRNA quantification.

Treatment of Acute Hepatitis C With Ledipasvir and Sofosbuvir in Patients With Hematological Malignancies Allows Early Re-start of Chemotherapy

Treatment of Acute Hepatitis C With Ledipasvir and Sofosbuvir in Patients With Hematological Malignancies Allows Early Re-start of Chemotherapy

Stevens-Johnson Syndrome in a Chronic Hepatitis C Patient Treated With Zepatier (Elbasvir/Grazoprevir)

Introduction: Stevens-Johnson syndrome (SJS) is an acute life threatening skin reaction involving mucocutaneous membranes. About 80 to 90% cases are drug induced. The recent introduction of new direct acting antiviral (DAA) agents has commenced a new era of safe and effective treatment of chronic Hepatitis C - previously known to be a difficult-to-treat lifelong infection. While SJS has been reported with the use of first generation DAA agents such as telaprevir, no known cases have been reported till date for patients on Zeptaier (Elbasvir/Grazosprevir combination therapy) for chronic Hepatitis C virus (HCV) infection. We report a case of SJS induced by Elbasvir/Grazosprevir in a patient with Chronic Hepatitis C and HIV.Figure: Mucocutaneous findings.Case Description: A 53-year old male with past history of chronic HCV and HIV presented with a 3-day history of fever, malaise, progressively worsening cutaneous symptoms which included- bilateral periorbital swelling and redness, painful oral ulcers and skin rash. Rash was concentrated on the upper body and was spreading caudally. Rash consisted of erythematous pruritic tender papules and vesicles. Patient had recently started taking Elbasvir/Grazosprevir to treat chronic HCV three weeks ago. He was also on Abacavir/dolutegravir/lamivudine (Triumeq) therapy for HIV with recent non-detectable HIV RNA levels and a CD4 count of 366. His pre-treatment HCV RNA count was 1,780,000 IU/mL (COBAS TaqMan HCV Test version 2.0). He had F3 fibrosis stage via FibroSure testing. Patient's presentation was attributed to early drug induced SJS from Zepatier use. Zepatier was discontinued and patient treated with steroids and antihistamines. He had complete resolution of his symptoms after two weeks. Discussion: Zepatier is a combination drug with Elbasvir, an NS5A inhibitor and Grasoprevir, an NS3/4A protease inhibitor, which was recently approved by the FDA for the treatment of chronic Hepatitis C in patients with genotype 1 and 4. This approval follows several clinical trials establishing the safety and efficacy of Elbasvir/Grasoprevir combination demonstrating a sustained virological response 12 weeks after completing treatment (SVR-12) greater than 92% and a minimal side effect profile. Most common side effects in different trials included headache, nausea and fatigue. In the C-EDGE trial, rash or pruritus was noted in 4% of patients on Elbasvir/Grasoprevir with Ribavirin regimen. However, no rash or pruritus was reported in patients on Elbasvir/ Grasoprevir regimen alone. To the best of our knowledge, serious adverse cutaneous reaction associated with Elbasvir/Grasoprevir therapy has not been previously reported. Possibility of adverse cutaneous drug reaction with Elbasvir/ Grasoprevir use has significant clinical implications. Patients with HIV seem to have a higher incidence of SJS than the general population. Clinicians need to be highly vigilant for cutaneous side effects while treating patients with HIV and HCV with Elbasvir/Grasoprevir combination for chronic HCV treatment. Because of significant mortality associated with SJS - early recognition, offending drug discontinuation and prompt treatment are warranted.

Analytical and clinical performance of the Hologic Aptima HCV Quant Dx Assay for the quantification of HCV RNA in plasma samples

BackgroundChronic hepatitis C virus (HCV) infection can be effectively treated with directly acting antiviral (DAA) therapy. Measurement of HCV RNA is used to evaluate patient compliance and virological response during and after treatment. ObjectivesTo compare the analytical performance of the Aptima HCV Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HCV Test v2.0 (CAPCTMv2) for the quantification of HCV RNA in plasma samples, and compare the clinical utility of the two tests in patients undergoing treatment with DAA therapy. Study designAnalytical performance was evaluated on two sets of plasma samples: 125 genotyped samples and 172 samples referred for quantification of HCV RNA. Furthermore, performance was evaluated using dilutions series of four samples containing HCV genotype 1a, 2b, 3a, and 4a, respectively. Clinical utility was evaluated on 118 plasma samples obtained from 13 patients undergoing treatment with DAAs. ResultsDeming regression of results from 187 plasma samples with HCV RNA >2 Log IU/mL indicated that the Aptima assay quantified higher than the CAPCTMv2 test for HCV RNA >4.9 Log IU/mL. The linearity of the Aptima assay was excellent across dilution series of four HCV genotypes (slope of the regression line: 1.00-1.02). The Aptima assay detected significantly more replicates below targeted 2 Log IU/mL than the CAPCTMv2 test, and yielded clearly interpretable results when used to analyze samples from patients treated with DAAs. ConclusionsThe analytical performance of the Aptima assay makes it well suited for monitoring patients with chronic HCV infection undergoing antiviral treatment.

Determination of drug resistance mutations of NS3 inhibitors in chronic hepatitis C patients infected with genotype 1

Direct-acting antiviral agents (DAA) such as NS3 protease inhibitors is the first class of drugs used for chronic hepatitis C (CHC) treatment. NS3 inhibitors (PI) with low genetic barrier have been approved to be used in the CHC genotype 1 infections, and in the treatment of compensated liver disease including cirrhosis together with pegile interferon and ribavirin. Consequently, the development of drug resistance during DAA treatment of CHC is a major problem. NS3 resistant variants can be detected before treatment as they can occurnaturally. The aim of this study was to investigate new and old generation NS3 inhibitors resistance mutations before DAA treatment in hepatitis C virus (HCV) that were isolated from CHC. The present study was conducted in 2015 and included 97 naive DAA patients infected with HCV genotype 1, who were diagnosed in Manisa and Kocaeli cities of Turkey. Magnetic particle based HCV RNA extraction and than RNA detection and quantification were performed using commercial real-time PCR assay QIASypmhony + Rotorgene Q/ArtusHCV QS-RGQ and COBAS Ampliprep/COBAS TaqMan HCV Tests. HCV NS3 viral protease genome region was amplified with PCR and mutation analysis was performed by Sanger dideoxy sequencing technique of NS3 protease codons (codon 32-185). HCV NS3 protease inhibitors; asunaprevir, boceprevir, faldaprevir, grazoprevir, pariteprevir, simeprevir and telaprevir were analysed for resistant mutations by Geno2pheno-HCV resistance tool. HCV was genotyped in all patients and 88 patients (n= 88/97, 91%) had genotype 1. Eight (n= 8/97, 8.2%) and 80 (n= 80/97, 82.4%) HCC patients were subgenotyped as 1a and 1b, respectively. Many aminoacid substitutions and resistance mutations were determined in 39/88 (44%) patients in the study group. Q80L, S122C/N, S138W were defined as potential substitutions (6/88 patients; 7%); R109K, R117C, S122G, I132V, I170V, N174S were described as potential resistance (34/88 patients; 39%); V36L, T54S, V55A, Q80H were characterized as resistance (7/88 patients; 8%) and Q80K, A156S were defined as high resistance (3/88 patients; 3%) mutations. Based on resistance and high resistance mutations, clinically significant mutations were defined in 10/88 (11%) of the patients. Our study shows that it is essential to analyse HCV NS3 protease inhibitors drug resistance before DAA treatment of CHC patients. On the other hand, our results pointed out that analysis of NS5A and NS5B genome region mutations may also be required in the near future.

The New Aptima HCV Quant Dx Real-time TMA Assay Accurately Quantifies Hepatitis C Virus Genotype 1-6 RNA

BackgroundSensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. Objectives and study designThe aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. ResultsThe limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). Conclusionsthe new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US.

Comparison of on-treatment HCV RNA during direct antiviral therapy using two different COBAS TaqMan HCV assays

BackgroundRepeated measurements of hepatitis C virus (HCV) RNA levels during antiviral therapy are recommended to monitor treatment efficacy and adherence. Throughout most direct antiviral agent (DAA) approval studies, HCV RNA cutoffs and endpoints were established with the COBAS TaqMan assay for use with the High Pure System (HPS/CTM). Different assays used in clinical practice may yield different quantitative results and possibly impact treatment decisions. ObjectivesThe concordance of the fully-automated COBAS AmpliPrep/COBAS TaqMan assay (CAP/CTM) with HPS/CTM and its ability to predict response to DAA-treatment with ledipasvir/sofosbuvir was assessed in cirrhotic patients with HCV genotype-1-infection who had failed prior treatment with protease inhibitor-based regimens. Study designSerum samples from patients (n=154) treated in the phase-2 SIRIUS-study were collected at baseline and during antiviral therapy (weeks 1–8), and were tested in parallel by both assays. ResultsThe mean difference between HPS/CTM and CAP/CTM at baseline (n=153) was 0.32 log10 IU/mL HCV RNA. Discordant results were observed in 12% of samples collected at treatment weeks 1–8, with the greatest differences observed at weeks 2 and 4 (14% and 29%, respectively, for undetectable HCV RNA). SVR rates were 96%–97% in the study and were not significantly different between patients with detectable vs. undetectable HCV RNA according to both assays at weeks 1–4 of antiviral therapy. ConclusionsCAP/CTM and HPS/CTM showed significantly different response rates during the early stages of ledipasvir/sofosbuvir treatment. However, on-treatment response was not predictive of SVR with either assay, indicating that determination of on-treatment HCV RNA levels may not be useful to guide treatment decisions.

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.

Efficacy and safety of 3-week response-guided triple direct-acting antiviral therapy for chronic hepatitis C infection: a phase 2, open-label, proof-of-concept study

To shorten the course of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, we examined the antiviral efficacy and safety of 3 weeks of response-guided therapy with an NS3 protease inhibitor and dual NS5A inhibitor-NS5B nucleotide analogue. In this open-label, phase 2a, single centre study, Chinese patients with chronic HCV genotype 1b infection without cirrhosis were randomly allocated by a computer program to one of three treatment groups (sofosbuvir, ledipasvir, and asunaprevir; sofosbuvir, daclatasvir, and simeprevir; or sofosbuvir, daclatasvir, and asunaprevir) until six patients in each group (1:1:1) achieved an ultrarapid virological response (plasma HCV RNA <500 IU/mL by day 2, measured by COBAS TaqMan HCV test, version 2.0). Patients with an ultrarapid virological response received 3 weeks of therapy. Patients who did not achieve an ultrarapid response were switched to sofosbuvir and ledipasvir for either 8 weeks or 12 weeks. The primary endpoint was the proportion of patients with a sustained virological response at 12 weeks (SVR12) after treatment completion, analysed in the intention-to-treat population. All patients who achieved an ultrarapid virological response were included in the safety analysis. This trial is registered with ClinicalTrials.gov, number NCT02470858. Between April 5, 2015, and April 15, 2015, 26 eligible patients were recruited. 12 patients were assigned to sofosbuvir, ledipasvir, and asunaprevir; six to sofosbuvir, daclatasvir, and simeprevir; and eight to sofosbuvir, daclatasvir, and asunaprevir. Six patients in each group achieved an ultrarapid virological response (18 [69%]). All patients with an ultrarapid virological response who were given 3 weeks of triple therapy achieved SVR12. The most common adverse events were fatigue (one [17%] of six patients receiving sofosbuvir, ledipasvir, and asunaprevir; one [17%] of six patients receiving sofosbuvir, daclatasvir, and simeprevir; and two [33%] of six patients receiving sofosbuvir, daclatasvir, and asunaprevir) and headache (one [17%] patient in each group). No patients experienced any serious adverse events. In this proof-of-concept study, all patients with chronic HCV without cirrhosis who achieved an ultrarapid virological response on triple direct-acting antiviral regimens by day 2 and received 3 weeks of treatment were cured, with excellent tolerability. By shortening the duration of therapy from the currently recommended 12 weeks to 3 weeks, we could drastically reduce the cost of therapy and the rate of adverse events. Further large-scale studies should be done to confirm our findings. Center for AIDS Research, National Institutes of Health, US Department of Energy, National Center for Research Resources and the Office of Research Infrastructure Programs, Cheng Si-Yuan (China-International) Hepatitis Research Foundation, and Humanity and Health Medical Group.

Clinical efficacy of the highly sensitive hepatitis C virus RNA quantitative assay in patients with relapse following interferon-based therapy with second-generation direct-acting antivirals.

For refractory chronic hepatitis C, interferon (IFN)-based triple-agent combination therapy with second-generation direct-acting antivirals (DAAs) has been established as the standard treatment method. The rate of decrease in the viral load and the negative conversion of hepatitis C virus (HCV) RNA in the early phase following treatment initiation are considered important factors for predicting the therapeutic outcome. In the present study, the Roche Cobas AmpliPrep/COBAS TaqMan (CAP/CTM) HCV v2.0 assay and the AccuGENE m-HCV RNA quantitative assay [Abbott RealTime HCV (ART) assay] were analyzed for their clinical efficacy and ability to predict therapeutic outcomes in the early phase in patients with relapse following IFN-based second-generation DAA therapy. Of the 56 patients who received IFN-based second-generation DAA therapy since December 2013, 6 achieved an end-of-treatment response (ETR), but subsequently experienced relapse. In these 6 patients, fluctuations in viral loads in the early phase detected by the CAP/CTM and ART assays were compared. At 4 weeks after treatment initiation, 4 of the 6 patients were diagnosed as negative by the CAP/CTM assay, whereas 2 of these 4 patients were not identified as negative by the ART assay. Of the 2 patients, one was signal-positive with an HCV RNA load <1.08 Log IU/ml, and the other patient had a viral load of 1.12 Log IU/ml. At 8 weeks after treatment initiation, 1 patient was found to be negative by the CAP/CTM assay, but signal-positive with a viral load <1.08 Log IU/ml by the ART assay. From 4 to 8 weeks after treatment initiation, 3 of the 6 patients appeared to be discrepant cases. In conclusion, of the 6 patients who achieved an ETR, 4 were determined to have achieved a rapid virological response (RVR) by the CAP/CTM assay, but may not have actually become negative. The ART assay is highly sensitive, has a wide measurement range, may be suitable for monitoring HCV RNA loads, and is expected to have an important role in providing a predictive marker for early therapeutic outcomes. In discrepant cases in which no RVR is proved by either assay, it was assumed important to consider continuation of treatment and to attempt to achieve a sustained virological response.

Impact of HCV kinetics on treatment outcome differs by the type of real-time HCV assay in NS3/4A protease inhibitor-based triple therapy

Repeated measurement of the HCV RNA level is essential for properly monitoring treatment efficacy. The aim of this study was to determine the utility of two HCV real-time assays in the evaluation of the impact of hepatitis C virus (HCV) kinetics on the outcome of triple therapy with NS3/4A protease inhibitors (PIs), telaprevir or simeprevir. This study consisted of 171 Japanese patients infected with HCV genotype 1. All 3266 serum samples taken during and post treatment were tested with both the COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HCV Test v2.0 and the Abbott RealTime (ART) HCV Test. Of the 2597 samples undetectable (lower limit of detection [<LOD]) for HCV RNA by the CAP/CTM assay from the on and post treatment, 400 (15.4%) (369 detectable/less than the lower limitation of quantification [<LLOQ] and 31 quantifiable) were detectable by the ART assay. HCV RNA < LOD within the first four weeks by ART was associated with sustained virological response (SVR) for the difficult-to-treat group that included patients with advanced fibrosis or prior partial/null response. In contrast, for the non-difficult-to-treat group, almost all of the late responders by ART achieved SVR, unlike by CAP/CTM. Despite HCV RNA being once < LOD by ART, 33.1% patients experienced the reappearance of residual HCV RNA (detectable/<LLOQ) during treatment. This event in the first 12 weeks (with PI-treatment period) was not related to treatment failure, however, relapse was observed in all patients with a reappearance of residual HCV RNA after 12 weeks (without PI-treatment period). The superior ability to detect low-level HCV RNA by ART could be useful for predicting SVR by difficult-to-treat patients in the early period and relapse in the late period.

Use of chimeric influenza viruses as a novel internal control for diagnostic rRT-PCR assays.

Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5′ untranslated region (5′ UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays.

Use of parenteral caffeinum natrio-benzoicum: an underestimated risk factor for HCV transmission in China.

BackgroundFuyu city in China has a high prevalence of hepatitis C virus (HCV) infection resulting in a high morbidity and mortality from chronic liver disease and hepatocellular carcinoma. This study was conducted to identify the risk factors for HCV infection in Fuyu city.MethodsRecruitment of study subjects involved a cross-sectional survey using non-random, convenience sampling. Information on demographic variables, risk factors for HCV infection, clinical manifestations, behavioral practices and family history was collected by administering a questionnaire. Anti-HCV antibody was detected using Abbott ARCHITECT i2000SR. HCV infection was confirmed by HCV-RNA testing by the Roche Taqman HCV test. Univariate and multivariate analyses were performed to identify the factors associated with HCV infection.ResultsOut of 3,228 persons that participated in the survey, 3,219 were enrolled in the study. The prevalence of HCV infection was 42.1 % (1355/3219). Among 734 patients with chronic HCV infection whose HCV-RNA genotyping was performed, genotype 1b was the most common (58.0 %), followed by genotype 2a (40.2 %), while co-infection with genotypes 1b and 2a was detected in 1.8 % of the subjects. On univariate analysis, male gender, older age, parenteral caffeinum natrio-benzoicum and share syringes (PCNBSS), and nine other factors were significantly associated with HCV infection. After adjusting for potential confounders, male gender, old age, cigarette smoking, lower education level, history of blood transfusion, blood donation, prior dental surgery, and PCNBSS were found to be independently associated with HCV infection.ConclusionsThe prevalence of HCV infection is likely to be high among residents in Fuyu and we observed that genotypes 1b and 2a dominated in the city. Our findings support the hypothesis that PCNBSS which became endemic in Fuyu city during 1970s-1980s is strongly associated with HCV positivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12889-015-2299-8) contains supplementary material, which is available to authorized users.

P11.23 Evaluation of hepatitis c virus in the rectum of hiv-infected men who have sex with men

Introduction An epidemic of Hepatitis C Virus (HCV) infection is occurring in HIV-infected men who have sex with men (MSM). Epidemiological studies suggest that sexual transmission is fueling the epidemic. Blood and semen have been considered as potential mechanisms of transmission, but there are still transmission circumstances that remain unexplained. Nothing is known about the presence of HCV in the rectal fluid of HCV-infected MSM. Methods Written informed consent was obtained from 45 HIV-infected MSM with HCV infection. A moistened swab was inserted atraumatically into the rectum, placed into transport medium, vortexed, and the supernatant analysed for HCV using AmpiPrep/COBAS TaqMan HCV test (Roche Diagnostics), lower limit of quantification 43 IU/mL, lower limit of detection 7 IU/mL. Results Successful virological analysis was performed on 43 rectal swabs. HCV was detected in 20 out of 43 (47%) specimens. The HCV viral load (VL) in the rectal fluid ranged from 10 IU/mL. Rectal HCV detection was associated with serum HCV VL >5 log 10 IU/mL (p = 0.011). The magnitude of paired rectal and serum HCV VLs were strongly correlated (correlation coefficient 0.688, p Conclusion This study provides the first documentation of the presence of HCV in non-blood rectal fluid. It is plausible that the combination of bathing of an inserted penis in rectal HCV, with the attendant friction of anal intercourse, could result in penetration of HCV into the inserted penis. The protection to the penis afforded by a condom would logically mitigate the risk of transmission by this mechanism. This study should inform public health policy concerning the primary prevention of sexually transmitted HCV. Disclosure of interest statement There are no competing or financial interests to disclose.