0000000000288857
AUTHOR
Martin Trippler
Performance of the COBAS AMPLICOR HCV MONITOR Test, Version 2.0, an Automated Reverse Transcription-PCR Quantitative System for Hepatitis C Virus Load Determination
ABSTRACT A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMP…
Selection of hepatitis B virus variants with aminoacid substitutions inside the core antigen during interferon-? therapy
The hepatitis B virus (HBV) core antigen carries many epitopes relevant for B and T cell response that show aminoacid variation during viral infection. In a longitudinal analysis, sequential serum samples of 15 patients that suffered from chronic HBV infection were collected before, during, and after high-dose IFN-α treatment. The HBV preCore/Core (preC/C) sequence of the selected samples in each patient was determined and analysed for sequence variations compared to the pretreatment sample. The positions of HBV core aminoacid substitutions were assigned to immunodominant B, CD4+ and CD8+ cell epitopes. Seventy-five percent of all aminoacid substitutions were found within immunodominant T a…
Clinical evaluation and applications of the Amplicor HBV Monitor™ test, a quantitative HBV DNA PCR assay
Viral load has emerged recently as a reliable marker of disease progression and therapeutic efficacy in chronic infections, including AIDS and hepatitis C. The clinical management of type B hepatitis could also be improved by monitoring viremia levels in patients with chronic liver disease undergoing anti-viral treatment. To address this question we evaluated the performance of a newly developed, quantitative PCR assay (Amplicor HBV Monitor test, Roche Diagnostic Systems) in the assessment of viremia changes over time in a group of 45 patients with chronic active hepatitis (CAH) who received interferon treatment. Of the 45 patients, 14 were HBsAg and anti-HBeAg positive and 31 HBsAg, HBeAg …
Interleukin-10 expression is autoregulated at the transcriptional level in human and murine kupffer cells
Interleukin 10 (IL-10) is known to downregulate immune responses. The regulation of IL-10 gene expression therefore determines the outcome of local immune reactions. We investigated time course and downregulation of IL-10 production in primary Kupffer's cells (KC), which are known to secrete IL-10 in response to endotoxin challenge. Human and murine KC were isolated by centrifugal elutriation and investigated for IL-10 gene expression by a two-step amplification procedure (reverse transcriptase-polymerase chain reaction [PCR] followed by T7-polymerase chain reaction). We show that IL-10 messenger ribonucleic acid (mRNA) showed a >450 fold increase in KC 2 hours after endotoxin challenge. IL…
Ligase Chain Reaction (LCR®) assay for semi-quantitative detection of HBV DNA in mononuclear leukocytes of patients with chronic hepatitis B
Summary. A ligase chain reaction (LCR®)-based approach to detect hepatitis B virus (HBV) DNA in peripheral blood mononuclear cells (PBMC) is described. Using this new amplification technique, we determined semi-quantitatively the amount of a short HBV S-gene fragment in PBMC lysates of 25 patients with different forms of chronic hepatitis (group A (n= 8), hepatitis B s antigen (HBsAg)+/hepatitis B e antigen (HBeAg)+; group B (n= 9), HBsAg+/HBeAg-; group C (n= 8), HBsAg-/HBeAg-). The LCR results were compared with the findings obtained with polymerase chain reaction (PCR) amplification of three distinct HBV gene regions (preS1/2, S and C) and related to the serological profiles of the patien…
HBV viral load within subpopulations of peripheral blood mononuclear cells in HBV infection using limiting dilution PCR.
Abstract Extrahepatic viral load in peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus (HBV) is still under debate. In this study, HBV infection rates and viral titers were examined within all PBMC subpopulations using limiting dilution-PCR (LD-PCR). PBMCs of patients with acute or chronic hepatitis B were separated by magnetic beads in monocytes, B-cells, CD4+ T-cells, CD8+ T-cells, and NK cells. Using two-round nested PCR, HBV-DNA sequences were detected in all patients examined within each PBMC subpopulation. The frequencies of HBV-positive cells and viral loads were calculated by Poisson analysis of HBV PCR results from serial dilutions of cells and cell lysat…
Rare pre-core stop-codon mutant nt. 1897 predominates over wide-spread mutant nt. 1896 in an unusual course of chronic hepatitis B
We present a patient with an unusual course of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B who had repeated reactivations of his disease progressing to cirrhosis with terminal liver failure. Each flare up presented like an acute hepatitis with very high titres of hepatitis B virus (HBV) and high inflammatory activity followed by rapid clearance of viraemia. The pre-core genome of HBV isolated from sera during 5 years of follow up was analysed. Direct sequencing of polymerase chain reaction (PCR) products derived from consecutive sera showed a rare pre-core stop-codon mutation at nucleotide (nt.) 1897 G --> A with an accompanying mutation nt. 1857 C --> T as well as a stop-cod…
Chronic hepatitis C: the viral load per liver cell before treatment as a new marker to predict long-term response to IFN-α therapy
Abstract Background/Aims: So far, there are no reliable parameters that can predict the long-term sustained response to treatment with interferon-α in patients with chronic hepatitis C. In this study, we have developed a semi-quantitative method to determine the viral load per liver cell and have correlated this factor with the outcome of hepatitis C patients treated with interferon-α. Methods: Hepatitis C virus RNA levels were measured in serum, peripheral blood mononuclear cells and liver cells of randomly chosen hepatitis C patients before treatment with interferon-α ( n =37). The number of cells present in the liver biopsies was determined by a polymerase chain reaction-based quantitati…
Posttransfusional, LKM-1-autoantibody-positive hepatitis C virus infection, cryoglobulinemia, and aplastic anemia.
Aplastic anemia is occasionally caused by viral hepatitis, hepatitis C virus being the most important factor. Pathogenetically, decreased bone marrow function, abnormalities of the bone marrow microenvironment, and immune-mediated suppression of hematopoiesis are important. Hepatitis C virus infection is associated with a variety of extrahepatic manifestations including autoimmune features like cryoglobulinemia, Sjogren's syndrome, and autoimmune hepatitis. Here we report the case of a 42-year-old man with aplastic anemia due to posttransfusional hepatitis C virus infection associated with cryoglobulinemia and LKM-1 autoantibodies. Following a triple immunosuppressive therapy, there was a c…