114 results on '"Zeichhardt, H."'
Search Results
2. Molecular surveillance of measles and rubella in the WHO European Region: new challenges in the elimination phase
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Santibanez, S., Hübschen, J.M., Ben Mamou, M.C., Muscat, M., Brown, K.E., Myers, R., Donoso Mantke, O., Zeichhardt, H., Brockmann, D., Shulga, S.V., Muller, C.P., O'Connor, P.M., Mulders, M.N., and Mankertz, A.
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- 2017
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3. Coxsackievirus B3 and adenovirus infections of cardiac cells are efficiently inhibited by vector-mediated RNA interference targeting their common receptor
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Fechner, H, Pinkert, S, Wang, X, Sipo, I, Suckau, L, Kurreck, J, Dörner, A, Sollerbrant, K, Zeichhardt, H, Grunert, H-P, Vetter, R, Schultheiss, H-P, and Poller, W
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- 2007
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4. Treatment of coxsackievirus-B3-infected BALB/c mice with the soluble coxsackie adenovirus receptor CAR4/7 aggravates cardiac injury
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Dörner, A., Grunert, H.-P., Lindig, V., Chandrasekharan, K., Fechner, H., Knowlton, K. U., Isik, A., Pauschinger, M., Zeichhardt, H., and Schultheiss, H.-P.
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- 2006
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5. UPDATE RiLiBÄK — IMPACT OF THE NEW GUIDELINE OF THE GERMAN MEDICAL ASSOCIATION ON THE DIAGNOSIS OF INFECTIOUS DISEASES PATHOGENS: T45 — Talk Zeichhardt
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Zeichhardt, H.
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- 2010
6. Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells
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Grunert, Hans-Peter, Wolf, Kai-Uwe, Langner, Klaus-Dieter, Sawitzky, Dirk, Habermehl, Karl-Otto, and Zeichhardt, H.
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- 1997
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7. Proteins occurring at, or near, the subunit interface of E. coli ribosomes
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Morrison, C. A., Garrett, R. A., Zeichhardt, H., and Stöffler, G.
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- 1973
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8. Neutralization of poliovirus by polyclonal antibodies requires binding of a single IgG molecule per virion
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Wetz, K., Willingmann, P., Zeichhardt, H., and Habermehl, K. -O.
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- 1986
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9. Location of proteins S5, S13 and S14 on the surface of the 30S ribosomal subunit from Escherichia coli as determined by immune electron microscopy
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Tischendorf, G. W., Zeichhardt, H., and Stöffler, G.
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- 1974
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10. Determination of the location of proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S ribosomal subunit of Escherichia coli by immune electron microscopy
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Tischendorf, G. W., Zeichhardt, H., and Stöffler, G.
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- 1974
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11. The accessibility of proteins of the Escherichia coli 30S ribosomal subunit to antibody binding
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Stöffler, G., Hasenbank, R., Lütgehaus, M., Maschler, R., Morrison, C. A., Zeichhardt, H., and Garrett, R. A.
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- 1973
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12. Ribosomal protein neighborhoods: I. S18 and S21 as well as S5 and S8 are neighbors
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Lutter, L. C., Zeichhardt, H., Kurland, C. G., and Stöffler, G.
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- 1972
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13. Performance evaluation of 70 hepatitis B virus (HBV) surface antigen (HBsAg) assays from around the world by a geographically diverse panel with an array of HBV genotypes and HBsAg subtypes.
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Scheiblauer, H., El-Nageh, M., Diaz, S., Nick, S., Zeichhardt, H., Grunert, H.-P., and Prince, A.
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ANTIGEN analysis ,HEPATITIS B treatment ,GENETIC polymorphisms ,MICROBIOLOGICAL assay ,ENZYME-linked immunosorbent assay - Abstract
Background and Objectives: This study was conducted by the International Consortium for Blood Safety (ICBS) to identify high-quality test kits for detection of hepatitis B virus (HBV) surface antigen (HBsAg) for the benefit of developing countries. Materials and Methods: The 70 HBsAg test kits from around the world were evaluated comparatively for their clinical sensitivity, analytical sensitivity, sensitivity to HBV genotypes and HBsAg subtypes, and specificity using 394 (146 clinical, 48 analytical and 200 negative) ICBS Master Panel members of diverse geographical origin comprising the major HBV genotypes A-F and the HBsAg subtypes adw2,4, adr and ayw1-4. Results: Seventeen HBsAg enzyme immunoassay (EIA) kits had high analytical sensitivity <0Æ13 IU ⁄ ml, showed 100% diagnostic sensitivity, and were even sensitive for the various HBV variants tested. An additional six test kits had high sensitivity (<0Æ13 IU ⁄ ml) but missed HBsAg mutants and ⁄ or showed reduced sensitivity to certain HBV genotypes. Twenty HBsAg EIA kits were in the sensitivity range of 0Æ13– 1 IU⁄ ml. The other eight EIAs and the 19 rapid assays had analytical sensitivities of 1 to >4 IU⁄ ml. These assays were falsely negative for 1–4 clinical samples and 17 of these test kits showed genotype dependent sensitivity reduction. Analytical sensitivities for HBsAg of >1 IU ⁄ ml significantly reduce the length of the HBsAg positive period which renders them less reliable for detecting HBsAg in asymptomatic HBV infections. Reduced sensitivity for HBsAg with genetic diversity of HBV occurred with genotypes ⁄ subtypes D⁄ ayw3, E⁄ ayw4, F⁄ adw4 and by S gene mutants. Specificity of the HBsAg assays was ‡99Æ5% in 57 test kits and 96Æ4–99Æ0% in the remaining test kits. Conclusion: Diagnostic efficacy of the evaluated HBsAg test kits differed substantially. Laboratories should therefore be aware of the analytical sensitivity for HBsAg and check for the relevant HBV variants circulating in the relevant population. [ABSTRACT FROM AUTHOR]
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- 2010
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14. Prevention of cardiac dysfunction in acute coxsackievirus B3 cardiomyopathy by inducible expression of a soluble coxsackievirus-adenovirus receptor.
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Pinkert S, Westermann D, Wang X, Klingel K, Dörner A, Savvatis K, Grössl T, Krohn S, Tschöpe C, Zeichhardt H, Kotsch K, Weitmann K, Hoffmann W, Schultheiss HP, Spiller OB, Poller W, and Fechner H
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- 2009
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15. Ribosomal protein neighborhoods.
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Lutter, L., Zeichhardt, H., Kurland, C., and Stöffler, G.
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- 1972
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16. First national external quality assessment scheme for avian influenza A virus (H5N1)
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Zeichhardt, H., Schweiger, B., Lindig, V., and Grunert, H.-P.
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- 2006
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17. Virologie: Ergebnisse und Aufdeckung von Testproblemen.
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Zeichhardt, H., Sawitzky, D., Stöffler-Meilicke, M., and Habermehl, K.-O.
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- 1994
18. A multicentre quality assessment study to monitor the performance of HIV-1 PCR
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Schweiger, B, Pauli, G, Zeichhardt, H, and Kücherer, C
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- 1997
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19. Do not blindly trust negative diagnostic test results!
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Buchta C, Zeichhardt H, Osterman A, Perrone LA, and Griesmacher A
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- Humans, Diagnostic Tests, Routine, Trust
- Abstract
Competing Interests: HZ declares that he was the majority owner and managing director of GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin and is the owner and managing director of IQVD GmbH, Institut für Qualitätssicherung, Berlin. LAP received support from the Donald B Rix Family Foundation. All other authors declare no competing interests.
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- 2024
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20. Hantavirus Disease Cluster Caused by Seoul Virus, Germany.
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Hofmann J, Ulrich RG, Mehl C, Drewes S, Esser J, Loyen M, Zeichhardt H, Schoppmeyer K, Essen L, Güthoff W, and Krüger DH
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- Animals, Rats, Disease Hotspot, Germany epidemiology, Europe, Seoul virus genetics, Orthohantavirus, RNA Viruses
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A cluster of 3 persons in Germany experienced hantavirus disease with renal insufficiency. Reverse transcription PCR-based genotyping revealed infection by Seoul hantavirus transmitted from pet rats. Seoul virus could be responsible for disease clusters in Europe, and infected pet rats should be considered a health threat.
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- 2024
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21. Correlation of SARS-CoV-2 RNA and nucleocapsid concentrations in samples used in INSTAND external quality assessment schemes.
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Valiente E, Falak S, Kummrow A, Kammel M, Corman VM, Macdonald R, and Zeichhardt H
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- Humans, SARS-CoV-2 genetics, Nucleocapsid, RNA, Viral genetics, COVID-19 diagnosis
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Objective: In routine clinical laboratories, severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the COVID pandemic, a wide range of antigen detection tests were also in high demand. We investigated the correlation between SARS-CoV-2 NCap antigen and N gene concentration by analyzing samples from several INSTAND external quality assessment (EQA) schemes starting in March 2021. The absolute N gene concentration was measured using reverse transcriptase digital PCR (RT-dPCR) as reference value. Moreover, the performance of five commercial ELISA tests using an EQA inactivated SARS-CoV-2 sample at different concentrations was assessed on the basis of these reference values., Results: Quantitative ELISA and RT-dPCR results showed a good correlation between SARS-CoV-2 NCap antigen and RNA concentration, but this correlation varies among SARS-CoV-2 isolates. A direct correlation between SARS-CoV-2 NCap antigen concentration and genome concentration should not be generally assumed., Conclusion: Further correlation studies between SARS-CoV-2 RNA and NCap antigen concentrations are needed, particularly in clinical samples and for emerging SARS-CoV-2 variants, to support the monitoring and improvement of antigen testing., (© 2023. BioMed Central Ltd., part of Springer Nature.)
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- 2023
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22. Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.
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Vierbaum L, Wojtalewicz N, Grunert HP, Zimmermann A, Scholz A, Goseberg S, Kaiser P, Duehring U, Drosten C, Corman V, Niemeyer D, Rabenau HF, Obermeier M, Nitsche A, Michel J, Puyskens A, Huggett JF, O'Sullivan DM, Busby E, Cowen S, Vallone PM, Cleveland MH, Falak S, Kummrow A, Schellenberg I, Zeichhardt H, and Kammel M
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- Chlorocebus aethiops, Animals, Humans, Pandemics, Vero Cells, Immunologic Tests, Sensitivity and Specificity, SARS-CoV-2, COVID-19 diagnosis, COVID-19 epidemiology
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The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 10
6 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed., (© 2023. Springer Nature Limited.)- Published
- 2023
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23. Classification of "Near-patient" and "Point-of-Care" SARS-CoV-2 Nucleic Acid Amplification Test Systems and a first approach to evaluate their analytical independence of operator activities.
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Buchta C, Zeichhardt H, Badrick T, Coucke W, Wojtalewicz N, Griesmacher A, Aberle SW, Schellenberg I, Jacobs E, Nordin G, Schweiger C, Schwenoha K, Luppa PB, Gassner UM, Wagner T, and Kammel M
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- Humans, Reproducibility of Results, Point-of-Care Systems, Nucleic Acid Amplification Techniques, SARS-CoV-2 genetics, COVID-19 diagnosis
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Background: European legislation defines as "near-patient testing" (NPT) what is popularly and in other legislations specified as "point-of-care testing" (POCT). Systems intended for NPT/POCT use must be characterized by independence from operator activities during the analytic procedure. However, tools for evaluating this are lacking. We hypothesized that the variability of measurement results obtained from identical samples with a larger number of identical devices by different operators, expressed as the method-specific reproducibility of measurement results reported in External Quality Assessment (EQA) schemes, is an indicator for this characteristic., Materials and Methods: Legal frameworks in the EU, the USA and Australia were evaluated about their requirements for NPT/POCT. EQA reproducibility of seven SARS-CoV-2-NAAT systems, all but one designated as "POCT", was calculated from variabilities in Ct values obtained from the respective device types in three different EQA schemes for virus genome detection., Results: A matrix for characterizing test systems based on their technical complexity and the required operator competence was derived from requirements of the European In Vitro Diagnostic Regulation (IVDR) 2017/746. Good EQA reproducibility of the measurement results of the test systems investigated implies that different users in different locations have no recognizable influence on their measurement results., Conclusion: The fundamental suitability of test systems for NPT/POCT use according to IVDR can be easily verified using the evaluation matrix presented. EQA reproducibility is a specific characteristic indicating independence from operator activities of NPT/POCT assays. EQA reproducibility of other systems than those investigated here remains to be determined., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter B. Luppa is member of the board of directors of INSTAND e.V., a non-profit organization, named as one of two EQA providers for Germany by the Deutsche Ärztekammer. Heinz Zeichhardt declares that he is co-chairman of the Joint Diagnostic Council of the Deutsche Vereinigung zur Bekaempfung der Viruskrankheiten e.V. (DVV e.V.) and Gesellschaft fuer Virologie (GfV e.V.) and is Advisor for the INSTAND External Quality Assessment (EQA) schemes in virus diagnostics. He is owner and managing director of IQVD GmbH - Institut fuer Qualitaetssicherung in der Virusdiagnostik, Berlin, and was majority owner and managing director of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin. He declares that he has no conflicts of interest with regard to the activities mentioned in relation to the publication. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)
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- 2023
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24. Design of external quality assessment schemes and definition of the roles of their providers in future epidemics.
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Buchta C, Zeichhardt H, Aberle SW, Camp JV, Görzer I, Weseslindtner L, Puchhammer-Stöckl E, Huf W, Benka B, Allerberger F, Mielke M, Griesmacher A, Müller MM, Schellenberg I, and Kammel M
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- Humans, Reproducibility of Results, SARS-CoV-2 genetics, Laboratories, Pandemics, COVID-19 diagnosis, COVID-19 epidemiology
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During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers., Competing Interests: Declaration of interests CB is chairman of the executive board of the European Organisation for External Quality Assurance Providers in Laboratory Medicine 2020–23. HZ was majority owner of Gesellschaft für Biotechnologische Diagnostik (until November, 2022) and is owner and managing director of Institut für Qualitätssicherung in der Virusdiagnostik. AG is president of the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)
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- 2023
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25. Ignoring SARS-CoV-2 testing performance during COVID-19.
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Buchta C, Zeichhardt H, Griesmacher A, Schellenberg I, and Kammel M
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- Humans, SARS-CoV-2, COVID-19 Testing, Sensitivity and Specificity, COVID-19
- Abstract
Competing Interests: HZ is majority owner of Gesellschaft für Biotechnologische Diagnostik; and owner and managing director of Institut für Qualitätssicherung in der Virusdiagnostik. All other authors declare no competing interests.
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- 2023
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26. Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.
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Vierbaum L, Wojtalewicz N, Kaufmann A, Goseberg S, Kaiser P, Grunert HP, Dühring U, Zimmermann A, Scholz A, Michel J, Nitsche A, Rabenau HF, Obermeier M, Schellenberg I, Zeichhardt H, and Kammel M
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- Humans, Monkeypox virus genetics, SARS-CoV-2 genetics, COVID-19, Mpox (monkeypox), Orthopoxvirus
- Abstract
Background: In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests., Methods: We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level., Results: 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each., Conclusion: The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: HZ declares that he was majority owner of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, and owner and managing director of IQVD GmbH - Institut fuer Qualitaetssicherung, Berlin. HPG declares that he was minority owner of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin. HZ and HPG declare that they were managing directors of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, during the study. This does not alter our adherence to PLoS One policies on sharing data and materials. All other authors have declared that no competing interests exist., (Copyright: © 2023 Vierbaum et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2023
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27. The Coronavirus Standards Working Group's roadmap for improved population testing.
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Mercer T, Almond N, Crone MA, Chain PSG, Deshpande A, Eveleigh D, Freemont P, Fuchs S, Garlick R, Huggett J, Kammel M, Li PE, Milavec M, Marlowe EM, O'Sullivan DM, Page M, Pestano GA, Suliman S, Simen B, Sninsky JJ, Sopchak L, Tato CM, Vallone PM, Vandesompele J, White TJ, Zeichhardt H, and Salit M
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- Coronavirus
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- 2022
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28. Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.
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Windsor WJ, Roell Y, Tucker H, Cheng CA, Suliman S, Peek LJ, Pestano GA, Lee WT, Zeichhardt H, Lamb MM, Kammel M, Wang H, Kedl R, Rester C, Morrison TE, Davenport BJ, Carson K, Yates J, Howard K, Kulas K, Walt DR, Dafni A, Taylor D, and Chu M
- Abstract
Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms., Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units., Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]., Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS., Competing Interests: GP and LP was employed by Biodesix, Inc. HZ and MK was employed by INSTAND e.V., IQVD GmbH, and GBD Gesellschaft fuer Biotechnologische Diagnostik mbH. HW was employed by Thermo Fisher Scientific. AD and DT were employed by company Oneworld Accuracy. The authors declare that this study received funding from the Bill & Melinda Gates Foundation. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Windsor, Roell, Tucker, Cheng, Suliman, Peek, Pestano, Lee, Zeichhardt, Lamb, Kammel, Wang, Kedl, Rester, Morrison, Davenport, Carson, Yates, Howard, Kulas, Walt, Dafni, Taylor and Chu.)
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- 2022
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29. An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1.
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Falak S, Macdonald R, Busby EJ, O'Sullivan DM, Milavec M, Plauth A, Kammel M, Zeichhardt H, Grunert HP, Kummrow A, and Huggett JF
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- Humans, RNA, RNA, Viral analysis, RNA, Viral genetics, Real-Time Polymerase Chain Reaction methods, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, HIV-1 genetics, Reverse Transcription
- Abstract
Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR., (Copyright © 2021. Published by Elsevier Inc.)
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- 2022
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30. The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.
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Milavec M, Pavšič J, Bogožalec Košir A, Jones GM, O'Sullivan DM, Devonshire AS, Van Heuverswyn F, Karczmarczyk M, Neeb J, Plauth A, Corbisier P, Schimmel H, Kummrow A, Neukammer J, Foy CA, Kammel M, Grunert HP, Zeichhardt H, and Huggett JF
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- Calibration, Humans, Real-Time Polymerase Chain Reaction methods, Reproducibility of Results, Cytomegalovirus genetics
- Abstract
A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from -0.8 log
10 to +0.8 log10 , the majority of dPCR results were between -0.2 log10 to +0.2 log10 . Only 4 out of 45 results exceeded this interval with the maximum deviation of -0.542 log10 . In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log10 from the target value, while more than 95% deviated less than ±0.4 log10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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31. Evaluation of the diagnostic capacities for emerging arboviral diseases in the international network MediLabSecure from 2014 to 2018 - Importance of external quality assessments.
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Mikaty G, Matheus S, Donoso Mantke O, McCulloch E, Zeichhardt H, and Manuguerra JC
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- Humans, Arbovirus Infections diagnosis, Arbovirus Infections epidemiology, Arboviruses, Chikungunya Fever, Zika Virus, Zika Virus Infection
- Abstract
Background: Emerging infectious diseases pose an increasing threat to all nations around the world, including to developed countries. By definition, because they are rare or unknown, public health systems are not well prepared against these emerging diseases. To be fully prepared, countries must have implemented surveillance systems to monitor rare or unusual sanitary events., Methods: The capacity of diagnostic laboratories is a key component of surveillance systems since they are in charge of identifying the pathogens responsible for outbreaks in a timely manner. The MediLabSecure project aims at implementing a comprehensive surveillance system for vector-borne diseases around the Mediterranean and Black Sea regions. From 2014 to 2018, the human-virology group of MediLabSecure notably supported the implementation of molecular diagnostic capacities for eight arboviruses and one coronavirus in 19 laboratories of its network through sharing of protocols and reagents, and technical training of the scientific staff of beneficiary laboratories., Results: We report the results of External Quality Assessments for four of these viruses to assess the efficiency of the diagnostic for these threats emerging in the geographic area. The results for these EQA demonstrate the success of the project in the implementation of diagnostic technics for the identification of Dengue, Chikungunya, Zika, and West Niles viruses in laboratories that did not have the capacity before. However, results also show that some work is still to be done to strengthen the newly acquired capacity., Conclusion: The MediLabSecure project deployed an effort to build an efficient capacity in identifying and survey the emergence of arboviruses in the Mediterranean area. Diagnostic technics were successfully implemented in many of the laboratories of the network, but the effort must be maintained over time to strengthen these capacities., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
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- 2022
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32. RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.
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Vierbaum L, Wojtalewicz N, Grunert HP, Lindig V, Duehring U, Drosten C, Corman V, Niemeyer D, Ciesek S, Rabenau HF, Berger A, Obermeier M, Nitsche A, Michel J, Mielke M, Huggett J, O'Sullivan D, Busby E, Cowen S, Vallone PM, Cleveland MH, Falak S, Kummrow A, Keller T, Schellenberg I, Zeichhardt H, and Kammel M
- Subjects
- COVID-19 epidemiology, COVID-19 virology, Genes, Viral, Germany epidemiology, Humans, Reproducibility of Results, COVID-19 diagnosis, COVID-19 Nucleic Acid Testing methods, Diagnostic Tests, Routine methods, RNA, Viral genetics, Reverse Transcriptase Polymerase Chain Reaction methods, SARS-CoV-2 genetics, Viral Load methods
- Abstract
SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements., Competing Interests: The work from SF and AK was partially funded by EURAMET in the project 18HLT03 SEPTIMET. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. AK is sitting in ISO/TC212/JWG 6 that is currently developing a standard for quality practice for detection of SARS-CoV-2. Work for this committee had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. TK is the owner of ACOMED statistik. He declares that he has received a payment from INSTAND e.V. for statistical consulting. He has received further payments from INSTAND e.V. in other projects. HZ declares that he is majority owner and managing director of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, and owner and managing director of IQVD GmbH - Institut fuer Qualitaetssicherung, Berlin. HPG declares that he is minority owner and managing director of GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have declared that no competing interests exist.
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- 2022
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33. The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.
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Evans D, Cowen S, Kammel M, O'Sullivan DM, Stewart G, Grunert HP, Moran-Gilad J, Verwilt J, In J, Vandesompele J, Harris K, Hong KH, Storey N, Hingley-Wilson S, Dühring U, Bae YK, Foy CA, Braybrook J, Zeichhardt H, and Huggett JF
- Subjects
- Belgium, Humans, RNA, Viral analysis, Reproducibility of Results, Republic of Korea, SARS-CoV-2, Sensitivity and Specificity, United Kingdom, COVID-19 diagnosis, COVID-19 Nucleic Acid Testing standards, Nucleic Acids analysis
- Abstract
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing., Methods: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq., Results: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI)., Conclusion: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic., (© American Association for Clinical Chemistry 2021.)
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- 2021
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34. Cautionary Note on Contamination of Reagents Used for Molecular Detection of SARS-CoV-2.
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Huggett JF, Benes V, Bustin SA, Garson JA, Harris K, Kammel M, Kubista M, McHugh TD, Moran-Gilad J, Nolan T, Pfaffl MW, Salit M, Shipley G, Vallone PM, Vandesompele J, Wittwer C, and Zeichhardt H
- Subjects
- COVID-19 virology, Diagnostic Errors, Humans, Limit of Detection, Nasopharynx virology, RNA, Viral genetics, RNA, Viral metabolism, Reverse Transcriptase Polymerase Chain Reaction, SARS-CoV-2 isolation & purification, Specimen Handling standards, COVID-19 diagnosis, Indicators and Reagents chemistry, SARS-CoV-2 genetics
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- 2020
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35. Novel multiple swab method enables high efficiency in SARS-CoV-2 screenings without loss of sensitivity for screening of a complete population.
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Schmidt M, Hoehl S, Berger A, Zeichhardt H, Hourfar K, Ciesek S, and Seifried E
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- COVID-19 virology, Germany, Humans, Nucleic Acid Amplification Techniques methods, Specimen Handling methods, SARS-CoV-2 pathogenicity
- Abstract
Background: In the pandemic, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time polymerase chain reaction is one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low-probability settings is proposed but may cause dilution and loss of sensitivity. Blood transfusion services had experience in performance of high throughput nucleic acid testing (NAT) analysis and can support the national health system by screening of the inhabitants for SARS-COV-2., Methods: We evaluated a new approach of a multiple-swab method by simultaneously incubating multiple respiratory swabs in a single tube. Analytical sensitivity was constant up to a total number of 50 swabs. It was consequently applied in the testing of 50 symptomatic patients (5-sample pools) as well as 100 asymptomatic residents of a nursing home (10-sample pools)., Results: The novel method did not cause false-negative results with nonsignificantly differing cycle threshold values between single-swab and multiple-swab NAT. In two routine applications, all minipools containing positive patient samples were correctly identified., Conclusions: The new method enables countries to increase the total number of testing significantly. The multiple-swab method is able to screen system relevant groups of employees frequently. The example in Germany shows that blood transfusion services can support general health systems with their experience in NAT and their high-throughput instruments. Screening of a huge number of inhabitants is currently the only option to prevent a second infection wave and enable exit strategies in many countries., (© 2020 The Authors. Transfusion published by Wiley Periodicals LLC. on behalf of AABB.)
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- 2020
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36. Digital PCR method for detection and quantification of specific antimicrobial drug-resistance mutations in human cytomegalovirus.
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Bogožalec Košir A, Cvelbar T, Kammel M, Grunert HP, Zeichhardt H, and Milavec M
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- Antiviral Agents pharmacology, Cytomegalovirus Infections virology, DNA, Viral genetics, Humans, Cytomegalovirus drug effects, Cytomegalovirus genetics, Drug Resistance, Viral genetics, Mutation, Real-Time Polymerase Chain Reaction methods
- Abstract
Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methods; e.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantification; and qPCR quantification is hampered by a lack of reference materials for standard curves. In recent years, digital PCR has been introduced, which provides absolute quantification without the need for reference materials for standard curves. Using digital PCR, we have developed a rapid, sensitive and accurate method for genotyping and quantification of the most prevalent mutations that cause human cytomegalovirus resistance to ganciclovir., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2020
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37. Results of cytomegalovirus DNA viral loads expressed in copies per millilitre and international units per millilitre are equivalent.
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Dimech W, Cabuang LM, Grunert HP, Lindig V, James V, Senechal B, Vincini GA, and Zeichhardt H
- Subjects
- DNA, Viral blood, Humans, World Health Organization, Cytomegalovirus, DNA, Viral analysis, Viral Load standards
- Abstract
Quantification of Cytomegalovirus (CMV) DNA is required for the initiation and monitoring of anti-viral treatment and the detection of viral resistance. However, due to the lack of standardisation of CMV DNA nucleic acid tests, it is difficult to set universal thresholds. In 2010, the 1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques was released. Since then CMV DNA viral load assays have been calibrated using this standard. Three external quality assessment (EQA) providers sent the same five samples to their participants and analysed the results to determine the equivalence of reporting CMV DNA results in international units per millilitre (IU/mL), and compared the difference in results reported in IU/mL with those reported in copies per millilitre (c/mL), and to determine the rate of adoption of IU/mL. About 78% of participants continue to report results in c/mL even though six of the 12 commercial assays are calibrated against the standard. The range of the results reported in IU/mL was less than those reported in c/mL indicating that the adoption of the WHO standard successfully improved the reporting of the CMV viral load. The variation in individual sample results reported by different assays, irrespective of whether in IU/mL or c/mL, is still great and therefore more standardisation of the assays is needed to allow the setting of treatment and monitoring thresholds. This study can act as a bench mark to determine rate of future adoption if reporting CMV DNA viral load results in IU/mL., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2018
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38. Soluble coxsackie- and adenovirus receptor (sCAR-Fc); a highly efficient compound against laboratory and clinical strains of coxsackie-B-virus.
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Pinkert S, Dieringer B, Diedrich S, Zeichhardt H, Kurreck J, and Fechner H
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- Coxsackie and Adenovirus Receptor-Like Membrane Protein genetics, Coxsackievirus Infections drug therapy, Coxsackievirus Infections virology, HeLa Cells, Humans, Immunoglobulin G pharmacology, Receptors, IgG, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins pharmacology, Solubility, Virus Replication drug effects, Antiviral Agents pharmacology, Coxsackie and Adenovirus Receptor-Like Membrane Protein chemistry, Coxsackie and Adenovirus Receptor-Like Membrane Protein pharmacology, Enterovirus B, Human drug effects, Immunoglobulin G genetics
- Abstract
Coxsackie-B-viruses (CVB) cause a wide variety of diseases, ranging from mild syndromes to life-threatening conditions such as pancreatitis, myocarditis, meningitis and encephalitis. Especially newborns and young infants develop severe diseases and long-term sequelae may occur among survivors. Due to lack of specific antiviral therapy the current treatment of CVB infection is limited to symptomatic treatment. Here we analyzed the antiviral activity of a soluble receptor fusion protein, containing the extracellular part of the coxsackievirus and adenovirus receptor (CAR) fused to the constant domain of the human IgG - sCAR-Fc - against laboratory and clinical CVB strains. We found a high overall antiviral activity of sCAR-Fc against various prototypic laboratory strains of CVB, with an inhibition of viral replication up to 3 orders of magnitude (99.9%) at a concentration of 2.5 μg/ml. These include isolates that are not dependent on CAR for infection and isolates that are resistant against pleconaril, the currently most promising anti-CVB therapeutic. A complete inhibition was observed using higher concentration of sCAR-Fc. Further analysis of 23 clinical CVB isolates revealed overall high antiviral efficiency (up to 99.99%) of sCAR-Fc. In accordance with previous data, our results confirm the strong antiviral activity of sCAR-Fc against laboratory CVB strains and demonstrate for the first time that sCAR-Fc is also highly efficient at neutralizing clinical CVB isolates. Importantly, during the sCAR-Fc inhibition experiments, no naturally occurring resistant mutants were observed., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
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39. Does proficiency testing improve the quality of hantavirus serodiagnostics? Experiences with INSTAND EQA schemes.
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Hofmann J, Grunert HP, Donoso-Mantke O, Zeichhardt H, and Kruger DH
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- Europe, Humans, Sensitivity and Specificity, Hantavirus Infections diagnosis, Health Services Research, Laboratory Proficiency Testing methods, Laboratory Proficiency Testing organization & administration, Serologic Tests methods
- Abstract
Hantavirus infections in Germany appear periodically with peak numbers every 2-3 years. The reported cases in the years 2007, 2010 and 2012 exceeded many times over those in the years in-between. In order to reveal faults of certain in vitro diagnostic assays (IVDs), to harmonize the performances of the individual assays and to improve the users' competence in interpreting the results, the National Consiliary Laboratory for Hantaviruses and INSTAND e.V. (Society for Promoting Quality Assurance in Medical Laboratories e.V.) established an external quality assessment (EQA) scheme for proficiency testing of hantavirus serodiagnostics. The first EQA scheme (pilot study) started in March 2009 with 58 participating laboratories from Germany and neighboring countries. Twice a year four serum samples were sent out to the participants to investigate whether the sample reflects an acute or past infection and to distinguish between infections with the hantavirus types Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV), both endemic in Central Europe. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied in the participating laboratories. An increasing number of laboratories participated, with a maximum of 92 in March 2014. When summarizing in total 2592 test results, the laboratories reached an overall specificity of 96.7% and a sensitivity of 95% in their detection of a hantavirus infection. A correct distinction between acute and past infections was forwarded in 90-96% of replies of laboratories. Exact serotyping (PUUV vs. DOBV) of the infection was reported in 81-96% of replies with the lowest accuracy for past DOBV infections; cross-reactivities between diagnostic antigens of the two viruses as well as persistent IgM titers in humans may interfere with exact testing. The EQAs revealed acceptable results for the serodiagnostic of hantavirus infection including serotyping but further improvement is still needed., (Copyright © 2015 Elsevier GmbH. All rights reserved.)
- Published
- 2015
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40. [Not Available].
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Rabenau HF, Bannert N, Berger A, Donoso Mantke O, Eberle J, Enders M, Fickenscher H, Grunert HP, Gürtler L, Heim A, Huzly D, Kaiser R, Korn K, Nick S, Kücherer C, Nübling M, Obermeier M, Panning M, and Zeichhardt H
- Published
- 2015
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41. Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells.
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Dutkiewicz M, Ojdowska A, Kuczynski J, Lindig V, Zeichhardt H, Kurreck J, and Ciesiołka J
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- 5' Untranslated Regions genetics, Base Sequence, Enterovirus B, Human drug effects, Enterovirus Infections genetics, Enterovirus Infections virology, Gene Silencing, HeLa Cells, Humans, Molecular Sequence Data, Nucleic Acid Conformation, Ribonuclease H genetics, Antiviral Agents pharmacology, Enterovirus B, Human genetics, Oligonucleotides, Antisense pharmacology, RNA, Small Interfering pharmacology, RNA, Viral genetics
- Abstract
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.
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- 2015
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42. Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses.
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Pavšič J, Devonshire AS, Parkes H, Schimmel H, Foy CA, Karczmarczyk M, Gutiérrez-Aguirre I, Honeyborne I, Huggett JF, McHugh TD, Milavec M, Zeichhardt H, and Žel J
- Subjects
- Bacterial Infections microbiology, Bacterial Load methods, Humans, Molecular Diagnostic Techniques methods, Reference Standards, Viral Load methods, Virus Diseases virology, Bacteria isolation & purification, Bacterial Load standards, Molecular Diagnostic Techniques standards, Viral Load standards, Viruses isolation & purification
- Abstract
Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
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- 2015
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43. Combination of RNA interference and virus receptor trap exerts additive antiviral activity in coxsackievirus B3-induced myocarditis in mice.
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Stein EA, Pinkert S, Becher PM, Geisler A, Zeichhardt H, Klopfleisch R, Poller W, Tschöpe C, Lassner D, Fechner H, and Kurreck J
- Subjects
- Animals, Antiviral Agents metabolism, Coxsackievirus Infections virology, Disease Models, Animal, Dose-Response Relationship, Drug, HeLa Cells, Humans, Male, Mice, Mice, Inbred BALB C, Myocarditis virology, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Receptors, Virus genetics, Receptors, Virus metabolism, Viral Load drug effects, Antiviral Agents pharmacology, Coxsackievirus Infections drug therapy, Enterovirus B, Human drug effects, Genetic Therapy methods, Myocarditis drug therapy, RNA Interference
- Abstract
Background: Coxsackievirus B3 (CVB3) is a major heart pathogen against which no therapy exists to date. The potential of a combination treatment consisting of a proteinaceous virus receptor trap and an RNA interference-based component to prevent CVB3-induced myocarditis was investigated., Methods and Results: A soluble variant of the extracellular domain of the coxsackievirus-adenovirus receptor (sCAR-Fc) was expressed from an adenoviral vector and 2 short hairpin RNAs (shRdRp2.4) directed against CVB3 were delivered by an adeno-associated virus (AAV) vector. Cell culture experiments revealed additive antiviral activity of the combined application. In a CVB3-induced mouse myocarditis model, both components applied individually significantly reduced inflammation and viral load in the heart. The combination exerted an additive antiviral effect and reduced heart pathology. Hemodynamic measurement revealed that infection with CVB3 resulted in impaired heart function, as illustrated by a drastically reduced cardiac output and impaired contractility and relaxation. Treatment with either sCAR-Fc or shRdRp2.4 significantly improved these parameters. Importantly, the combination of both components led to a further significant improvement of heart function., Conclusions: Combination of sCAR-Fc and shRdRp2.4 exerted additive effects and was significantly more effective than either of the single treatments in inhibiting CVB3-induced myocarditis and preventing cardiac dysfunction., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2015
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44. Adenine nucleotide translocase 1 expression affects enterovirus infection in human and murine hearts.
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Kühl U, Ebermann L, Lassner D, Klingel K, Klumpe I, Winter J, Zeichhardt H, Schultheiss HP, and Dörner A
- Subjects
- Adenine Nucleotide Translocator 1 biosynthesis, Animals, Disease Models, Animal, Disease Progression, Enterovirus Infections metabolism, Enterovirus Infections virology, Female, Humans, Male, Mice, Mice, Inbred C57BL, Middle Aged, Myocarditis metabolism, Myocarditis virology, Adenine Nucleotide Translocator 1 genetics, Enterovirus Infections genetics, Gene Expression Regulation, Myocarditis genetics, RNA genetics
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- 2014
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45. Protease-activated receptor-2 regulates the innate immune response to viral infection in a coxsackievirus B3-induced myocarditis.
- Author
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Weithauser A, Bobbert P, Antoniak S, Böhm A, Rauch BH, Klingel K, Savvatis K, Kroemer HK, Tschope C, Stroux A, Zeichhardt H, Poller W, Mackman N, Schultheiss HP, and Rauch U
- Subjects
- Animals, Antibodies, Viral analysis, Biopsy, Blotting, Western, Disease Models, Animal, Enterovirus Infections virology, Gene Expression Regulation, Immunohistochemistry, Male, Mice, Mice, Inbred C57BL, Myocarditis metabolism, Myocarditis virology, Myocardium pathology, RNA genetics, Real-Time Polymerase Chain Reaction, Receptor, PAR-2 genetics, Toll-Like Receptor 3 biosynthesis, Toll-Like Receptor 3 genetics, Enterovirus B, Human immunology, Enterovirus Infections immunology, Immunity, Innate, Myocarditis immunology, Myocardium enzymology, Receptor, PAR-2 biosynthesis
- Abstract
Objectives: This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)-induced myocarditis., Background: An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNβ)., Methods: To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 10(5) plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry., Results: Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNβ than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNβ expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNβ antibody. Overexpression of PAR2 reduced the basal IFNβ expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNβ expression and left ventricular ejection fraction., Conclusions: PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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46. Spiegelzymes: sequence specific hydrolysis of L-RNA with mirror image hammerhead ribozymes and DNAzymes.
- Author
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Wyszko E, Szymański M, Zeichhardt H, Müller F, Barciszewski J, and Erdmann VA
- Subjects
- Animals, Base Sequence, COS Cells, Chlorocebus aethiops, DNA, Catalytic blood, DNA, Catalytic chemistry, Deoxyribonucleases metabolism, Humans, Hydrolysis, Models, Molecular, Nucleic Acid Conformation, RNA chemistry, RNA Stability, RNA, Catalytic blood, RNA, Catalytic chemistry, Ribonucleases metabolism, DNA, Catalytic metabolism, RNA metabolism, RNA, Catalytic metabolism
- Abstract
In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.
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- 2013
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47. The mitochondrial respiratory chain has a critical role in the antiviral process in Coxsackievirus B3-induced myocarditis.
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Ebermann L, Wika S, Klumpe I, Hammer E, Klingel K, Lassner D, Völker U, Erben U, Zeichhardt H, Schultheiss HP, and Dörner A
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- Animals, Apoptosis, Disease Resistance, Electron Transport Complex I physiology, Heart virology, Male, Mice, Mice, Inbred C57BL, Oxidative Stress, Viral Load, Coxsackievirus Infections immunology, Electron Transport physiology, Enterovirus B, Human, Mitochondria, Heart physiology, Myocarditis immunology
- Abstract
Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5 ± 1.4 × 10(4) plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4 ± 0.8 × 10(7) p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.
- Published
- 2012
- Full Text
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48. Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line.
- Author
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Pinkert S, Klingel K, Lindig V, Dörner A, Zeichhardt H, Spiller OB, and Fechner H
- Subjects
- Animals, Cell Survival, Cells, Cultured, Coxsackievirus Infections pathology, Coxsackievirus Infections virology, Enterovirus B, Human pathogenicity, Humans, Male, Mice, Mice, Inbred BALB C, Rats, Virulence, Biological Evolution, Enterovirus B, Human genetics, Enterovirus B, Human growth & development, Myocytes, Cardiac virology
- Abstract
Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.
- Published
- 2011
- Full Text
- View/download PDF
49. Transactivation of human parvovirus B19 gene expression in endothelial cells by adenoviral helper functions.
- Author
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Pozzuto T, von Kietzell K, Bock T, Schmidt-Lucke C, Poller W, Zobel T, Lassner D, Zeichhardt H, Weger S, and Fechner H
- Subjects
- Adenovirus E1A Proteins metabolism, Adenovirus E4 Proteins metabolism, Cells, Cultured, Humans, Parvovirus B19, Human genetics, Transcription, Genetic, Adenoviruses, Human genetics, Endothelial Cells virology, Gene Expression Regulation, Viral, Parvovirus B19, Human physiology, Trans-Activators metabolism, Transcriptional Activation, Viral Proteins metabolism
- Abstract
Human parvovirus B19 (B19V) DNA is highly prevalent in endothelial cells lining up intramyocardial arterioles and postcapillary venules of patients with chronic myocarditis and cardiomyopathies. We addressed the question of a possible stimulation of B19V gene expression in endothelial cells by infection with adenoviruses. Adenovirus infection led to a strong augmentation of B19V structural and nonstructural proteins in individual endothelial cells infected with B19V or transfected with an infectious B19V genome. Transactivation was mostly mediated at the level of transcription and not due to adenovirus-mediated induction of second-strand synthesis from the single-stranded parvoviral genome. The main adenoviral functions required were E1A and E4orf6, which displayed synergistic effects. Furthermore, a limited B19V genome replication could be demonstrated in endothelial cells and adenovirus infection induced the appearance of putative dimeric replication intermediates. Thus the almost complete block in B19V gene expression seen in endothelial cells can be abrogated by infection with other viruses., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
50. Rapid construction of adeno-associated virus vectors expressing multiple short hairpin RNAs with high antiviral activity against echovirus 30.
- Author
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Rothe D, Wajant G, Grunert HP, Zeichhardt H, Fechner H, and Kurreck J
- Subjects
- Base Sequence, CD55 Antigens genetics, Cell Line, Tumor, Echovirus Infections therapy, Echovirus Infections virology, Enterovirus B, Human physiology, HEK293 Cells, HeLa Cells, Humans, RNA, Viral genetics, Transfection, Virus Replication, Dependovirus genetics, Enterovirus B, Human genetics, Genetic Vectors, RNA Interference, RNA, Small Interfering genetics
- Abstract
RNA interference has proven to be a powerful tool to inhibit viruses. For the prevention of viral escape, multiple short hairpin RNAs (shRNAs) will have to be employed. This article describes a rapid procedure for the generation of shRNA expression cassettes by parallel cloning as well as a simple strategy for the combination of selected units. After delivery of the shRNA expression cassettes with adeno-associated virus vectors, inhibition of echovirus 30 as well as silencing of an important cellular cofactor of virus replication were achieved. The procedure has the potential to be generally applicable for silencing of multiple endogenous targets or viruses.
- Published
- 2010
- Full Text
- View/download PDF
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