1. Avidity determination of IgG in diagnosis of tick-born encephalitis Hana Zelená Jiří Januška Jan Raszka Virology department, National Reference Laboratory for Arboviruses, Institute of Public Health, Ostrava, Czech Republic

2. Diagnosis of tick-born encephalitis 1 st phase (fever): direct detection -PCR -virus isolation from blood 2 nd phase (neurological): indirect detection -IgG, IgM ELISA, IFA -Complement fixation test (CFT) -Virus neutralizing antibodies (VNA )

3. Dynamics of diagnostic markers in tick- born encephalitis

5. Avidity of IgG antibodies Avidity is a measure of the strenght of the antibody-antigen interactions, it increases with their binding affinity and with their valence Avidity reflects the maturity of the antibodies. Low avidity antibodies are synthetized during the primary infection, in time avidity gradually increases. High avidity antibodies are produced by memory B-cells during the secondary infection or reactivation or infection in people that were vaccinated. Low avidity occures also in immunosupression.

6. Usefullness of IgG avidity measurement Rubella CMV VZV Toxoplasmosis VCA-EBV HIV Viral hepatitis WNV

7. Dynamics of diagnostic markers in tick- born encephalitis

8. Principle of IgG avidity measurement Test of the strenght of antigen-antibody interaction by incubation with denaturing agent (urea) Low avidity antibodies dissociate from complexes and are washed away. High avidity antibodies withstand urea treatment and remain bound to the antigen.

9. Protocol for the determination of anti- TBEV IgG avidity Anti-TBEV IgG test kit is based on indirect ELISA. Patient serum is tested in two parallel wells. During the 1 st incubation step antibodies found in the serum sample bind to the antigen. One of the two wells is incubated with urea solution (8 mol/L) for 5 minutes, while the other well remains empty. Absorbance ratio between the two parallel wells is calculated at the end of the test (the well with urea/the well without urea)

10. IgG avidity measurement results and interpretation Avidity (%) = absorbance of the well with urea/absorbance of the well without urea AvidityResultInterpretation <40%low avidity Recent infection (< 3 weeks) 40-60%„gray zone“ >60%high avidity Infection in the past, infection in people that were vaccinated

11. IgG avidity in time (patients)

12. Dynamics of anti-TBEV antibody levels (patient with typical TBE)

13. Anti-TBEV positive samples (april to september 2010) 29 samples IgG+ IgM+ 59 samples IgG+ IgM-

14. Pitfalls in TBE serology – relevance of avidity testing Infection in vaccinated people -min. 4-fold increase in CFT and VNA – must be paired samples -IgM positive or negative -high IgG avidity Elevated IgM pesistence after primary infection (up to 1 year) -IgG and IgM positive -high IgG avidity Atypical or subclinical course of the disease -IgM and IgG positive -low IgG avidity Early disappearance of IgM in primary infection – low IgG avidity

15. Conclusion Avidity determination of IgG is a complementary tool that makes serological diagnosis of tick-born encephalitis more accurate It increases the testing reliability in acute infections, posses high importance in cases of: -atypical course of the disease -atypical antibody response -infection in vaccinated people Interpretation of serological result that is complemented with IgG avidity measurement has clearly higher validity.