Evaluation of the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test

The performance of the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM) was evaluated. The limit of detection was 9.5 IU/mL using the 3rd International Standard. Serial dilutions of each genotype demonstrated good reproducibility and linearity. Correlation with samples previously tested with the Roche Analyte Specific Reagent (ASR) was very good, with the CAP/CTM assay measuring 0.24 log IU/mL higher on average than ASR. Genotype inclusivity evaluated in the CAP/CTM, ASR, and Siemens Versant HCV RNA 3.0 Assay (bDNA) assay using a commercially available panel showed higher measurements than ASR or bDNA. The differences in observed CAP/CTM and ASR results for genotype 3 patient samples were significantly different (P < 0.05) from those for both genotype 1 and 2 samples. Common inhibitory substances had no more than a 0.25 log IU/mL affect. Overall, the automated CAP/CTM assay exhibits excellent sensitivity, reproducibility, and dynamic range. Its performance is compatible with its use in guiding therapy using direct acting antivirals such as boceprevir and telaprevir.     

慢性丙型肝炎患者血清基因型与HCV RNA的相关性研究

目的 研究丙型肝炎患者HCV基因型的分布,探讨基因型在性别上的分布以及基因型与HCV RNA病毒载量的相关性.方法 收集2010年5-12月来自40家医院的206例丙型肝炎患者的血清标本,采用瑞士罗氏公司生产的定量PCR试剂(罗氏试剂)对进行HCV RNA检测,应用雅培公司生产的Abbott RealTime HCV GenotypeⅡ试剂(雅培试剂)对206例丙型肝炎患者的血清标本进行基因分型,分析基因型在性别上的分布以及HCV基因型与HCV RNA病毒载量的相关性.结果 206份HCV RNA阳性血清标本中HCV1型(未具体分1a和1b型)占3.4%(7/206)、1a型占1.0%(2/206)、1b型占59.7%(123/206)、2型占15.5%(32/206)、3型占13.1%(27/206)、6型占2.9%(6/206)、1/6混合型占2.4%(5/206)、2/4混合型占0.5%(1/206),未分型占1.5%(3/206).132例基因1型和65例非基因1型(2型、3型和6型)患者HCV基因型在性别上的分布差异无统计学意义(x2=0.000,P＞0.05).188例患者不同基因型之间血清HCV RNA病毒载量差异有统计学意义(F=3.371,P＜0.05).将197例HCV单基因型患者按地区分为东、南、西、北、中5组,基因型1型与非基因1型在地区分布上差异无统计学意义(x2=5.840,P＞0.05).结论 丙型肝炎病毒感染以1b型为主,其次为基因2型.基因型在性别上的分布没有差异.基因1b型HCV RNA病毒载量高于基因3型,基因2型HCV RNA病毒载量高于基因3型,基因6型HCV RNA病毒载量高于基因3型.     

Evaluation of automated RNA-extraction technology and a qualitative HCV assay for sensitivity and detection of HCV RNA in pool-screening systems

BACKGROUND: The objective of this study was the evaluation of NAT technology for the detection of HCV RNA in plasma pools according to the recommendations of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool). STUDY DESIGN AND METHODS: Serial dilutions of both the EUROHEP standard (3,800 genome equivalents [geq]/mL; HCV genotype 1) and the World Health Organization (WHO) international standard (100,000 IU/mL; HCV genotype 1) were made in S/D plasma (ESPEP plasma, OctaPharma), which was nonreactive in serologic tests. Serial dilutions of plasma (2 mL) were used for extraction of HCV RNA with an automated version of a nucleic acid isolation method (NucliSens Extractor, Organon Teknika). HCV RNA was co-extracted from 2 mL of plasma, together with 84 copies of an in vitro-synthesized single-strand RNA serving as internal extraction control (IC) to monitor the efficiency of extraction and PCR. Amplification and detection of both HCV RNA and IC RNA were performed with an automated PCR system and a qualitative HCV assay (COBAS Amplicor 2.0 HCV, Roche Diagnostics). RESULTS: A cutoff value of 16 geq per mL (10/10 runs [100% hit rate]) was found by using the EUROHEP standard, whereas the WHO international standard had a cutoff value of approximately 12 IU per mL (10/10 runs [100% hit rate]). The IC had a cutoff value of approximately 17.5 copies per mL (6/6 runs [100% hit rate]). Forty-two copies per mL of IC RNA were found in 282 of 284 runs (99% hit rate). The negative controls (ESDEP plasma) were negative in all experiments. Experiments with pool sizes of 12, 24, 48, and 96 using serial dilutions of the WHO international standard revealed a cutoff value of 8 IU per mL (100% hit rate). The EUROHEP standard and the WHO international standard were detected with a 50 percent detection endpoint of 5.2 geq per mL and 1.5 IU per mL, respectively. CONCLUSION: This test system (NucliSens Extractor, and the COBAS Amplicor 2.0 HCV assay) revealed a high sensitivity for HCV RNA; considering the proposed requirements for sensitivity of NAT assays for the detection of HCV RNA in donor plasma, pool sizes of about 400 donors are possible. These endpoint results indicated that 1 IU is equal to about 3.4 geq.     

Early on-treatment viral load and baseline METAVIR score: improved prediction of sustained virological response in HCV genotype 1 patients

BACKGROUND: On-treatment HCV viral load during early therapy with pegylated interferon (PEG-IFN) and ribavirin is highly predictive of sustained virological response (SVR). We sought to provide further refinement of this prediction through an extensive evaluation of the effect of HCV viral loads at weeks 4, 8 and 12 on SVR, including analysis by liver disease stage grouping. METHODS: A total of 309 patients with genotype 1 chronic HCV and recent liver biopsy enrolled in the CHARIOT study received 180 μg of PEG-IFN-α2a weekly with 1,000/1,200 mg of ribavirin daily. The probability of an SVR was estimated using baseline METAVIR fibrosis stage and HCV viral loads at weeks 4, 8 and 12. RESULTS: HCV RNA was undetectable in 27.5%, 50.3% and 62.6% of patients at weeks 4, 8 and 12, respectively. SVR was 80.0%, 76.8% and 72.4% among patients with undetectable HCV RNA at weeks 4, 8 and 12, respectively. SVR decreased in a progressive fashion with increasing HCV viral loads at each early time point, but was similar for patients with HCV viral load <15 IU/ml, 15-100 IU/ml and 100-1,000 IU/ml. The effect of fibrosis stage on SVR was modest for patients with HCV viral load <1,000 IU/ml at week 4, but more marked for those with week 4 HCV viral load >1,000 IU/ml, and all HCV viral load categories at weeks 8 and 12. CONCLUSIONS: A combination of baseline fibrosis stage and on-treatment HCV viral load at early time points provides improved estimates for treatment response in patients with chronic HCV genotype 1.     

Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV

BACKGROUND: Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti-HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated. STUDY DESIGN AND METHODS: Blood donations (n = 251,737) were screened for HCV RNA over 4 years. RNA extraction, amplification, and detection were done by two commercial HCV PCR kits (HCV Cobas Amplicor and HCV Cobas Amplicor 2.0, Roche Diagnostics). Screening was done by pool testing with a maximum pool size of 40 serum samples. RESULTS: Three donations out of 251,737 were HCV RNA positive and anti-HCV negative. ALT levels of these donations were 271, 32, and 10 U per L. The HCV infection of a fourth HCV RNA-positive donor could not be identified by routine, second-generation HCV EIA (Abbott Diagnostika). In this case, two previous donations were also HCV RNA positive, and three second-generation test systems (Abbott) could not detect anti-HCV, whereas third-generation anti-HCV screening assays detected antibody with different sensitivity. The first HCV RNA-positive donation was identified only by the HCV ELISA 3.0 (Ortho Diagnostic Systems). The results of confirmatory assays like RIBA HCV 3.0 (Ortho) and Matrix (Abbott) indicate a restricted immune response to NS3 only. CONCLUSION: HCV RNA detection by PCR can be carried out routinely in blood donor screening without significant delay of release of the components. The residual risk of transmission can be reduced by identification of early infection, which can lead to an improved safety of blood components. RNA screening can also be advantageous in cases of incomplete or lack of antibody response to HCV.     

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.     

Increased sensitivity of the Roche COBAS AMPLICOR HCV test, version 2.0, using modified extraction techniques

Processing modifications were made to the COBAS AMPLICOR HCV version 2.0 assay to enhance sensitivity. Two methods of specimen concentration, centrifugation ("ultraspin") and cationic detergent plus silica membrane ("ultracolumn"), were compared to the standard method. The effect of these changes on assay sensitivity and specificity was examined using commercial hepatitis C virus (HCV) preparations. The limits of detection (LOD, defined as detection of HCV RNA in >/= 95% of replicates) of genotype 1a were 50, 12, and 6 by standard method, ultraspin and ultracolumn, respectively. For genotype 1b, the LOD was 25 IU/ml, 12 IU/ml, and 3 IU/ml; for 2b, it was 50, 12, and 3; for 3a, it was 25, 12, and 1.5; for 4 it was 18, 4, and 2; for 5a, it was 38, 9, and 2; and for 6a it was 47, 6, and 3. No false positives were detected after ultraspin when controls containing high or low HCV concentrations were alternated with normal human plasma. Plasmas in which HCV RNA was not detected by the standard assay were re-tested with modified methods to assess the effect of altered processing in clinical specimens. Three of 152 specimens with no detectable HCV RNA by the standard method were positive by ultraspin and 2 of 109 were positive by ultracolumn, suggesting that these methods may increase assay sensitivity in clinical specimens.     

High sustained virologic response rate after interferon monotherapy in Japanese hepatitis C patients with a low HCV RNA titer and/or HCV genotype 2. A prospective study

OBJECTIVE: Hepatitis C virus (HCV) RNA titer and HCV genotype are considered to be major determinants of the outcome of interferon monotherapy. To clarify whether interferon monotherapy is really effective in patients with the appropriate viral parameters, we prospectively examined these parameters and treated the patients with interferon monotherapy. METHODS: Sixty-four patients with an HCV RNA titer <100 kIU/ml and/or HCV genotype 2 were enrolled in the study. Eighteen patients with an HCV RNA titer >100 kIU/ml and genotype 1 were also enrolled as controls. All patients were treated with 10 megaunits of interferon-alpha2b every day for 2 weeks and then 3 times a week for 24 weeks. RESULTS: Of the 64 patients with either HCV RNA <100 kIU/ml and/or genotype 2, seven dropped out from the study. Of the remaining 57 who completed the treatment, 48 (84%) showed a virologic sustained response. In contrast, only 4 of the 18 patients (22%) with HCV RNA >100 kIU/ml and genotype 1 were virologic sustained responders (p < 0.001). CONCLUSION: Our current study showed that the patients with HCV RNA <100 kIU/ml and/or HCV genotype 2 are good candidates for interferon monotherapy.     

Performance characteristics of a quantitative hepatitis C virus RNA assay using COBAS AmpliPrep total nucleic acid isolation and COBAS taqman hepatitis C virus analyte-specific reagent

Performance characteristics of a hepatitis C virus (HCV) RNA quantification assay comprised automated specimen extraction [COBAS AmpliPrep (CAP) using total nucleic acid isolation reagents (TNAI)], and real-time polymerase chain reaction [COBAS TaqMan 48 HCV with analyte-specific reagents (CTM48)] were determined. CAP TNAI/CTM48 performed linearly from approximately 2.0 to at least 6.7 log10 IU/ml for HCV genotypes (Gts) 1, 2, and 3. The limit of detection for the World Health Organization International Standard was 23 IU/ml. Variabilities ranged from 1.3 to 2.1%. Excellent quantitative agreement was observed in clinical samples using CTM48 and two different methods for HCV RNA extraction (CAP TNAI and BioRobot M48; regression line slope, 0.98; y-intercept, 0.11; R2, 0.98; mean difference, 0.003). Good agreement was also observed between CAP TNAI/CTM48 and COBAS Amplicor Monitor (regression line slope, 0.94; y-intercept, 0.08; R2, 0.96), although HCV RNA concentrations were on average greater by COBAS Amplicor Monitor (mean difference -0.27 log10 IU/ml). Better overall agreement was observed for Gt 1 than non-Gt 1 specimens when comparing extraction and quantification methods; however, no consistent genotype-dependent quantification bias was observed. These data suggest that CAP TNAI/CTM48 offers an alternative method for the quantification of HCV in plasma samples.     

深圳献血者中HCV感染自然清除与病毒血症特征分析

目的测定我国深圳地区献血者中的丙型肝炎病毒(hepatitis C virus,HCV)感染者自然清除和病毒血症,即自然康复与慢性感染者比率及其人群特征,为丙型肝炎防治研究提供数据。方法对深圳献血者中抗-HCV初筛阳性的血清标本采用2种EIA方法进行抗体再测定,以定量PCR方法测定病毒载量,并采用巢式PCR对核酸进行确认,进而将标本分为3种HCV感染状态,即病毒自然清除(RNA-/Ab+)、病毒血症(RNA+/Ab+)和假阳性(RNA-/Ab-),通过统计学方法分析3种感染状态献血者在临床信息(性别、年龄)、ALT、抗-HCV的差异。HCV RNA阳性标本通过分析5'-NCR序列进行基因分型。结果 152份初筛抗-HCV阳性的标本中,病毒自然清除标本45份、病毒血症51份、假阳性56份。50份HCV定量PCR阳性标本病毒载量范围从[(12.6～2.43)×106]IU/ml(中位值2.54×104IU/ml)。36份进行基因分型的标本包括47.2%基因1型、5.6%基因2型、19.4%基因3型和27.8%基因6型。慢性感染组标本的年龄及抗-HCV水平(S/CO值)显著高于病毒自然清除和假阳性组(χ2=7.812,P0.05;χ2=90.865,P0.01)。结论深圳地区献血者HCV感染病毒自然清除率约为46.9%。HCV基因型中1型为主要,6型也占有较高比例。年龄小、女性献血者更易于自然清除病毒。病毒血症即慢性感染者HCV抗体水平显著升高,其在HCV自然康复过程中的作用还有待进一步研究。     

HIV-1感染者中HCV混合感染情况分析

目的：调查我国不同地区、通过不同传播途径感染人类获得性免疫缺陷病毒I型（HIV－1）患中丙型肝炎病毒（HCV）的流行情况及不同亚型的分布。方法：采用酶联免疫吸附试验（ELISA）检测抗－HIV－1并以蛋白印迹试验（Western blot,WB)进行确认。采用DNA分支放大（bDNA)技术检测HIV－1病毒载量，采用荧光抗体流式细胞检测技术（FACs)作CD4和CD8细胞计数。抗－HCV检测采用ELISA方法。HCV基因亚型的测定采用实时（real-time)聚合酶链反应（PCR）方法。结果：共检测了239例HIV－1感染，抗－HCV阳性率为56．9％（136／239），其中经不同传播途径感染HCV的阳性率分别为：静脉注毒：42．7％（58／136）；经血液：53．7％（73／136）；性接触途径：3．7％（5／36）。静脉注毒（云南和新疆）HCV感染以1，3，4亚型最多，而输血人群（河南省）感染的HCV以1，2亚型为主。结论：HIV－1感染中存在HCV混合感染，我国HCV基因亚型以1型为主。     

The magnitude of week 4 HCV RNA decay on pegylated interferon/ribavirin accurately predicts virological failure in patients with genotype 1

BACKGROUND: Current stopping rules during pegylated interferon (peg-IFN)/ribavirin (RBV) treatment rely on week 12 HCV RNA response, but earlier identification of non-responders offers clinical and economic advantages. AIMS AND METHODS: To evaluate, among 129 HCV-genotype-1-infected, treatment-naive patients receiving peg-IFN/RBV, the feasibility of predicting treatment failure using receiver operating characteristics (ROC) curves after measuring week 4 HCV RNA decreases, and to assess baseline predictors of not achieving sustained virological response (SVR). RESULTS: Peg-IFN-alpha2b was used in 84.5% of patients. Fifty-three (41%) reached SVR. The best cutoff value of HCV RNA decrease at week 4 to predict non-SVR corresponded to 1 log10 IU/ml: sensitivity and negative predictive value: 100%; specificity: 64%; positive predictive value: 66%; ROC curve area: 0.91 (95% confidence interval [CI]: 0.86-0.96). By applying this threshold, treatment could have been discontinued at week 4 in 64% of virological non-responders (49/76). By univariate analysis, baseline HCV RNA > 800,000 IU/ml (P = 0.029), older age (P = 0.011), and higher aspartate aminotransferase (AST) levels (P = 0.005) or AST/alanine aminotransferase ratio values (P = 0.04) were associated with failure. After multivariate analysis, only baseline HCV RNA >800,000 IU/ml (odds ratio [OR]: 2.12; 95% CI: 1.005-4.488; P = 0.048) and higher AST levels (OR: 1.01; 95% CI: 1.003-1.024; P = 0.011) remained statistically significant. CONCLUSIONS: The lack of > or = log10 IU/ml decrease in baseline HCV RNA at week 4 was 100% predictive of treatment failure, independently associated with HCV RNA > 800,000 IU/ml and higher AST levels.     

Performance comparison of new generation HCV core antigen test versus HCV RNA test in management of hepatitis C virus infection

The study has evaluated the performance of HCV core antigen (Cag) test by comparing HCV RNA PCR assay which is considered the gold standard for management of HCV infection.Totally, 132 samples sent for HCV RNA (real-time PCR) test were included in the study. Anti-HCV antibody test and HCV Cag test were performed by chemiluminescent enzyme immunoassay (CMEI).Anti-HCV test was positive in all samples. HCV RNA was detected in 112/132 (84.8%) samples, and HCV Cag in 105/132 (79.5%). The most common HCV genotype was genotype 1 (86%). Considering the HCV RNA test as gold standard; the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Cag test were found to be 93.75%, 100%, 100%, 74.07% and 94.69%, respectively, and paired test results were detected as highly concordant. A high level of correlation was seen between HCV RNA and Cag tests, however, the concordance between the two tests appeared to be disrupted at viral loads lower than 10³ IU/mL. On the contrary, the correlation reached significance for the values higher than 10³ IU/mL. Viral loads were in the 17–2500 IU/mL range for the negative results for Cag test. Pearson's correlation coefficient revealed a considerably high correlation.The concordance between HCV RNA and Cag tests was disrupted under a viral load lower than 10³ IU/mL. Therefore, it would be appropriate to consider cost effectiveness, advantages and limitations of the HCV RNA and Cag tests during the decision on which method to use for patient management.     

国产HCV RNA分型试剂盒检测结果分析

目的分析丙型肝炎病毒(HCV)RNA分型试剂盒检测深圳市抗-HCV阳性献血者结果。方法收集2014-2015年158份深圳市抗-HCV阳性献血者血液样本,应用聚合酶链反应(PCR)-荧光探针法进行HCV RNA定量检测,病毒载量1.0×103 IU/mL的样本经HCV RNA分型试剂盒检测HCV基因型,分析不同基因型所占比例,病毒基因型与载量之间的相关性。结果 158份抗-HCV阳性献血者PCR-荧光探针法检出HCV RNA阳性样本54例,病毒载量1.0×103 IU/mL的45例,全部得到分型结果,HCV 1b型、2型、3型、6型分别占57.78%(26/45)、6.67%(3/45)、8.89%(4/45)、26.67%(12/45)。单因素方差分析结果显示,1b型与2型病毒载量差异有统计学意义(F=2.861,P0.05);不同性别间HCV RNA定量检测结果及抗-HCV S/CO值结果差异有统计学意义(P0.05);不同年龄段各基因型分布比例经Fisher精确检验,差异有统计学意义(P0.05)。结论 HCV 1b型、6型仍为深圳市无偿献血人群感染HCV的主要基因型,而HCV 2型和3型比例有所减少。