Amplicor Hepatitis C Virus Monitor 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.

The Different Distribution of Hepatitis C Virus Genotypes in Eastern Black Sea Region of Turkey

Hepatitis C virus (HCV) has infected about 170-200 million people worldwide and HCV genotypes have been related with a particular geographic distribution. The information of genotypes in chronic hepatitis C is critical for the decision of the pharmaceutical regimen, and for the therapeutic outcome. The aim of the study was determining the dispersion of hepatitis C Virus genotype in our area. The study was conducted on 42 patients who referred from various clinics infected with HCV. Serum HCV RNA levels were quantified by using the COBAS AMPLICOR HCV Monitor 2.0. An 851 bp long fragment spanning codons 63 to 347 of the RNA-dependent RNA polymerase in the NS5b part of the HCV genome was amplified. PCR amplifications were conducted in a BioRad DNA Engine using Quantitect SYBR Green PCR mix. Purified PCR products expressively sequenced with the ABI PRISM 310 Genetic Analyzer appliance utilizing DYEnamic ET Terminator Cycle Sequencing Kit. The most prevalent genotype was type 1b (90.4%) in our study. In the other types, type 3 and 4 were detected. We believe that distribution of HCV genotype in this region should be followed strictly due to difference from reports, which reported other parts of Turkey.

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.

A new sensitive and automated chemiluminescent microparticle immunoassay for quantitative determination of hepatitis C virus core antigen

A new sensitive and automated chemiluminescent assay was developed for the quantitative determination of hepatitis C virus (HCV) core antigen (Ag) in human sera or plasma: the Abbott ARCHITECT ® HCV Ag test. The assay sensitivity was determined by testing 10 commercial HCV seroconversion panels. Without exception, a positive result for HCV core Ag was observed before anti-HCV detection, resulting in an average reduction in the period between exposure and detection of 35.8 days. Both HCV core Ag and HCV RNA were detected in the panels at the same time, indicating equivalent sensitivity and detectability. A total of 197 HCV specimens comprising genotypes 1a, 1b, 2a, 2b, 3a, 3k, 4a, 5a and 6a were evaluated. Among these, 196 (99.5%), 191 (97%) and 193 (98%) were reactive using the HCV Ag, the immunoradiometric HCV Ag and the Amplicor HCV Monitor 2 assays, respectively. A comparison with the Amplicor HCV Monitor 2 showed a correlation coefficient ( r) of 0.74. The specificity of the assay was established at 99.8% by testing 5403 specimens from US volunteer blood donors, hospitalized patients and individuals with medical conditions unrelated to HCV infection, in addition to specimens containing potentially interfering substances.

Evaluation of the Real-Q HCV Quantification Kit

Background: Hepatitis C virus (HCV) RNA quantification is necessary for predicting the therapeutic response and assessing treatment results in patients with chronic HCV infection. Recently, real-time PCR technology for HCV RNA quantification displayed good linearity within the dynamic range. Thus, it is gradually replacing branched-DNA (bDNA) and PCRhybridization assays. In this study, we evaluated the performance of the Real-Q HCV quantification kit (biosewoom. Inc., Seoul, Korea) developed in Korea. Methods: We evaluated the HCV quantification kit for detection limit, specificity, linearity, accuracy, and recovery rate of HCV RNA standard material. The results were analyzed for a correlation with those of Cobas Amplicor HCV Monitor 2.0. Results: The HCV quantification kit showed a high recovery rate of HCV RNA standard material of various concentrations and amplication of HCV RNA equally in all genotypes. Hepatitis B virus and human immunodeficiency virus showed no cross-reactivity with HCV. Within-run and between-run coefficients of variation (CV) were 9.52∼15.84% and 9.40∼17.53%, respectively. Between-day coefficients of variation were 11.62∼18.04%, and detection limit was 44 IU/mL. It showed a good correlation with Cobas Amplicor HCV Monitor 2.0 (R=0.8954). Conclusion: The Real-Q HCV quantification kit showed a good specificity, sensitivity, linearity, and accuracy; therefore, we propose that it is fully adequate for monitoring antiviral therapy in patients with chronic HCV infection. (Korean J Clin Microbiol 2009;12:7277)

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.

Development and evaluation of a sensitive enzyme-linked oligonucleotide-sorbent assay for detection of polymerase chain reaction-amplified hepatitis C virus of genotypes 1–6

A high-throughput polymerase chain reaction (PCR)-based enzyme-linked oligonucleotide-sorbent assay (ELOSA) was developed for use in the diagnostic testing of serum from patients who may be infected with different hepatitis C virus (HCV) genotypes. Twelve genotype-specific 5′-aminated DNA-coated probes were designed based on the variable 5′-untranslated region sequences of the HCV genotypes 1–6. Using 100 clinical serum samples, the performance of the PCR-ELOSA method was compared with Roche's COBAS Amplicor HCV Monitor V2.0 assay and the VERSANT HCV genotype assay (LiPA), and the overall agreement was 99% at the level of HCV genotypes with a detection range of 2.0 × 10 2 to 1.0 × 10 7 IU/ml for PCR-ELOSA. The PCR-ELOSA was more comprehensive as demonstrated by the fact that ∼20% of the samples with different subtypes could be discriminated by this method but not by LiPA. In addition, the PCR-ELOSA system showed high accuracy (CV ≤ 6.36%) and even higher reproducibility (CV ≤ 5.55%). Thus, this novel PCR-ELOSA system provides a sensitive and versatile alternative to current HCV detection assays.

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.

Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays

A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and <2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.

Detection and quantitation of HCV RNA using real‐time PCR after automated sample processing

There is considerable evidence that the loss of hepatitis C virus (HCV) RNA during the first 3 months of treatment with pegylated interferon plus ribavirin is a prognostic marker of response to therapy. Real-time polymerase chain reaction (PCR) assays for quantifying HCV RNA in plasma or serum are now commercially available. The extraction of HCV RNA can also be automated. This report analyses the performance of the COBAS Ampliprep-COBAS Taqman 48 (CAP/CTM) real-time PCR assay and compares this new test with the COBAS Amplicor HCV Monitor v 2.0 assay (CAM). CAP/CTM was 100% specific. The assay was linear across a wide range of HCV RNA concentrations without sample dilution. The intra-assay variation was 0.3-3.3% and the interassay variation was 1.5-6.7%. A total of 118 clinical samples with different HCV genotypes were assayed using both methods. The results obtained using the two methods were well correlated (r = 0.89, P < 0.001). The mean difference [CAP/CTM-CAM] was 0.17 log IU/ml and it was not influenced by the HCV genotype or by the subtype. It is concluded that the new CAP/CTM system is adequate for quantifying HCV RNA in clinical practice.

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.

Comparison of Performance Characteristics of Three Real-Time Reverse Transcription-PCR Test Systems for Detection and Quantification of Hepatitis C Virus

We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log(10) IU/ml with a linear dynamic range of 1.0 to 7.0 log(10) IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log(10) IU/ml with a linear dynamic range of 2.0 to 7.0 log(10) IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy-six clinical specimens (50 with detectable levels of HCV RNA and various titers and genotypes) were tested, and results were compared to those of the COBAS Amplicor HCV Monitor v2.0. Good correlation was obtained for the Roche RUO-MPLC and Abbott ASR-Q (R(2) = 0.84 and R(2) = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation (R(2) = 0.79) and agreement were poor for Roche ASR-HP, with bias relative to concentration and genotype. Roche ASR-HP underestimated HCV RNA for genotypes 3 and 4 as much as 2.19 log(10) IU/ml. Our study demonstrates that Roche RUO-MPLC and Abbott ASR-Q provided acceptable results and agreed sufficiently with the COBAS Amplicor HCV Monitor v2.0.

Comparison of the Bayer VERSANT HCV RNA 3.0 and the Roche COBAS Amplicor HCV Monitor, Version 2.0, assays in HCV genotype 4 infection

Prediction of treatment response is clinically important in chronic hepatitis C virus (HCV) genotype 4 infection. Early viral kinetics is useful in this respect for genotype 1 but interpretation is dependent on assay linearity and reproducibility. The VERSANT HCV RNA 3.0 (bDNA-3.0) and the COBAS Amplicor HCV Monitor 2.0 (HCM-2.0) have been widely used quantitative assays. We wanted to comparatively evaluate the two tests in a large genotype 4 sample. Genotyping was performed by NS5b sequencing. Viral load was tested in parallel in 32 patients at least six times on antiviral therapy with interferon alpha (IFNalpha). Totally, 198 samples within a quantitative range from undetectable to about 7 x 10(6) IU/mL (bDNA-3.0) were obtained and compared. Twenty-two samples with viral load above 500 000 IU/mL tested by HCM-2.0 were 1:100 diluted and retested. Quantitative values were fitted to a third order polynomial (M = 0.118303 + 1.07503 x V+ 0.0112128 x V(2) - 0.0055504 x V(3); M...HCM-2.0, V...bDNA-3.0, both log IU/mL) showing progressive nonlinearity of HCM-2.0 above 100 000 IU/mL but better clinical sensitivity with respect to bDNA-3.0. Dilution lead to a gain of at least a factor of 2.7 and thus, overestimation compared with bDNA-3.0. Deviation from linearity and overestimation upon dilution by HCM-2.0 are similar with HCV genotype 4, compared with other HCV genotypes. Differences in test performance were not detected for subtypes but for individual patients possibly related to specific quasi-species patterns. The interpretation of viral kinetic data becomes difficult due to overestimation upon dilution of baseline values by HCM-2.0.

Multicenter Evaluation of the New Abbott RealTime Assays for Quantitative Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

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.