Amplicor Hepatitis C Virus Monitor Test Research Articles

Analysis of Hepatitis C Virus NS5A Region in Patients with Cirrhosis Using an Ultra-Deep Pyrosequencing Method.

HCV (Hepatitis C Virus) is genetically more diverse than HBV and HIV (Human Immunodeficiency Virus) and exists as quasispecies within infected individuals. This is due to the lack of efficient proofreading of the viral RNA-dependent RNA polymerase. Consequently, quasispecies emerge depending on the mutation rate of the viral polymerase, which may display a high level of genetic variability in a population. In infected individuals, HCV replicates and circulates as quasispecies composed of a complex mixture of different but closely related genomes that undergoes continuous change due to competitive selection and cooperation between arising mutants. The aim of this study is to investigate mutations in the NS5A region as a whole, including ISDR, PKRBD, IRRDR, and V3 of HCV genotype 1b cirrhosis patients being naive and nonresponders, treated with IFN (interferon) + ribavirin (RBN) by using an ultra-deep pyrosequencing method (UDPS). During the study, five patients (four females, and one male, mean age 59.8 ± 11 years) with HCV related cirrhosis were analyzed. Three patients received IFN + RBN for six months, but two patients did not receive any therapy. HCV-RNA concentrations in patients' sera were determined using a COBAS AMPLICOR HCV MONITOR Test, Version 2.0. Genotyping was performed by using a commercial reverse hybridization method, Line Probe Assay. The quasispecies for the NS5A region were investigated using UDPS. All five patients were HCV genotype 1b (Mean Child-Pugh score 7.2 ± 1.9, 2 pts Child A, 2 pts Child B, and one pt Child C) but only one patient had hepatocellular carcinoma (HCC). A total of 19 different mutations were detected in each of the five patients (ranging from 3 to 6 mutations per patient). In all five patients, several mutations in the ISDR and PKR-BD regions were detected. On the other hand, mutations in the V3 and IRRDR regions were only detected in one patient. UDPS is a new sequencing technology and a very sensitive method in detection of quasispecies with low frequency NS5A region mutations. These mutations may affect the antiviral response and development of HCC. However, further studies in larger number of patients should be conducted to clarify this hypothesis.

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.

Response to peginterferon-alpha 2b and ribavirin in Japanese patients with chronic hepatitis C genotype 1.

Patient age and gender may be associated with response to peginterferon alpha plus ribavirin, the current standard of care (SOC) for chronic hepatitis C genotype 1. We queried whether there was an association between age, gender, and treatment response to SOC in Japanese patients infected with hepatitis C virus (HCV) genotype 1. Between 2006 and 2009, HCV-infected Japanese patients treated with peginterferon alpha-2b plus ribavirin for 48weeks were enrolled. Patients were allocated into four groups according to age and gender, and epidemiological data and treatment outcomes were retrospectively analyzed. HCV RNA was measured with COBAS AMPLICOR HCV Monitor Test v. 2.0. The overall sustained virological response (SVR) rate was 49.8%: patients aged ≤65 and >65years, 50.9 and 44.0%, respectively; male and female, 56.5 and 39.0%. SVR rates of SOC against HCV genotype-1 females aged >65years (19.0%) were inferior to those in males aged >65years (57.8%) in Japan. Multivariate logistic regression analysis showed that SVR was attained independently of adherence 80/80/80 in all groups. Adherence to medication is also a key factor for the eradication of HCV in patients aged >65years. As the SVR rate of patients aged ≤65years was similar to that of patients aged >65years, SOC could be useful for treating some of the elderly patients.

Response to Peginterferon-alfa 2b and Ribavirin in Japanese Patients with Chronic Hepatitis C Genotype 2

The current standard treatment for patients infected with hepatitis C virus (HCV) of genotype 2 is the combination of peginterferon (PEG-IFN) plus ribavirin (RBV) for 24weeks. We assessed the sustained virological response (SVR) rates in HCV genotype 2-infected Japanese patients in relation to the duration of treatment. Between 2006 and 2009, among 147 patients with HCV genotype 2-infection in Chiba Prefecture, 138 consecutive patients were finally enrolled. Twenty-one, 97 and 20 patients were treated with PEG-IFN-alfa 2b plus RBV for 16, 24 and 48weeks, respectively. Epidemiological data and treatment outcomes were retrospectively evaluated. HCV RNA was measured with COBAS AMPLICOR HCV Monitor Test v. 2.0. The overall SVR rate was 82.6% (114 of 138): treatment-naïve patients, 86.4% (89 of 103); patients with history of previous treatment, 71.4% (25 of 35). Patients treated for 16, 24 and 48weeks obtained SVR rates of 66.6% (14 of 21), 86.5% (84 of 97) and 80.0 (16 of 20), respectively. The SVR rates of PEG-IFN-alfa 2b plus RBV in Japanese patients were similar to those in previous studies. Combination treatment for 24weeks for some patients infected with HCV genotype 2 may be superior to that for 16weeks. More precise patient selection will be needed to shorten the combination treatment.

Hepatitis C virus RNA detection in different semen fractions of HCV/HIV-1 co-infected men by nested PCR

Objective The aim was to evaluate, by nested PCR, the prevalence of hepatitis C virus (HCV) RNA in seminal plasma in different semen fractions of HCV/HIV-1 co-infected men. Study design This study enrolled 16 HCV/HIV-1 infected men. A total of 16 seminal samples and 16 blood samples were tested for the presence of HCV-RNA. HCV-RNA in blood plasma was quantified by Amplicor HCV Monitor Test version 2.0 and HCV-RNA detection in seminal plasma, non-spermatozoa cells (NSCs), spermatozoa pellet and swim-up was investigated by nested PCR. Results Thirteen blood plasma samples were positive for HCV-RNA. HCV-RNA was detectable in seminal plasma and in non-sperm cells, but not detectable in spermatozoa samples, neither before nor after swim-up. One of the two patients whose seminal plasma tested positive at nested PCR had undetectable HCV virus in blood plasma. Conclusions HCV-RNA can be found in seminal plasma and non-sperm cells but not in spermatozoa before and after swim-up. We observed HCV-RNA in the semen of an aviremic man. According to these findings we suggest that sperm washing should be performed for each semen sample of HCV patients before assisted reproduction techniques.

Insulin Resistance Is Associated With Advanced Liver Fibrosis and High Body Mass Index in HIV/HCV-Coinfected Patients

Insulin Resistance Is Associated With Advanced Liver Fibrosis and High Body Mass Index in HIV/HCV-Coinfected Patients

Abbott RealTime Hepatitis C Virus (HCV) and Roche Cobas AmpliPrep/Cobas TaqMan HCV Assays for Prediction of Sustained Virological Response to Pegylated Interferon and Ribavirin in Chronic Hepatitis C Patients

Two commercial real-time PCR assays are currently available for sensitive hepatitis C virus (HCV) RNA quantification: the Abbott RealTime HCV assay (ART) and Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM). We assessed whether the two real-time PCR assays were more effective than Roche Cobas Amplicor HCV Monitor test, v.2.0 (CAM) for prediction of the sustained virological response (SVR) to pegylated interferon (PEG-IFN) plus ribavirin (RBV) in chronic hepatitis C. Sixty patients chronically infected with HCV genotype 1b (37 males and 23 females, 53 +/- 12 years of age) were treated with PEG-IFNalpha2b plus RBV for 48 weeks. Stored specimens at nine time points for each patient (at baseline, on treatment, and 24 weeks after treatment) were tested by the two real-time PCR assays and CAM. Twenty-six (43.3%) patients reached SVR. The positive predictive values (PPVs) for SVR of undetectable HCV RNA at week 12 by CAM, ART, and CAP/CTM were 74.3%, 88.0%, and 95.2%, respectively. An undetectable HCV RNA level by CAM, ART, and CAP/CTM correctly predicted SVR at week 4 in 100%, 100%, and 100% of patients, at weeks 5 to 8 in 91.7%, 100%, and 100% of patients, at weeks 9 to 12 in 55.6%, 75%, and 87.5% of patients, and at weeks 13 to 24 in 0%, 26.7%, and 40% of patients, respectively. Of 16 patients who relapsed after treatment, HCV RNA was detectable in 2 patients at the end of treatment by CAP/CTM but undetectable by ART and CAM. HCV RNA tests using ART and CAP/CTM are considered to be more effective at predicting SVR than CAM, and the PPV for SVR was slightly higher in CAP/CTM than in ART.

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.

Improved COBAS TaqMan Hepatitis C Virus Test (Version 2.0) for Use with the High Pure System: Enhanced Genotype Inclusivity and Performance Characteristics in a Multisite Study

We have evaluated the COBAS TaqMan hepatitis C virus (HCV) test (version 2.0) for use with the High Pure system (HCVHPS V2), a new, revised real-time reverse transcription-PCR assay developed to improve the genotype quantitation of version 1.0 (HCVHPS V1). Revisions were made in the wash buffer and in the reverse transcription temperature. The genotype inclusivity of HCVHPS V2 was evaluated at three different sites, using HCVHPS V2, HCVHPS V1, and the COBAS AMPLICOR HCV MONITOR test (version 2.0) (CAHCM). The fully automated COBAS Ampliprep/COBAS TaqMan HCV test was also used in one of the participating laboratories. The mean differences in HCV RNA values between HCVHPS V2 and CAHCM and between HCVHPS V2 and HCVHPS V1 ranged from -0.21 to 0.13 log and from 0.24 to 1.27 log, respectively, with >0.5-log differences for genotypes 2, 3, 4, and 5. With a NIBSC panel of HCV genotypes 1 through 6, the measured HCVHPS V2 values were within 0.25 log of the nominal values for all 6 genotypes. When serial dilutions of genotype-specific clinical HCV specimens were tested, the assay showed a limit of detection between 10 and 20 IU/ml and a linear range of 25 IU/ml to 3.91 x 10(8) IU/ml. Clinical and analytical specificities of 100% were demonstrated with 100 HCV-seronegative specimens as well as with 12 non-HCV members of Flaviviridae and 22 additional microorganisms. These data indicate that HCVHPS V2 is a robust and accurate test for the quantitation of all six HCV genotypes and useful in monitoring viral load in all clinical HCV specimens.

DETERMINATION OF HEPATITIS C VIRUS GENOTYPES BY MELTING-CURVE ANALYSIS OF QUANTITATIVE POLYMERASE CHAIN REACTION PRODUCTS

Hepatitis C virus (HCV) is the major causative agent of non-A and non-B viral hepatitis. Factors associated with disease progression following HCV infection include the viral genotype, the patient’s alcohol consumption, and viral load. In this study, the COBAS AMPLICOR HCV MONITOR test, a commercially available quantitative assay for HCV RNA, was used for HCV genotyping analysis. Amplification products obtained from 100 HCV-positive cases were subjected to real-time polymerase chain reaction (PCR) typing using a single pair of fluorescence resonance energy transfer (FRET) probes and melting-curve analysis. Of 100 samples tested, two inhibited the PCR, two samples yielded discrepancies between our results and the reference laboratory results, and the remaining samples provided correct typing. The present report suggests that HCV genotypes can be determined rapidly with FRET probes directly from COBAS AMPLICOR MONITOR test PCR products.

Determination of hepatitis C virus genotypes by melting-curve analysis of quantitative polymerase chain reaction products

Hepatitis C virus (HCV) is the major causative agent of non-A and non-B viral hepatitis. Factors associated with disease progression following HCV infection include the viral genotype, the patient's alcohol consumption and viral load. In this study, the COBAS AMPLICOR HCV MONITOR test, a commercially available quantitative assay for HCV RNA, was used for HCV genotyping analysis. Amplification products obtained from 100 HCV-positive cases were subjected to real-time polymerase chain reaction (PCR) typing using a single pair of fluorescence resonance energy transfer (FRET) probes and melting-curve analysis. Of 100 samples tested, two inhibited the PCR, two samples yielded discrepancies between our results and the reference laboratory results and the remaining samples provided correct typing. The present report suggests that HCV genotypes can be determined rapidly with FRET probes directly from COBAS AMPLICOR MONITOR test PCR products.

Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA

Since October 1997, an international standard for hepatitis C virus (HCV) nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection. A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits. Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture's series. The standard calibration curve with the series of lyophilized serum showed an excellent correlation (R(2) > 0.98), slope and intercept that were similar to those from the manufacture's series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample, lower coefficients of variation between kits from different manufactures were obtained. The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection.

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.

Pegylated Interferon Alfa-2b Plus Ribavirin in the Retreatment of Interferon-Ribavirin Nonresponder Patients

Inadequate data are available about retreatment of nonresponders to interferon (IFN) and ribavirin. Thus, this study evaluated the efficacy and tolerability of a 48-week therapy with pegylated IFN-alpha-2b plus high-dose ribavirin in patients who have failed to respond to the combination. Treatment up to 48 weeks also in patients who have failed to clear hepatitis C virus (HCV) RNA by week 24 was also evaluated. One hundred forty-one patients who previously did not respond to IFN and ribavirin, 86% with genotype 1 or 4 infection, 52% with high viral load (>800.000 IU/mL), 22% with cirrhosis, were retreated with pegylated IFN-alpha-2b 1.5 microg/kg per week and ribavirin 1000-1200 mg/day for 48 weeks and followed up for 24 weeks. By intent-to-treat analysis, 20% of patients achieved a sustained virologic response (SVR). SVR of genotype 1 patients was 19%. Independent predictors of SVR were low gamma-glutamyltransferase levels (OR, 22.9; 95% CI: 6.6-79.6) and low viral load (OR, 3.8; 95% CI: 1.1-12.6). Twelve (23%) out of 51 patients who were HCV RNA positive after 24 weeks of therapy achieved a late virologic response (after week 24) and 5 (10%) of them, all with genotype 1, achieved an SVR. Genotype was not associated with response (P = .2) or with early response (P = .3). Retreatment with pegylated IFN-alpha-2b and ribavirin of multi-experienced and "difficult to treat" nonresponder patients produced a very promising SVR. Accurate selection of patients, such as those with low viral load and low gamma-glutamyltransferase levels, and prolongation of therapy beyond 24 weeks also in HCV RNA-positive patients may further increase the rate of SVR.

Evaluation of the Invader Assay for Genotyping Hepatitis C Virus

The Invader 1.0 assay (Invader HCV Genotyping Assay, version 1.0; Third Wave Technologies, Inc., Madison, WI) has been developed for the rapid differentiation of hepatitis C virus (HCV) genotypes 1 to 6 based on sequence variation within the HCV 5' noncoding (NC) region. In the present study, we evaluated the compatibility of Invader 1.0 with the COBAS MONITOR (COBAS AMPLICOR HCV MONITOR Test, version 2.0; Roche Molecular Systems, Inc., Branchburg, NJ), COBAS AMPLICOR (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche Molecular Systems, Inc.), and COBAS TaqMan (COBAS TaqMan HCV Test; Roche Molecular Systems, Inc.) assays. The minimum HCV RNA titers required for successful HCV genotyping (>/=90% success rate) were 1,000 IU/ml for COBAS MONITOR, 100 IU/ml for COBAS AMPLICOR, and 10 IU/ml for COBAS TaqMan. Invader 1.0 results obtained from unpurified COBAS TaqMan amplification products of 111 retrospectively selected clinical serum specimens (genotypes 1 to 6, with virus titers ranging from 15.1 to 2.1 x 10(7) IU/ml) showed 98% concordance with results obtained from the TRUGENE HCV 5' NC Genotyping Kit (Bayer HealthCare LLC, Tarrytown, NY), used in conjunction with COBAS AMPLICOR. Although the assay is sensitive, accurate, and easy to perform, additional optimization of the Invader 1.0 interpretive software (Invader Data Analysis Worksheet) may be necessary to reduce potential misidentification of HCV genotypes in low-titer specimens. In summary, Invader 1.0 is compatible with a variety of commercially available PCR-based HCV 5' NC region amplification assays and is suitable for routine HCV genotyping in clinical laboratories.

P.142 Evaluation of the sensitivity of the enhanced ARCHITECT anti-HCV assay on well characterized samples from early seroconversion and seroreversion

Background: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. It is associated with the development of end-stage liver disease and hepatocellular carcinoma. Studies have shown that patients infected with different genotypes of HCV may respond to interferon–ribavirin therapy differently and thus HCV genotype information is very important in helping physicians to better managing their patients. Objectives: Compare the end results of HCV typing of the two commercially available tests. Study design: TRUGENE Genotyping test (Visible Genetics) was used to analyze clinical specimens obtained from North America. The 5′ NC was amplified with the Roche COBAS Amplicor HCV Monitor Test. Amplification products were blinded and genotyped by the TRUGENE HCV 5′NC method. Genotype results were compared with those obtained by the reverse hybridization based INNO-LiPA HCV II (Innogenetics) assay. Additional sequencing of the NS5B region was done to resolve discrepancies. Results and conclusions: Among the total of 110 consecutively collected serum specimens submitted for HCV genotyping, 108/110 could be typed by the sequencing method and 107/110 were typable by LiPA HCV II method. Our experiences with the tests suggest that at type level, HCV genotype results are 100% concordant between the two tests studied for those 106 specimens successfully typed by both methods. More sensitive amplification, such as qualitative PCR, is needed to test specimens with viral load lower than 20 000 IU/ml. Both tests can be easily adapted by a clinical diagnostic laboratory.

Benefit of Hepatitis C Virus Core Antigen Assay in Prediction of Therapeutic Response to Interferon and Ribavirin Combination Therapy

A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) and ribavirin. HCV core Ag negativity could predict SVR on day 1 (sensitivity = 100%, specificity = 85.0%, accuracy = 86.4%), whereas RNA negativity could predict SVR on day 7 (sensitivity = 100%, specificity = 87.2%, accuracy = 88.6%). None of the patients who had detectable serum core Ag or RNA on day 14 achieved SVR (specificity = 100%). The predictive accuracy on day 14 was higher by RNA negativity (93.2%) than that by core Ag negativity (75.0%). The combined predictive criterion of both viral load decline during the first 24 h and basal viral load was also predictive for SVR; the sensitivities of Lumipulse-Ag and Amplicor-M were 45.5 and 47.6%, respectively, and the specificity was 100%. Amplicor-M had better predictive accuracy than Lumipulse-Ag in 2-week disappearance tests because it had better sensitivity. On the other hand, estimates of kinetic parameters were similar regardless of the detection method. Although the correlations between Lumipulse-Ag and Amplicor-M were good both before and 24 h after IFN administration, HCV core Ag seemed to be relatively lower 24 h after IFN administration than before administration. Lumipulse-Ag seems to be useful for detecting the HCV concentration during IFN therapy; however, we still need to understand the characteristics of the assay.

Evaluation of total Hepatitis C virus core antigen quantification / Evaluierung eines Tests zur Quantifizierung des Hepatitis C Kern Antigens

Abstract Infections with hepatitis C virus (HCV) are pandemic. The WHO estimates a worldwide prevalence of 3%. For monitoring of chronic hepatitis C, assays for direct virus detection are employed. In this study, an improved version of the Ortho® trak-C assay™ (Ortho-Clinical Diagnostics, Raritan, NJ) for detection of the HCV core antigen was evaluated. Precision, interassay, and intraassay variation were determined and a total of 115 clinical specimens were investigated. When precision of the Ortho® trak-C assay™ was evaluated, the assay gave positive results in those samples above the expected detection limit. Interassay coefficients of variation ranged between 6–25%, while intra-assay coefficients of variation were found to be between 19%. When clinical samples were tested with the Ortho trakC assay and compared with results obtained with the Cobas Ampli Prep/Cobas Amplicor HCV Monitor Test (Roche Molecular Systems, Inc., Branchburg, NJ), 66% of all positive values were found to be within a +-0.5 log difference of the level obtained with the molecular assay. In conclusion, the Ortho® trak-C™ assay showed a good test performance and proved to be suitable for the routine-diagnostic laboratory.

Performance attributes of the LCx ® HCV RNA quantitative assay

The LCx ® HCV RNA quantitative assay (Abbott Laboratories, North Chicago, IL) is designed to use competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and microparticle enzyme immunoassay (MEIA), in combination with a modified Qiagen sample preparation method, to measure the level of hepatitis C virus (HCV) in human plasma and serum. The assay provides quantitative results in international units (IU) of HCV RNA/ml, in copies of HCV RNA/ml, or their log (base 10) equivalents. A conversion study determined that 1 IU equals 4.3 copies. The LCx HCV assay detected HCV RNA transcripts representative of genotypes 1–6 with near equal efficiency. The assay did not cross-react with high concentrations of 21 potentially cross-reactive microorganisms or with 100 HCV-negative specimens. The lower limit of detection was demonstrated to be 23 IU/ml. The LCx assay had similar sensitivity to the Roche Amplicor HCV (version 2.0) qualitative assay when used to test panels containing 6, 12, 23, and 47 IU/ml. The assay linear range was shown to extend from 23 to 2.3 million IU/ml. The intra-assay standard deviation (S.D.) was ≤0.066 log IU/ml for the four HCV positive samples tested, while for the same samples the observed inter-assay S.D. was ≤0.075 log IU/ml. The overall mean assay quantitation value for seven HCV-positive WHO-standardized Acrometrix NAP linearity panel members was within 0.06 log IU/ml of the mean assigned value. The assay was demonstrated to correlate acceptably against the Roche Amplicor HCV monitor test (version 2.0). These data suggest that the assay is standardized appropriately against the WHO standard across its linear range and can be used for quantitation of HCV. In addition, with a sensitivity of 23 IU/ml, the assay can be used to determine if post-therapy viral clearance has occurred.

Quantitative Assay of Hepatitis C Virus RNA Using an Automated Extraction System for Specific Capture with Probes and Paramagnetic Particle Separation

A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log(10) (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R(2) = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.