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

3. 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

4. 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

5. 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

6. 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)

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

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

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

10. 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 % total32 %

11. 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

12. HAV Kit Availabiliy 1.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

13. 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