| 1. |
- Herrmann, Björn, et al.
(författare)
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Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus
- 2004
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 42:5, s. 1909-14
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Tidskriftsartikel (refereegranskat)abstract
- A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
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| 2. |
- Westman, Gabriel, et al.
(författare)
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Application of a Multiplex Herpesvirus Immunoassay in Alzheimer’s Disease
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Human herpesviruses have previously been implicated in the pathogenesis of Alzheimer's disease (AD) but whether they are causal, facilitating or confounding factors is yet to be established. Here, we present an application of a suspension matrix immunoassay (SMIA) allowing multiplex detection of IgG levels related to five human herpesviruses. Furthermore, we analysed peripheral blood mononuclear cells with a subtype specific HHV-6 PCR. The level of HHV-6 antibodies was lower in AD subjects (n=51) than in non-demented controls (ND, n=52), whereas there were no differences in HSV, VZV, CMV or EBV antibody levels between groups. AD and ND subjects presented with comparable DNA levels in PBMC and all positive samples were of subtype B. The SMIA protocol, applied to herpesvirus antigens, could provide an useful addition to the scientific arsenal. Whether HHV-6 is a factor in AD remains a hypothesis for future studies.
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| 3. |
- Westman, Gabriel, et al.
(författare)
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Decreased HHV-6 IgG in Alzheimer's Disease
- 2017
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Ingår i: Frontiers in Neurology. - : Frontiers Media SA. - 1664-2295. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Human herpesviruses have previously been implicated in the pathogenesis of Alzheimer's disease (AD) but whether they are causal, facilitating, or confounding factors is yet to be established. A total of 50 AD subjects and 52 non-demented (ND) controls were analyzed in a multiplex assay for IgG reactivity toward herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6). The HHV-6 IgG reactivity was significantly lower in AD subjects compared to ND controls, whereas there were no differences in HSV, VZV, or CMV antibody levels between the groups. Analysis of peripheral blood mononuclear cells with a subtype-specific HHV-6 PCR revealed no signs of reactivation, as AD and ND subjects presented with comparable HHV-6 DNA levels in PBMCs, and all positive samples were of subtype B. Whether HHV-6 is a factor in AD remains to be elucidated in future studies.
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| 4. |
- Yun, Zhibing
(författare)
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Nucleic acid-based detection and characterization of hepatitis C virus
- 1995
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nucleic acid-based methods were developed to detect and characterize the HCV genome in immunocompetent and immunodeficient patents of whom some were IFN-a treated. HCV RNA was found in most sera (95%), livers (97%) and PBMC (100%) of patients with confirmed chronic hepatitis, but also in sera (21%) of patients with indeterminate confirmatory antibody test, especially those with c22 antigen reactivity. Minus-stranded HCV RNA was found in almost all livers, but also in PBMC (53%) and sera (35%). The quantification assay, based on competitive PCR, was found to be sensitive and suitable to follow the viral load during IFN-c therapy. The pretreatment HCV RNA level was lower in patients with sustained response than in those with no or non-sustained response. HCV was misclassified as genotype Ib in mixed infections by a widely used type-specific PCR due to mispriming of one primer. Concordant results were found between phylogenetic analysis of short core and NS5 sequences. Using a direct sequencing assay, the mutation rate of the hyper variable region I (HVRI) was as high as O.1-0.2 substitutions/genome site/year in immunocompetent patients. During 15 months IFN-o~ therapy, the heterogeneity and mutation rate of HVRI decreased significantly. The heterogeneity and the mutation rate of patients with a gamma-globulinemia or AIDS was substantially lower or absent. As determined by both direct sequencing and single strand poly-morphism analysis, the heterogeneity was decreased in liver transplantat patients with advanced immunosuppression during the early post-LTx period and increased when the dosages of the immuno-suppressive drugs were decreased. By our developed nucleic acid-based assays, important information was obtained. Thus, HCV replicates not only in the liver, but probably also in PBMC. The pretreatment HCR RNA level seems to be predictive for the response to IFN-c-therapy. Phylogenetic analysis of short core and NS5 sequences is suitable for HCV genotyping. Both selection of HCV strains and general suppression of the viral replication may occur during IFN-c~therapy. An intact immune response is crucial for the development of a high heterogeneity in HVR 1, although also other factors may contribute in shaping the viral population.
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