AMPLICOR Hepatitis C Virus Test Research Articles

The evaluation of Recombinant Immunoblot assay (RIBA) and HCV-RNA test results in patients with low titer Anti-HCV positivity

Objectives: Laboratory diagnosis of hepatitis C virus (HCV) infection is based on the detection of anti-HCV antibodies by enzyme immunoassay (EIA) or chemiluminescence immunoassay (CIA) techniques. However, a consensus related to the problem of low titer (Serum/Cut-off; S/C= 1.0) anti-HCV antibodies is still lacking. The study attempts to evaluate the clinical status of the patients with low titer anti-HCV antibodies detected by third generation anti-HCV tests during February 2013- May 2014 retrospectively. Methods: Serum samples were studied by Advia Centaur XP autoanalyser (Bayer-Siemens, Germany) for anti-HCV, and line immunoassay (Inno-LIATM HCV Score, Innogenetics, Belgium) for anti-HCV confirmatory test, Cobas AmpliPre/Cobas AMPLICOR HCV Test (Roche diagnostics, Switzerland) for HCV RNA. Results: A total of 55.631 serum samples were studied, and 55 of them were anti-HCV positive of which with low antibody levels (sample/cutoff [S/CO]). S/CO values ranged from 1.15 to 6.15. Seventeen (31%) of patients who have low antibody levels were defined as positive and 2 (4%) patients were intermittent and 36 (65%) patients were negative with line immunoassay. HCV-RNA was not detected in any of the samples. Conclusions: It is thought that antibody positivity must be verified in cases of recurrent reactivity when considering the cost-effectiveness of molecular tests. In the study was concluded that the use of molecular tests would be appropriate diagnosis, and the effectiveness of treatment if necessary after evaluation of patients with biochemical analysis. J Clin Exp Invest 2014; 5 (4): 553-556

Prevalence of Hepatitis C Virus in the Population of Albania for the Period 2007-2010

BACKGROUND: Hepatitis C is a blood-borne, infectious, viral disease that is caused by a hepatotropic virus called Hepatitis C virus (HCV).AIM: The aim of this study is to determine the prevalence of active HCV infection (HCV–RNA) in the cases that were anti-HCV positive.MATERIAL AND METHODS: Plasma of 301 high-risk for HCV infection consecutive from University Hospital Centre “Mother Theresa” Tirana-Albania, during January 2007 to December 2010 was included in this study. To identify the presence of HCV RNA, the samples were examined by Cobas Amplicor HCV test (qualitative method).RESULTS: From 301 samples analyzed in total, 214 of them resulted positive for the presence of HCV-RNA's, corresponding to a prevalence of 71.1%, with 95% CI interval [65.8 - 75.9] for value of χ2 = 52.7 p value <0.0001. Divide by the sex 56% were males and 44% females, with statistically significant difference between them for value χ2 =4306 p value=0.0380. Among the age groups the highest prevalence was observed in the age groups > 25 years with a significant difference with other age groups for p value <0.001.CONCLUSION: Among tested samples, 71.1 % were confirmed to be positive for HCV –RNA infections. The prevalence of male was highest compared to female. For males and females infected the prevalence was highest in the age group of > 25 years.

HCV RNA detection in HCV antibody-positive patients with the COBAS® AmpliPrep/COBAS® TaqMan® HCV test, v2.0 in comparison with FDA-approved nucleic acid tests

BackgroundAnalysis of hepatitis C virus (HCV) RNA levels is critical for assessing the efficacy of antiviral therapy and the achievement of a sustained virologic response. Objective and study designThis study evaluated the clinical performance of the COBAS® AmpliPrep/COBAS® TaqMan® HCV quantitative test, version 2.0 (TAQMAN v2.0) with the COBAS® AmpliPrep/COBAS® TaqMan® HCV quantitative test, version 1.0 (TAQMAN v1.0), the VERSANT® HCV qualitative assay (VERSANT), and the COBAS® AMPLICOR HCV test, v2.0 (AMPLICOR) qualitative test for the detection of HCV RNA in serum or EDTA plasma from patients who are or have been infected with HCV and carry HCV antibodies. ResultsA total of 277 participants were evaluable for the percent agreement analysis of the TAQMAN v2.0 with the VERSANT and with the AMPLICOR. The overall percent agreement between the TAQMAN v2.0 and the VERSANT or the AMPLICOR was 99.3% (95% CI: 97.4%, 99.8%) or 98.9% (95% CI: 96.9%, 99.6%), respectively. The overall percent agreement between the TAQMAN v2.0 and the TAQMAN v1.0 when 267 of the original samples were assessed was 98.9% (95% CI=96.7%, 99.6%). ConclusionThe TAQMAN v2.0 demonstrated high correlation with the previously approved HCV RNA quantitative and qualitative tests.

Performance of Molecular Methods for Hepatitis C Virus Diagnosis: Usefulness among Chronic Cases and during the Course of Infection

Molecular methods are essential to define hepatitis C virus (HCV) infection. This study was conducted to evaluate the performance of molecular qualitative and quantitative methods for HCV RNA among chronic patients and individuals during the course of HCV infection. Single serum samples were obtained from 82 HCV infected individuals where six of them donated serial serum samples (n = 52) during the course of HCV infection. Qualitative (in-house RT-nested PCR and COBAS AMPLICOR HCV Test v2.0 and TMA) and quantitative (COBAS AMPLICOR HCV Monitor Test v2.0 and bDNA) techniques were employed. TMA presented the highest rate (87.8%) of HCV detection among qualitative tests and it was the most sensitive for HCV RNA detection during the early and late phases of HCV infection. HCV RNA was quantified among 56 samples and significant correlation was observed between the two assays (r 0.92; p < 0.0001). It is concluded that both quantitative methods can be used among chronic and acute HCV cases, but TMA was the most efficient for HCV qualitative detection among chronic cases and in the early and late phases of HCV infection.

Pegylated interferon-alfa-2a monotherapy in patients infected with HCV genotype 2 and importance of rapid virological response

BackgroundPegylated (PEG)-interferon (IFN)-alfa-2a plus ribavirin (RBV) therapy for 24 weeks is now a standard treatment protocol for patients with hepatitis C virus (HCV) genotype 2. As RBV cannot be used in certain situations, we examined whether PEG-IFN-alfa-2a monotherapy for 24 weeks or less would be sufficient to obtain a sustained virological response (SVR) in patients infected with HCV genotype 2.MethodsForty-nine consecutive patients with HCV genotype 2 received PEG-IFN-alfa-2a (180 μg/week) subcutaneously without oral RBV for 8-64 weeks. HCV RNA level was determined by COBAS AMPLICOR HCV Test, v2.0.ResultsHCV RNA was equal to or less than 100 KIU/mL (defined as low viral load) in 15 of 49 patients, and the remaining 34 had HCV RNA above 100 KIU/mL (defined as high viral load). All 15 patients with low viral load achieved rapid virological response (RVR; HCV RNA negative at week 4), and also achieved SVR with an average treatment duration of 17.1 weeks. The 34 patients with high viral load were treated for 33.7 weeks on average, and 19 of them (55.9%) achieved RVR. The SVR rates of these patients were significantly higher in those with RVR than without RVR (16/19 vs. 6/15 p = 0.0074).ConclusionPEG-IFN-alfa-2a monotherapy for 24 weeks or less might be sufficient to treat selected patients with HCV genotype 2, especially those with low viral load and becoming negative for HCV RNA by week 4 of treatment.

Uncommon Hepatitis B Virus and/or Hepatitis C Virus Occult Infection in HIV-Positive Patients With Abnormal Level of Hepatic Enzyme

Elevated liver enzyme levels, detected through liver function test (LFT), are markers of liveror biliary tract diseases. Alanine aminotransferase (ALT) is a more specific indicator of liverinflammation. These abnormal findings are very frequent in HIV infected patients and theirdiagnosis and management may be difficult because of intricacies of the pathogenic mechanisminvolved

The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection System. The primers and probe were designed to target the 5'-untranslated region of the hepatitis C viral genome. A second heterologous probe assay was developed for the detection of the haemagglutinin gene of phocine distemper virus (PDV) and was used as an internal control. A semi-automated HCV extraction method was also implemented using the ABI PRISM 6100 Nucleic Acid PrepStation. The HCV assay was optimised as a qualitative singleplex RT-PCR assay with parallel testing of the target and internal control. The assay results (n = 200) were compared to the COBAS AMPLICOR HCV Test v2.0 assay. The assay demonstrated a high rate of sensitivity (99%), specificity (100%) and an acceptable limit of detection (LOD) of 100 IU/ml. The development of a qualitative multiplex assay for the simultaneous detection of HCV and internal control indicates the same high rates of sensitivity and specificity. This sensitive real-time assay may prove to be a valuable method for the detection of HCV.

Peginterferon Alfa-2a and Ribavirin for 24 Weeks in Hepatitis C Type 1 and 4 Patients With Rapid Virological Response

This analysis reports the rate of sustained virological response (SVR) in patients infected with hepatitis C virus (HCV) genotype 1 or 4 who were assigned to 24 weeks of treatment with pegylated interferon (peginterferon) alfa-2a 180 mug/wk plus ribavirin 1000/1200 mg/day after achieving a rapid virological response (RVR; HCV RNA level <50 IU/mL) at week 4 in a prospective trial investigating response-guided therapy. Non-RVR patients with an early virological response were randomized to 48 or 72 weeks of therapy (this is a still-ongoing trial). A total of 150 of 516 patients (29%) had an RVR, 143 of whom completed 24 weeks of treatment. Younger patients, leaner patients, and those with an HCV RNA level </=400,000 IU/mL and HCV genotype 4 infection were more likely to achieve an RVR; however, among patients with an RVR, no baseline factor predicted SVR. The SVR rate was 80.4% (115/143; 95% confidence interval [CI], 72.9-86.6) in patients who completed 24 weeks of treatment. The SVR rate was 86.7% (26/30; 95% CI, 69.3%-96.2%) in patients infected with genotype 4 and 78.8% in those infected with genotype 1 (89/113; 95% CI, 70.1%-85.9%; intent to treat: 89/120; 74.2%; 65.4-81.7%). Treatment was well tolerated. This prospective study confirms that a 24-week regimen of peginterferon alfa-2a plus ribavirin 1000/1200 mg/day is appropriate in genotype 1 and 4 patients with a low baseline HCV RNA level who achieve an RVR by week 4 of therapy.

Large Intestine Histopathology of Pegylated-Interferon-Alpha Plus Ribavirin Treated Chronic Hepatitis C Patients

Large Intestine Histopathology of Pegylated-Interferon-Alpha Plus Ribavirin Treated Chronic Hepatitis C Patients

Factors influencing a low rate of hepatitis C viral RNA clearance in heroin users from Southern China

To study the virological and host factors influencing hepatitis C infection outcomes in heroin users in southern China. HCV RNA and associated factors were analyzed among 347 heroin users from Guangxi Zhuang Autonomous Region, southern China who were hepatitis C virus (HCV) EIA positive for two or more consecutive visits. Using the COBAS AMPLICOR HCV TEST, a remarkably low HCV RNA negative rate of 8.6% was detected. After multivariate logistic regression analysis, HCV RNA clearance was significantly associated with the presence of HBsAg (OR = 8.436, P < 0.0001), the lack of HIV-1 infection (OR = 0.256, P = 0.038) and age younger than 25 (OR = 0.400, P = 0.029). Our study suggests HCV infection among Chinese heroin users results in high levels of viral persistence even amidst factors previously found to enhance viral clearance. Prospective studies of a possible genetic component within the Chinese population and the pathogenicity of non-genotype 1 HCV infections are needed.

A multi–Chinese blood center study testing serologic‐negative donor samples for hepatitis C virus and human immunodeficiency virus with nucleic acid testing

A multi-blood center study was conducted to evaluate a human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) multiplex nucleic acid testing (NAT) donor screening test and to determine the residual risk for HIV-1 and HCV infection. A commercially available HIV-1 and HCV assay (Procleix, Chiron Corp.) was used for simultaneous detection of HIV-1 RNA and HCV RNA on 89,647 unlinked donor samples. NAT was performed with pools of 16 samples that had passed all routine screening tests. Single-donor NAT was performed for samples that had been disqualified by any reactive screening test result(s). Anti-HCV (Ortho third-generation HCV enzyme immunoassay [EIA]), alanine aminotransferase, and HCV NAT (Roche COBAS Amplicor HCV test) confirmatory tests were used for HCV EIA-nonreactive, HCV NAT-reactive samples. Three HCV NAT yield cases and no HIV-1 yield cases were detected. The yield rate for HCV NAT was 3.4 per 10(5) (95 percent confidence interval [CI], 0.7-9.8). The estimated incidence rate for HCV is 24.2 per 100,000 person-years (95% CI, 3.4-88.0). If minipool NAT is added to routine donor screening, the residual risk for HCV is estimated to be reduced to 1 in 20.4x10(4) (95% CI, 1 in 5.2x10(4)-1 in 165.5x10(4)). The residual risk for transfusion-transmitted HCV infection is still relatively high in China. Incorporating NAT technology into blood donor screening would be estimated to reduce the residual risk of HCV infections eightfold over current EIA screening.

Qualitative Application of COBAS AMPLICOR HCV Test Version 2.0 Assays in Patients with Chronic Hepatitis C Virus Infection and Comparison of Clinical Performance with Version 1.0

The objective of this research was to investigate the clinical performance of COBAS AMPLICOR hepatitis C virus (HCV) test version 2.0 Assays (CA V2.0). Eight serial samples with standard HCV ribonucleic acid (RNA) concentration and 10 times serial dilution of the 500 IU/mL samples were tested in triplicate by CA V2.0 (the limit of detection was 50 IU/mL). HCV RNA was investigated with CA V2.0 in 220 specimens from 100 chronic hepatitis C (CHC) patients, 60 chronic hepatitis B patients, and 60 healthy blood donors. The sensitivity was 99% and the specificity was 98.3%. Sera of 84 naïve CHC patients receiving standard interferon plus ribavirin for 24 weeks were tested by CA V2.0 and CA V1.0 at weeks 2, 4 and 8. The positive detection rates of CA V2.0 were significantly higher than CA V1.0 at week 2 (60.7% vs. 51.2%; p < 0.01) and week 8 (27.4% vs. 21.4%; p < 0.05). At weeks 2, 4 and 8, the positive predictive values were 90.91%, 83.02% and 78.69% with CA V2.0, and 90.24%, 82.14% and 72.73% with CA V1.0. The negative predictive values were 58.82%, 77.42% and 86.96% with CA V2.0, and 67.44%, 82.14% and 83.33% with CA V1.0. However, there was no significant difference between CA V2.0 and CA V1.0 for predicting sustained virologic response.

Chronic Coinfection With Hepatitis B and Hepatitis C Viruses in an Italian Population of HIV-Infected Patients

Chronic Coinfection With Hepatitis B and Hepatitis C Viruses in an Italian Population of HIV-Infected Patients

Chronic Hepatitis C in Patients With Persistently Normal Alanine Transaminase Levels

Many patients with chronic hepatitis C virus (HCV) have persistently normal serum alanine transaminase (ALT) levels. We compared characteristics of chronic hepatitis C patients with patients with normal and elevated ALT levels using data from 3 randomized phase III trials of peginterferon alfa-2a (40 kDa). The characteristics of 480 patients with normal ALT values (on >or=3 occasions without any increases in ALT level over a 6- to 18-month period) and 1993 patients with elevated ALT levels were compared. Sixty-eight of the 480 patients with normal ALT levels were randomized to no treatment and monitored for 72 weeks. More patients with normal ALT levels than patients with elevated ALT levels were women (59% vs 32%; P<.01). The serum HCV RNA titer was significantly lower in patients with normal ALT levels (P<.01 vs in patients with elevated ALT levels). Patients with normal ALT levels had significantly lower inflammation and fibrosis scores on liver biopsy examination than patients with elevated ALT levels, but almost two-thirds had portal fibrosis and 10% had bridging fibrosis. No correlation between baseline ALT activity, HCV RNA level, and liver histology was observed in patients with normal ALT levels. During the 72-week follow-up period, ALT activity elevated above the upper limit of normal in 53% of the untreated patients with normal levels of ALT. None became HCV RNA undetectable. Chronic hepatitis C patients with normal ALT levels should be evaluated in a similar manner as patients with elevated ALT levels because they are at risk for developing significant liver disease. The decision to treat with peginterferon alfa and ribavirin should be based on multiple factors, rather than on ALT levels alone.

Rapid Genotyping of Hepatitis C Virus by Primer-Specific Extension Analysis

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5' untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n=312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n=190), 12.8% type 2 (n=40), 20.2% type 3 (n=63), 0.3% type 4 (n=1), and 0.3% other (n=1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.

Fully automated detection of hepatitis C virus RNA in serum and whole-blood samples.

In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.

Clinical Evaluation of an Automated Nucleic Acid Isolation System

Nucleic acid isolation is among the most technically demanding and labor-intensive procedures performed in molecular diagnostic laboratories. Although considerable progress has been made in automating the amplification and detection steps of nucleic acid amplification assays, sample preparation is still performed manually in many laboratories. Various automated systems have been developed to streamline these procedures, ranging from versatile robotic stations to systems dedicated to specific amplification platforms or formats. The MagNA Pure LC system (Roche Applied Science, Indianapolis, IN) uses robotics, precision pipettors, and magnetic glass particles to purify DNA, RNA, mRNA, or total nucleic acid from various sample types. The samples are dissolved and simultaneously stabilized by incubation with a buffer containing denaturing agents and proteinase K. Nucleic acids are bound to the surface of the magnetic glass particles, and several washing steps remove the unbound substances. The purified nucleic acids are then eluted in a low-salt buffer, with elution volumes ranging from 50 to 100 μL. The instrument can process up to 32 samples in 1.5 h; it can also automate PCR set-up and transfer the purified nucleic acids directly into a wide variety of reaction vessels, including LightCycler capillary tubes (Roche Applied Science), 96-well microplates, standard PCR tubes, and COBAS AMPLICOR amplification rings (Roche Diagnostics). We developed and verified MagNA Pure LC (software Ver. 2.1) protocols for use with a quantitative test for cytomegalovirus (CMV) DNA (COBAS AMPLICOR MONITOR CMV Test) (1) and a qualitative test for hepatitis C virus (HCV) RNA (AMPLICOR HCV Test version 2.0) (2). The MagNA Pure LC total nucleic acid reagent set was used to recover CMV DNA and HCV RNA from EDTA plasma. The starting sample volume in each case was 200 μL. The total nucleic acid isolation reagent set and instrument were used as recommended by the manufacturer with the following …

Advances in the Molecular Diagnosis of Hepatitis C and Their Clinical Implications

Advances in the Molecular Diagnosis of Hepatitis C and Their Clinical Implications

Real-time detection system for quantitation of hepatitis C virus RNA: a comparison with the other three methods

Real-time detection (RTD) system for quantitation of hepatitis C virus (HCV) was developed. Its sensitivity and usefulness were compared with the other three commercially available methods for quantitation of HCV. The sera of 166 patients positive for serum HCV RNA by Amplicor HCV test were assessed. HCV was detected in 78.5% (128/163) by branched DNA assay, in 88.8% (111/125) by HCV core protein assay, in 94.5% (156/165) by Amplicor HCV Monitor test, and in 97.0% (161/166) by the RTD system. The values of viral load by the RTD system were significantly well correlated with those obtained by the other three methods. In the 50 patients treated by interferons (IFNs), the range which predicts the highest sustained response rate was less than 0.5 Meq/ml for branched DNA assay (sustained response rate: 57.9% (11/19)), less than 1 kcopies/ml for Amplicor HCV Monitor test (85.7% (6/7)), and less than 10 4 copies/ml for RTD system (100% (7/7)). None of the patients with greater than or equal to 2.8 Meq/ml by branched DNA assay ( n=14), greater than or equal to 250 kcopies/ml by Amplicor HCV Monitor test ( n=19), or greater than or equal to 2×10 6 copies/ml by RTD system ( n=16) obtained sustained response. In conclusion, RTD system was demonstrated to be the most sensitive method for quantitation of HCV, and useful for the prediction of sustained response to IFN therapy.

Genotyping of hepatitis C virus isolates using CLIP sequencing.

Determination of hepatitis C virus (HCV) genotypes and subtypes has become increasingly important for the clinical management and prognosis of HCV infections. The aim of the present study was to assess the specificity and reliability of a newly developed, commercially available HCV genotyping kit (TRUGENE HCV 5'NC genotyping kit). This technique utilizes PCR fragments previously generated by the diagnostic Roche AMPLICOR HCV test, which are subsequently subjected to simultaneous PCR amplification and direct sequencing (CLIP sequencing) of the 5' noncoding region (5'NCR). HCV isolates from 100 randomly chosen patients were genotyped by both the TRUGENE HCV 5'NC genotyping kit and DNA enzyme immunoassay (DEIA). Typing results obtained by both methods were in complete concordance in 91% of the cases. HCV RNA from the samples with discordant genotype assignment in both assays was additionally amplified with primers from the HCV core and NS5B regions. Phylogenetic analysis of the obtained sequences supported the results obtained from DEIA in six cases and CLIP sequencing in two cases. In the former six cases, the TRUGENE HCV 5'NC genotyping kit could not correctly differentiate between subtypes of genotypes 1 and 2 due to the high conservation of the 5'NCR. However, since there was not any misclassification between HCV genotypes 1 and non-1 types, the results obtained with this system are, in general, reliable and can be used in clinical practice. The TRUGENE HCV 5'NC genotyping kit in our hands proved to be a fast and convenient technique that might be an attractive option for HCV genotyping in laboratories already using the Roche AMPLICOR HCV test for diagnostic reverse transcription-PCR.