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Biomedical subjects

W K Roth

Publications and source records attributed to W K Roth.

At least 37 records · Page 2Linked to original sources
PubMed2000

Hepatocellular proliferation in patients with chronic hepatitis C and persistently normal or abnormal aminotransferase levels.

BACKGROUND/AIMS: Some patients chronically infected with the hepatitis C virus (HCV) have persistently normal alanine aminotransferase (ALT) levels while progressive liver damage is observed histologically. In the present study, we compared the rate of proliferation, apoptosis, and necrosis in liver biopsy specimens of patients with persistently normal or elevated ALT levels. METHODS: Fourteen patients with persistently normal and 14 age- and sex-matched patients with elevated ALT levels were enrolled. Proliferation was detected using anti-Ki 67 in 10-microm liver biopsy specimens of the patients. Apoptosis was measured by TUNEL-assay and by monoclonal anti-M30 directed against caspase-cleaved cytokeratin 18 filaments. RESULTS: The mean number of anti-Ki 67 positive hepatocytes was lower in patients with persistently normal aminotransferases (3.1 +/- 2.8/10(3) vs 10.8 +/- 8.8/10(3) hepatocytes, p<0.0011) and was correlated with serum ALT (r=0.86, p<0.01) and aspartate aminotransferase levels (r=0.83, p<0.01). The rate of apoptosis detected by TUNEL assay was low and not different between patients with persistently normal and elevated aminotransferases. Staining with anti-M30 revealed a granular staining pattern and showed a trend towards higher cell death rates in patients with elevated aminotransferase levels (apoptotic hepatocytes with >75% staining: 3.97 +/- 6.24/10(3) hepatocytes vs 13.65 +/- 19.41/10(3) hepatocytes; p=0.08). CONCLUSIONS: Patients with chronic hepatitis C and normal aminotransferases have significantly lower hepatocyte proliferation rates and show a trend towards lower apoptosis rates compared with patients with elevated aminotransferases.

Adult↗
PubMed2000

Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening.

BACKGROUND: High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing. STUDY DESIGN AND METHODS: Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template-derived competitive internal control sequence designed through site-directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single-donor and single-patient testing and by spiking sample mini-pools. Three seroconversion panels were tested. RESULTS: Sensitivity is high, reaching 300 HBV genomes per mL of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors. CONCLUSION: The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.

Blood Donors↗
PubMed2000

Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response.

Defined regions of hepatitis C virus (HCV) envelope 2 (E2), PePHD, and nonstructural 5A (NS5A) protein (PKR-binding domain) have been shown to interact with interferon alfa (IFN-alpha)-inducible double-stranded RNA-activated protein kinase (PKR) in vitro, suggesting a possible mechanism of HCV to evade antiviral effects of IFN-alpha. The clinical correlation between amino acid mutations within the E2 PePHD or the NS5A PKR-binding domain and response to antiviral treatment in HCV-3a-infected patients is unknown. Thirty-three patients infected with HCV-3a isolates were treated with IFN-alpha with or without ribavirin. The carboxyterminal half of E2 and of the NS5A gene were sequenced. Sixteen patients achieved a sustained virological response (SR), 6 patients an end-of-treatment response with relapse thereafter (ETR), and 11 patients were nonresponders (NR). Within the PePHD of the E2 protein 0.5 (range, 0-2) mutations were observed in SR patients, whereas the number of mutations in ETR or NR patients was 0.2 (0-1). Quasispecies analyses showed almost no heterogeneity. The mean number of mutations within the PKR-binding domain of the NS5A protein was 1.6 (range, 0-4) in SR patients, 1 (0-2) in ETR patients, and 1.6 (0-3) in NR patients. Patients with higher numbers of mutations within the E2 or NS5A region showed a trend towards lower pretreatment viremia. Phylogenetic and conformational analyses of E2 or NS5A sequences allowed no differentiation between sensitive and resistant isolates. However, mutations within the E2 PePHD in SR patients were frequent, and hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 were exclusively observed in sustained virological responders.

Adult↗
PubMed2000

Mutations in the protein kinase-binding domain of the NS5A protein in patients infected with hepatitis C virus type 1a are associated with treatment response.

An interaction of the hepatitis C virus (HCV) NS5A protein with the interferon (IFN)-alpha-inducible double-stranded RNA-activated protein kinase (PKR) was demonstrated in vitro. The clinical correlation between amino acid mutations within the HCV NS5A region and response to antiviral treatment is controversial. Thirty-two patients chronically infected with HCV-1a, who were treated with IFN-alpha with or without ribavirin, were studied. The carboxy-terminal half of HCV NS5A was sequenced and was investigated by phylogenetic and conformational analyses. Eight patients achieved a sustained virologic response. An end-of-treatment response but relapse thereafter was observed among 8 patients, whereas 16 patients were nonresponders. The median number of mutations within the PKR-binding domain but not within the previously described IFN sensitivity-determining region was significantly higher for patients with sustained (3 mutations [range, 1-5]) or end-of-treatment (4 mutations [range, 1-5]) virologic response than for nonresponders (2 mutations [range, 0-3]) (P=.0087). Phylogenetic and conformational analyses of NS5A sequences allowed no differentiation between sensitive and resistant strains.

Adult↗
PubMed2000

Antiviral effect of human recombinant interleukin-12 in patients infected with hepatitis C virus.

The heterogeneity of hepatitis C virus (HCV) is due to the continuous and high replication rate, the low fidelity of the RNA-dependent RNA polymerase, and the immune surveillance of the host. Interleukin-12 (IL-12) plays a central role in mounting an effective cellular immune response directed towards elimination of intracellular pathogens. The effect of IL-12 on hepatitis C viremia and the HCV quasispecies population is unknown. In this study, 12 patients (9 males, 3 females; mean age: 44 +/- 11 years), all virological non-responders to previous IFN-alpha treatment, received recombinant human IL-12 s.c. once weekly for 10 weeks stratified to three dose schedules (0.03 microg/kg, 0.1 microg/kg, and 0.5 microg/kg body weight, respectively). Fourteen IFN-alpha non-responders and 14 untreated patients served as age- and sex-matched controls. Serum HCV RNA concentrations and HCV quasispecies distribution were measured serially by quantitative reverse transcription - polymerase chain reaction and single strand conformation polymorphism analysis of the hypervariable region of the second envelope gene, respectively. Serum ALT and median HCV RNA levels before treatment (52.7 +/- 21.7 U/L; 2.6 x 10(6) copies/mL) showed no significant changes during IL-12 treatment (57.3 +/- 58.8 U/L and 3.2 x 10(6) copies/mL, 50.3 +/- 46.2 U/L and 3.1 x 10(6) copies/mL, and 46.8 +/- 35.3 U/L and 3.9 x 10(6) copies/mL at weeks 1, 4, and 10, respectively). Similar results were observed in 14 IFN-alpha non-responders and 14 untreated patients. However, changes in HCV quasispecies occurred in 10/12 (83%) and 9/14 (64%) patients treated with interleukin-12 and interferon-alpha, respectively, but only in 3/14 (21%) untreated subjects (P < 0.003 and P < 0.03). These results imply that interleukin-12 exerts only limited antiviral activity against certain HCV quasispecies in vivo.

Adult↗
PubMed2000

NAT and viral safety in blood transfusion.

BACKGROUND AND OBJECTIVES: Nucleic acid testing was introduced at our blood transfusion service in order to reduce the diagnostic preseroconversion window for transfusion relevant viruses. MATERIALS AND METHODS: Up to 96 donor samples were pooled overnight by two Tecan Genesis RSP 150 pipettors. Pools were centrifuged at 48,000 g for one hour to enrich viruses. Viral nucleic acids were extracted from centrifugation pellets using modified Qiagen viral RNA kit. HIV and HBV sequences were amplified by in house TaqMan PCR's with patented primers and probes for HBV. HCV PCR was performed by Roche Amplicor V2.0. "Nadis" software was developed for pooling, resolving positive pools and data communication with main frame computer. RESULTS: PCR testing was introduced in January 1997 for HCV HBV and HIV-1 for all plasma products and for labile components at the 21st of April 1997. Throughput increased from 2000 to 4000 samples per day. PCR testing is done in parallel to serological testing and products are released eight hours after pooling is completed. Until January 2000, 1,078,940 donations have been tested in 13,274 pools. A total of seven PCR only positives were identified (3 HCV, 3 HBV, 1 HIV). CONCLUSION: The yield of PCR testing for transfusion relevant viruses confirms theoretical estimates on the residual risk of antibody screening.

Blood Donors↗
PubMed1999

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

Enzyme Activation↗
PubMed1999

Feasibility and efficacy of routine PCR screening of blood donations for hepatitis C virus, hepatitis B virus, and HIV-1 in a blood-bank setting.

BACKGROUND: Despite sensitive antibody-based blood-donor screening, a residual risk of transfusion-transmitted viral infections exists. Only direct monitoring by sensitive nucleic-acid tests would provide data accurately to measure the risk and to assess risk-reduction procedures. We investigated the feasibility and efficacy of routine screening of donors for hepatitis C virus (HCV), hepatitis B virus (HBV), and HIV-1 by PCR. METHODS: For PCR testing, individual donor plasma samples were pooled (96x100 microL) overnight by two automatic pipetting machines. Viruses were concentrated by centrifugation and nucleic acids were extracted. HCV PCR was done on the Cobas Amplicor system (Hoffmann-La Roche, Mannheim, Germany). HBV and HIV-1 sequences were amplified by single (non-nested) in-house PCRs and detected by agarose-gel electrophoresis. Detection limits were 1000-5000 genome equivalents/mL in the donor blood. FINDINGS: PCR testing was done in parallel to antibody screening with a maximum throughput of 3000 samples in 7-8 h. Positive samples were identified 1-2 days later. 111 of 373,423 donations (107 of 4500 pools) were PCR and antibody/antigen-confirmed positive. We found one HCV PCR-positive antibody-negative donation with normal alanine aminotransferase and one HCV PCR-positive donation with an elevated alanine aminotransferase (100 IU), which was negative in the AxSYM 2.0 and Matrix 1.0, but positive after control in the Abbott Prism test (Abbott GmbH, Wiesbaden, Germany). INTERPRETATION: PCR is a suitable and fast blood-donor screening procedure and contributes to a reduction in viral transmission by transfusion of blood components. In our selected donor population, the yield of detected contaminated donations from donors in the time window in which they are highly infectious but do not have any symptoms or detectable antigen and antibody concentrations (diagnostic window), confirms theoretical estimates.

Blood Banks↗
PubMed1999

Improved correlation between multiple mutations within the NS5A region and virological response in European patients chronically infected with hepatitis C virus type 1b undergoing combination therapy.

BACKGROUND/AIMS: Studies from Japan showed that HCV-1b isolates with at least four amino acid changes within NS5A2209-2248 compared with the prototype sequence HCV-J are more sensitive to interferon than isolates with a prototype sequence. However, the data were not unequivocally confirmed in studies from other geographical areas. These discrepancies may be explained by differences in the prevalence of multiple mutations within the NS5A2209-2248 and/or the treatment efficacy. METHODS: In the present study, we therefore investigated the correlation between NS5A2209-2248 sequences of HCV-1b isolates and sustained virological response in 72 European patients treated with 3x6 MU interferon-a per week with (n = 26) and without (n = 46) ribavirin (1000-1200 mg/day). Serum HCV RNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the NS5A2209-2248 region was analyzed by sequencing of PCR products or individual clones. RESULTS: Compared with HCV-1b prototype sequences, 19 patients (26%) had no amino acid changes (prototype), 47 patients (65%) had 1-3 mutations (intermediate type) and six patients (8%) had at least 4 mutations in the NS5A2209-2248 region (mutant type). Nine of the 12 patients with sustained virological response were infected with an intermediate type HCV-1b, the remaining three patients revealed a mutant type HCV-1b. A sustained virological response was achieved in three of four patients with a mutant type HCV-1b treated with interferon-alpha and ribavirin, but in none of the mutant type HCV-1b infected patients treated with interferon-a alone. Quasispecies analysis of HCV in the NS5A2209-2248 region showed only minor heterogeneity of the amino acid sequence. CONCLUSIONS: The prevalence of mutant type HCV-1b isolates in European patients is low. In patients treated with combination therapy interferon-a and ribavirin, a correlation between mutant type HCV-1b isolates and sustained virological response was observed. The discrepancies between previous studies appear to be related to the efficacy of antiviral treatment and to the low prevalence of mutant type HCV-1b isolates in Western countries.

Adult↗
PubMed1999

Peutz-Jeghers syndrome: molecular analysis of a three-generation kindred with a novel defect in the serine threonine kinase gene STK11.

The Peutz-Jeghers syndrome, phenotypically characterized by mucocutaneous pigmentation and hamartomatous polyposis, is an autosomal dominant disease with variable expression and incomplete penetrance. Moreover, affected patients are at increased risk for gastrointestinal and other malignancies. Recently, a mutated gene encoding abnormal forms of the novel serine threonine kinase STK11 has been identified as a genetic cause of Peutz-Jeghers syndrome. Here, we report the molecular analysis of the STK11 gene in a patient with Peutz-Jeghers syndrome, which in exon 1 revealed a guanine (G) insertion in the 5 G repeat of codons 51-53. The insertion leads to a frameshift with a premature TGA stop codon 324 bp downstream in codon 162, predicting the expression of a truncated protein without kinase activity. This heterozygous germline mutation was also found in the affected father and in one affected sister of the index patient, but not in any phenotypically unaffected family member or in unrelated control subjects. In DNA isolated from microdissected hamartomatous polyps of the index patient, exon 1 of the STK11 gene could not be amplified suggesting that both alleles of STK11 exon 1 were lost in the hamartomatous polyps. Identification of a STK11 gene mutation in an index patient offers the possibility of a predictive diagnosis, and initiation of specific screening programs in the genetically affected kindred.

Adult↗
PubMed1998

Quantification of the initial decline of serum hepatitis C virus RNA and response to interferon alfa.

Although several virus- and host-related predictive factors for the response to interferon alfa (IFN-alpha) have been defined in patients with chronic hepatitis C, no pretreatment parameter can definitely predict the response to antiviral treatment. Assessment of the initial response by quantification of serum hepatitis C virus RNA before and 4 weeks after initiation of therapy may be a clinically applicable and reliable parameter to predict long-term response. Therefore, the aims of the present study were to test the predictive value of a decline in HCV RNA of at least 3 log in the first 4 weeks of treatment (deltaHCV RNA) in patients treated with 3 x 10(6) units of recombinant IFN-alpha2a (rIFN-alpha2a) three times per week subcutaneously and to compare deltaHCV RNA with other established predictive factors, such as HCV genotype and pretreatment viremia. Serum HCV RNA was measured by a validated quantitative reverse transcription-polymerase chain reaction (RT-PCR). Geno/subtyping of HCV was performed by direct sequencing of the nonstructural (NS) 5B region of PCR-amplified isolates and subsequent phylogenetic analysis. Stable HCV RNA levels (deltaHCV RNA < or = 1 log) within the first 4 weeks of IFN-alpha treatment were present in 42 of 70 patients. A decline in HCV RNA levels between 1 to 3 log and more than 3 log was observed in 9 (13%) and 19 patients (27%), respectively. In 21 of 70 patients (30%), HCV RNA was not detectable at the end of 12 months' treatment. Three of 26 patients (11%) with a pretreatment viremia of < or = 10(6) copies/mL (all HCV subtype 3a) and 6 of 44 patients (14%) with a pretreatment viremia of > 10(6) copies/mL (HCV subtypes 1b, 2a, 2c, 3a [two patients], and 4) achieved a virological sustained response to interferon-alpha2a treatment. All patients with a virological sustained response had an initial deltaHCV RNA of more than 3 log. In a stepwise discriminant-function analysis, the initial deltaHCV RNA was confirmed as the strongest predictor of virological sustained response (P < .0001). In conclusion, the data of the present study suggest that IFN-alpha treatment can be terminated after 4 weeks in patients with a decrease in HCV RNA levels of less than 3 log, when apparent HCV eradication is considered the therapeutic target. The predictive value of deltaHCV RNA clearly exceeds the significance of HCV genotype and pretreatment viremia as predictors of successful IFN-alpha treatment.

Adult↗
PubMed1998

Hepatitis C virus dynamics in vivo: effect of ribavirin and interferon alfa on viral turnover.

Treatment of patients with chronic hepatitis C with recombinant interferon alfa (rIFN-alpha) can cause a decrease of serum transaminases and hepatitis C virus (HCV) RNA. Recent trials evaluating combination therapy of IFN-alpha and ribavirin suggested a potential synergistic effect. From serial measurements of serum HCV RNA concentrations following treatment-induced perturbation of the balance between virus production and clearance, we compared the antiviral efficacy of both IFN-alpha alone and IFN-alpha in combination with ribavirin. Chronically HCV-infected patients were treated with either 3 x 3 MU or 3 x 6 MU rIFN-alpha per week or 3 x 6 MU rIFN-alpha plus 14 mg/kg of body weight ribavirin per day. The time-dependent HCV RNA concentrations during antiviral treatment were analyzed by iterative least-squares regression. After initiation of antiviral therapy, HCV RNA declined exponentially below the detection limit of the reverse-transcription polymerase chain reaction assay (1,000 HCV RNA molecules per milliliter) in 10 of 26 (39%), 10 of 19 (53%), and 10 of 18 patients (56%) treated with 3 x 3 MU, 3 x 6 MU rIFN-alpha without and with ribavirin, respectively. Viral clearance from serum was faster in patients treated with 3 x 6 MU rIFN-alpha (t1/2 = 0.23 +/- 0.15) compared with patients treated with 3 x 3 MU rIFN-alpha per week (0.67 +/- 0.36 days) (P < .004). However, half-lives of viral clearance were similar in patients treated with rIFN-alpha or rIFN-alpha plus ribavirin. For virus release from infected hepatocytes, absence and presence of ribavirin yielded half-lives of t1/2 = 2.54 +/- 2.10 and t1/2 = 1.99 +/- 1.70, respectively, indicating that ribavirin does not significantly inhibit HCV production. In conclusion, the data of the present study indicate that higher rIFN-alpha doses accelerate viral clearance from serum. Ribavirin (14 mg/kg/d), however, lacks synergistic antiviral effects in the treatment of chronic hepatitis C with 3 x 6 MU rIFN-alpha per week.

Adult↗
PubMed1998

Effect of ribavirin on virus load and quasispecies distribution in patients infected with hepatitis C virus.

BACKGROUND/AIMS: The combination of ribavirin and interferon alfa has potent synergistic effects in the treatment of chronic hepatitis C. The antiviral mechanism of ribavirin is unknown. We investigated whether a transient initial antiviral effect of ribavirin was sufficient to improve the response to interferon. METHODS: Fifteen HCV-infected patients (ten male, five female; mean age 45.4+/-11.0 years) treated with ribavirin (1000-1200 mg) and 17 untreated patients with chronic hepatitis C (11 male, six female; mean age 45.6+/-9.9 years) were investigated. All patients were either non-responders to (n=19) or relapsed after (n=13) previous interferon treatment. Serum HCV-RNA concentrations and HCV quasispecies distribution were serially measured over 4 weeks by quantitative reverse transcription-polymerase chain reaction and single-strand conformation polymorphism analysis, respectively. RESULTS: In six of the 15 patients treated with ribavirin, but in none of the controls, serum alanine aminotransferase levels declined by at least 30%. Pretreatment HCV-RNA levels ranged from 5.0x10(5)-5.0x10(7) copies/ml. After initiation of ribavirin treatment, minor (0.5-1.0 log) or no changes (<0.5 log) in total hepatitis C viremia were observed in ten and five patients, respectively. In HCV-infected patients without treatment 7/17 patients had minor and 10/17 no changes in viremia. Polymerase chain reaction amplification of the hypervariable region-1 of HCV was successful in 13/15 treated and in 17/17 untreated patients. Changes in HCV quasispecies according to the single-strand conformation polymorphism band pattern occurred in only one patient treated with ribavirin and in three of the untreated patients. CONCLUSIONS: Ribavirin monotherapy has no initial antiviral effect on total hepatitis C viremia nor on HCV quasispecies. Unlike the rapid emergence of antiviral drug-resistant strains in HIV-infected patients, no viral escape phenomena are observed in HCV-infected patients treated with ribavirin.

Adult↗
PubMed1998

Clinical evaluation of autoantibodies to p53 protein in patients with chronic liver disease and hepatocellular carcinoma.

In hepatocellular carcinoma (HCC) of patients from the Western hemisphere, mutations in the p53 tumour suppressor gene are present in up to 37% of cases. Conformational change and cellular accumulation can initiate an immune response with generation of circulating autoantibodies to p53 protein. In the present study, we investigated 711 consecutive patients with chronic liver disease to evaluate the sensitivity and specificity of autoantibodies to p53 protein as a serological marker for HCC. Detection of p53 autoantibodies was performed using an enzyme-linked immunosorbent assay with immobilised recombinant p53 protein. Liver cirrhosis was present in 259 patients (36.4%) and a HCC was diagnosed in 75 patients (10.6%). Autoantibodies to p53 protein were detectable in 15 of 377 patients with chronic liver disease (4.0%) and in 10 of 259 patients presenting with liver cirrhosis (3.9%). All 25 p53 autoantibody-positive/HCC-negative patients were carefully investigated and no underlying malignancy was clinically detected, suggesting that elevated p53 antibody levels may not exclusively be detectable in patients with malignant disease. In patients with clinically manifest HCC, p53 autoantibodies were detected in 17 of 75 cases, thus resulting in a sensitivity of 22.7% and a specificity of 96.1%. In contrast, assessment of serum alpha-fetoprotein (AFP) resulted in a sensitivity and specificity of 69.3 and 91.8% (AFP > 20 ng/ml) and 53.3 and 99.1% (AFP > 100 ng/ml) for the detection of HCC, respectively. The data of the present study reveal that the presence of p53 autoantibodies in patients with chronic liver disease is not completely specific for HCC. Moreover, we obtained no direct evidence that p53 autoantibody formation precedes the clinical diagnosis of HCC. However, serological screening for HCC might be improved by combining AFP and p53 autoantibody assays.

Adolescent↗
PubMed1998

Quantification of hepatitis C virus RNA by RT-PCR in comparison to the branched DNA method.

INTRODUCTION: Quantification of hepatitis C virus (HCV) serum viremia levels is an important measure to predict and monitor response to interferon-alpha therapy as well as to predict clinical outcome. We were interested to compare the most widely used HCV quantification methods, quantitative RT-PCR (qRT-PCR) and the branched DNA (bDNA) method, with respect to sensitivity and reliability. RESULTS: In the present study, 101 serum samples from patients chronically infected with HCV were simultaneously quantified by an in-house reverse transcriptase (RT)-PCR assay and the branched DNA Quantiplex HCV RNA kit 1.0 and 2.0. The concentration of HCV RNA molecules/ml serum ranged from 1.5 x 10(4) to 1.0 x 10(8) as assessed by our quantitative (q)RT-PCR. Serum concentrations of HCV-genotype 1 were significantly higher when measured with bDNA 1.0 compared to bDNA 2.0 and qRT-PCR, whereas measurements by qRT-PCR and bDNA 2.0 were concordant. Measurements of genotypes 2 and 3 by bDNA 2.0 were significantly higher than by bDNA 1.0. Significantly lower measurements were obtained for genotype 3 by qRT-PCR in comparison to bDNA 2.0. CONCLUSION: Despite subtype-specific differences, there is clinically sufficient agreement in measurements between qRT-PCR and bDNA of HCV RNA concentrations. However, with respect to the therapeutic monitoring of the individual patient, kits and methods should not be exchanged.

DNA, Viral↗
PubMed1998

[GB virus-C/hepatitis G virus--discovery, epidemiology, diagnosis and clinical relevance].

Recently, two isolates of a new virus, designated GB virus C (GBV-C) and hepatitis G virus (HGV) were identified. Genomic organization place them in the family Flaviviridae, which includes HCV. A classification of GBV-C/HGV strains into at least three genotypes (West Africa, Europe/North America, Asia) has been proposed. GBV-C/HGV has a positive-stranded, linear RNA genome possessing a large open reading frame that encodes a single large polyprotein. As a possible genomic defect no core protein has yet been identified in GBV-C/HGV isolates. Whether GBV-C/HGV can be classified as a hepatotropic virus with replication in the liver is under discussion. The prevalence in the general population is high (2%). GBV-C/HGV is transmitted parenterally e.g., by transfusion of blood and blood products. Furthermore, cases of vertical and horizontal transmission are reported. Detection of GBV-C/HGV infection is exclusively possible by reverse transcription polymerase chain reaction (RT PCR). E2-specific antibodies are associated with loss of detectable GBV-C/HGV RNA and appear to indicate recovery from GBV-C/HGV infection. An acute icteric state with transient elevation of aminotransferases in association with GBV-C/HGV infection may exist. These cases are rare and approximately only 0.3% of persons with acute viral hepatitis are infected with GBV-C/HGV alone. Whether GBV-C/HGV is a cause of fulminant hepatic failure has not yet been proven. Persistent viremia of the GBV-C/HGV infection is frequent but is not related to chronic liver disease. Since no other chronic diseases associated with GBV-C/HGV infection could yet be identified, chronic GBV-C/HGV infections appear to be without clinical significance. GBV-C/HGV and hepatitis B or C virus (HBV, HCV) coinfection seem not to interfere with the outcome of HBV-respective HCV-related liver disease. The cause of the majority of cases with non-A-E viral hepatitis remains unknown and other yet undiscovered hepatic viruses must exist.

Cross-Sectional Studies↗