Diagnose van hepatitis B en hepatitis C

1. Diagnose van hepatitis B en hepatitis C Prof. Patrick Goubau UCL Antwerpen 28/02/2005

2. Blood of chronic hepatitis B carrier in electronic microscopy HBs Ag Dane particles

4. Replication of Hepatitis B virus circular partly double stranded viral DNA particle Covalently closed circular DNA cell Pregenomic RNA Reverse Transcriptase particle circular partly double stranded viral DNA

5. HBV: genomic structure

6. Amino acids specifying determinants of HBsAg Magius & Norder, Intervirol. 1995;38:24-34 Position Aminoacid Specificity 122 Lys d Arg y 127 Pro w1*/w2 Thr w3 Leu/Ile w4 160 Lys w Arg r * w1 also requires Arg122, Phe134 and/or Ala 159

7. Geographical distributionof genotypes and serotypes Magius & Norder, Intervirol. 1995;38:24-34 Genotype A Northwestern Europe, Central Africa B Indonesia, China, Vietnam C Korea, China, Japan, Polynesia,Vietnam D Mediterranean area, India E West Africa F American Natives,Polynesia

8. F Genotypes of HBV in Belgian children, 1999 D DA AI GM WS 100 KrMe VC DeAy 98.4 72.6 CM 99.7 DL VA RR BM 100 PI TA LP A 100 KM DV E C LS B

9. Acute hepatitis B symptoms anti-HBe HBeAg anti-HBc Total Titre anti-HBs IgM anti-HBc HBsAg weeks 0 4 8 12 16 24 28 32 52 100 20 36

10. HBeAg anti-HBe HBsAg anti-HBc total Titre IgM anti-HBc 0 4 8 12 16 20 24 32 36 28 52 years weeks Chronic hepatitis B

11. -29 aa 183 aa 1 HBeAg precursor P aa -10 aa 1 aa 149 AUG AUG mature HBeAg aa 183 aa 1 HBcAg HBV core et antigène e

12. -29 aa 183 aa 1 HBeAg precursor P G1896A aa -10 aa 1 aa 149 AUG AUG mature HBeAg aa 183 aa 1 HBcAg HBV precore and promotor mutants

13. HBV transmission • Mother => child: essentially if HBeAg positive • Horizontal • child => child • Institutions for mentally handicaped • Sexual • Parenteral • Transfusion, transplantation • Nosocomial and iatrogenic • Shared siringes among drug addicts

14. Geographic Distribution of Chronic HBV Infection Prévalence AgHBs 8% - High 2-7% - Medium <2% - Low

15. Transmission patterns of HBV

16. Evolution of HBV infection • Acute infection : mortality 0,5 – 1% • Chronic carriership: • Favourable evolution: negative for Ag HBe • Unfavourable evolution: AgHbe pos or precore mutant • Chronic hepatitis and fibrosis • Cirrhosis • Primary hepatocarcinoma (possible without cirrhosis) • Cure: often persistance of hepatic DNA Cave liver transplants!

17. Outcome of Hepatitis B Virus Infection by Age at Infection 100 100 80 80 60 60 Chronic Infection Chronic Infection (%) Symptomatic Infection (%) 40 40 20 20 Symptomatic Infection 0 0 1-6 months 7-12 months Older Children and Adults Birth 1-4 years Age at Infection

18. Age at Aquisition of Acute and Chronic HBV Infection United States, 1989 Estimates (4% ) Perinatal (24%) (4%) Children (12%) (1-10 yrs) (8%) Adolescent (6%) Adult (59%) Adult (83%) Acute HBV Infections Chronic HBV Infections

19. Treatment of chronic hepatitis B with interferon αMeta-analysis of 15 studies (837 patients) Wong et al. 1993

20. Treatment options for HBV • Interferon and pegylated interferon • Lamivudine (3TC): frequent resistance (YMDD) • Adefovir: remains active on strains resistant to lamivudine

21. LAMIVUDINE • Incidence of detectable serum variant HBV after: • 1 yr = 24% • 2 yr = 42% • 3 yr = 53% Lai NEJM 1998; Liaw Gastroenterology 2000; Leung Hepatology 2001 ADEFOVIR • Incidence of detectable serum variant HBV after: • 48 weeks = 0% • 96 weeks= <2% Angus Gastroenterology 2003

22. Peg IFN α-2b with/without lamivudineJanssen et al. Lancet, 365:123-129, 2005 • 52 weeks of treatment • 26 weeks post-treatment follow-up • Pegylated interferon α-2b with / without lamivudine (3TC) • Combined therapy: n=130 • Monotherapy: n=136

23. Peg IFN α-2b with/without lamivudineJanssen et al. Lancet, 365:123-129, 2005

24. Peg IFN α-2b with/without lamivudineJanssen et al. Lancet, 365:123-129, 2005

25. Acute hepatitis B symptoms anti-HBe HBeAg anti-HBc Total Titre anti-HBs IgM anti-HBc HBsAg weeks 0 4 8 12 16 24 28 32 52 100 20 36 Hepatitis B diagnostics

26. Isolated anti-HBc antibody • Post-acute window phase • Years later: loss of anti-HBs antibody • False positive result Check hepatitis C!!

27. Post-immunization control Vaccine schedule Month 0, 1, 6-12 Month 0, 1, 2, 12 serological control 1-2 months later Anti-HBs ≥ 10 IU/l 5% non-responders in adults

28. HBeAg anti-HBe HBsAg anti-HBc total Titre IgM anti-HBc 0 4 8 12 16 20 24 32 36 28 52 years weeks Hepatitis B diagnostics Chronic hepatitis B High viral replication Low viral replication or precore mutant =>test DNA

29. HBV quantitative DNA test • Indications: • institution of therapy in chronic HBsAg + patients, • follow-up of chronic hepatitis B treatment- acute rebound in chronic hepatitis B • Conditions: • - abnormal liver tests, particularly ALT- HBsAg positivity- maximum twice a year per patient

30. HBV quantitative DNA test • Limits: • Maximal sensitivity: few responders • Sensitivity 103 to 105 IU/ml (IU X ± 6 = copies) most useful for follow-up of treatment • Is it sufficient for pre-core mutants?

31. HBV qualitative DNA test Prior telephone contact with the laboratory is necessary. The test may be useful if infection is doubtful: e.g. if- isolated positive anti-HBcore- seronegative chronic hepatitis

32. Real time PCRwith sybr green fluorescence

33. Real time PCR with use of probes

35. Real time PCR: advantages • No contamination • Rapid results • Standardized procedures • Automation possible • Quantitative results and wide dynamic range

36. 180 204 236 Lamivudine rt L180M rt M204V rt M204I rt N236T Adefovir Protein S 832 178 336 680 RNAse H TP Spacer Reverse Transcriptase 1 Domains F A B C D E SNLSWLSLDVSAAFYHI VLGFRKIPMGVGLSPFLLAQFTSAICS AFSYMDDVVLG SLGIHLNPNKTT LNFMGYVIGSW 241 200 210 230 75 91 163 189 247 257

37. Envelope proteins E1 (gp31) E2 (gp70) Nucleoprotein - Core (p21) RNA genome Virus de l’hépatite C (HCV)

38. HCV genome and processing C E1 E2 NS2 NS3 NS4 NS5 5’UTR 3’UTR signal peptidase serine protease p21 gp31 gp70 p7 p23 p70 p8 p27 p58 p68 capsid envelope protease protease helicase NTPase RNA polymerase

39. Genotypes of HCV

40. HCV Clinical évolution Infection 80% 20% Resolution Chronic infection 1/3 1/3 Subclinical 1/3 Moderate disease Severe disease fibrosis cirrhosis liver failure

41. Risk history in HCV infected Mother to child 1% Sexual 5% Unknown 34% Parenteral 60%

42. Prevalence of HCV infection • Weak 0.5% • Scandinavia, Australia, Canada, Switzerland • Intermediate 1% • Belgium, USA, western Europe • High >2% • Third world, Asia • Very high >10% • Selected areas: Nile delta, southern Cameroon

43. Antibody negative window in acute hepatitis C • Second generation EIA: 82 days • Third generation EIA: 52 days Kupek E.J., J Vir Hepat 2001; 8: 78-82.

44. Retreatment of hepatitis CVirological clearance Davis et al. 1998

45. Treatment of chronic hepatitis CVirological clearance McHutchinson et al. 1998; Poynard et al. 1998

46. Innolipa genotyping of HCV

49. Detection de l’ARN de l’HCV ROCHE Amplicor HCV Amplicor HCV Monitor HCV Taqman Bayer Versant TMA bDNA Abbott LCX IU/ml 0 10 101 102 103 104 105 106 107 108

50. HCV qualitative assaysother indications • Determination of a vertical transmission: infection of a baby of a HCV-RNA positive mother, test to be performed on blood of the baby at 6 months of age. • Detection of HCV-RNA in a HCV positive patient with chronic hepatitis with at least 2 x elevated transaminases at 6 months interval. • Confirmation of a HCV infection, in the absence of anti-HCV, in immunosuppressed patients (renal dialysis, HIV, treatment,...