Detectable Hepatitis C Virus RNA Research Articles

Performance evaluation of the Hologic Aptima HCV Quant Dx assay for detection of HCV RNA from dried blood spots

BackgroundThe availability of effective direct-acting antiviral therapy for hepatitis C virus (HCV) has led to a need for simplified diagnostic pathways. Barriers to treatment uptake, specifically in people who inject drugs and in remote and resource limited settings, may be overcome by utilizing novel collection methods, such as dried blood spots (DBS). However, there are currently no registered assays for HCV RNA testing from DBS samples. ObjectivesTo evaluate the sensitivity and specificity of the Aptima HCV Dx Quant assay for HCV RNA detection in DBS samples Study design107 paired venepuncture and DBS samples from HCV antibody positive individuals were analyzed for HCV RNA on the Aptima HCV Dx Quant and Roche CAP/CTM (gold standard) HCV assays. Results78% (n=83) had detectable HCV RNA in plasma. Sensitivity of the Aptima assay for HCV RNA detection in DBS was 96.4% (95% CI 89.8–99.3%) and specificity was 95.8% (95% CI 78.8–99.9%). Sensitivity for HCV RNA detection in DBS using a quantitative threshold of ≥15 IU/mL in plasma was 95.1% (95% CI 88%–98.7%) and specificity was 96.0% (95% CI 79.7%–99.9%). The sensitivity of HCV RNA detection in DBS using a quantitative threshold of ≥1000 IU/mL (based on a clinically relevant threshold) was 100% (95% CI 95.3–100%) and specificity was 100% (95% CI 88.4–100%). ConclusionsOur data indicates that the Aptima HCV Dx Quant can detect active HCV infection from a DBS sample with good sensitivity and specificity, particularly when using a threshold of ≥1000 IU/mL.

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

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

Detection Of Hepatitis C Virus (Hcv) Infection And Its Genotype In Patients At Hepatology Outpatient Clinic, Dr Soetomo General Hospital, Surabaya.

Hepatitis C virus (HCV) is an RNA virus that can cause liver inflammation (hepatitis) and has the potential to become chronic and can progress to liver cirrhosis and hepatocellular carcinoma. Detection of HCV RNA infection, and genotype/subtype of HCV was performed on 70 blood sera of patients at the Hepatology Outpatient Clinic of Dr Soetomo General Hospital, Surabaya, Indonesia. Detection of HCV infection was carried out by Anti-HCV determination using enzyme immunoassay (EIA) technique, detection of HCV RNA by Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique based on genome regions NS5b and 5’UTR, followed by electrophoresis with agarose gel. In positive PCR results, HCV genotype/subtype was determined by direct sequencing method using ABI 310 sequencer and sequencing results were analyzed by comparing the products with previously published HCV nucleotides. Sera were obtained from 41 (58.6%) male and 29 (41.4%) female patients. Anti-HCV was found positive in 17/70 (24.29%) patients and 16/17 (94.1%) was proved to contain HCV RNA when determined by RT-PCR technique. Patients with positive HCV RNA have the potential to transmit HCV infection. From the genotype/subtype analysis of sequencing results we obtained 2/16 (12,5%), 3/16 (18,75%) and 6/16 (37,5%), 1/16 (6,25%), 1/16 (6, 25%), 2/16 (12,5%), 1/16 (6,25%) HCV genotypes 1, 2, and HCV subtypes 1b, 1c, 2a, 3a, 3k respectively.Conclusion: In patients who went to the Hepatology Outpatient Clinic, Dr. Soetomo General Hospital Surabaya, we found positive Anti-HCV was 24,29%. In 94,1% of patients with positive Anti-HCV, HCV RNA was still detected and HCV genotype 1 with subtype 1b were still dominant HCV subtypes.

Prevalence and genotyping pattern of hepatitis C virus among patients on maintenance hemodialysis at five centers in Pune, India

BackgroundWorldwide prevalence of Hepatitis C virus (HCV) infection hemodialysis (HD) ranges from 1 to 84.6% with serious complications. Assessment of prevalence, risk factors, and genotyping of HCV infection in patient on HD was carried out at Pune, India. MethodsA total of 250 patients on HD from five HD centers were recruited and tested for anti-HCV antibody using third-generation enzyme-linked immunosorbent assay (ELISA). Qualitative HCV RNA detection was carried out by nested reverse transcriptase polymerase chain reaction (RT-PCR). Genotyping and sequencing were carried out using the BigDye Terminator cycle sequencing ready reaction kit. ResultsMean age of patients was 47.3 years. Forty-seven cases out of a total of 250 were reactor for HCV antibody. Overall prevalence rate was 18.8% ranging from 6.7% to 35.6% in the five centers. Of total, 44.1% of females and 13.5% of males were HCV infected. The mean duration of HD in HCV-infected patients was 6.03 years. Prevalence was higher in patients aged > 5 years on HD with higher number of blood transfusions. Thirty-six cases were positive for HCV RNA. Only one HCV RNA was detected among the 203 anti-HCV negative samples. Discordance between antibody and HCV RNA positivity was noted. Seventeen infected cases had changed dialysis centers four times. Thirteen cases were HBsAg positive, of which six cases were coinfected with HCV. Thirty-seven samples were genotyped. ConclusionThe predominant genotype was 1a (54.1%) followed by 1b (43.2%) and 3a (2.7%). Highest prevalence of HCV (35.6%) and intracenter PNI of 99.3% of genotype 1b (84.6%) in center 3 indicates a possible nosocomial transmission.

Distribution of Hepatitis C Virus Genotypes and Related Risk Factors Among Iranian Blood Donors: A Penalized Logistic Regression

Background: The hepatitis C virus (HCV) is a blood born virus and the major cause of liver diseases worldwide. Distribution of HCV genotypes varies depending on geographical regions and routes of infection. Knowledge regarding the distribution of HCV genotypes and related risk factors plays an essential role in the control of HCV infection in the community. Objectives: The current study aimed at determining the current distribution of HCV genotypes and related risk factors among Iranian blood donors. Methods: In the current analytical, cross sectional study, 106 HCV-infected blood donors with detectable HCV RNA over the country were interviewed by trained physicians through a post-donation questionnaire on demographic, medical, and risk history from November 2015 to May 2017. The hepatitis C virus genotype was determined by sequencing of a segment of non-structural 5B region in HCV genome. Penalized logistic regression model was used for statistical analysis through STATA software. Results: Hepatitis C virus genotype was determined in all subjects, and the genotype 3a was the most frequent (65, 61.32%), followed by 1a (31, 29.25%), and 1b (10, 8.49%). Based on the multivariable analysis results, tattooing (adjusted odds ratio: 2.76; 95% confidence interval: 1.03 - 7.37) was associated with HCV genotype 3a. Conclusions: According to the results, it seems that changes in molecular epidemiology of HCV infection and replacement of HCV genotype 1a with 3a, characterized by an increase in genotype 3a and decrease in genotype 1a have occurred over the last decade among Iranian blood donors. Tattooing was an independent risk factor for HCV infection by genotype 3a.

Molecular diagnosis of occult hepatitis C virus infection in Iranian injection drug users.

Occult HCV infection (OCI) has been described as the presence of hepatitis C virus (HCV) genomic RNA in hepatocytes and/or peripheral blood mononuclear cell (PBMC) specimens and the lack of HCV genomic RNA and anti-HCV antibodies (Abs) in plasma samples. Injection drug users (IDUs) are the most important high-risk group for infection with blood-borne viruses, particularly HCV. The purpose of this study was to determine the presence of OCI in IDUs. A prospective cross-sectional study of 126 consecutive Iranian IDUs was performed from March 2017 to January 2018. PBMCs were separated from blood samples from the participants, and after extraction of the viral RNA from the plasma and PBMC specimens, HCV RNA was detected in the samples using RT-nested PCR by amplification of the 5'-NTR of HCV. HCV genotyping was carried out using restriction a fragment length polymorphism (RFLP) assay. The viral RNA was amplified using RT-nested PCR with specific primers for the NS5B gene, and the PCR products were sequenced to confirm the results obtained by HCV RNA detection and HCV genotyping. Out of the 126 IDUs studied, 105 (83.3%) were negative for anti-HCV Abs and HCV RNA in plasma samples, whereas HCV RNA was detected in the PBMC samples of six (5.7%) participants, indicating that these individuals had OCI. Moreover, HCV genomic RNA was detected in PBMC samples from five (23.8%) of the 21 IDUs studied who were positive for anti-HCV Abs and negative for HCV genomic RNA in plasma specimens. These IDUs also had OCI. The HCV genotypes in the PBMC samples from the subjects with OCI were determined. Six (54.5%) subjects were infected with HCV subtype 3a, and five (45.5%) were infected with HCV subtype 1a. This study showed that 8.7% of the Iranian IDUs had OCI, and therefore, a study focusing on the diagnosis of OCI in these individuals can be valuable and informative.

Hepatitis C Virus Diagnostics: The Road to Simplification.

Hepatitis C Virus Diagnostics: The Road to Simplification.

Hepatitis C Follow-Up After Hospital Discharge

Introduction: A sustained virologic response against hepatitis C virus (HCV) is now viable with development of direct-acting antiviral regimens. Although social factors, co-morbidities and insurance may determine if a patient is treated against HCV, it is imperative to follow-up with gastroenterology (GI) for management strategies. We aimed to determine if patients with chronic hepatitis C established care with GI after hospital discharge. Methods: Retrospective chart review of 62 patients with confirmed or reported history of chronic hepatitis C was performed. Confirmation was established with detection of HCV RNA. Patients admitted for both GI-related conditions as well as non GI-associated complaints were included. Presence of cirrhosis, portosystemic encephalopathy (PSE), esophageal varices (EV) and ascites was evaluated. Follow-up with GI after discharge was the primary outcome. Secondary outcomes included 30-days re-admissions, mortality, and GI evaluation during their inpatient stay. Results: Of the 62 patients, 46 (74.2%) were males. 49 (79.0%) patients were confirmed to have HCV but the rest only reported a prior history of chronic hepatitis C. Median age was 54 years and average length of stay was 8.3 days. 9 (14.5%) patients were readmitted to the hospital within 30 days. 22 (35.5%) reported concurrent alcohol use and 24 (38.7%) admitted illicit drug use. Cirrhosis was present in 22 (35.5%), PSE in 12 (19.3%), EV in 10 (16.1%) and ascites in 12 (19.3%) patients. 41 (66.1%) were admitted for non-GI related complaints. At discharge, 35 (56.4%) were referred to GI but only 16 (25.8%) followed with GI as outpatient. Of those that did see GI after discharge, 13 (81.2%) were within first three months of discharge. 9 (14.5%) patients died either during hospitalization or after discharge. Conclusion: Most patients with reported history of hepatitis C did not have cirrhosis and their inpatient admissions were not related to GI problems. Consequently, most of these patients did not see GI as an outpatient. With the change in insurance approval trends for treatment of chronic hepatitis C, this population in initial stages or without cirrhosis would benefit from earlier evaluation by GI to facilitate management before the onset of cirrhosis and its associated complication. Future avenues include introducing prompts in the electronic health record system at the time of discharge to refer patients to GI as well as contacting patients within 30 days of discharge to remind of outpatient visits.

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.

Hepatitis virus (HCV) diagnosis and access to treatment in a UK cohort

BackgroundAs direct acting antiviral (DAA) therapy is progressively rolled out for patients with hepatitis C virus (HCV) infection, careful scrutiny of HCV epidemiology, diagnostic testing, and access to care is crucial to underpin improvements in delivery of treatment, with the ultimate goal of elimination.MethodsWe retrospectively studied microbiology records from a large UK teaching hospital in order to compare the performance of HCV screening and diagnostic tests (antibody, antigen and HCV RNA detection). Having described a local cohort of adults with active HCV infection, we investigated the proportion who attended hospital appointments, were prescribed direct acting antiviral (DAA) therapy, and cleared HCV RNA following treatment.ResultsOver a total time period of 33 months between 2013 and 2016, we tested 38,509 individuals for HCV infection and confirmed a new diagnosis of active HCV infection (HCV-Ag + and/or HCV RNA+) in 353 (positive rate 0.9%). Our in-house HCV-Ab screening test had a positive predictive value of 87% compared to repeat HCV-Ab testing in a reference laboratory, highlighting the potential for false positives to arise using this test. HCV-Ag had 100% positive predictive value compared to detection of HCV RNA. There was a strong correlation between quantitative HCV-Ag and HCV RNA viral load (p < 0.0001). Among the cases of infection, genotype-1 and genotype-3 predominated, the median age was 37 years, 84% were male, and 36% were in prison. Hepatology review was provided in 39%, and 22% received treatment. Among those who received DAA therapy with 12 weeks of follow-up, 93% achieved a sustained virologic response (SVR12).ConclusionsHCV-Ag performs well as a diagnostic test compared to PCR for HCV RNA. Active HCV infection is over-represented among men and in the prison population. DAA therapy is successful in those who receive it, but a minority of patients with a diagnosis of HCV infection access clinical care. Enhanced efforts are required to provide linkage to clinical care within high risk populations.

Clinical significance of detectable and quantifiable HCV RNA at the end of treatment with ledipasvir/sofosbuvir in GT1 patients.

AASLD/IDSA treatment guidelines for hepatitis C virus (HCV) infection state that testing for quantitative HCV RNA can be considered at the end of antiviral treatment (EOT) with interferon-free regimens. However, it remains unclear how to respond to a detectable or even quantifiable HCV RNA result. The aim of this study was to analyse the frequency and predictive value of detectable and quantifiable HCV RNA results at the EOT in patients with HCV genotype 1 infection treated with ledipasvir (LDV) and sofosbuvir (SOF)±ribavirin (RBV) in a large real-world cohort. A retrospective analysis of the DHC-R (Deutsches Hepatitis C-Register, German Hepatitis C-Registry) cohort was performed including all patients who were treated with LDV/SOF±RBV and in whom HCV RNA testing was done with either the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) or the Abbott RealTime HCV assay (ART). The frequency of detectable HCV RNA at the EOT was 7% in this real-world study involving 471 patients. Furthermore, 3% of the patients (n=14/471) even had quantifiable viral load at the EOT. Detectable and quantifiable results were more frequent if the ART was used for testing. However, SVR was achieved by 32/33 patients (97%) with detectable and even by all 14 patients (100%) with quantifiable HCV RNA results at the EOT. Detectable and even quantifiable HCV RNA results are quite frequent if highly sensitive HCV RNA assays are used. However, treatment prolongation is not indicated, as SVR rates remain high in these patients.

The hepatitis C cascade of care in people who inject drugs in Dar es Salaam, Tanzania.

SummaryThe World Health Organisation has recently called for hepatitis C virus (HCV) elimination and has identified people who inject drugs (PWID) as a key population to scale‐up screening and linkage to care. This study reports the cascade of care for HCV in PWID attending the largest opioid substitution treatment (OST) clinic in Dar‐es‐Salaam, Tanzania. Between February 2011 and March 2016, HCV serology for all PWID registered at the Muhimbili National Hospital OST clinic, Dar‐es‐Salaam were obtained from records. In 2015, consecutive HCV‐seropositive PWID were invited to undergo a clinical evaluation including epidemiological questionnaire, liver stiffness measurement (Fibroscan) and virological analysis (HCV RNA viral load and genotyping). During the study period, 1350 persons registered at the OST clinic: all had a HCV serology including 409 (30%) positive results. Among the HCV‐seropositive individuals, 207 (51%) were active attenders and 153 (37%) were enrolled for clinical assessment: 141 (92%) were male, median age: 38 years (IQR 34‐41), and 65 (44%) were co‐infected with HIV; 116 patients (76%) had detectable HCV RNA, with genotypes 1a (68%) and 4a (32%); 21 (17%) had clinically significant fibrosis (≥F2) and 6 (5%) had cirrhosis (F4). None were offered HCV treatment. Chronic hepatitis C among PWID enrolled in the OST centre in Dar‐es‐Salaam is frequent, but its continuum of care is insufficient; integration of HCV diagnosis and treatment should form a part of OST intervention in PWID in Tanzania.

Hepatitis C Virus Elimination is Not Attained in a Certain of Immunocompetent Patients Who Achieved Sustained Viral Response to Direct-Acting Antiviral Agents

Background: Direct-acting antiviral agents (DAAs) have opened a new era for treating chronic hepatitis C virus (HCV) infection. However, whether DAA induced SVR attains real cure and complete eradication of HCV RNA is unknown, because occult HCV infection (OCI), as defined the detection of HCV RNA in hepatocytes or peripheral blood mononuclear cells (PBMC) in absence of serum of HCV RNA, may occur despite SVR and the clinical significance is yet to be established. We thus investigated the prevalence and provisional outcome of OCI in patients who achieved spontaneous or therapy-induced resolution of hepatitis C. Methods: Chronic HCV subjects from three tertiary hospitals who had achieved serum viral clearance, including 60 of DAAs treatment, 50 of pegylated interferon plus ribavirin (PR) treatment, and 30 of spontaneous recovery, were subjected to detect HCV RNA in hepatocytes by in suit hybridization as well as in PBMC by PCR. Liver function testes, HCV genotypes, and paired liver biopsies of pre- and post-treatment for each patient were acquired for comparison analysis. Findings: 16 of 140 subjects (11·4%) were found to have OCI, with the highest (15·0%) rate in DAA-based treated subjects, 10·0% in PR treated subjects and 6·7% in spontaneously resolved cases. Rates of OCI were 16·7% and 11·1% among patients with PR-free regimen and PR-based regimen, respectively. The existence of intermediate negative strand of HCV genome in all PBMCs of OCI patients suggested ongoing viral replication. The occurrence of OCI is more frequent in patients with genotype 3 HCV but less in patients with genotype 1. No correlation between baseline HCV viral load, interleukin-28B genotype, baseline transaminases, post transaminases and occurrence of OCI was found. While notably, OCI patients had more advanced liver fibrosis and active inflammation at both baseline and post- treatment. In addition, fibrosis scores were significantly decreased after SVR only in patients without OCI (P <0·001), but not in OCI subjects (P = 0·385). Patients with OCI have less incidence of fibrosis regression than in those without OCI after SVR. Importantly, we found two cases of HCV reactivation in OCI patients during 48 weeks to three-year follow up. Interpretation: HCV RNA can persist as ongoing replicative virus after treatment-induced or spontaneous serum resolution of hepatitis C, with highest incidence in patients with PR-free regimen induced SVR. The existence of OCI is associated with persistent pathological abnormality and likely to result in HCV relapse. Funding Statement:This study is supported by grants from National Natural Science Foundation of China (NNSFC) (No. 81673654) (to J.Zhao); NNSFC (No. 31770186) (to Y. Wang); the National S&T Major Project for Infectious Diseases (No. 2017ZX10302201001007). Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: This study was done in accordance with the ethical principles of the Declaration of Helsinki as revised in 2013 and was approved by the Ethics Committee by each institution’s human research committee. Informed consent was obtained from all participants prior to enrollment. All patients were recruited from three tertiary hospitals, Peking University People’s Hospital, the first Hospitalof Jilin University and Beijing 302 hospital.

Observed increases in CD4:CD8 and TIGIT expression during sofosbuvir-based therapy in a longitudinal hepatitis C cohort

Abstract Chronic hepatitis C virus (HCV) infection reduces immune function through exhaustion and repertoire reduction. Sofosbuvir-based direct-acting antiviral (DAA) therapies offer &amp;gt;90% sustained virologic response (SVR) for HCV infection, but the extent of immune recovery and relationship with clinical factors are unknown. The longitudinal Alaska Hepatitis C Cohort provides an opportunity to evaluate immune recovery following sofosbuvir-based DAA and determine the influence of an extensive clinical and laboratory history. Forty-two persons (genotype 1; mean age 50 ± 12 years; 69% female) received 8 or 12-week sofosbuvir-based DAA therapy. Mean HCV RNA detection date was 2007 (range: 1994–2017). Pre-treatment HCV RNA levels averaged 4.9×106 IU/L (range: 4.9×104–2.4×107), were not detectable in any person after 4-weeks of therapy, and all achieved SVR. 18% (n=7) of persons had a fibrosis score of ≥F2 (moderate fibrosis to cirrhosis). Peripheral blood mononuclear cells (PBMCs) were evaluated at four time points: Pre-treatment (Pre), 4-weeks treatment (4wk), End of Treatment (EOT), and SVR post 12-weeks EOT (SVR-12) by flow cytometry to evaluate memory, exhaustion, and functional immune markers. Initial analysis revealed a significant increase in the CD4:CD8 from 4wk to EOT (p=0.007). Regulatory T-cells significantly increased at EOT and SVR-12 compared to 4wk (p=0.04 and 0.03, respectively). TIGIT showed a significant increasing trend in T-cells and NK cells throughout sampling timeline. Preliminary analysis indicates sofosbuvir-based DAA supports immune recovery to some extent. Additional analysis of immune recovery markers, determination of estimated date of HCV infection, and correlation of morbidity factors continues.

Development and clinical validation of the Genedrive point-of-care test for qualitative detection of hepatitis C virus

ObjectiveRecently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live...

HCV Seroreactivity and Detection of HCV RNA in Hepatitis Cases

Background: Hepatitis C virus (HCV) known to be associated with wide variety of liver pathology. It is less studied in India as compared to western region. Methods: Suspected patients sera screened for HCV by ELISA and confirmed with reverse transcription polymerase chain reaction (RT-PCR) along with routine investigations and liver profile. All HCV positive patients were undergone liver biopsy. Results: All 24 HCV ELISA reactive and two ELISA indeterminate sera are confirmed by RT- PCR. The liver biopsy of these patients showed normal picture (19.2%), Acute hepatitis (11.5%), Chronic hepatitis (23.7%), Cirrhosis (34.72%), Hepato-cellular carcinoma (HCC) (15.38%). ALT levels were not significant. Conclusion: All the suspected HCV cases need to be confirmed for HCV by RT-PCR.

Detection of Anti-Hepatitis C virus and Hepatitis C virus RNA in Dried Blood Spot Specimens using Whatman no. 1 Filter Paper

Purpose: Dried blood spot (DBS) specimen simplifies blood collection, processing, storage and shipment and may reduce the cost of testing for hepatitis C virus (HCV) infection. We wanted to see if DBS using a cheap filter paper is reliable alternative to serum for detection of anti-HCV and HCV RNA. Materials and Methods: At a tertiary care hospital in Northeast India, we collected 91 paired DBS and serum specimens from patients at risk of HCV infection from July 2014 to June 2015. DBS was collected on Whatman No. 1 filter paper. After processing, the specimens were subjected to anti-HCV detection by a third-generation Enzyme-Linked Immunosorbent Assay (ELISA). The reactive DBS and serum specimens were further subjected to HCV RNA detection by polymerase chain reaction. The results were analysed in paired screen-positive study design. Results: Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens. There was statistically significant (P < 0.0001) correlation between the optical density values of DBS and serum specimens (Pearson r = 0.9181, 95% confidence interval: 0.8781–0.9453). HCV RNA was detected in 5/9 (55.6%) reactive DBS and 9/10 (90.0%) reactive serum specimens. There was no correlation between HCV RNA levels in the DBS and the serum specimens. The relative sensitivity rate and the relative false-positive rate of DBS anti-HCV ELISA were 0.89 and 1.00, respectively. Conclusions: DBS using Whatman No. 1 filter paper is quite reliable as serum for detection of anti-HCV. It can be useful in effective surveillance. However, it is not suitable for confirmation of chronic HCV infection.

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release

AIMTo explore hepatitis C virus (HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODSA series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and cell lysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets (LDs) with NS5A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5A was analyzed further.RESULTSThe plasmids named JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/NS5A, and mJFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units (FFUs)/mL. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) were critical adaptive mutations. The effect of NS5A (C2274R, I2340T, and V2440L), p7 (H781Y), and NS4B (N1931S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5A proteins or the phosphorylation of NS5A.CONCLUSIONIn this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/mL, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.

Epidemiological features of chronic hepatitis C infection caused by remunerated blood donors: A nearly 27-year period survey.

AIMTo understand the prevalence of hepatitis C virus (HCV) infection in blood donors over a nearly 27-year interval and to explore the factors that affect the outcome of HCV infection.METHODSA retrospective and cross-sectional study was conducted. The participants, mostly plasma donors, were selected from three administrative villages in the Jiangsu province in Eastern China. A questionnaire was administered among the villagers who had a history of blood donation from the late 1980s to the early 1990s. All participants underwent physical examination, liver B-ultrasonography, and liver stiffness measurement. In addition, 10 mL of blood was collected from each participant to measure simple liver function parameters (albumin, alanine aminotransferase, aspirate aminotransferase), blood factors (platelet), and for hepatitis B surface antigen, antiHCV, and antihuman immunodeficiency virus detection. HCV RNA detection, HCV genotyping, and other tests were carried out in antiHCV-positive patients.RESULTSAfter a median of 27 years (25-31 years) from the last blood donation to the time of survey, a total of 1694 participants were investigated, and the antiHCV-positive individuals were categorized into three groups: blood donors (n = 12, 3.3%), plasma donors (n = 534, 68.5%), and mixed donors (n = 324, 58.8%). A total of 592 (68.05%) patients had detectable HCV RNA, and 91.9% had genotype 1b. A total of 161 (27.2%, 161/592) patients with chronic HCV were considered to have cirrhosis with a liver stiffness measurement level higher than 12 kPa. Multiple logistic (binary) regression analysis results showed that platelet and IgG levels were associated with cirrhosis.CONCLUSIONThe nearly 27-year interval investigation revealed that chronic hepatitis C infection is a very serious public health problem in Eastern China. Plasma donation and subsequent return of blood cells to the donor are the main causes of hepatitis C infection. The main HCV genotype is 1b. Nearly 28% of cases progressed to cirrhosis. Age, especially over 60 years, and regular drinking habits were risk factors associated with cirrhosis.

Evaluation of the Xpert HCV Viral Load Finger-Stick Point-of-Care Assay.

Point-of-care hepatitis C virus (HCV) RNA testing is advantageous, enabling diagnosis of active infection in a single visit. This study evaluated the sensitivity and specificity of the Xpert HCV Viral Load Finger-Stick assay (Xpert HCV VL FS) for HCV RNA detection (finger-stick) and the Xpert HCV Viral Load assay (plasma) compared with the Abbott RealTime HCV Viral Load assay by venepuncture. Plasma and finger-stick capillary whole-blood samples were collected from participants in an observational cohort in Australia. Of 223 participants enrolled, HCV RNA was detected in 40% of participants (85 of 210) with available Xpert HCV Viral Load testing. Participants receiving HCV therapy were excluded from subsequent analyses (n = 16). Sensitivity of the Xpert HCV Viral Load assay for HCV RNA quantification in plasma collected by venepuncture was 100.0% (95% confidence interval [CI] 96.9%-100.0%) and specificity was 100.0% (95% CI, 94.4%-100.0%). Sensitivity of the Xpert HCV VL FS assay for HCV RNA quantification in samples collected by finger-stick was 100.0% (95% CI, 93.9%-100.0%) and specificity was 100.0% (95% CI, 96.6%-100.0%). The Xpert HCV VL FS test can accurately detect active infection from a finger-stick sample in 1 hour allowing single-visit HCV diagnosis.