RealTime HCV Research Articles

La valoración de RNA del virus de la hepatitis C realizada a las 12 semanas de finalizar el tratamiento predice la respuesta virológica sostenida en enfermos con coinfección por VIH y VHC

The aim of this study was to assess whether measurement of hepatitis C virus RNA (HCV-RNA) at 12 weeks post-treatment could predict sustained virological response (SVR) to antiviral therapy for chronic hepatitis C (pegylated interferon alfa-2a and ribavirin) in HIV-co-infected patients. HIV-HCV co-infected patients were included if they completed a full course of anti-HCV therapy, achieved an end-of-treatment response and complied with the week +12 and +24 post-treatment follow-up schedule for serum HCV-RNA determination (Real-time HCV (Abbott, Wiesbaden, Germany) (lower limit of detection, 12 IU/ml). Forty out of 66 patients (61%) showed an end-of-treatment response. They were assessed in a follow-up visit at +12 and at +24 weeks post-treatment. Serum HCV-RNA was undetectable in 28 of them at +12 weeks, and 100% of these remained undetectable at 24 weeks post-treatment (the gold standard of (SVR). The positive predictive value was 100% (95% confidence interval, 98.21-100%). Post-treatment follow-up to identify virological relapse could be shortened to 12 weeks, providing a new definition of sustained virological response.

Correlation between genotype and HCV RNA in chronic hepatitis C patients

Objective To determine the distribution of HCV genotypes in patients with chronic hepatitis C,study the distribution of genotype in different gender and the relationship between genotypes and serum HCV-RNA levels.Methods Two hundred and six cases of HCV RNA positive patients(all with relevant clinical data) receiving pegylated interferon therapy were collected from May to December 2010.HCV RNA was detected in 206 hepatitis C patients from 40 hospitals in China by Roche Cobas AmpliPrep/Cobas TaqMan HBV test,and genotype was determined by Abbott RealTime HCV G enotype Ⅱ .The distribution of genotypes in the gender was analyzed by chi-square test analysis.The relationship between genotypes and serum HCV RNA levels was detected by single factor analysis and two independent sample t test analysis.Results There were seven different subtypes of HCV in 206 samples,including genotype 1,7 cases(3.4% ,7/206); genotype 1a,2 cases(1.0%,2/206); genotype 1b,123 cases (59.7 %,123/206); genotype 2,32 cases(15.5 %,32/206); genotype 3,27 cases(13.1%,27/206); genotype 6,6 cases(2.9% ,6/206) ;genotype 1/6,5 cases(2.4% ,5/206) ;genotype 2/4,1 cases(0.5%,1/206).There was no significant difference between HCV genotype and gender in 132 cases with genotype 1 and 65 cases with non-genotype 1(genotype 2,3,6) (x2 = 0.000,P ＞ 0.05).There was significant association between quantity of HCV RNA and genotype in 188 patients with HCV(F = 3.371,P＜ 0.01).The 197 patients with HCV single genotype were divided into five groups in terms of region(East,South,West,North and Center).There was no significant difference between HCV genotype 1 and non-genotype 1 in the five groups(x2 = 5.840,P ＞ 0.05).Conclusions It is suggested that HCV 1 b is the most prevalent type in China,followed by HCV 2.There is no significant difference between HCV genotype and gender.The levels of HCV RNA with genotype 1b are significantly higher than those with genotype 3.The levels of HCV RNA with genotype 2 are significantly higher than those with genotype 3.The levels of HCV RNA with genotype 6 are significantly higher than those with genotype 3. Key words: Hepacivirus; RNA; Genotype; Polymerase chain reaction

Distribution of hepatitis C virus genotypes among different exposure categories in the State of Pará, Brazilian Amazon

Epidemiological studies concerning HCV genotypic distribution in the Brazilian Amazon are scarce. Thus, this study determined the patterns of distribution of HCV genotypes among different exposure categories in the State of Pará, Brazilian Amazon. A cross-sectional study was conducted on 312 HCV-infected individuals belonging to different categories of exposure, who were attended at the HEMOPA, CENPREN and a private hemodialysis clinic in Belém. They were tested for HCV antibodies using an immunoenzymatic test, RNA-HCV, using real-time PCR and HCV genotyping through phylogenetic analysis of the 5' UTR. The population groups were epidemiologically characterized according to data collected in a brief interview or medical consultation. Genotype 1 predominated in all the different categories of HCV exposure. HCV genotypic distribution among blood donors comprised genotypes 1 (94%) and 3 (6%). All patients with chronic hematologic diseases had HCV genotype 1. The genotypic distribution in illicit-drug users comprised genotypes 1 (59.6%) and 3 (40.4%). In patients under hemodialysis, genotypes 1 (90.1%), 2 (3.3%), and 3 (6.6%) were detected. Finally, the frequency of genotypes 1 and 3 was significantly different between the groups: BD and DU, PUH and DU, PUH and PCHD and PCHD and DU. The genotypic frequency and distribution of HCV in different categories of exposure in the State of Pará showed a predominance of genotype 1, regardless of the possible risk of infection.

Comparable performance of TMA and Real-Time PCR in detecting minimal residual hepatitis C viraemia at the end of antiviral therapy

Background Antiviral therapy for chronic hepatitis C may cause transient on-treatment response followed by post-treatment relapse. Objectives We have compared the prognostic value for post-treatment relapse of minimal hepatitis C residual viraemia detected at end-of-therapy by transcription mediated assay (TMA) and by Abbott RealTime PCR HCV assay. Study design Minimal residual viraemia was investigated in 202 HCV patients who had completed a full course of Pegylated Interferon (PEG-IFN) plus ribavirin and were HCV-RNA negative by conventional PCR in two separate serum samples obtained during the last week of therapy and the results were then correlated with post-treatment outcome. Results Minimal residual viraemia was detected in 22/202 (11%, 95% CI: 7–16%) and in 28/202 (13.8%, 95% CI 10–19%) patients by TMA and by Abbott RealTime HCV assay, respectively, with 92% concordant results. Post-treatment relapse was seen in 81.8% (95% CI: 60–93%) of TMA positive and in 82.1% (95% CI: 64–92%) of Abbott RealTime HCV assay positive cases compared to 16.6% (95% CI: 12–23%) of TMA negative and 17.2% (95% CI: 12–23%) of Abbott RealTime HCV assay negative patients. Conclusions These results indicate that TMA and the Abbott RealTime HCV assay have comparable sensitivity and specificity in identifying minimal residual viraemia at the end of antiviral therapy, with excellent predictive value for post-treatment relapse.

Construction of HCV-producing cell model based on self-cleaving ribozyme

To construct a stable HCV-producing cell model for anti-HCV drug research. The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy. HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha. We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.

A multicenter evaluation of the Abbott RealTime HCV genotype II assay

Viral genotype is an important determinant of the therapeutical outcome of the chronic hepatitis C and is useful in clinical practice to determine the duration of treatment1.While the viral type shows a clear association with therapeutic success, there is currently no evidence to that effect for HCV subtype, whose value is thus confined to epidemiological studies.The Abbott RealTime HCV Genotype II assay, through the use of Minor Groove Binder probes (MGB) is able to distinguish genotypes 1 to 6 (target 5’-UTR region) and subtypes 1a and 1b (NS5B region). In four different Italian centers a comparison between the Abbott RealTime HCV Genotype II assay and the Versant HCV Genotype 2.0 (LIPA) has been performed.A total of 143 non selected samples with the request of HCV genotyping have been analysed. 141/143 samples (98.6%) have provided reportable results with both tests (2 indeterminates with LIPA). Concordance at the type level was 96.5% (136/141). Considering the 136 concordant samples, the distribution was as follows: type 1 = 61 (44.9%), 2 = 36 (26.5%), 3 = 21 (15.4%), 4 = 17 (12.5% ) 5 = 1 (0.7%). Both assays assigned subtype in 56/61 (91.8%) samples of genotype 1 (3 and 2 samples only provided the type for LIPA and Abbott, respectively) and 50/56 (89.3%) had concordant subtype. It is worth to note that 4 of the 5 samples with discordant subtype Abbott 1a/LiPA 1b came from the only center that used for LIPA the 5-UTR amplicon, loosing the benefit of the core region which has been introduced in the version 2 of the test to improve the accuracy in distinguishing between 1a and 1b.There was only one discordant sample at type level (Abbott 4, LIPA 1b) which after sequencing and phylogenetic analysis was resolved as type 4. Four mixed infections were detected, 3 with the Abbott test (two 1a+4 and one 1b+3) and 1 with the LIPA test (1a+3). In all cases the comparison test showed a single genotype 1 infection. The new Abbott RealTime HCV Genotype II assay showed a high correlation with the Versant HCV Genotype 2.0 assay (LIPA).The automation platform m2000 system (Abbott), together with objective interpretation and digital archiving results may be particularly advantageous for the laboratory.

Génotypage du virus de l’hépatite C : comparaison du test Abbott RealTime HCV Genotype II au séquençage de la région NS5B

Purpose of the study Hepatitis C virus genotyping is needed for treatment decision and monitoring. The results of a genotyping assay based on real-time PCR and TaqMan chemistry were compared with the results of NS5B region sequencing. Materials and methods One hundred and two sera (genotypes 1–6) were tested. Amplification and detection of viral RNA were performed with the Abbott RealTime HCV Genotype II assay targeting 5′non-coded region (5′NC) for the identification of genotypes 1 to 6 and NS5B, for 1a and 1b subtypes detection. Sequencing of 5′NC fragment was used to resolve discrepant results. Results No indeterminate results were obtained. Concordance with NS5B sequencing was 93% (95 on 102), 96% at the genotype level (98 on 102) and 93% for genotype 1 subtyping (40 on 43). Discordant genotyping results were a 2f subtype identified as 5, a 6a typed as 1, a 3a identified as a 1-3 co-infection and a 4r identified as a 1-4 co-infection. Discordant subtyping results were 2 1b subtypes only typed as 1 and a 1e identified as 1a. Conclusion Abbott RealTime HCV Genotype II assay is a rapid, automated and simple to interpret method for HCV genotyping. It allows the detection of possible mixed infections which might have a negative impact on therapeutic response. However, the discrepant results found in this small series underline the need for assay optimization.

PVI-3 Performance evaluation of the Abbott RealTime HCV genotype II kit using clinical samples of Korean patients

PVI-3 Performance evaluation of the Abbott RealTime HCV genotype II kit using clinical samples of Korean patients

Clinical evaluation of the COBAS Ampliprep™/COBAS TaqMan™ for HCV RNA quantitation in comparison with the branched‐DNA assay

Diagnosis and monitoring of HCV infection relies on sensitive and accurate HCV RNA detection and quantitation. The performance of the COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ), a fully automated, real-time PCR HCV RNA quantitative test was assessed and compared with the branched-DNA (bDNA) assay. Clinical evaluation on 576 specimens obtained from patients with chronic hepatitis C showed a good correlation (r = 0.893) between the two test, but the CAP/CTM scored higher HCV RNA titers than the bDNA across all viral genotypes. The mean bDNA versus CAP/CTM log10 IU/ml differences were -0.49, -0.4, -0.54, -0.26 for genotype 1a, 1b, 2a/2c, 3a, and 4, respectively. These differences reached statistical significance for genotypes 1b, 2a/c, and 3a. The ability of the CAP/CTM to monitor patients undergoing antiviral therapy and correctly identify the weeks 4 and 12 rapid and early virological responses was confirmed. The broader dynamic range of the CAP/CTM compared with the bDNA allowed for a better definition of viral kinetics. In conclusion, the CAP/CTM appears as a reliable and user-friendly assay to monitor HCV viremia during treatment of patients with chronic hepatitis. Its high sensitivity and wide dynamic range may help a better definition of viral load changes during antiviral therapy.

Evaluation of the analytical performance of the new Abbott RealTime RT-PCRs for the quantitative detection of HCV and HIV-1 RNA

Despite FDA approval and CE marking of commercial tests, manufacturer independent testing of technical aspects is important. To evaluate the analytical performance of the new Abbott RealTime HCV and HIV-1 viral load tests. Sensitivity, specificity and inter-/intra-assay variation were investigated. The HCV and HIV-1 assays were compared with Siemens bDNA 3.0 and Roche Cobas Monitor 2.0, respectively, on diagnostic samples. Lower isolation volumes on the M1000 gave minor but statistically significant lower quantitative values. Minor differences were observed in the lower limit of detection relative to the specification given by the manufacturer. Inter-/intra-assay coefficients of variations ranged from 0.31 to 4.75 between 5.0 x 10(4) and 5.0 x 10(2) copies/mL. Both the HCV and HIV-1 Abbott RealTime tests did not show a geno-/sub-type dependent under-quantification on WHO reference panels, quality control panels or clinical specimens. The Abbott RealTime HIV-1 viral load assay detected subtype O whereas several other systems failed to detect this subtype. The technical aspects of the HCV and HIV-1 RealTime viral load assays on the M2000 system make it attractive for use in routine diagnostic settings.

P1734 Performance evaluation of the QIAGEN EZ1 DSP virus kit with Abbott RealTime HIV-1, HCV and HBV assays

P1734 Performance evaluation of the QIAGEN EZ1 DSP virus kit with Abbott RealTime HIV-1, HCV and HBV assays