Hepatitis C Virus RNA Quantification Research Articles

Efficacy and Safety of Sofosbuvir/Velpatasvir Plus Ribavirin in Patients with Hepatitis C Virus-Related Decompensated Cirrhosis.

A fixed-dose combination of sofosbuvir/velpatasvir (SOF/VEL) plus weight-based ribavirin (RBV) for 12 weeks is recommended for the treatment of patients with hepatitis C virus (HCV)-associated decompensated cirrhosis. However, large global studies, while confirming the effectiveness of SOF/VEL in a broad range of patients, often exclude these patients. This Phase 2, single-arm, open-label study in adult patients with HCV-associated decompensated cirrhosis in France and the USA aimed to provide further data on the safety and efficacy of SOF/VEL plus RBV for 12 weeks in this population. Patients were treated with a fixed-dose combination of SOF 400 mg/VEL 100 mg plus weight-based RBV once daily for 12 weeks. The inclusion criteria were chronic HCV infection (≥6 months), quantifiable HCV RNA at screening, Child-Turcotte-Pugh class B or C cirrhosis, and liver imaging within 6 months of Day 1 to exclude hepatocellular carcinoma. Among 32 patients who initiated treatment, 78.1% achieved sustained virologic response 12 weeks after the end of treatment (SVR12). Failure to achieve SVR12 was due to non-virologic reasons (investigator discretion, n = 1; death, n = 6). All 25 patients in the per-protocol population achieved SVR12 and all but one achieved sustained virologic response 24 weeks after the end of treatment. Adverse events (AEs) were as expected for a patient population with advanced liver disease. All Grade 3-4 and serious AEs and deaths were deemed unrelated to treatment. In patients with HCV-associated decompensated cirrhosis, SOF/VEL plus RBV achieved high SVR12 rates and was generally well tolerated.

Impact of storage time in dried blood samples (DBS) and dried plasma samples (DPS) for point-of-care hepatitis C virus (HCV) RNA quantification and HCV core antigen detection.

The scale-up of hepatitis C virus (HCV) diagnosis and treatment requires affordable and simple tools to improve access to care, especially in low- and middle-income settings with limited infrastructure or high-risk populations. Dried blood and plasma samples (DBS and DPS) are useful alternative for hepatitis C detection in settings lacking adequate infrastructure. We evaluated the performance of DBS and DPS vs plasma in a point-of-care HCV RNA quantitative assay (Xpert HCV Viral Load-Cepheid), and compared HCV core antigen (HCVcAg) detection by the Architect HCV core antigen assay (Abbott) in DBS vs serum. The dried samples were stored at room temperature for different storage times to reproduce the time from sampling to testing in settings with centralized diagnosis or when testing mobile populations. HCV RNA quantification in DBS and DPS presented 100% sensitivity and specificity and a high correlation for up to 3 months of storage. HCV viremia showed a mean decrease of 0.5 log10 IU/mL (DBS) and 0.3 log10 IU/mL (DPS) for storage times up to 1 month. Architect HCVcAg detection presented high sensitivity/specificity (96%/100%) in DBS tested immediately after sampling, decreasing to 86% sensitivity after 7 days of storage. However, sensitivity increased when an optimized cut-off was applied for each storage time. We conclude that DBS and DPS are suitable samples for HCV RNA detection and quantification, being DPS more reliable for shorter storage times. DBS can be also used for HCVcAg qualitative detection and the sensitivity can be increased when adjusting the cut-off values. IMPORTANCE Hepatitis C infection remains a global burden despite the effectiveness of antivirals. In the WHO roadmap to accomplish HCV elimination by 2030, HCV diagnosis is one of the main targets. However, identifying patients in resource-limited settings and high-risk populations with limited access to healthcare remains a challenge and requires innovative approaches that allow decentralized testing. The significance of our research is in verifying the good performance of dried samples for HCV diagnosis using two different diagnostics assays and considering the effect of room temperature storage in this sample format. We confirmed dried samples are an interesting alternative for HCV screening and reflex testing in resource-limited settings or high-risk populations.

HCV prevalence and treatment outcomes among drug users in an outpatient center for drug addiction in Northern Italy.

We aimed at evaluating hepatitis C virus (HCV) prevalence and treatment referral outcomes in a large population of drug users in Northern Italy. Each participant underwent a quick capillary blood test. Positive participants underwent HCV RNA quantification. HCV RNA positive subjects were referred to treatment and evaluated immediately at the end of treatment and at 3 and 6 months after treatment. Of the 636 participants tested, 244 were positive. Intravenous drug use was more frequent among subjects who tested positive for HCV antibodies (99%). Among subjects who tested positive, 68% were HCV-RNA positive while 32% were negative. Among people referred to treatment, nearly 30% did not show up while 70% completed the treatment with success. Over 99% of people who started direct-acting antiviral agent (DAA) have a sustained response. We observed a significant higher prevalence of HCV positive subjects among people who inject drugs (99%) and we observed a high success rate for HCV treatment engagement. Rapid testing for HCV represents a potential tool for HCV screening among high-risk groups.

HCV Core-antigen Assay as an Effective Alternative to HCV RNA Quantification in Patients With Hepatitis C.

Hepatitis C virus (HCV) core antigen (Ag) test has been increasingly applied as an effective alternative to conventional molecular tests allowing rapid and affordable diagnosis, which is of paramount relevance to achieve global elimination of HCV infection. ARCHITECT® HCV Ag test was evaluated in comparison with HCV RNA quantification test (CAP/CTM) to calculate its sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and to determine their correlation level. Its performance, according to low and high viral load values and in different treatment stages [during treatment (T), at the end of the therapeutic protocol (EOT) and when sustained virological response (SVR) was evaluated]. In total, 145 samples were included. Considering CAP/CTM, the sensitivity, specificity, PPV and NPV of the HCV-Ag test were 88.9%, 99.1%, 97.0% and 96.4%, respectively, and the correlation among tests was high (r=0.890), with only five discordant results. A decrease in sensitivity was found for low viral load values (<1,000 IU/ml), but the opposite was verified for high viral concentrations (≥1,000 IU/ml). A good agreement was verified for the T and EOT groups (k=0.789 and k=0.638) and an excellent agreement in the SVR group (k=1.000). HCV-Ag seems to be an effective alternative that can be routinely combined with other faster and more accessible tests (e.g., HCV antibody tests) for the identification of new HCV infections in suspected patients, eventually reserving the molecular techniques for samples with discordant results.

HCV elimination in a Swiss opioid agonist therapy programme – a cohort study

In opioid agonist therapy (OAT) programmes, chronic hepatitis C is highly prevalent and directly observed therapy guarantees optimal adherence. Since 2017, all patients with chronic hepatitis C in Switzerland can be treated with pangenotypic direct-acting antivirals irrespective of liver fibrosis stage. Until the end of 2021, however, prescription was restricted to infectious disease specialists, gastroenterologists and certain addiction specialists. Difficult venous access after long-term intravenous drug use and, in the case of referral to a specialist, difficulties keeping appointments are barriers to HCV diagnosis and treatment. To assess whether minimally invasive point-of-care tests and a "test-and-treat / vaccinate on-site" approach can improve human immunodeficiency virus (HIV) / hepatitis C virus (HCV) screening, HCV treatment uptake and immunity against hepatitis A/B. Since September 2018, an infectious disease specialist and a study nurse performed 4-weekly visits in the OAT programme "HAG" (heroin dispensation of the canton Aargau), offering HIV/HCV antibody rapid testing (20 min) and HCV RNA quantification (GeneXpert®, 60 min) from capillary blood, noninvasive liver fibrosis assessment (Fibroscan®, 5-10 min) and HCV treatment prescription on-site. Recommended venous blood draws for HAV/HBV serology and HAV/HBV vaccinations were performed by the staff of the "HAG". Project performance was assessed by annual cross-sectional chart review. Of the 128 patients registered in April 2018, 79 (62%) were still present in May 2021. With 72 newly registered, a total of 200 patients could be assessed, of whom 129 (65%) were still present in May 2021. Between April 2018 and May 2021, the proportion ever tested for HIV antibodies increased from 79% (101/128) to 91% (117/129), the proportion ever tested for HCV antibodies from 83% (106/128) to 93% (120/129) and the proportion of those HCV antibody positive ever tested for HCV RNA tested from 89% (47/53) to 98% (56/57). The proportion with adequate HCV management (last HCV antibody test ≤1 year ago, if HCV antibody negative or last HCV RNA test ≤1 year ago, if HCV antibody-positive and RNA-negative) improved from 23% ([15 + 15]/128) to 80% ([55 + 48]/129). Overall, HCV treatment uptake increased from 60% (21/35) to 92% (55/60) and HCV RNA prevalence among the HCV antibody positives decreased from 38% (18/47) to 7% (6/84). Between 2018 and 2021, 19 non-cirrhotic chronic hepatitis C patients were successfully treated on-site (18 sustained virological responses [SVR] 12, 1 SVR4), with excellent adherence (≥93%) and, so far, no reinfection. The proportion with known HAV/HBV serostatus increased from 38%/51% to 64%/76%. Immunity against HAV/HBV improved from 19%/23% to 50%/57%. Capillary blood point-of-care tests and a "test-and-treat / vaccinate on-site" approach remove crucial barriers to diagnosis and treatment, making hepatitis elimination in OAT programmes achievable. A high fluctuation rate requires HIV/HCV/HAV/HBV testing at admission, but also allows more patients to be screened.

Comparing RT-qPCR and Hepatitis C Virus Antigen Detection Assay for Detecting Active Infection in Blood Donors in Fars Province, Iran

Background: Immunoassay is still used to detect hepatitis C virus (HCV) antibodies in donated blood in many developing countries. However, an immunoblotting confirmation test is needed to confirm positive results. Objectives: We compared the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of nucleic acid testing and HCV core antigen (HCVcAg) detection in the serum samples of blood donors with HCV antibodies to determine active infection. Methods: Overall, 90 serum samples from blood donors referred to Fars Blood Transfusion Organization, Iran during March 2017-March 2019 and initially tested for HCV antibodies were included in the study. Enzyme immunoassays were used to detect the HCV antigen and anti-HCV antibody. A commercial reverse transcription-polymerase chain reaction (RT-PCR) kit was used to quantify HCV RNA. The HCV genotypes were also determined by DNA sequencing. In order to compare the HCVcAg detection method with the RT-qPCR reference method, sensitivity, specificity, performance, PPV, and NPV were calculated. Results: Out of 90 serum samples, 73 were positive for anti-HCV antibody, and 17 sera were negative. The HCV RNA was detected in 60 (82%) of anti-HCV antibody-positive samples, whereas the HCVcAg test detected HCV antigen in 54 (74%) of the samples, indicating a significant correlation between the two assays (r = 0.86). The overall sensitivity and specificity for HCVcAg detection method were 93.85% [95% confidence interval (CI): 84.99 - 98.3%] and 100% (95% CI: 94.64 - 100%), respectively. Based on the statistical analysis, the accuracy of the antigen detection test was 94.83% (95% CI: 87.26 - 98.58%). Moreover, the agreement between HCV RNA detection using RT-qPCR and HCVcAg detection was 97.78% (kappa value: 0.94). Conclusions: The sensitivity and specificity of HCVcAg detection in blood donors were ideal compared to the RT-qPCR reference method. However, the method should be tested on more HCV antibody-positive and -negative samples. Furthermore, our study revealed a significant association between the number of RT-qPCR-positive cases and the cases diagnosed by the HCVcAg detection method for screening and detecting active HCV infection in blood donors.

Diagnostic Accuracy of Assays Using Point-of-Care Testing or Dried Blood Spot Samples for the Determination of Hepatitis C Virus RNA: A Systematic Review.

Finger-stick point-of-care and dried blood spot (DBS) hepatitis C virus (HCV) RNA testing increases testing uptake and linkage to care. This systematic review evaluated the diagnostic accuracy of point-of-care testing and DBS to detect HCV RNA. Bibliographic databases and conference presentations were searched for eligible studies. Meta-analysis was used to pool estimates. Of 359 articles identified, 43 studies were eligible and included. When comparing the Xpert HCV Viral Load Fingerstick assay to venous blood samples (7 studies with 987 samples), the sensitivity and specificity for HCV RNA detection was 99% (95% confidence interval [CI], 97%-99%) and 99% (95% CI, 94%-100%) and for HCV RNA quantification was 100% (95% CI, 93%-100%) and 100% (95% CI, 94%-100%). The proportion of invalid results following Xpert HCV Viral Load Fingerstick testing was 6% (95% CI, 3%-11%). When comparing DBS to venous blood samples (28 studies with 3988 samples) the sensitivity and specificity for HCV RNA detection was 97% (95% CI, 95%-98%) and 100% (95% CI, 98%-100%) and for HCV RNA quantification was 98% (95% CI, 96%-99%) and 100% (95% CI, 95%-100%). Excellent diagnostic accuracy was observed across assays for detection of HCV RNA from finger-stick and DBS samples. The proportion of invalid results following Xpert HCV Viral Load Fingerstick testing highlights the importance of operator training and quality assurance programs.

HCV RNA quantification in capillary dried blood spots with the Xpert® HCV Viral Load test for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection.

For patients with difficult venous access after long-term intravenous drug use, rapid point-of-care hepatitis C virus (HCV) RNA quantification in capillary whole blood with the Xpert® HCV Viral Load Fingerstick (VL FS) test (60 minutes) is a convenient and reliable method for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection. However, an expensive GeneXpert® system must be available on site. In decentralised settings with a low case-load, dried blood spot (DBS) testing might be an alternative. Between December 2019 and January 2021, patients with an indication for HCV RNA quantification and informed consent provided 100 µl capillary whole blood each for on-site Xpert® HCV VL FS testing (reference) and DBS testing in the laboratory. For the latter, 100 µl blood, collected with an EDTA Minivette®, were transferred to a Whatman® 903 filter card. After drying for at least 1 hour, the DBS sample was packed into a sealable plastic bag with desiccant and sent to the central laboratory of our hospital, where it was stored at -20°C. For HCV RNA extraction, the whole DBS was cut out with an 18-mm puncher and transferred into 1.3 ml guanidinium thiocyanate-containing buffer (provided by Cepheid®). After mixing and incubating at room temperature for 2-3 hours, 1 ml supernatant was analysed with the Xpert® HCV VL test (105 minutes) (filter paper absorbs 0.3 ml). Of 109 paired samples from 67 patients, 38 (34.9%) were positive with the Xpert® HCV VL FS test. Sensitivity and specificity of DBS testing were 89.5% (34/38; 95% confidence interval [CI] 75.9-95.8%) and 97.2% (69/71; 95% CI 90.3-99.2%), respectively. The six (5.5%) discordant results (four false negative, two false positive) all were observed in samples with HCV RNA detectable below the limit of quantification after 2-8 weeks of pan-genotypic direct-acting antiviral treatment or 5 weeks after acute hepatitis C in a patient clearing HCV spontaneously. Quantifiable results (n = 30; 16 genotype 1, 7 genotype 3, 4 genotype 4, 1 genotype 1a and 3a, 2 unknown; HCV RNA range: 2.74-6.66 log IU/ml) correlated well (R2 = 0.981). On average, uncorrected DBS test results were 1.30 ± 0.14 log IU/ml lower than Xpert® HCV VL FS test results (~42 μl instead of the expected 1000 μl plasma used). Storage of DBS samples at room temperature for 7 days before freezing reduced HCV RNA by 0.29 ± 0.12 log IU/ml. HCV RNA can reliably be quantified with the Xpert® HCV VL test in capillary dried blood spot samples. Thus, access to capillary HCV RNA quantification for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection can be extended to decentralised settings with a low case load.

Hepatitis C Virus Core Antigen as an Alternative to RNA in the Assessment of Response to Treatment with Direct-acting Oral Antivirals

Background: Affordable and effective diagnostic and treatment monitoring algorithms are urgently needed to achieve the global elimination of hepatitis C virus (HCV) infection. Methods: A total of 274 patients were treated with direct-acting antivirals (DAAs) in the Spanish Hospital of Albacete between 2004 and 2020. This study compared the enzyme-immunoassay technique for HCV core antigen (HCVcAg) with the determination of RNA of HCV (HCV RNA) by polymerase chain reaction (PCR) in monitoring treatment with DAA, setting the lower limit of detection of HCVcAg &lt; 3 fmol/L and RNA &lt; 10 IU/mL. In all cases, the P value of differences associated with the contrast test was less than or equal to 0.05. Results: We evaluated the viral loads of our patients before treatment, during their treatment, and after its completion. The HCV RNA quantification at diagnosis was 2309327 IU/mL. The mean HCVcAg load was 5972 fmol/L. There was a strong correlation between HCVcAg levels and RNA levels with a Spearman rho of 0.832 (P &lt; 0.01). The HCVcAg sensitivity at diagnosis was 99%, but the specificity could not be calculated because there were no true negatives or false positives at this point. Twelve weeks after treatment, in patients with treatment failure, we obtained a mean of 19084 IU/mL for RNA, while for HCVcAg, the mean was 103 fmol/L. At this time point, we also found a strong correlation between HCVcAg levels and HCV RNA levels with a Spearman rho of 0.775 (P &lt; 0.01). Finally, the virological cure was achieved in 99% of our patients. The results for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 100%, 99.87%, 86.33%, and 100%, respectively. Conclusions: HCVcAg determination is an excellent alternative to HCV RNA in the assessment of treatment response. This is particularly relevant in lower- and middle-income countries and resource-limited settings where the high cost of labor, equipment, and reagents can prohibit molecular testing.

The performance of hepatitis C virus (HCV) antibody point-of-care tests on oral fluid or whole blood and dried blood spot testing for HCV serology and viral load among individuals at higher risk for HCV in South Africa.

Background and AimsTo enhance screening and diagnosis in those at‐risk of hepatitis C virus (HCV), efficient and improved sampling and testing is required. We investigated the performance of point‐of‐care (POC) tests and dried blood spots (DBS) for HCV antibody and HCV RNA quantification in individuals at higher risk for HCV (people who use and inject drugs, sex workers and men who have sex with men) in seven South African cities.MethodsSamples were screened on the OraQuick HCV POC test (471 whole blood and 218 oral fluid); 218 whole blood and DBS paired samples were evaluated on the ARCHITECT HCV antibody (Abbott) and HCV viral load (COBAS Ampliprep/COBAS TaqMan version 2) assays. For HCV RNA quantification, 107 dB were analyzed with and without normalization coefficients.ResultsPOC on either whole blood or oral fluid showed an overall sensitivity of 98.5% (95% CI 97.4‐99.5), specificity of 98.2% (95% CI 98.8‐100) and accuracy of 98.4% (95% CI 96.5‐99.3). On the antibody immunoassay, DBS showed a sensitivity of 96.0% (95% CI 93.4‐98.6), specificity of 97% (95% CI 94.8‐99.3) and accuracy of 96.3% (95% CI 93.8‐98.8). A strong correlation (R 2 = 0.90) between viral load measurements for DBS and plasma samples was observed. After normalization, DBS viral load results showed an improved bias from 0.5 to 0.16 log10 IU/mL.ConclusionThe POC test performed sufficiently well to be used for HCV screening in at‐risk populations. DBS for diagnosis and quantification was accurate and should be considered as an alternative sample to test. POC and DBS can help scale up hepatitis services in the country, in light of our elimination goals.

Architect HCV Ag assay is an efficient alternative tool to RNA qRT-PCR quantification for assessing viral load in HCV infection

Objective To evaluate the performance characteristics of the automated Architect hepatitis C virus (HCV) core Ag assay versus HCV RNA by PCR among Egyptian patients and to assess its use for valuable clinical workup. Background HCV diagnosis by conventional anti-HCV assays has high rate of false positivity, false negativity, and a limited sensitivity for detection. Although HCV RNA assays are a reliable method for HCV diagnosis, they need technical skills and may also have false-positive results because of contamination. Moreover, the test is time consuming and more expensive. In contrast, the HCV core antigen test detects circulating HCV core antigen and identifies individuals who are actively infected with HCV. A commercialized test (the Architect HCV core antigen test) is supposed to have a sensitivity to detect ∼0.06 pg/ml and consequently a significant increase in sensitivity over the previous assay and a stronger correlation with HCV RNA testing. Patients and methods A descriptive, cross-sectional study was conducted on 60 HCV antibody-positive patients attending the outpatient clinic of Tropical Medicine Department, Al-Hussein University Hospital, Cairo, Egypt. The patients were classified into four groups based on the level of HCV viremia: group A included five patients with PCR below detection limit (12 IU/ml), group B included 39 patients with low viremia (&lt;100 000 IU/ml), group C included 13 patients with moderate viremia (100 000–10 000 000 IU/ml), and group D included three patients with high viremia (&gt;10 000 000 IU/ml). Each case was subjected to thorough clinical evaluation, HCV RNA quantification by Abbott Real Time HCV assay, and HCV Ag quantification by Architect HCV core antigen test. Results HCV Ag was found to be negative only in five of 55 HCV RNA-positive patients who had low level of viremia. The levels of HCV Ag showed a good correlation with those from the HCV RNA quantification (r=0.913, P≤0.001). Regarding HCV core antigen/HCV RNA ratio, it was not fixed for all patients. In most of them, each 1 pg/ml core Ag was equal to ∼10 000 IU/ml of RNA. Conclusion The Architect HCV Ag assay could be used as an alternative tool to HCV RNA PCR quantification in assessing viral load in HCV infection, and it has the advantages of lower cost, easy testing, and rapid reporting.

Improving Diagnosis of Hepatitis C Virus Infection Using Hepatitis C Core Antigen Testing in a Resource-Poor Setting.

INTRODUCTION: We compared the hepatitis C virus (HCV) core antigen test with the HCV RNA assay to confirm anti-HCV results to determine whether the HCV core antigen test could be used as an alternative confirmatory test to the HCV RNA test. METHODS:Sera from 156 patients were analyzed for anti-HCV and HCV core antigen using a chemiluminescent microparticle immunoassay (Architect i2000SR) and for HCV RNA using the artus HCV RG RT-PCR Kit (QIAGEN) in a Rotor-Gene Q instrument. RESULTS: The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 77.35%, 100%, 100%, and 89.38%, respectively. HCV core antigen levels showed a good correlation with those from HCV RNA quantification (r =0.872). However, 13 samples with a viral load of less than 4000 IU/mL were negative in the HCV core antigen assay. All gray-zone reactive samples were also RNA positive and were positive on repeat testing.CONCLUSIONS: The Architect HCV core antigen assay is highly specific and has an excellent positive predictive value. At the present level of sensitivity (77%), the study is still relevant in a low-income setting in which most of the HCV-positive patients would go undiagnosed, since HCV RNA testing is not available and/or not affordable. HCV core antigen testing can also help determine the true burden of infection in a population, considering the fact that almost 50% of the anti-HCV positive cases are negative for HCV RNA.

The Biochemical investigation and analysis to determine a potent biomarker for HCC and HCV infections

The communicable Hepatitis C Virus (HCV) infection causes global health problems and deaths due to transmission by blood and body fluids and general symptoms include loss of appetite, fatigue, fever, abdominal pain, swelling, jaundice, and dark urine.This study contains a detailed study about the potent biochemical biomarkers for the diagnosis and detection of HCV infection. It includes the biochemical profiling relating to the HCV infection among both the genders of the human population is included in this paper.96 (5ml) EDTA blood samples of suspected HCV infected patients were collected from the different hospitals in Dehradun, Uttarakhand, India. The RNA extraction was performedwith the help of QIAamp Viral RNA mini kit (Cat. no. 52904). The HCV RNA quantification was achieved using the machine named Rotor Gene Q5 Plex Real-Time PCR and master mix was prepared by the Artus Amplification Kit from Qiagen.Out of 96 samples collected for HCV RNA biochemical profiling, 58 cases i.e., were found with the heavy load of virus and the study had 38 cases in which target virus was not detected. The biochemical investigation was performed on 96 cases with a high viral load of more than 34 IU/ml (≥34 IU/ml). The cases belonging to age 21-40 and 41-60 years were found to be most prominent among 35 and 37 cases respectively due to the repeated exposure to infected blood and the age group of 0-20 years was found to be least susceptible due to the protective antibody titer against HCV due to the vaccination in early age.

Detection and Quantification of HCV-RNA by RT-PCR among Anti-HCV Positive Patients

Introduction: The most common contemporary strategy to diagnose chronic hepatitis C virus (HCV) infection consists of initial screening with an HCV enzyme immunoassay (EIA) antibody test followed by supplemental testing of positive screening tests with a quantitative HCV RNA assay to confirm the positive EIA and to determine whether they have active or resolved hepatitis C infection.&#x0D; Objectives: To detect and quantify HCV-RNA by real-time polymerase chain reaction (real-time PCR) among anti-HCV positive patients and to identify the socio demographic factors among these patients.&#x0D; Materials and Methods: This was a descriptive type of cross-sectional study which was conducted in Combined Military Hospital and Armed Forces Institute of Pathology, Dhaka cantonment. A total of 108 anti-HCV positive patients by enzyme-linked immunosorbent assay (ELISA), who were clinically suspected and advised for anti-HCV test, were selected randomly for the study and subjected to do HCV-RNA analysis during the period of October 2016 to September 2017.&#x0D; Results: Out of 108 anti-HCV positive patients by ELISA, HCV-RNA was detected in 72 (66.7%) cases with mean value of HCV RNA quantification was 2013323.95±2695207.41 (IU/ ml). Majority of anti-HCV positive patients (29.6%) belonged to 51-60 years age group with male predominance (58.33%). It was observed that 43.52% patients came from middle income group family, 31.48% came from poor and 25.0% came from high income group family. Risk factor for HCV infected population was found maximum in dialysis patients (47.37%), followed by blood transfusion (13.89%), Injecting drug User (IDUs) (12.04%), surgery &amp; intervention (9.26%) and sexual transmission (1.85%). Mean alanine aminotransferase (ALT) was found 67.30±44.99 U/L among HCV-RNA detected patients (p&lt; 0.05).&#x0D; Conclusion: The quantification of HCV RNA by RT-PCR will be helpful to rationalize the treatment, enhance antiviral responses and mitigate mortalities of HCV infected patients.&#x0D; Journal of Armed Forces Medical College Bangladesh Vol.15 (1) 2019: 84-86

Evaluation of the Aptima HCV Quant Dx Assay for Hepatitis C Virus RNA Detection from Fingerstick Capillary Dried Blood Spot and Venepuncture-Collected Samples.

Simplified diagnostic strategies are needed increase hepatitis C virus (HCV) testing to determine active infection and link people into treatment. Collection methods such as dried blood spots (DBS) have advantages over standard phlebotomy, especially within marginalized populations. We evaluated the diagnostic performance of the Aptima HCV Quant assay for the quantification and detection of HCV RNA from paired DBS and venepuncture samples. Specimens were collected from participants enrolled in an Australian observational study. We compared HCV RNA detection from DBS against venepuncture samples (gold standard). One hundred sixty-four participants had paired samples and HCV RNA was detected in 45 (27% [95% confidence interval, 21%-35%]) by the Aptima assay in venepuncture samples. Sensitivity of the Aptima assay for HCV RNA quantification from DBS (≥10 IU/mL in plasma) was 100% and specificity was 100%. Sensitivity for HCV RNA detection from DBS was 95.6% and specificity was 94.1%. A small bias in plasma over DBS was observed with good agreement (R2 = 0.96). The Aptima HCV Quant assay detects active infection from DBS samples with acceptable diagnostic performance and is clinically comparable to plasma. These data will strengthen the case for the registration of a DBS kit insert claim, enabling future clinical utility.

Sofosbuvir-Based HCV Treatment in Maintenance Hemodialysis Patients: A Single-Center Study

There is scant data on use of sofosbuvir containing direct-acting antiviral (DAA) regimens in maintenance hemodialysis (MHD) patients infected with hepatitis C virus (HCV). Recent data have focused on possible safe use of sofosbuvir in MHD patients. The aim of this study was to study the efficacy and safety of the sofosbuvir-based combination DAA in HCV-infected naive patients on MHD. Before starting treatment HCV viral load, genotype, liver function tests, and liver ultrasound were performed in all patients. HCV RNA quantification was assessed at baseline, at end of therapy, at 12 weeks, and 24 weeks after stopping therapy. Forty-three MHD patients (67.4% male, with no cirrhosis) with HCV infection were included in the study. Fifteen patients were diabetics. Thirteen patients received multiple transfusions. None of the patients had baseline cirrhosis. Average vintage of dialysis was 6.2 years. At 24-week follow-up none of the patients relapsed. Full dose sofosbuvir was used in combination with ledipasvir (N = 40, 100% genotype 1) and valpatasvir (N = 3, 2 genotype 1, 1 genotype 3). Sustained virological response (HCV RNA < 12 IU/mL) at 12 and 24 weeks after stopping treatment was seen in 100% of the patients. Full-dose sofosbuvir-based DAA therapy is highly effective for MHD patients with HCV infection.

Evaluation of the cobas® HCV test for quantifying HCV RNA in dried plasma spots collected using the cobas® Plasma Separation Card

ObjectiveThis study evaluated the performance of the cobas® hepatitis C virus (HCV) Test for use on the cobas® 6800/8800 Systems for the detection and quantification of HCV RNA collected using the cobas® Plasma Separation Card (PSC) compared with plasma samples. MethodsWhole EDTA-venous blood was collected from 50 HCV-positive donors and 140 μL from each donor was spotted onto a PSC and stored either frozen or at ambient temperature. These were compared with matched EDTA-plasma samples. The limit of detection (LoD) of the assay for PSC samples was determined using serial dilutions of the Roche HCV secondary calibration standard. The stability of the PSC samples across a range of timepoints was also assessed. ResultsThe mean titer difference between ambient and −10 °C storage of PSC samples was 0.04 log10 IU/mL (95% CI: 0.00, 0.07). The mean titer difference between frozen PSC samples and EDTA plasma samples was -1.59 log10 IU/mL (95% CI: -1.66, -1.53) and between ambient PSC samples and EDTA samples was -1.64 log10 IU/mL (95% CI: -1.70, -1.57). Correlation between PSC samples and EDTA plasma was linear in both cases (frozen: slope = 1.039, intercept=-1.839, R2 = 0.89; ambient: slope = 1.012, intercept=-1.712, R2 = 0.88). The LoD of the cobas® HCV Test using the PSC was 866 IU/mL (95% CI: 698, 1153 IU/mL) and 408 IU/mL (95% CI: 336, 544 IU/mL) using an optimized Assay Specific Analysis Package. ConclusionsPSC samples correlate well with plasma viral load and demonstrate a LoD below 1000 IU/mL and good stability at ambient temperature for 28 days. A correction factor would allow quantification of HCV viral RNA load from samples collected using a PSC.

In-field evaluation of Xpert® HCV viral load Fingerstick assay in people who inject drugs in Tanzania.

BackgroundAlthough novel hepatitis C virus (HCV) RNA point‐of‐care technology has the potential to enhance the diagnosis in resource‐limited settings, very little real‐world validation of their utility exists. We evaluate the performance of HCV RNA quantification using the Xpert® HCV viral load Fingerstick assay (Xpert® HCV VL Fingerstick assay) as compared to the World Health Organisation pre‐qualified plasma Xpert® HCV VL assay among people who inject drugs (PWID) attending an opioid agonist therapy (OAT) clinic in Dar‐es‐Salaam, Tanzania.MethodsBetween December 2018 and February 2019, consecutive HCV seropositive PWID attending the OAT clinic provided paired venous and Fingerstick samples for HCV RNA quantification. These were processed onsite using the GeneXpert® platform located at the Central tuberculosis reference laboratory.ResultsA total of 208 out of 220 anti‐HCV‐positive participants recruited (94.5%) had a valid Xpert® HCV VL result available; 126 (61%; 95% CI 53.8‐67.0) had detectable and quantifiable HCV RNA. About 188 (85%) participants had paired plasma and Fingerstick whole blood samples; the sensitivity and specificity for the quantification of HCV RNA levels were 99.1% and 98.7% respectively. There was an excellent correlation (R 2 = .95) and concordance (mean difference 0.13 IU/mL, (95% CI −0.9 to 0.16 IU/mL) in HCV RNA levels between plasma samples and Fingerstick samples.ConclusionThis study found excellent performance of the Xpert® HCV VL Fingerstick assay for HCV RNA detection and quantification in an African‐field setting. Its clinical utility represents an important watershed in overcoming existing challenges to HCV diagnosis, which should play a crucial role in HCV elimination in Africa.

Effects of free patchy ends in ssDNA and dsDNA on gold nanoparticles in a colorimetric gene sensor for Hepatitis C virus RNA.

The presence or absence and nature of the free patchy ends in DNA sequences has a decisive effect on the performance of colorimetric sensors based on the use of gold nanoparticles (Au NPs). The authors have designed two unmodified gene probes (probe 1: a 19-mer; probe 2: an 18-mer). They are complementary to either half of a 37-mer target derived from the conserved region of Hepatitis C Virus (HCV) RNA. Each probe has further been modified with 10-mer poly(A) and thiol-functionalized 10-mer poly(A) at the 5' positions. Nine combinations of probe and HCV RNA were then designed to investigate the effect of free patchy ends on the stability of citrate-modified Au NPs against salt-induced aggregation which lead to color change from red to blue. The aggregation of Au NPs can be monitored by ratiometric spectroscopy at wavelengths of 520 and 700nm. The differentiation between HCV RNA and control has also been studied by varying the concentration of probe and analyte. The particle size and zeta potentials were determined before and after aggregation. It is demonstrated that the change in surface charge density of the Au NPs governs the critical coagulation concentration of NaCl. The method presented here can be used to quantify HCV RNA in the 370nM to 3μM concentration range, and the detection limit is 500nM. The results obtained with Au NPs that are chemically non-conjugated with the oligonucleotides have been found to be valuable in rationally devising the design rules for rapid and efficient colorimetric sensing of oligonucleotides. Graphical abstract Schematic representation of the nine combinatorial pairs of oligonucleotides that vary in the length of patchy ends and their position to unearth their effect in rapid gold nanoparticle-based colorimetric gene sensing without time-consuming and expensive thiol-conjugation step.

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.