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Full characterization of HAV RNA window period positive blood donations in Germany

Full characterization of HAV RNA window period positive blood donations in Germany. SoGAT XVIII Bethesda. Hepatitis A Virus. Picornavirus Non-enveloped First isolated in 1979 Natural host: human Stable: heat and acid-resistant Inactivated by high temperature, formalin, chlorine

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Full characterization of HAV RNA window period positive blood donations in Germany

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  1. Full characterization of HAV RNA window period positive blood donations in Germany SoGAT XVIII Bethesda

  2. Hepatitis A Virus • Picornavirus • Non-enveloped • First isolated in 1979 • Natural host: human • Stable: heat and acid-resistant • Inactivated by high temperature, formalin, chlorine • Transmission: fecal-oral • Replication: liver (hepatocytes) • Peak viremia 10-12 days after infection • Self-limited illness: asymptomatic or acute fulminant hepatitis

  3. HAV Pathogenesis • Incubation period of 15-50 days • Prodromal illness is nonspecific • Age-related severity: • Children <6 years: 70% of infections are asymptomatic • Older children and adults >70% of cases result in acute hepatitis with jaundice

  4. HAV Recent Developments in Germany • RT-PCR based tests for the presence of HAV RNA were • introduced in Germany in 2000. • Systems: • In house • Artus based technology transfer • Technology: • Real time RT PCR • LightCyler/ABI Prism

  5. HAV Kit Adaptation HAV positive blood donation (in house test) • Artus/Roche negative • Inhouse 4+/4- • Enterovirus etc. negative • Contamination/unspecific? • What to do? • Clinical positive (2 weeks later)

  6. HAV Kit Adaptation Tests • 7 real time PCRs spanning the HAV genome • 6 negative • 1 positive (ct 28) • Long range PCR • Sequencing • Culture

  7. HAV Kit Adaptation • Real time pos. HAV Genotype III • Full length pos. HAV Genotype IIIA • Cell culture pos. And the Kit ??

  8. HAV Kit Adaptation • New Kit (with CE mark) • Alignment (conserved region) • Sensitivity • Specificity • Robustness • Positive Controls • Precision

  9. HAV Kit Adaptation • Results: New Kit (with CE mark) • Alignment • VP4 highest homology • Sensitivity (with new primer and probes) • 183 IU/ml (WHO Int. Std/Qiagen Extr.) • Specificity • all available strains (in vitro) • 100% (HAV Ab neg.) • weak crossreactivity with Enterovirus 71 • Precision • intra-assay 28 % • inter-assay 31 % • total 32 %

  10. HAV Kit Adaptation • Results: New Kit (with CE mark) • End of 2003 Kit release • Detection of: • 1 food associated HAV-RNA positive blood donation • 4 travel related HAV-RNA positive blood donation • only 1 would have been detected with the old system • All Genotype I

  11. HAV Kit • Availabiliy • As IVD • Worldwide • 2. For blood screening • LC-PCR based Systems (via Roche starting in Juli/August 2005) • ABI Prism based Systems (via Abbott) • Country specific restrictions may apply

  12. HAV Kit • Acknowledgments • The German Red Cross Blood Transfusion Service of Baden Württemberg-Hessen, PCR Lab in Frankfurt • The German Red Cross Blood Transfusion Center West in Hagen • Blood Center of the German Red Cross NSTOB, Institute Springe • Department of Virology, University of Bremen

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