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Tulaeva I, Lehmann F, Goldmann N, Dubovets A, Trifonova D, Tulaev M, Cornelius C, Weber M, Focke-Tejkl M, Karaulov A, Henning R, Springer DN, Wiedermann U, Glebe D, Valenta R. The PreS-Based Recombinant Vaccine VVX001 Induces Hepatitis B Virus Neutralizing Antibodies in a Low-Responder to HBsAg-Based HBV Vaccines. Vaccines (Basel) 2024; 12:1123. [PMID: 39460290 PMCID: PMC11511130 DOI: 10.3390/vaccines12101123] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2024] [Revised: 09/20/2024] [Accepted: 09/23/2024] [Indexed: 10/28/2024] Open
Abstract
Background: Approximately 10-20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A-H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.
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Affiliation(s)
- Inna Tulaeva
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
- Laboratory of Immunopathology, Department of Clinical Immunology and Allergology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia
| | - Felix Lehmann
- Institute of Medical Virology, National Reference Center for Hepatitis B Viruses and Hepatitis D Viruses, German Center for Infection Research (DZIF), Partner Site Giessen-Marburg Langen, Justus Liebig University, 35390 Giessen, Germany; (F.L.)
| | - Nora Goldmann
- Institute of Medical Virology, National Reference Center for Hepatitis B Viruses and Hepatitis D Viruses, German Center for Infection Research (DZIF), Partner Site Giessen-Marburg Langen, Justus Liebig University, 35390 Giessen, Germany; (F.L.)
| | - Alexandra Dubovets
- Laboratory of Immunopathology, Department of Clinical Immunology and Allergology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia
| | - Daria Trifonova
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
- Laboratory of Immunopathology, Department of Clinical Immunology and Allergology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia
| | - Mikhail Tulaev
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
| | - Carolin Cornelius
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
| | - Milena Weber
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
| | - Margarete Focke-Tejkl
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
| | - Alexander Karaulov
- Laboratory of Immunopathology, Department of Clinical Immunology and Allergology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia
| | | | | | - Ursula Wiedermann
- Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Dieter Glebe
- Institute of Medical Virology, National Reference Center for Hepatitis B Viruses and Hepatitis D Viruses, German Center for Infection Research (DZIF), Partner Site Giessen-Marburg Langen, Justus Liebig University, 35390 Giessen, Germany; (F.L.)
| | - Rudolf Valenta
- Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; (I.T.)
- Laboratory of Immunopathology, Department of Clinical Immunology and Allergology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia
- NRC Institute of Immunology FMBA of Russia, 115552 Moscow, Russia
- Karl Landsteiner University of Health Sciences, 3500 Krems, Austria
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2
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IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection. Viruses 2022; 14:v14040727. [PMID: 35458456 PMCID: PMC9027621 DOI: 10.3390/v14040727] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Revised: 03/28/2022] [Accepted: 03/28/2022] [Indexed: 02/04/2023] Open
Abstract
The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.
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Teh CP, Chook JB, Ngeow YF, Tong TYK, Tee KK, Bong JJ, Mohamed R. Primer and probe conservation issue in the quantification of hepatitis B virus DNA. Rev Med Virol 2020. [DOI: 10.1002/rmv.2182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Affiliation(s)
- Chye Phing Teh
- Department of Biological Sciences School of Science and Technology Sunway University Petaling Jaya Selangor Malaysia
- Department of Medical Sciences School of Healthcare and Medical Sciences Sunway University Petaling Jaya Selangor Malaysia
| | - Jack Bee Chook
- Department of Medical Sciences School of Healthcare and Medical Sciences Sunway University Petaling Jaya Selangor Malaysia
| | - Yun Fong Ngeow
- Department of Pre‐Clinical Sciences Faculty of Medicine and Health Sciences Universiti Tunku Abdul Rahman Kajang Malaysia
| | - Tommy Yuh Koon Tong
- Department of Biological Sciences School of Science and Technology Sunway University Petaling Jaya Selangor Malaysia
| | - Kok Keng Tee
- Department of Medical Microbiology Faculty of Medicine University of Malaya Kuala Lumpur Malaysia
| | - Jan Jin Bong
- Sunway Medical Centre Petaling Jaya Selangor Malaysia
| | - Rosmawati Mohamed
- Department of Medicine Faculty of Medicine University of Malaya Kuala Lumpur Malaysia
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4
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Rasche A, Sander AL, Corman VM, Drexler JF. Evolutionary biology of human hepatitis viruses. J Hepatol 2019; 70:501-520. [PMID: 30472320 PMCID: PMC7114834 DOI: 10.1016/j.jhep.2018.11.010] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Revised: 11/09/2018] [Accepted: 11/10/2018] [Indexed: 02/06/2023]
Abstract
Hepatitis viruses are major threats to human health. During the last decade, highly diverse viruses related to human hepatitis viruses were found in animals other than primates. Herein, we describe both surprising conservation and striking differences of the unique biological properties and infection patterns of human hepatitis viruses and their animal homologues, including transmission routes, liver tropism, oncogenesis, chronicity, pathogenesis and envelopment. We discuss the potential for translation of newly discovered hepatitis viruses into preclinical animal models for drug testing, studies on pathogenesis and vaccine development. Finally, we re-evaluate the evolutionary origins of human hepatitis viruses and discuss the past and present zoonotic potential of their animal homologues.
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Affiliation(s)
- Andrea Rasche
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, 10117 Berlin, Germany,German Center for Infection Research (DZIF), Germany
| | - Anna-Lena Sander
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, 10117 Berlin, Germany
| | - Victor Max Corman
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, 10117 Berlin, Germany,German Center for Infection Research (DZIF), Germany
| | - Jan Felix Drexler
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, 10117 Berlin, Germany; German Center for Infection Research (DZIF), Germany.
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5
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Owusu M, Bonney JK, Annan AA, Mawuli G, Okyere K, Mutocheluh M, Aryeequaye J, Adjei NK, Afihene M, Spangenberg K, Sylverken J, Owusu-Dabo E, Drosten C, Adu-Sarkodie Y. Aetiology of viral hepatitis among jaundiced patients presenting to a tertiary hospital in Ghana. PLoS One 2018; 13:e0203699. [PMID: 30208084 PMCID: PMC6135398 DOI: 10.1371/journal.pone.0203699] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Accepted: 08/24/2018] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Viral hepatitis continues to play significant role in causing morbidity and mortality in sub-Saharan Africa. Apart from the few population based studies available, not many have investigated the burden of these viruses in jaundiced patients. Among the few studies, hepatitis E is the least studied among jaundiced patients. This study was aimed at describing the frequency, distribution and risk of the different hepatitis viruses among jaundiced patients reporting to the second largest teaching hospital in Ghana. METHODS From November, 2015 to April, 2016, a cross-sectional study was conducted among jaundiced patients attending the Komfo Anokye Teaching Hospital. Between 3-5 ml of blood was collected from each patient and screened for viral hepatitis agents using both serologic and molecular-based assays. RESULTS In the 155 patients recruited, hepatitis B was the most prevalent [54.2% (95% CI = 46.0%-62.2%)] followed by hepatitis E [32.9% (95% CI = 25.6-40.9%)]. Most cases of hepatitis E occurred as co-infections with hepatitis B (18%), with the predominant clinical feature being hepatocellular carcinoma. Risk factor variable analysis showed middle and older aged individuals were more at risk of hepatitis B exposure whereas younger age groups (<18 years) were more at risk of hepatitis E virus infection. CONCLUSION Hepatitis viruses are still important in the viral aetiology of jaundice in Ghana. Hepatitis B and hepatitis E co-infections could play significant roles in causing severe disease. A more aggressive approach needs to be adopted in order to reduce the morbidity and mortality associated with hepatitis causing viruses in Ghana and other developing countries.
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Affiliation(s)
- Michael Owusu
- Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
- * E-mail:
| | - Joseph Kofi Bonney
- Department of Virology, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
| | - Augustina Angelina Annan
- Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
- Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
| | - Gifty Mawuli
- Department of Virology, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
| | - Kennedy Okyere
- Department of Clinical Microbiology, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
| | - Mohamed Mutocheluh
- Department of Clinical Microbiology, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
| | - Juliana Aryeequaye
- Department of Virology, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
| | | | - Mary Afihene
- Department of Medicine, Komfo Anokye Teaching Hospital, Kumasi, Ghana
| | - Kathryn Spangenberg
- Department of Family Medicine, Komfo Anokye Teaching Hospital, Kumasi, Ghana
| | - Justice Sylverken
- Department of Child Health, Komfo Anokye Teaching Hospital, Kumasi, Ghana
| | - Ellis Owusu-Dabo
- Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
- Department of Global Health, School of Public Health,Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
| | - Christian Drosten
- Institute of Virology, Charité - Universitätsmedizin Berlin, Berlin, Germnany
| | - Yaw Adu-Sarkodie
- Department of Clinical Microbiology, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
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6
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Mühlemann B, Jones TC, Damgaard PDB, Allentoft ME, Shevnina I, Logvin A, Usmanova E, Panyushkina IP, Boldgiv B, Bazartseren T, Tashbaeva K, Merz V, Lau N, Smrčka V, Voyakin D, Kitov E, Epimakhov A, Pokutta D, Vicze M, Price TD, Moiseyev V, Hansen AJ, Orlando L, Rasmussen S, Sikora M, Vinner L, Osterhaus ADME, Smith DJ, Glebe D, Fouchier RAM, Drosten C, Sjögren KG, Kristiansen K, Willerslev E. Ancient hepatitis B viruses from the Bronze Age to the Medieval period. Nature 2018; 557:418-423. [PMID: 29743673 DOI: 10.1038/s41586-018-0097-z] [Citation(s) in RCA: 128] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2017] [Accepted: 04/06/2018] [Indexed: 12/16/2022]
Abstract
Hepatitis B virus (HBV) is a major cause of human hepatitis. There is considerable uncertainty about the timescale of its evolution and its association with humans. Here we present 12 full or partial ancient HBV genomes that are between approximately 0.8 and 4.5 thousand years old. The ancient sequences group either within or in a sister relationship with extant human or other ape HBV clades. Generally, the genome properties follow those of modern HBV. The root of the HBV tree is projected to between 8.6 and 20.9 thousand years ago, and we estimate a substitution rate of 8.04 × 10-6-1.51 × 10-5 nucleotide substitutions per site per year. In several cases, the geographical locations of the ancient genotypes do not match present-day distributions. Genotypes that today are typical of Africa and Asia, and a subgenotype from India, are shown to have an early Eurasian presence. The geographical and temporal patterns that we observe in ancient and modern HBV genotypes are compatible with well-documented human migrations during the Bronze and Iron Ages1,2. We provide evidence for the creation of HBV genotype A via recombination, and for a long-term association of modern HBV genotypes with humans, including the discovery of a human genotype that is now extinct. These data expose a complexity of HBV evolution that is not evident when considering modern sequences alone.
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Affiliation(s)
- Barbara Mühlemann
- Center for Pathogen Evolution, Department of Zoology, University of Cambridge, Cambridge, UK
| | - Terry C Jones
- Center for Pathogen Evolution, Department of Zoology, University of Cambridge, Cambridge, UK.,Institute of Virology, Charité, Universitätsmedizin Berlin, Berlin, Germany
| | | | - Morten E Allentoft
- Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark
| | - Irina Shevnina
- Archaeological Laboratory, Faculty of History and Law, A. A. Baitursynov Kostanay State University, Kostanay, Kazakhstan
| | - Andrey Logvin
- Archaeological Laboratory, Faculty of History and Law, A. A. Baitursynov Kostanay State University, Kostanay, Kazakhstan
| | - Emma Usmanova
- Saryarka Archaeological Institute, Karaganda State University, Karaganda, Kazakhstan
| | | | - Bazartseren Boldgiv
- Department of Biology, School of Arts and Sciences, National University of Mongolia, Ulaanbaatar, Mongolia
| | - Tsevel Bazartseren
- Laboratory of Virology, Institute of Veterinary Medicine, Mongolian University of Life Sciences, Ulaanbaatar, Mongolia
| | | | - Victor Merz
- Pavlodar State University, Pavlodar, Kazakhstan
| | - Nina Lau
- Centre for Baltic and Scandinavian Archaeology, Schleswig, Germany
| | - Václav Smrčka
- Institute for History of Medicine and Foreign Languages of the First Faculty of Medicine, Charles University, Prague, Czech Republic
| | | | - Egor Kitov
- N. N. Miklouho-Maklay Institute of Ethnology and Anthropology, Russian Academy of Sciences, Moscow, Russia
| | - Andrey Epimakhov
- South Ural Department, Institute of History and Archaeology UBRAS, South Ural State University, Chelyabinsk, Russia
| | - Dalia Pokutta
- Department of Archaeology and Classical Studies, Stockholm University, Stockholm, Sweden
| | | | - T Douglas Price
- Department of Historical Studies, University of Gothenburg, Gothenburg, Sweden
| | - Vyacheslav Moiseyev
- Department of Physical Anthropology, Peter the Great Museum of Anthropology and Ethnography, Saint-Petersburg, Russia
| | - Anders J Hansen
- Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark
| | - Ludovic Orlando
- Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark.,Laboratoire d'Anthropobiologie Moléculaire et d'Imagerie de Synthèse, CNRS UMR 5288, Université de Toulouse, Université Paul Sabatier, Toulouse, France
| | - Simon Rasmussen
- Department of Bio and Health Informatics, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Martin Sikora
- Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark
| | - Lasse Vinner
- Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark
| | - Albert D M E Osterhaus
- Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany
| | - Derek J Smith
- Center for Pathogen Evolution, Department of Zoology, University of Cambridge, Cambridge, UK
| | - Dieter Glebe
- Institute of Medical Virology, Justus Liebig University of Giessen, Giessen, Germany.,National Reference Centre for Hepatitis B and D Viruses, German Center for Infection Research (DZIF), Giessen, Germany
| | - Ron A M Fouchier
- Department of Viroscience, Erasmus Medical Centre, Rotterdam, The Netherlands
| | - Christian Drosten
- Institute of Virology, Charité, Universitätsmedizin Berlin, Berlin, Germany.,German Center for Infection Research (DZIF), Braunschweig, Germany
| | - Karl-Göran Sjögren
- Department of Historical Studies, University of Gothenburg, Gothenburg, Sweden
| | | | - Eske Willerslev
- Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark. .,Cambridge GeoGenetics Group, Department of Zoology, University of Cambridge, Cambridge, UK. .,Wellcome Trust Sanger Institute, Hinxton, UK.
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7
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Santos APDT, Levi JE, Lemos MF, Calux SJ, Oba IT, Moreira RC. An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA. Mem Inst Oswaldo Cruz 2016; 111:134-40. [PMID: 26872342 PMCID: PMC4750454 DOI: 10.1590/0074-02760150415] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2015] [Accepted: 01/15/2016] [Indexed: 12/14/2022] Open
Abstract
This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.
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Affiliation(s)
| | - José Eduardo Levi
- Laboratório de Virologia, Instituto de Medicina Tropical, Universidade de São Paulo, São Paulo, SP, Brasil
| | - Marcilio Figueiredo Lemos
- Núcleo de Doenças Sanguíneas e Sexuais, Centro de Virologia, Instituto Adolfo Lutz, São Paulo, SP, Brasil
| | - Samira Julien Calux
- Núcleo de Doenças Sanguíneas e Sexuais, Centro de Virologia, Instituto Adolfo Lutz, São Paulo, SP, Brasil
| | - Isabel Takano Oba
- Núcleo de Doenças Sanguíneas e Sexuais, Centro de Virologia, Instituto Adolfo Lutz, São Paulo, SP, Brasil
| | - Regina Célia Moreira
- Núcleo de Doenças Sanguíneas e Sexuais, Centro de Virologia, Instituto Adolfo Lutz, São Paulo, SP, Brasil
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8
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Wu X, Wang J, Song J, Li J, Yang Y. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification. J Virol Methods 2016; 235:152-157. [PMID: 27283884 DOI: 10.1016/j.jviromet.2016.06.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Revised: 06/04/2016] [Accepted: 06/06/2016] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.
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Affiliation(s)
- Xuping Wu
- The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University, Nanjing, China
| | - Jianfang Wang
- The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University, Nanjing, China
| | - Jinyun Song
- The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University, Nanjing, China
| | - Jiayan Li
- The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University, Nanjing, China
| | - Yongfeng Yang
- The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University, Nanjing, China.
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9
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Schmidt M, Jimenez A, Mühlbacher A, Oota S, Blanco L, Sakuldamrongpanich T, Schennach H, Seifried E. Head-to-head comparison between two screening systems for HBsAG, anti-HBc, anti-HCV and HIV combination immunoassays in an international, multicentre evaluation study. Vox Sang 2015; 109:114-21. [PMID: 25899479 DOI: 10.1111/vox.12259] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2014] [Revised: 01/19/2015] [Accepted: 01/19/2015] [Indexed: 01/05/2023]
Abstract
BACKGROUND Mandatory screening of blood donations for hepatitis B and hepatitis C viruses and human immunodeficiency viruses 1 and 2 requires assays with exceptional sensitivity and specificity. This study reports the results from a direct head-to-head comparison of the Elecsys HBsAG II, Elecsys Anti-HBc, Elecsys Anti-HCV II and Elecsys HIV combi PT immunoassays with the respective ABBOTT PRISM/Architect instrument immunoassays in a multicentre blood bank evaluation study. STUDY DESIGN AND METHODS Assay validation was performed in the blood screening laboratories of four blood bank centres in Austria, Germany, Spain and Thailand, where both first-time donor samples (approximately 6000 donors) and repeat donor samples (approximately 14,000 donors) were screened. RESULTS Of all screened donor samples, 93 (0.46%) were confirmed to be positive using assays from both manufacturers. The specificity of all immunoassays was >99.5% and was comparable between first-time and multiple-time donors. A direct comparison between the assays from Roche and ABBOTT according to Bland and Altman analysis demonstrated equivalent quality. CONCLUSIONS These results suggest that the Elecsys immunoassays for HBV, HCV and HIV infection, with a comparative sensitivity of 100% and a specificity exceeding the common technical specification threshold of >99.5%, meet the stringent performance criteria stipulated for blood donor screening for these infectious agents. Significant differences in the specificity between first-time and repeat donors were not detectable.
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Affiliation(s)
- M Schmidt
- DRK-Blutspendedienst Baden-Württemberg, Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt, Germany
| | - A Jimenez
- Centro de Hemoterapia y Hemodonación Castilla y León, Valladolid, Spain
| | - A Mühlbacher
- Zentralinstitut fuer Bluttransfusion und Immunologie Abteilung der Tilak Universitätsklinik LKH Innsbruck, Innsbruck, Austria
| | - S Oota
- National Blood Centre, Thai Red Cross Society, Bangkok, Thailand
| | - L Blanco
- Centro de Hemoterapia y Hemodonación Castilla y León, Valladolid, Spain
| | | | - H Schennach
- Zentralinstitut fuer Bluttransfusion und Immunologie Abteilung der Tilak Universitätsklinik LKH Innsbruck, Innsbruck, Austria
| | - E Seifried
- DRK-Blutspendedienst Baden-Württemberg, Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt, Germany
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Changotra H, Sehajpal PK. An improved method for the isolation of hepatitis B virus DNA from human serum. INDIAN JOURNAL OF VIROLOGY : AN OFFICIAL ORGAN OF INDIAN VIROLOGICAL SOCIETY 2013; 24:174-9. [PMID: 24426273 DOI: 10.1007/s13337-013-0155-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Accepted: 07/26/2013] [Indexed: 10/26/2022]
Abstract
Studies show that hepatitis B virus (HBV) DNA isolation methods vary in their efficiency to extract DNA from serum samples. The purpose of the present study was to develop an improved method for isolation of HBV DNA and compare it with commonly used HBV DNA isolation protocols. In order to develop HBV DNA isolation protocol, serum samples were collected from patients and screened for the presence of hepatitis B surface antigen, hepatitis B e antigen and HBV DNA. Highly viremic samples were pooled and used to compare commonly used HBV DNA isolation methods; namely alkaline lysis, microwave treatment, organic, inorganic with modified inorganic method. DNA isolated by these methods was detected qualitatively by polymerase chain reaction and quantitatively with competitive polymerase chain reaction (cPCR). The modified inorganic method gave maximum yield of HBV DNA followed by inorganic, organic, microwave treatment and alkaline lysis method. Our data also demonstrated a critical role of proteinase K in HBV DNA isolation. DNA isolation method described here, in combination with a reproducible and sensitive quantitative technique would further help in accurate classification of HBV infected patients, designing suitable drug regimen for treatment and monitoring antiviral treatment as well as emergence of drug resistant mutants.
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Affiliation(s)
- Harish Changotra
- Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, 173234 Himachal Pradesh India
| | - Prabodh K Sehajpal
- Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, 143005 India
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11
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Xu J, Cao L, Su L, Dong N, Yu M, Ju J. A new accurate assay for Coxsackievirus A 16 by fluorescence detection of isothermal RNA amplification. J Virol Methods 2013; 193:459-62. [PMID: 23872269 DOI: 10.1016/j.jviromet.2013.07.013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2012] [Revised: 06/30/2013] [Accepted: 07/10/2013] [Indexed: 11/24/2022]
Abstract
Coxsackievirus A 16 (CA16) is one of the most common causes of hand, foot, and mouth disease (HFMD) worldwide. Without a vaccine or antiviral drug early, rapid, and accurate detection is critical for preventing and controlling HFMD. A simultaneous amplification and testing (SAT) assay was developed for detecting CA16 based on isothermal RNA amplification with fluorescence using standard, real-time PCR equipment. Primers and probes were designed to target the VP1 region of CA16. Virus strains and clinical specimens were used to evaluate the diagnostic performance characteristics of the assay. The assay detected as few as 10 copies of CA16 RNA transcripts. Using real-time PCR plus sequencing as the reference standard, the sensitivity and specificity of the SAT-CA16 assay were 100% and 99.2%, respectively. These findings indicate that SAT-CA16 is a rapid and reliable method for detecting CA16.
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Affiliation(s)
- Jin Xu
- Laboratory Medicine Center, Children's Hospital of Fudan University, Shanghai 201102, China.
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12
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Abstract
Serologic testing for hepatitis B virus (HBV) surface antigen (HBsAg) and antibody to HBV core antigen (anti-HBc) has historically been the foundation of blood screening, while HBV nucleic acid testing (NAT) was recently developed to detect HBsAg-negative, anti-HBc-negative blood units donated during early acute infection. Comparison data on seroconversion panels using HBsAg assays of varying sensitivities and pooled- or single-sample NAT, along with viral load estimates corresponding to HBsAg assay detection limits, have provided information on the theoretical benefits of NAT relative to HBsAg. Model-derived estimates have generally been predictive of the yields of DNA-positive, HBsAg-negative window period blood units detected in a number of studies from Europe, Japan, and the US. Studies indicate that the added benefit of pooled-sample NAT is relatively small in areas of low endemicity, with greater yields in areas highly endemic for HBV. Single-sample NAT would offer more significant early window period closure and could prevent a moderate number of residual HBV transmissions not detected by HBsAg assays; however, no fully automated single-sample HBV NAT systems are currently available.Even single-sample HBV NAT may not substitute for anti-HBc screening, as indicated by studies of donors with isolated anti-HBc who have extremely low DNA levels undetectable by standard single-sample NAT and who have been associated with transfusion-transmitted HBV. Moreover, HBsAg testing may still be needed even in the setting of combined anti-HBc and NAT screening. HBsAg-positive units from donors in the chronic stage of infection may contain very low or intermittently detectable DNA levels that single-sample NAT would miss. Although such donors are usually anti-HBc reactive and would be interdicted by anti-HBc screening, some lack anti-HBc. Extensive parallel testing will be needed to determine whether single-sample NAT in combination with anti-HBc might be sufficient to detect all the infectious donors currently interdicted by HBsAg testing. In countries that do not screen for anti-HBc, HBsAg testing would be the only means of detecting donations from chronically infected individuals with low/intermittently detectable DNA, since even single-donor NAT would not identify these potentially infectious blood units. In the future, the current fully automated HBsAg assays may incorporate significant sensitivity improvements, and automated single-sample HBV NAT may become a reality. Each country will need to develop its blood screening strategy based on HBV endemicity, yields of infectious units detected by different serologic/NAT screening methods, and cost effectiveness of test methods in ensuring blood safety.
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Affiliation(s)
- Mary C Kuhns
- Abbott Diagnostics, Abbott Park, Illinois 60064, USA.
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Jothikumar P, Hill V, Narayanan J. Design of FRET-TaqMan probes for multiplex real-time PCR using an internal positive control. Biotechniques 2009; 46:519-24. [DOI: 10.2144/000113127] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
The multiplexing capabilities with different fluorescent dyes are limited in real-time PCR instruments equipped with one excitation source. Considering this limitation, a design was developed to create a triple-labeled probe as an internal positive control (IPC) that utilizes a combination of the fluorescence resonance energy transfer (FRET) and TaqMan techniques. The IPC probe, labeled with FAM and Cy5.5 fluorophores at the 5′ end and Black Hole Quencher (BHQ) at the 3′ end, enabled Cy5.5 emission through energy transfer from the FAM fluorophore. The second, target-specific TaqMan assay in the multiplex used a FAM- and BHQ1-labeled probe at the 5′ and 3′ ends, respectively. Thus, one excitation source was used to generate two different fluorescence emissions (FAM and Cy5.5) that were measured in two separate channels by the real-time PCR instrument. This method can facilitate the development of a low-cost portable handheld real-time PCR instrument capable of multiplex real-time PCR assays using a single excitation source.
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Affiliation(s)
| | - Vincent Hill
- Centers for Disease Control and Prevention, National Center for Zoonotic, Vector-borne, and Enteric Diseases, Division of Parasitic Diseases, Atlanta, GA, USA
| | - Jothikumar Narayanan
- Centers for Disease Control and Prevention, National Center for Zoonotic, Vector-borne, and Enteric Diseases, Division of Parasitic Diseases, Atlanta, GA, USA
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14
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Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, Drosten C. Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma. PLoS Negl Trop Dis 2009; 3:e422. [PMID: 19381285 PMCID: PMC2667260 DOI: 10.1371/journal.pntd.0000422] [Citation(s) in RCA: 149] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2008] [Accepted: 03/26/2009] [Indexed: 01/19/2023] Open
Abstract
Introduction Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR. Methodology/Principal Findings Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year. Conclusions/Significance PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring. Bilharzia (schistosomiasis) occurs in the tropics and subtropics and is one of the most important parasite diseases of humans. It is caused by flukes residing in the vessels of the gut or bladder, causing fever, pain, and bleeding. Bladder cancer or esophageal varices may follow. Diagnosis is difficult, requiring detection of parasite eggs in stool, urine, or gut/bladder biopsies. In this paper, we introduce a fundamentally new way of diagnosing bilharzia from the blood. It has been known for almost 20 years that patients with cancer have tumor-derived DNA circulating in their blood, which can be used for diagnostic purposes. During pregnancy, free DNA from the fetus can be detected in motherly blood, which can be used for diagnosing a range of fetal diseases and pregnancy-associated complications. We found that parasite DNA can be detected in the same way in the blood of patients with bilharzia. In patients with early disease, diagnosis was possible earlier than with any other test. DNA could be detected in all patients with active disease in our study. Patients after treatment had significantly lower parasite DNA concentrations and turned negative 1–2 years after treatment. Future studies should implement the method in large cohorts of patients and should define criteria for the confirmation of the success of treatment by comparing the concentration of fluke DNA before and after therapy.
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Affiliation(s)
- Dominic Wichmann
- Sektion Infektiologie und Tropenmedizin, I. Medizinische Klinik, Universitätsklinikum Eppendorf, Hamburg, Germany
| | - Marcus Panning
- Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
| | - Thomas Quack
- Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University, Giessen, Germany
| | - Stefanie Kramme
- Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
| | - Gerd-Dieter Burchard
- Sektion Infektiologie und Tropenmedizin, I. Medizinische Klinik, Universitätsklinikum Eppendorf, Hamburg, Germany
| | - Christoph Grevelding
- Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University, Giessen, Germany
| | - Christian Drosten
- Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
- * E-mail:
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15
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Low-density macroarray for rapid detection and identification of Crimean-Congo hemorrhagic fever virus. J Clin Microbiol 2009; 47:1025-30. [PMID: 19225100 DOI: 10.1128/jcm.01920-08] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis which occurs throughout Africa, Eastern Europe, and Asia and results in an approximately 30% fatality rate. A reverse transcription-PCR assay including a competitive internal control was developed on the basis of the most up-to-date genome information. Biotinylated amplification products were hybridized to DNA macroarrays on the surfaces of polymer supports, and hybridization events were visualized by incubation with a streptavidin-horseradish peroxidase conjugate and the formation of a visible substrate precipitate. Optimal assay conditions for the detection of as few as 6.3 genome copies per reaction were established. Eighteen geographically and historically diverse CCHF virus strains representing all clinically relevant isolates were detected. The feasibility of the assay for clinical diagnosis was validated with acute-phase patient samples from South Africa, Iran, and Pakistan. The assay provides a specific, sensitive, and rapid method for CCHF virus detection without requiring sophisticated equipment. It has usefulness for the clinical diagnosis and surveillance of CCHF infections under limited laboratory conditions in developing countries or in field situations.
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16
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Orientia tsutsugamushi bacteremia and cytokine levels in Vietnamese scrub typhus patients. J Clin Microbiol 2009; 47:586-9. [PMID: 19144812 DOI: 10.1128/jcm.00997-08] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Scrub typhus, caused by the intracellular bacterium Orientia tsutsugamushi, is a major cause of febrile illness in the Asia/Pacific region. Here, we implemented a novel real-time PCR and determined the relation of DNA target gene concentration with serum cytokine levels. The limit of detection of the novel real-time PCR was 1,062 DNA copies per ml of EDTA whole blood. Specificity was excellent as determined on a panel of blood- and skin-borne bacteria, including Rickettsia spp. as well as healthy Vietnamese blood donors. Bacterial DNA concentrations after 9 to 12 days from symptoms onset were significantly higher than in earlier or later periods (P<0.05). Significantly higher concentrations of gamma interferon (IFN-gamma) and interleukin-10 (IL-10) occurred during the acute phase of disease (<10 days from onset) as opposed to the convalescent phase (P<0.05). No significant differences were observed between the acute and the convalescent phases for tumor necrosis factor alpha (TNF-alpha) and IL-1beta concentrations. Regression analysis of DNA concentrations and cytokine levels identified a significant positive relationship for IL-10 (P<0.0182) but not for IFN-gamma, TNF-alpha, and IL-1beta. In conclusion, proinflammatory cytokines and IL-10 were differentially related to human bacteremia. They may thus be induced by different constituents of O. tsutsugamushi. As a future prospect in a clinical diagnostic laboratory, quantitative real-time PCR may serve as a reliable tool to monitor therapy and to detect treatment failure.
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17
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Bilder CR, Tebbs JM. Bias, efficiency, and agreement for group-testing regression models. J STAT COMPUT SIM 2009; 79:67-80. [PMID: 20046956 DOI: 10.1080/00949650701608990] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Group testing involves pooling individual items together and testing them simultaneously for a rare binary trait. Whether the goal is to estimate the prevalence of the trait or to identify those individuals that possess it, group testing can provide substantial benefits when compared to testing subjects individually. Recently, group-testing regression models have been proposed as a way to incorporate covariates when estimating trait prevalence. In this paper, we examine these models by comparing fits obtained from individual and group testing samples. Relative bias and efficiency measures are used to assess the accuracy and precision of the resulting estimates using different grouping strategies. We also investigate the agreement of individual and group-testing regression estimates for various grouping strategies and the effects of group size selection. Depending on how groups are formed, our results show that group-testing regression models can perform very well when compared to the analogous models based on individual observations. However, different grouping strategies can provide very different results in finite samples.
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18
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Diagnostics of tick-borne rickettsioses in Germany: A modern concept for a neglected disease. Int J Med Microbiol 2008. [DOI: 10.1016/j.ijmm.2007.11.009] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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19
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Hourfar MK, Walch LA, Geusendam G, Dengler T, Janetzko K, Gubbe K, Frank K, Karl A, Löhr M, Sireis W, Seifried E, Schmidt M. Sensitivity and specificity of Anti-HBc screening assays--which assay is best for blood donor screening? Int J Lab Hematol 2008; 31:649-56. [PMID: 18673399 DOI: 10.1111/j.1751-553x.2008.01092.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMérieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.
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Affiliation(s)
- M K Hourfar
- Institute of Transfusion Medicine and Immunohematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany
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Panning M, Laue T, Olschlager S, Eickmann M, Becker S, Raith S, Courbot MCG, Nilsson M, Gopal R, Lundkvist A, di Caro A, Brown D, Meyer H, Lloyd G, Kummerer BM, Gunther S, Drosten C. Diagnostic reverse-transcription polymerase chain reaction kit for filoviruses based on the strain collections of all European biosafety level 4 laboratories. J Infect Dis 2008; 196 Suppl 2:S199-204. [PMID: 17940950 PMCID: PMC7109892 DOI: 10.1086/520600] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
A network of European biosafety level 4 laboratories has designed the first industry-standard molecular assay for all filoviruses species, based on the strain collections of all participants. It uses 5 optimized L gene primers and 3 probes, as well as an internal control with a separate detection probe. Detection limits (probit analysis, 95% detection chance) were as follows: Zaire ebolavirus, 487 copies/mL of plasma; Sudan ebolavirus Maleo, 586 copies/mL; Sudan ebolavirus Gulu, 1128 copies/mL; Cote d'Ivoire ebolavirus, 537 copies/mL; Reston ebolavirus, 4546 copies/mL; Lake Victoria marburgvirus Musoke, 860 copies/mL; and Lake Victoria marburgvirus Ravn, 1551 copies/mL. The assay facilitates reliable detection or exclusion screening of filovirus infections.
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Affiliation(s)
- Marcus Panning
- Bernhard-Nocht Institute for Tropical Medicine, Hamburg 20359, Germany
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Panning M, Kramme S, Petersen N, Drosten C. High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation. Med Microbiol Immunol 2006; 196:41-50. [PMID: 17093976 DOI: 10.1007/s00430-006-0029-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2006] [Indexed: 10/23/2022]
Abstract
Human infections with Bacillus anthracis have become rare but in cases of intentional release, masses of samples would have to be expected. Current PCR assays for anthrax are appropriate for use in single cases, but they have not been formulated for high throughput screening. This article describes a high throughput real-time PCR for anthrax, including automated sample preparation without the need for pre-culturing of samples. The assay detects single copies of target gene. An internal control monitors the whole assay including sample preparation. The limit of detection in blood was 1,066 (95%CI, 741-1,739) copies/ml, corresponding to 4.4-32.3 organisms/ml. Using spore preparations, 20 colony-forming units (CFU) per sample could be detected reliably (0.8 CFU per PCR). The extraction procedures depleted viable spores from solution by factors of 10,000 (automated procedure) and >100,000 (conventional column procedure). One hundred and ten clinical and environmental specimens were retested, 50 of them sampled during a period of heightened anthrax awareness in 2001. A widely used assay yielded two false positive results (cross-reaction with B. cereus), while the new assay tested all samples negative. The internal control operated stable in all clinical samples. The assay is capable of testing for anthrax in the high throughput mode.
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Affiliation(s)
- Marcus Panning
- Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht Str. 74, 20359 Hamburg, Germany
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22
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Schmidt M, Nübling CM, Scheiblauer H, Chudy M, Walch LA, Seifried E, Roth WK, Hourfar MK. Anti-HBc screening of blood donors: a comparison of nine anti-HBc tests. Vox Sang 2006; 91:237-43. [PMID: 16958836 DOI: 10.1111/j.1423-0410.2006.00818.x] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
BACKGROUND AND OBJECTIVES Since voluntary introduction of hepatitis B virus (HBV) minipool nucleic acid amplification technology (NAT) at the German Red Cross, the expected residual risk of a transfusion-associated HBV infection has been estimated to be 1 : 500,000 - about 10 times higher than for human immunodeficiency virus (HIV) or hepatitis C virus (HCV) infection. Donors demonstrating chronic positivity for antibody to hepatitis B core antigen (anti-HBc), negativity for hepatitis B surface antigen (HBsAg) and polymerase chain reaction (PCR)-negative with a low virus load are a major cause of this increased risk. MATERIALS AND METHODS Ten-thousand blood donors from our blood-donation centre were screened for anti-HBc using the current PRISM HBc and the new PRISM HBcore assay to evaluate the diagnostic sensitivity and specificity of these tests. PRISM HBc- or PRISM HBcore-reactive samples were further analysed using seven additional tests for anti-HBc, two tests for antibody to hepatitis B surface antigen (anti-HBs), one test for antibody to hepatitis B envelope antigen (anti-HBe) and three HBV NAT assays. RESULTS From a total of 10,000 donors, nine and 14 samples were reactive only in the PRISM HBc and the PRISM HBcore, respectively, whereas 165 samples were reactive in both anti-HBc assays. Further analysis of these 188 anti-HBc-reactive specimens in a total of nine different anti-HBc assays revealed concordant results for 162 (86.2%) specimens. Sample cut-off values for anti-HBc were significantly (P < 0.01) lower for anti-HBc-only reactive samples compared with specimens that were also reactive for anti-HBs or anti-HBe. CONCLUSIONS Both PRISM anti-HBc assays revealed that approximately 1.8% of non-prescreened blood donors from Germany were reactive for anti-HBc. Although sensitivity was comparable between both assays, specificity was increased significantly with the PRISM HBcore. High anti-HBc sample cut-off values were indicative for reactivity in other HBV parameters and for concordant results in the nine different anti-HBc assays. Look-back investigations are necessary to estimate the infection risk both of anti-HBc-only positive and of anti-HBc/anti-HBs-positive blood transfusions.
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Affiliation(s)
- M Schmidt
- Institute of Transfusion Medicine and Immunohematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany.
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Drosten C, Panning M, Drexler JF, Hänsel F, Pedroso C, Yeats J, de Souza Luna LK, Samuel M, Liedigk B, Lippert U, Stürmer M, Doerr HW, Brites C, Preiser W. Ultrasensitive monitoring of HIV-1 viral load by a low-cost real-time reverse transcription-PCR assay with internal control for the 5' long terminal repeat domain. Clin Chem 2006; 52:1258-66. [PMID: 16627558 PMCID: PMC7108179 DOI: 10.1373/clinchem.2006.066498] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
BACKGROUND Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. METHODS We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n = 1186), South Africa (n = 130), India (n = 44), and Germany (n = 127). RESULTS The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (> 95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n = 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n = 431 samples), 51.7% vs 65% positives; Amplicor Ultrasensitive vs LTR (n = 133), 81.2% vs 82.7%; BioMerieux NucliSens HIV-1 QT (n = 453), 60.5% vs 65.1%; Bayer Versant 3.0 (n = 433), 57.7% vs 55.4%; total (n = 1450), 59.0% vs 63.8% positives. Intra-/interassay variability at medium and near-negative concentrations was 18%-51%. The quantification range was 50-10,000,000 IU/mL. Viral loads for subtypes A-D, F-J, AE, and AG yielded mean differences of 0.31 log(10) compared with Amplicor in the 10(3)-10(4) IU/mL range. HIV-1 N and O were not detected by Amplicor, but yielded up to 180 180.00 IU/mL in the LTR assay. Viral loads in stored samples from all countries, compared with Amplicor, NucliSens, or Versant, yielded regression line slopes (SD) of 0.9 (0.13) (P < 0.001 for all). CONCLUSIONS This method offers all features of commercial assays and covers all relevant genotypes. It could allow general monitoring of antiretroviral therapy in resource-limited settings.
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Ameen R, Sanad N, Al-Shemmari S, Siddique I, Chowdhury RI, Al-Hamdan S, Al-Bashir A. Prevalence of viral markers among first-time Arab blood donors in Kuwait. Transfusion 2006; 45:1973-80. [PMID: 16371052 DOI: 10.1111/j.1537-2995.2005.00635.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND The aim of this study was to assess the effect of blood donation modes on the prevalence of viral markers among Arab first-time blood donors in Kuwait. STUDY DESIGN AND METHODS Donor ethnic background was classified as Kuwaiti nationals and non-Kuwaiti Arabs. A total of 26,874 donors were screened in 2002 for the following viral markers: hepatitis C virus antibody (anti-HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), human immunodeficiency virus-1 and -2 antibody (anti-HIV-1 and -2), HIV p24, and human T lymphotropic virus-I and -II antibody (anti-HTLVI/II). All samples positive for the presence of anti-HBc were tested for anti-HBs. Among these donors, 12,798 were first-time donors of which 74 percent were replacement and 26 percent were volunteers. RESULTS The prevalence of HCV among replacement donors was significantly higher than the volunteer group. The difference between the two modes of blood donations, however, was not significant for HBsAg. The prevalence of anti-HCV among Kuwaiti national and non-Kuwaiti Arab first-time donors was 0.8 and 5.4 percent, respectively, whereas the prevalence of HBsAg was 1.1 and 3.5 percent, respectively, with the difference being significant at a p level of <0.0001. The difference observed for prevalence of anti-HBc among Kuwaiti national and non-Kuwaiti Arab donors (17 and 33.3%, respectively) was significant (p < 0.0001). Among first-time donors, 13.7 percent were positive for the presence of anti-HBs, indicating that 13.7 percent of the total Arab donor population might have had a previous infection and possible immunity to hepatitis B virus (HBV). CONCLUSION A high prevalence of HBV and HCV was found among non-Kuwaiti Arab donors. The prevalence of anti-HCV was only significantly higher among replacement versus volunteer first-time donors. Therefore, there is a need to develop a strategic plan that incorporates the diverse background of the blood donors living in Kuwait.
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Affiliation(s)
- Reem Ameen
- Medical Laboratory Sciences Department and the Health Information and Administration Department, Faculty of Allied Health Sciences, and the Medicine Department, Faculty of Medicine, Kuwait University, Kuwait.
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Mueller J, Gessner M, Remberg A, Hoch J, Zerlauth G, Hanfland P. Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA. Clin Chem Lab Med 2005; 43:827-33. [PMID: 16201892 DOI: 10.1515/cclm.2005.139] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.
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Affiliation(s)
- Jens Mueller
- Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany.
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Hourfar MK, Schmidt M, Seifried E, Roth WK. Evaluation of an automated high-volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment. Vox Sang 2005; 89:71-6. [PMID: 16101686 DOI: 10.1111/j.1423-0410.2005.00649.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
BACKGROUND AND OBJECTIVES Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. MATERIALS AND METHODS The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9.6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. RESULTS Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9.6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. CONCLUSIONS The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study.
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Affiliation(s)
- M K Hourfar
- Institute of Transfusion Medicine and Immunohaematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany
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Heitmann A, Laue T, Schottstedt V, Dotzauer A, Pichl L. Occurrence of hepatitis A virus genotype III in Germany requires the adaptation of commercially available diagnostic test systems. Transfusion 2005; 45:1097-105. [PMID: 15987353 DOI: 10.1111/j.1537-2995.2005.04372.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
BACKGROUND A blood donation, obtained in 2003 in Germany during the preseroconversion diagnostic window period of a hepatitis A virus (HAV) infection, tested HAV-negative by commercially available HAV reverse transcription-polymerase chain reaction (RT-PCR) detection assays. STUDY DESIGN AND METHODS The virus responsible for this infection was identified as HAV genotype IIIA by characterization of the nearly complete genome sequence. RESULTS Thereby, this HAV variant, which was named strain HMH, was detected in Germany for the first time. Because the commercially available HAV RNA detection systems failed to detect this genotype, a real-time RT-PCR kit was developed that allows quantification and detection of all HAV genotypes. The first nearly full-length nucleotide sequence so far available for HAV genotype IIIA is also provided. CONCLUSION This case demonstrates that owing to the genetic variability of HAV, constant monitoring and adaptation of the diagnostic nucleic acid assays are required to guarantee the safety of blood and blood products.
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Burggraf S, Reischl U, Malik N, Bollwein M, Naumann L, Olgemöller B. Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay. J Clin Microbiol 2005; 43:1564-9. [PMID: 15814966 PMCID: PMC1081397 DOI: 10.1128/jcm.43.4.1564-1569.2005] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.
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Affiliation(s)
- Siegfried Burggraf
- Labor Becker, Olgemöller und Kollegen, Führichstrasse 70, D-81671 München, Germany.
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Bennett RS, Nezworski J, Velayudhan BT, Nagaraja KV, Zeman DH, Dyer N, Graham T, Lauer DC, Njenga MK, Halvorson DA. Evidence of avian pneumovirus spread beyond Minnesota among wild and domestic birds in central North America. Avian Dis 2005; 48:902-8. [PMID: 15666873 DOI: 10.1637/7208-051804r] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
To detect avian pneumovirus (APV) in central North America, nasal turbinates or choanal deft tissues from domestic turkeys and wild birds were examined for the presence of APV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas serum samples from domestic turkeys were analyzed for APV antibodies by enzyme-linked immunosorbent assay (ELISA). In 2002, the seroprevalence of disease in domestic turkeys in Minnesota remained high (42.3% of the flocks). In addition, there is evidence the disease has spread to turkey flocks in North Dakota (8.2%), South Dakota (7%), Iowa (10%), and Wisconsin (8.6%) as detected by RT-PCR and/or ELISA. House sparrows and ring-billed gulls sampled in Minnesota and snow geese from Saskatchewan, Canada, were found to harbor APV RNA. Sequence analysis of wild bird APV strains showed high amino acid sequence identity among wild bird isolates (<97%) and between wild bird and turkey viral isolates (93.2%-99.3%). This study demonstrated that APV infections were present in domestic turkey flocks and wild birds outside the state of Minnesota; however, the role of wild birds in spreading APV to domestic turkeys remains unclear.
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Affiliation(s)
- R S Bennett
- Department of Veterinary and Biomedical Sciences, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA
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Schmidt M, Roth WK, Meyer H, Seifried E, Hourfar MK. Nucleic acid test screening of blood donors for orthopoxviruses can potentially prevent dispersion of viral agents in case of bioterrorism. Transfusion 2005; 45:399-403. [PMID: 15752158 DOI: 10.1111/j.1537-2995.2005.04242.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
BACKGROUND Microbiologic agents such as variola virus (VAR) are very attractive for terrorism. As a result of international collaboration under the WHO eradication campaign, smallpox was declared eradicated in 1980. Therefore, the immunization programs were discontinued worldwide. Because most people are now immunologically naive, VAR is considered to be a potential threat agent or bioterrorist weapon. Real-time polymerase chain reaction (PCR) followed by melting analysis was developed for fast and safe analysis and allows differentiation of VAR from other orthopoxviruses (OPVs) like vaccinia or camelpox virus. STUDY DESIGN AND METHODS A RealArt Orthopox LC PCR kit (Artus GmbH) was used to amplify OPV sequences from blood donor samples. A total of 31,500 blood donor samples were tested in minipools of up to 96 samples. To evaluate the sensitivity of the assay, routine donor minipools (90 +/- 6 samples per pool) were spiked with vaccinia virus used as positive control. RESULTS Specificity was 100 percent because none of 31,500 blood donors was positive for the presence OPV. The detection limit of the assay was 10.6 copies per PCR procedure. Therefore, a sensitivity of 1590 copies per mL was calculated. Overall, 0.28 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION The RealArt Orthopox LC PCR kit enables reliable detection of OPV DNA in viremic blood donor samples, even at the beginning of the disease when patients present minor clinical symptoms, and could be implemented in our routine screening procedure immediately. Thus, the assay could potentially help to prevent dispersion of viral agents by blood transfusion in case of bioterrorism.
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Affiliation(s)
- Michael Schmidt
- Institute of Transfusion Medicine and Immunohematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany.
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Chen YX, Huang AL, Qi ZY, Guo SH. Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA. World J Gastroenterol 2004; 10:2666-9. [PMID: 15309716 PMCID: PMC4572190 DOI: 10.3748/wjg.v10.i18.2666] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR).
METHODS: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography, and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigenin-labeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigenin-labeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed.
RESULTS: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay. After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis. Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle. The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P < 0.01) between fluorescent qPCR and dot-blot hybridization.
CONCLUSION: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.
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MESH Headings
- Alkaline Phosphatase
- Animals
- DNA Probes
- DNA, Viral/analysis
- DNA, Viral/blood
- Digoxigenin
- Ducks
- Hepadnaviridae Infections/blood
- Hepadnaviridae Infections/diagnosis
- Hepadnaviridae Infections/virology
- Hepatitis B Virus, Duck/genetics
- Hepatitis B Virus, Duck/isolation & purification
- Hepatitis, Viral, Animal/blood
- Hepatitis, Viral, Animal/diagnosis
- Hepatitis, Viral, Animal/virology
- Polymerase Chain Reaction
- Sensitivity and Specificity
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Affiliation(s)
- Ya-Xi Chen
- Institute of Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing, 400010, China.
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Panning M, Asper M, Kramme S, Schmitz H, Drosten C. Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay. Clin Chem 2004; 50:702-8. [PMID: 14962998 DOI: 10.1373/clinchem.2003.026781] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Abstract
Background: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes.
Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction.
Results: Strain differentiation results showed little interassay variability (CV, 0.4–0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with ≥95% probability (235–849 genome copies/mL of plasma).
Conclusions: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.
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Affiliation(s)
- Marcus Panning
- Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
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Abstract
BACKGROUND In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. METHODS Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe-control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. RESULTS To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. CONCLUSIONS A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.
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Affiliation(s)
- Siegfried Burggraf
- Labor Becker, Olgemöller und Kollegen, Führichstrasse 70, 81671 München, Germany.
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Drosten C, Nippraschk T, Manegold C, Meisel H, Brixner V, Roth WK, Apedjinou A, Günther S. Prevalence of hepatitis B virus DNA in anti-HBc-positive/HBsAg-negative sera correlates with HCV but not HIV serostatus. J Clin Virol 2004; 29:59-68. [PMID: 14675872 DOI: 10.1016/s1386-6532(03)00090-8] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BACKGROUND Hepatitis B virus (HBV) DNA often remains detectable in serum despite clinical recovery and loss of HBsAg. OBJECTIVE To study whether coinfection with HIV and HCV influence the chance of detecting HBV DNA in sera with markers of past hepatitis B. STUDY DESIGN AND RESULTS The test panel included 160 anti-HBc-positive/HBsAg-negative sera collected in the diagnostic setting. The following parameters were determined in the sera: anti-HIV (32% positive), anti-HCV (34% positive), HCV RNA (18% positive), and anti-HBs (37% positive). A highly sensitive PCR (90%-detection limit 100 copies/ml) amplifying the terminal protein (TP) region of HBV was established and HBV DNA was detected in 12.5% of the samples. In 70% of these samples, the HBV DNA concentration was below 500 copies/ml as measured by real-time PCR in the S gene. Logistic regression analysis revealed that the chance of detecting HBV DNA was increased by a positive HCV serostatus (odds ratio 5.0, 95%-CI 1.6-15.7), whereas HIV coinfection (odds ratio 2.0, 95%-CI 0.7-5.8), anti-HBs (odds ratio 0.9, 95%-CI 0.3-2.6), and HCV RNA status (odds ratio 0.4, 95%-CI 0.1-1.7) had no statistically significant influence. In contrast, the chance of detecting HCV RNA in the subgroup of anti-HCV-positive sera was increased by HIV coinfection (odds ratio 4.5, 95%-CI 1.2-17.4). Sequencing of the TP PCR products revealed neither a specific phylogenetic origin of the circulating HBV DNA nor clustering of uncommon mutations in the TP region. CONCLUSIONS The prevalence of HBV DNA in serum of anti-HBc-positive/HBsAg-negative subjects correlates with HCV rather than HIV serostatus.
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Affiliation(s)
- Christian Drosten
- Department of Virology, Bernhard-Nocht-Institute of Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany
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MOTSON GRAHAMR, FLEMING JEANS, BROOKER SALLY. POTENTIAL APPLICATIONS FOR THE USE OF LANTHANIDE COMPLEXES AS LUMINESCENT BIOLABELS. ADVANCES IN INORGANIC CHEMISTRY 2004. [DOI: 10.1016/s0898-8838(03)55007-3] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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Drosten C, Panning M, Kramme S. Detection of Mycobacterium tuberculosis by Real-Time PCR Using Pan-Mycobacterial Primers and a Pair of Fluorescence Resonance Energy Transfer Probes Specific for the M. tuberculosis Complex. Clin Chem 2003; 49:1659-61. [PMID: 14500592 DOI: 10.1373/49.10.1659] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Christian Drosten
- Bernhard-Nocht Institute of Tropical Medicine, National Reference Centre for Tropical Infections, 20359 Hamburg, Germany.
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Schulz A, Mellenthin K, Schönian G, Fleischer B, Drosten C. Detection, differentiation, and quantitation of pathogenic leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay. J Clin Microbiol 2003; 41:1529-35. [PMID: 12682141 PMCID: PMC153923 DOI: 10.1128/jcm.41.4.1529-1535.2003] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation of Leishmania organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes Leishmania organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of >or=95% at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow, skin, and liver). The Leishmania donovani complex, the Leishmania brasiliensis complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0 degrees C, respectively). All three groups could be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient of variation [CV], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR is utilized for therapy monitoring.
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Affiliation(s)
- Alexandra Schulz
- National Reference Center for Tropical Infections. Leishmaniasis Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
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Stöcher M, Leb V, Berg J. A convenient approach to the generation of multiple internal control DNA for a panel of real-time PCR assays. J Virol Methods 2003; 108:1-8. [PMID: 12565148 DOI: 10.1016/s0166-0934(02)00266-5] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Real-time polymerase chain reaction (PCR) assays allow convenient detection and quantitation of virus-derived nucleic acids in clinical specimens. When specimens are assayed for the presence of virus-derived nucleic acids against external standards, sample adequacy is not monitored. This can be achieved by using internal controls that are co-amplified with the virus-specific DNA in competitive PCR. Each of the various real-time PCR assays in a routine clinical laboratory requires its specific internal control. In order to complement a panel of virus-specific real-time PCR assays with internal controls, a convenient approach is described to generate the several internal controls within single DNA fragment. By applying composite primer technology, PCR primer sequences used in real-time PCR assays were added in 5' and 3' of a stretch of heterologous DNA during consecutive preparative PCRs. The heterologous DNA was used for internal control specific detection by e.g. FRET-hybridisation probes. The presented example of such a multiple internal control DNA contained five internal controls for five competitive LightCycler-PCR assays. All five PCR products derived from the multiple internal control DNA were detected with a single pair of specific FRET-hybridisation probes. The example described proved useful in real-time PCR assays specific for the detection of EBV-, CMV-, VZV- HSV-, and HBV-DNA on the LightCycler instrument. This methodology should enable laboratories to conveniently complement their panel of existing real-time PCR assays with a single multiple internal control DNA.
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Affiliation(s)
- Markus Stöcher
- Dept of Lab Med (Zentral-Labor), Institute of Laboratory Medicine, General Hospital Linz, Allgemeines Krankenhaus Linz, Krankenhausstrasse 9, A-4020, Linz, Austria
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Abstract
This review addresses the diagnostics of viral hemorrhagic fevers (VHFs). In the first part, an overview is given on classical methods of VHF diagnostics as well as novel molecular diagnostic tools. Currently available polymerase chain reaction (PCR) assays for diagnosis of VHF are summarized and discussed. In the second part, VHF diagnostics are described in particular for Lassa fever, yellow fever, and Crimean-Congo hemorrhagic fever, based on cases that were imported into or occurred within Europe. The third part is focussed on important differential diagnoses of VHF.
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Affiliation(s)
- Christian Drosten
- Department of Virology, Bernhard-Nocht-Institute of Tropical Medicine, Bernhard-Nocht Strasse 74, 20359 Hamburg, Germany
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Wang JT, Lee CZ, Chen PJ, Wang TH, Chen DS. Transfusion-transmitted HBV infection in an endemic area: the necessity of more sensitive screening for HBV carriers. Transfusion 2002; 42:1592-7. [PMID: 12473140 DOI: 10.1046/j.1537-2995.2002.00274.x] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND By NAT, HBV DNA is occasionally detectable in blood donors with past HBV infection but negative for HBsAg. Whether or not these donors can cause transfusion-transmitted HBV infections is uncertain. STUDY DESIGN AND METHODS To determine whether or not donors with past HBV infection but negative for HbsAg can cause HBV transfusion-transmitted infections, recipients followed for blood transfusion in a university medical center in Taiwan were studied. HBV DNA and serologic markers were tested in donors and recipients. RESULTS Of 1,038 enrolled recipients, 910 completed the 6-month post-transfusion follow-up visit. Of these, only 39 patients (4.3%) tested negative on the pretransfusion sample for HBsAg, anti-HBs, anti-HBc, and HBV DNA by PCR. These 39 HBV-naive recipients had been transfused with blood from 147 donations for which stored samples were available for HBV DNA testing by PCR; 11 of these HBsAg-negative samples tested positive for HBV DNA and anti-HBc. Two of the 11 patients who received the HBV-DNA-positive donations (18%) became positive for HBV DNA, and one seroconverted to anti-HBc and finally to anti-HBs, with a mild transient elevation of serum ALT activities. Based on the one confirmed case of HBV transmission, a projection was made that approximately 200 post-transfusion HBV infections could occur in one million units of transfused blood in Taiwan. CONCLUSIONS In HBV-endemic areas like Taiwan, where blood donors are screened for HBsAg only, the risk of transfusion-transmitted HBV appears to be substantial. Implementation of NAT for blood screening in these settings warrants consideration.
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Affiliation(s)
- Jin-Town Wang
- Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan
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41
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Bennett RS, McComb B, Shin HJ, Njenga MK, Nagaraja KV, Halvorson DA. Detection of avian pneumovirus in wild Canada (Branta canadensis) and blue-winged teal (Anas discors) geese. Avian Dis 2002; 46:1025-9. [PMID: 12495069 DOI: 10.1637/0005-2086(2002)046[1025:doapiw]2.0.co;2] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Choanal cleft swab samples from 770 wild Canada geese (Branta canadensis) and 358 blue-winged teal (Anas discors), captured for relocation or banding, were examined for the presence of avian pneumovirus (APV) RNA by reverse transcription (RT)-polymerase chain reaction (PCR) and for virus isolation. The swab samples were pooled into groups of 5 or 10. Sixty eight of 102 (66.7%) pooled goose samples were RT-PCR positive for APV RNA. Thirteen of 52 (25.0%) pooled blue-winged teal samples were RT-PCR positive for APV RNA. APV RNA-positive samples were inoculated onto chick embryo fibroblasts (CEF) and QT-35 cells. Infectious APV was isolated from five Canada goose pooled samples in CEF and from one Canada goose pool in QT-35 cells but not from blue-winged teal.
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Affiliation(s)
- R S Bennett
- Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA
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42
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Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmitz H, Günther S. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol 2002; 40:2323-30. [PMID: 12089242 PMCID: PMC120575 DOI: 10.1128/jcm.40.7.2323-2330.2002] [Citation(s) in RCA: 442] [Impact Index Per Article: 19.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >or=95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.
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43
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Roth WK, Weber M, Petersen D, Drosten C, Buhr S, Sireis W, Weichert W, Hedges D, Seifried E. NAT for HBV and anti-HBc testing increase blood safety. Transfusion 2002; 42:869-75. [PMID: 12375659 DOI: 10.1046/j.1537-2995.2002.00128.x] [Citation(s) in RCA: 90] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Routine HBV PCR screening of blood donations to our institutes was introduced in January 1997 to complete the NAT screening program for transfusion-relevant viruses. Testing was successively extended to customer transfusion services with a total of 1,300,000 samples tested per year. STUDY DESIGN AND METHODS Minipools of 96 blood donation samples were formed by automatic pipettors. HBsAg-reactive samples were included. HBV particles were enriched from the minipools by centrifugation. Conventional and in-house TaqMan PCRs were successively applied for HBV amplification. Sensitivity reached 1000 genome equivalents per mL for each individual donation. Confirmatory single-sample and single-sample enrichment PCRs were established with sensitivities of 300 and 5 to 10 genome equivalents per mL, respectively. RESULTS After screening of 3.6 million donor samples, 6 HBV PCR-positive, HBsAg-negative donations were identified. Two samples were from infected donors who had not seroconverted and four were from chronic anti-HBc-positive low-level HBV carriers. Retesting by single-sample PCR of 432 samples confirmed positive for HBsAg identified 37 donations that were negative in minipool PCR. Donor-directed look-back procedures indicated that no infected donor who had not yet seroconverted was missed by minipool PCR. However, recipient-directed look-back procedures revealed two anti-HBc-positive recipients of HBsAg-negative minipool PCR-negative, anti-HBc-positive and single-sample PCR-positive blood components. After testing randomly selected 729 HBsAg-negative minipool PCR-negative, anti-HBc-positive donors by single-sample enrichment PCR, 7 were identified with < or = 10 HBV particles per mL of donor plasma. CONCLUSION Minipool PCR testing after virus enrichment was sensitive enough to identify HBsAg-negative donors who had seroconverterd and HBsAg-negative, anti-HBc-positive chronic HBV carriers. HBV NAT in conjunction with anti-HBc screening would reduce the residual risk of transfusion-transmitted HBV infection.
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Affiliation(s)
- W Kurt Roth
- Institute of Transfusion Medicine and Immunohematology German Red Cross, Hesse, Frankfurt
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44
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Vieth S, Manegold C, Drosten C, Nippraschk T, Günther S. Sequence and phylogenetic analysis of hepatitis B virus genotype G isolated in Germany. Virus Genes 2002; 24:153-6. [PMID: 12018706 DOI: 10.1023/a:1014572600432] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Genotype G of hepatitis B virus (HBV) has only recently been discovered. This report describes the full-length sequence of genotype G HBV (designated 235/01) isolated in Germany from a chronic HBV carrier. The patient was hepatitis B e antigen-positive, had high HBV DNA levels of about 10(10) copies/ml serum, lacked a measurable anti-HBc response, and was coinfected with human immunodeficiency virus type 1. Genome 235/01 showed characteristic genotype G-specific features: stop codons at codon 2 and 28 of the pre-C region and insertion of 36 nucleotides at the 5' end of the C gene. It was nearly identical (< or = 99.7% identity) to both genotype G genomes (B1-89 and FR1 from France) described so far, suggesting either close epidemiological link among European genotype G isolates or high genetic stability of genotype G.
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Affiliation(s)
- Simon Vieth
- Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany
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45
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Chandra S, Groener A, Feldman F. Effectiveness of alternative treatments for reducing potential viral contaminants from plasma-derived products. Thromb Res 2002; 105:391-400. [PMID: 12062540 DOI: 10.1016/s0049-3848(02)00044-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
An issue of great importance and continuing concern with regard to all products derived from human plasma is their safety from potential contaminants in the source material from which they are purified. Since viral contaminants are a major safety consideration with these products, a number of different methods, including dry heating, vapor heating, filtration and nanofiltration, ultraviolet and gamma irradiation, pasteurization, solvent/detergent (S/D) treatment, sodium thiocyanate treatment, and chromatography (immunoaffinity, metal chelation, affinity, and ion exchange), have been developed to remove or inactivate potentially contaminating viruses. Pasteurization and S/D treatment have emerged as the dominant viral inactivation methods. Results summarized in this review demonstrate that pasteurization is the broadest and most rigorous currently available method for removal of potential viral contaminants from plasma-derived products. S/D treatment requires control over a large number of manufacturing parameters and has no ability to inactivate nonlipid-enveloped viruses. Pasteurization requires control over only a small number of manufacturing variables, is easily monitored, and remains effective even if deviations are encountered from specified protein and stabilizer concentrations and temperature. In addition, pasteurization is effective against a wide range of lipid- and nonlipid-enveloped viruses.
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46
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Drosten C, Seifried E, Roth WK. TaqMan 5'-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening. J Clin Microbiol 2001; 39:4302-8. [PMID: 11724836 PMCID: PMC88540 DOI: 10.1128/jcm.39.12.4302-4308.2001] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >or=95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.
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Affiliation(s)
- C Drosten
- Institute of Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service, Frankfurt am Main, Germany
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47
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Gobbers E, Oosterlaken TA, van Bussel MJ, Melsert R, Kroes AC, Claas EC. Efficient extraction of virus DNA by NucliSens Extractor allows sensitive detection of hepatitis B virus by PCR. J Clin Microbiol 2001; 39:4339-43. [PMID: 11724842 PMCID: PMC88546 DOI: 10.1128/jcm.39.12.4339-4343.2001] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The system has been validated for the isolation of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNAs from human samples in combination with nucleic acid sequence-based amplification- and reverse transcription-PCR-based methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extraction from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared with the Qiagen extraction method, and the impact of the sample volume used in the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume resulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sample in GuSCN at 80 degrees C (10 min) and at 37 degrees C (30 min) allowed detection of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilution panels. Sample lysis in GuSCN at room temperature and proteinase K treatment prior to use of the extractor were less efficient procedures. All clinical samples that were PCR positive after Qiagen extraction and/or that were HBsAg positive were also PCR positive after extractor isolation. HBV DNA, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single sample, allowing reliable detection of viral genomes.
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Affiliation(s)
- E Gobbers
- Organon Teknika, Boxtel, The Netherlands
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