COBAS TaqMan Hepatitis C Virus Research Articles

Effectiveness of the HCV blood screening strategy through eighteen years of surveillance of HCV infection in blood donors in France.

The question of maintaining blood screening based on both Hepatitis C virus (HCV) infection antibodies (Ab) and Nucleic Acid Testing (NAT) has been raised in several countries. The French blood donor surveillance database was used to address this issue. In France, HCV-NAT was implemented in mini pools (MP) in 2001 and in individual testing (ID) in 2010. HCV-positive donations are further investigated including detection of RNA with an alternative polymerase chain reaction assay: Amplicor HCV v2.0 (Roche; LOD95 50 IU/mL) from 2001 to 2006 and CobasTaqMan (CTM) HCV 2.0 assay (Roche; LOD95 9.3 IU/mL) since 2007. From 2001 to 2018, 3,058/48.8 million donations were confirmed HCV positive: 64.4% were Ab+/NAT+, 35.1% Ab+/NAT- and 0.5% Ab-/NAT+. From 2001 to 2018, the NAT yield decreased from 0.65 per million donations to 0, and NAT+ donations dropped from 77% to 46% of the total of HCV donations. 2,491/3,058 were further tested for HCV-RNA: 1,032 (816 NAT+, 216 NAT-) with Amplicor and 1,459 (897 NAT+, 562 NAT-) with CTM. Four (3 MP and 1 ID-NAT, 0.5%) of the 778 NAT negative donations had low viral loads. The decline in HCV-NAT yield cases raises the question of the relevance of NAT. Conversely, the increase in Ab+/NAT-donors, suggesting a growing number of resolved infections, argue for Ab discontinuation. In our experience, at least 0.5% of Ab+/NAT-donations had low RNA level when retested. Although the risk of viral transmission by such donations is probably low, the uncertainty associated with their infectivity goes against the removal of Ab in blood screening in our country.

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.

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.

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.

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.

Reply to Harrington et al

To the Editor—Harrington et al raise 3 important points in a detailed analysis of pooled data from 12 registrational trials of interferon-free, direct-acting antiviral (DAA) therapies [1]. First, the authors point out that hepatitis C virus (HCV) RNA was detected at the end of treatment in only 0.3% (12/3671) of patients who achieved sustained virological response 12 (SVR12) in the larger trials compared to 29% (28/96) in our studies [2]. As suggested by Harrington et al, our use of the more sensitive Abbott RealTime HCV assay could have resulted in higher rates of detectable viremia. Conversely, using the Roche COBAS TaqMan HCV test v1.0, only 3.1% (3/96) of patients who achieved SVR12 in our studies had a detectable viral load at the end of therapy. These differences may also be a result of varying treatment durations among the trials: over one-third of our patients were treated for 6 weeks, whereas the majority of subjects from the pooled data were treated for 12–24 weeks. Longer durations of therapy may certainly result in higher rates of viral suppression by the end of treatment.

Performance evaluation of the nanoquant real-time HCV assay for quantitation of hepatitis C virus RNA by real-time RT-PCR

Accurate quantitation of Hepatitis C virus (HCV) RNA level is essential for measurement of active viremia in chronic HCV-infected patients undergoing antiviral therapy. In Vietnam, two molecular assays based on real-time reverse transcription-polymerase chain reaction (RT-PCR) technique are widely used to quantify HCV RNA level: Cobas Taqman HCV (Roche) and Abbott RealTime HCV (Abbott). Recently, a third assay has been introduced for HCV RNA quantitation: NanoQuant real-time HCV. In this study, performance characteristics of this assay were evaluated using methods for validation of newly developed molecular assays for infectious diseases. Evaluating results showed that NanoQuant real-time HCV was able to quantify HCV in a linear range from 102 to 108 IU/mL with the detection limit of 28.2 IU/mL. None of the other viruses, bacteria, fungi and human genetic materials showed cross-reactivity with HCV. The assay gave no positve result with HCV-negative sera. No interference in the performance of the assay was observed in the presence of inhibitory endogenous and exogenous substances. Intra-assay and interassay coefficients of variation were 4.18% and 9.18%, respectively. All of HCV genotypes (1a, 1b, 2a, 2b, 3a, 6a, 6e, 6f) which are prevalent in Vietnam were detected by the assay. HCV quantitation results showed a good correlation with those determined with Cobas Taqman HCV (R2=0.94). These results indicate that NanoQuant real-time HCV is reliable for monitoring HCV RNA level in the treatment of patients with chronic HCV infection.

Distribution of hepatitis C virus genotypes among patients with chronic hepatitis C infection in Akdeniz University Hospital, Antalya, Turkey: a five-year evaluation

Hepatitis C virus (HCV) is one of the major causes of chronic hepatitis. It is important to know the genotypes of HCV in the decision of the HCV related chronic hepatitis therapy. The aim of this study was to evaluate the HCV genotypes determined at the Microbiology Laboratory of Akdeniz University Hospital, and to evaluate the changes in the distribution of the genotypes within the last five years. A total of 422 blood samples from HCV-RNA positive chronic hepatitis C patients (219 male, 203 female; age range: 8-79 yrs, mean age 46.3 ± 15.5 yrs) which were sent to our laboratory for genotyping between 2009-2013 period, were analyzed retrospectively. HCV-RNA extractions were performed in an automated system (EZ1 Virus Mini Kit v2.0, Qiagen, Germany), and a commercial reverse hybridization line probe-based assay (LIPA; GEN-C RT-PCR, Italy) was carried out for genotyping, For viral load determinations, a real-time PCR method (Cobas TaqMan HCV, Roche Diagnostics, Germany) was used. Demographic data of the patients were obtained from the hospital information systems and electronic patients' files. Out of the 422 patients, genotype 1b was detected in 63.3% (n= 267), genotype 1a in 14.7% (n= 62), genotype 3a in 11.1% (n= 47), genotype 2b in 0.9% (n= 4), genotype 4e in 0.2% (n= 1). The subtypes couldn't be determined for 5.4% (n= 23), 2.6% (n= 11) and 1.4% (n= 6) of the patients infected with genotype 1, 2 and 4, respectively. One (0.2%) patient, was coinfected with genotype 1 and 4. Of the patients, 40 were foreign-born (16 cases from Russia; 4 of each from Ukraine and Georgia; 3 of each from Turkmenistan, Kyrgyzstan, and Germany; one of each from Tajikistan, Azerbaijan, Uzbekistan, Chechnya, Moldova, Switzerland and Romania) and among these patients genotype 3a (19/40; 47.5%) was the most common genotype followed by genotype 1b (17/40; 42.5%). Median values of HCV viral load were 668.500 IU/ml (range: 2.000-9.630.000) in the whole group; while it was 732.000 IU/ml (range: 2.000-9.630.000) in patients infected with genotype 1 and 444.000 IU/ml (range: 2.650- 8.330.000) in patients infected with the other genotypes (p> 0.05). Patients infected with genotype 1 were found to be older than those infected with other genotypes (47 ± 15.7 and 39.5 ± 12.2, respectively; p< 0.001). Among patients infected with different genotypes, there was no statistically significant difference in terms of genders (p> 0.05). In conclusion, the determination of HCV genotypes is of crucial importance for treatment decision-making of chronic HCV infection. Besides, it also allows monitoring the changes in the epidemiology of HCV. In this study, although genotype 1b was determined as the most common HCV genotype, the detection of other genotypes was remarkable. This finding was attributed to the presence of many foreign national people in Antalya region which was a high capacity tourism area in Turkey.

Oral direct-acting antiviral therapy to prevent reinfection of the liver graft after liver transplantation for hepatitis C virus-related cirrhosis

Hepatitis C virus (HCV) remains the most common indication for orthotopic liver transplantation (OLT) and universally reinfects the new liver graft; as a result, patients with HCV have poorer outcomes than patients undergoing OLT for non–HCV-related cirrhosis.1 Recently, new direct-acting antiviral (DAA) agents have been approved for genotype 1 HCV infections in combination with peginterferon/ribavirin (RBV).2, 3 However, these therapies may be associated with morbidity and mortality when they are given to patients with advanced liver disease.4 We report a sustained response with an oral DAA/RBV combination without interferon in a 61-year-old male with HCV genotype 1b (interleukin-28B TT) who underwent OLT at our center for HCV-related cirrhosis. He presented to our center for an OLT evaluation with a Model for End-Stage Liver Disease (MELD) score of 18 as well as ascites and encephalopathy, and interferon and RBV had previously failed in 1999. Once listed, he requested therapy with peginterferon/RBV and a DAA agent while he was awaiting OLT. Because of his ascites, encephalopathy, and MELD score, this was not offered. However, his HCV RNA viral level was found to be low at 887 IU/mL (Cobas TaqMan HCV assay), so telaprevir (TVR; 750 mg every 8 hours) and RBV (1200 mg daily) were initiated. At week 1 of therapy, his HCV RNA level was <43 IU/mL (target not detected). Because of gastrointestinal side effects at week 1 (nausea and diarrhea), he was converted to boceprevir (BOC; 800 mg every 8 hours) with RBV and underwent OLT 9 weeks after the initiation of therapy with a MELD score of 19. There were no additional episodes of decompensation, and he did not require transfusions, although an RBV dose reduction to 600 mg was required. Just before surgery, he took BOC (800 mg) as well as RBV (400 mg). Twenty-four weeks after OLT, he remained a sustained responder (Fig. 1). Recent reports have demonstrated that a sustained virological response (SVR) can be achieved in HCV patients without interferon, although no studies to date have included patients with decompensated cirrhosis.5 However, to our knowledge, this represents the first successful cure of HCV in which DAA therapy without interferon was used to suppress the virus before OLT in a patient with decompensated liver disease in whom interferon was contraindicated. It should be emphasized that this case was unique because of the very low viral level and the more favorable genotype 1b infection. In addition, our patient was able to undergo OLT with a MELD score of 19, which is a level below the national median MELD score for deceased transplants in 2011.6 These factors allowed the combination of TVR and RBV followed by BOC with RBV to achieve viral suppression without the emergence of viral resistance. Because BOC may be administered for up to 44 weeks, our patient could have continued to receive BOC with RBV if no donor had been available, although it is unknown whether a breakthrough would have occurred with a longer duration of therapy. The vast majority of patients with genotype 1 infections who present for listing for OLT will have a viral level significantly higher than our patient's level (typically 104−106 IU/mL), and this level in our opinion would contraindicate our approach in this report because of the risk of treatment futility and resistance with nonstructural protein 3 protease inhibitors and RBV. However, more effective, all-oral DAA combinations with high barriers to resistance are currently being studied that can achieve high SVR rates in patients with high viral levels, and we believe the approach of DAA combinations without interferon may serve as a model for preventing reinfection of the liver graft and improving the survival of HCV-infected individuals who require OLT. Paul Y. Kwo, M.D.1 A. Joseph Tector, M.D., Ph.D.2 Departments of 1Medicine and 2Transplant SurgeryIndiana University School of MedicineIndianapolis, IN

IL-28B polymorphisms and treatment response in hepatitis C virus patients with persistently normal alanine aminotransferase

To examine the association between the interleukin 28B (IL-28B) genotype and treatment response in hepatitis C virus (HCV)-infected patients with persistently normal alanine aminotransferase (PNALT). We compared the treatment response of HCV-infected patients with PNALT to that of patients with non-PNALT. Between February 2010 and April 2013, 278 patients infected with HCV were enrolled in this study. All of the patients were treated with peginterferon-alpha 2a or 2b plus ribavirin. In addition, 180 μg of peginterferon alpha-2a or 1.5 μg/kg peginterferon alpha-2b per week plus weight-based ribavirin (600-1000 mg/d) were typically administered for 24 wk to HCV genotype 2-infected patients or for 48-72 wk to HCV genotype 1-infected patients. In all of the patients, the IL-28B rs8099917 genotype was determined using a TaqMan single-nucleotide polymorphism assay. HCV RNA was measured using the COBAS TaqMan HCV test. Female patients were dominant in the PNALT group (P < 0.0001). Among 72 HCV genotype 1-infected patients with PNALT, the early virologic response (EVR) rates (P < 0.01) and the sustained virologic response (SVR) rates (P < 0.01) were higher in patients with the IL-28B TT genotype than in those with the IL-28B TG/GG genotype. In HCV genotype 1-infected patients with PNALT, multivariate logistic-regression analysis showed that SVR was independently predicted by the IL-28B rs8099917 TT type (P < 0.05) and having an EVR (P < 0.01). The IL-28B rs8099917 TT genotype strongly correlated with treatment response in HCV genotype 1-infected Asian patients with PNALT. The IL-28B genotype may be useful for selecting HCV genotype 1-infected patients with PNALT who should receive interferon-based treatment.

Platelet count and sustained virological response in hepatitis C treatment

To examine the epidemiological data, hematological safety and treatment responses of peginterferon-alpha 2a plus ribavirin therapy for hepatitis C. Between March 2008 and February 2011, 196 hepatitis C virus (HCV) genotype 1 infected Japanese (127 treatment-naive and 69 treatment-experienced patients) patients treated with peginterferon-alpha 2a plus ribavirin were enrolled. We examined the epidemiological data and treatment responses were retrospectively analyzed in terms of hematological safety. HCV RNA was measured by the COBAS TaqMan HCV test. Overall sustained virological response (SVR) rates of treatment-naive and treatment-experienced patients were 56% and 39%, respectively. Multivariate logistic regression analysis showed that SVR was attained independently of early virological response in both treatment-naive and treatment-experienced patients. SVR rates did not differ between the pretreatment hemoglobin < 13 g/dL and ≥ 13 g/dL groups. However, in treatment-naive patients, the SVR rate of the pretreatment platelet count < 130000/µL group was significantly lower than that of the pretreatment platelet count ≥ 130000/µL group. Attention should be paid to potential thrombocytopenia in the treatment of chronic hepatitis C patients.

Risk factors for Hepatitis C virus (HCV) transmission and efficiency of antiviral therapy in chronically infected patients

Background: This study monitored the prevalence and genotype distribution of HCV in patients suspected for infection as well as risk factors for infection and transmission of HCV in northeast Croatia. Methods: From January 2009 to December 2011, 405 plasma specimens of patients suspected for HCV infection were tested by the COBAS TaqMan HCV test v2.0 (Roche Diagnostics) ; 77 HCV- positive samples were genotyped using the Linear Array HCV Genotyping test (Roche) ; 33 HCV- positive patients with unfavorable liver tests were further treated with anti-viral therapy and monitored at weeks 28 and 53. Risk factors for HCV infection and transmission were analyzed from patient-filled questionnaires (age, sex, risk behavior etc.) and their clinical data (age at initial HCV infection, degree of liver inflammation and fibrosis). Results: Out of 405 tested patients, 198 (48.8%) were HCV-positive and the highest prevalence of infection was found in men and women between 30 and 40 years of age. HCV genotype distribution in northeast Croatia was similar to other European regions: G1 was the most common (66.6%), followed by G3 (22.1%), G4 (10.4%), and G2 (1.3%) ; genotypes G5 and G6 were not detected. Viral therapy evaluated at week 53 demonstrated that 58 % of patients were virus- free, 12 % had lower viral load while 12 % showed no change in viral load. Moreover, HCV G1-infected patients showed a delayed response to antiviral therapy when compared to G3 and G4-infected patients: 31 % G1-, 40 % G3- and 50 % G4-infected patients were virus-free at week 28 ; 58 % G1-, 60 % G3- and 50 % G4-infected patients were virus- free at week 53. Significant risk factors for HCV infection and transmission in northeast Croatia include history of blood transfusion, surgery before the year 1993, and body piercing in 31-40 year-old men (p<0.05). Conclusion: Our results strengthen the importance of early testing for HCV infection in men showing the symptoms of chronic hepatitis (muscle pain, appetite loss, history of jaundice) since they are statistically more often intravenous drug, piercing, and tattoo users then women in our region.

Quantification of hepatitis C virus in patients treated with peginterferon‐alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test

Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG-IFN-alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non-SVR patients with greater accuracy.

Clearance of hepatitis C virus RNA from serum in HIV/hepatitis C virus coinfection indicates eradication from peripheral blood mononuclear cells

The objectives of this study are to determine the frequency of hepatitis C virus (HCV) RNA persistence in peripheral blood mononuclear cells (PBMCs) of HIV-positive patients with clearance of the virus from serum and to identify the presence of any ongoing replication. This is a prospective cross-sectional study. HIV antibody-positive individuals with previous exposure to HCV, but not current infection with HCV, were recruited. Blood was taken to allow identification of HCV RNA in both serum and PBMCs. Intracellular HCV was extracted using the QIAamp RNA Blood MiniKit. Reverse transcriptase-PCR was performed using a modification of the COBAS TaqMan HCV Test for use with the high pure system. Twenty-six HIV-positive individuals were recruited to the study. All had previously been infected with HCV. Six individuals had spontaneously cleared HCV, 10 had achieved sustained virological response following 24 weeks of pegylated interferon and ribavirin for acute HCV, and 10 had achieved sustained virological response following standard pegylated interferon and ribavirin therapy for chronic HCV. None demonstrated HCV RNA persistence in either serum or PBMCs. Our findings lend support to the view that clearance of HCV RNA from serum in HIV/HCV coinfection indicates eradication from PBMCs. Thus, absence of serum HCV RNA 6 months after the end of therapy can be used as a marker of treatment success for interferon-based therapies. However, the advent of small molecule HCV inhibitors may require us to rethink our definitions of response and cure.

Dried Blood Spot for Hepatitis C Virus Serology and Molecular Testing

We investigated the performance of dried blood spots (DBS) in hepatitis C virus (HCV) diagnosis using modified commercial tests. Paired DBS and serum samples were collected from 200 patients: 100 patients with anti-HCV antibodies (anti-HCV), including 62 patients with detectable serum HCV RNA, and 100 patients without anti-HCV. The DBS sample consisted of three drops of approximately 50 microL of whole blood applied to a paper card, which was then stored at -20 degrees C within 48 hours of collection. Using the Ortho HCV 3.0 enzyme-linked immunosorbent assay kit on DBS, we observed both a specificity and sensitivity of 99% in detecting anti-HCV. HCV RNA was detected on DBS in 60/62 (97%) patients with detectable serum HCV RNA, which was then successfully quantified in 55 samples (89%) using the Cobas TaqMan HCV test. A good correlation was observed between the DBS HCV RNA concentration and the serum level (r(2) = 0.95; P < 0.001). HCV genotyping was successfully performed on DBS samples, with a full concordance between the 14 paired DBS and serum samples (genotypes 1-4). This study presents DBS as a reliable alternative to serum specimens for detecting anti-HCV, quantifying HCV RNA and genotyping HCV. DBS may increase the opportunities for HCV testing and treatment follow-up in hard-to-reach individuals.

Evolution of Plasma Hepatitis C Virus Load in Patients Coinfected by HIV and Hepatitis C Virus Started on a Protease Inhibitor-Containing Antiretroviral Regimen, Agence Nationale de Recherches sur le SIDA CO8 APROCO-COPILOTE Cohort

Evolution of Plasma Hepatitis C Virus Load in Patients Coinfected by HIV and Hepatitis C Virus Started on a Protease Inhibitor-Containing Antiretroviral Regimen, Agence Nationale de Recherches sur le SIDA CO8 APROCO-COPILOTE Cohort

Comparison of Performance Characteristics of Three Real-Time Reverse Transcription-PCR Test Systems for Detection and Quantification of Hepatitis C Virus

We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log(10) IU/ml with a linear dynamic range of 1.0 to 7.0 log(10) IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log(10) IU/ml with a linear dynamic range of 2.0 to 7.0 log(10) IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy-six clinical specimens (50 with detectable levels of HCV RNA and various titers and genotypes) were tested, and results were compared to those of the COBAS Amplicor HCV Monitor v2.0. Good correlation was obtained for the Roche RUO-MPLC and Abbott ASR-Q (R(2) = 0.84 and R(2) = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation (R(2) = 0.79) and agreement were poor for Roche ASR-HP, with bias relative to concentration and genotype. Roche ASR-HP underestimated HCV RNA for genotypes 3 and 4 as much as 2.19 log(10) IU/ml. Our study demonstrates that Roche RUO-MPLC and Abbott ASR-Q provided acceptable results and agreed sufficiently with the COBAS Amplicor HCV Monitor v2.0.

Efficacy of Antiviral Therapy on Hepatitis C Recurrence After Liver Transplantation: A Randomized Controlled Study

Recurrence of hepatitis C virus (HCV) infection is a relevant problem of liver transplantation programs. We evaluated the effect of antiviral therapy on disease progression in 81 HCV-infected liver transplantation recipients. Patients with mild hepatitis C recurrence (fibrosis stage F0 to F2, n = 54) were randomized to no treatment (group A, n = 27) or peginterferon alfa-2b/ribavirin for 48 weeks (group B, n = 27). Patients with severe recurrence (F3 to F4, cholestatic hepatitis) were treated (group C, n = 27). All patients (n = 81) underwent a liver biopsy at baseline and after follow-up; paired hepatic venous pressure gradient (HVPG) measurements were available in 51 patients. Thirteen (48%) patients of group B and 5 (18.5%) of group C achieved sustained virological response. Liver fibrosis progressed > or =1 stage in 40 (49%) of 81 patients: 19 (70%) of group A versus 7 (26%) of group B (P = .001) and in 14 (54%) of group C. HVPG increased (6.5 to 13 mm Hg, P < .01) in patients in whom fibrosis worsened, whereas it decreased (5 to 3.5 mm Hg, P = .017) or remained unchanged in those with fibrosis improvement or stabilization, respectively. The only variable independently associated with fibrosis improvement/stabilization was treatment (odds ratio [OR] =3.7, 95% confidence interval [CI] 1.3 to 10, P = .009). Among treated patients, alanine aminotransferase (ALT) normalization and viral clearance were independently associated with histological or hemodynamic improvement/stabilization (OR 5.3, 95% CI 1.5 to 18, P < .01; OR 7.4, 95% CI 1.4 to 38, P = .01; respectively). Our data demonstrate that in liver transplantation recipients, antiviral therapy slows disease progression (particularly in sustained virological responders), as shown by its effects on liver histology and on HVPG.