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

SoGAT XVIIIBethesda

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

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

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

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)

HAV Kit Adaptation

Tests

• 7 real time PCRs spanning the HAV genome• 6 negative• 1 positive (ct 28)

• Long range PCR• Sequencing • Culture

HAV Kit Adaptation

• Real time pos. HAV Genotype III• Full length pos. HAV Genotype IIIA• Cell culture pos.

And the Kit ??

HAV Kit Adaptation

New Kit (with CE mark)

• Alignment (conserved region)

• Sensitivity

• Specificity

• Robustness

• Positive Controls

• Precision

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 %

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

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

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