Hepatitis A, B, C, D, E, G,… diagnostic tools and their use

1. Hepatitis A, B, C, D, E, G,…diagnostic tools and their use Geert Leroux-Roels Laboratorium voor Klinische Biologie UZ Gent

2. Human Hepatitis VirusesDiscovery and characteristics

3. Human Hepatitis VirusesDiagnostic tools

4. Epidemiology of HAV

5. Hepatitis A virus – diagnostic tools • Serology • Anti-HAV IgM acute HAV infection • Anti-HAV Totaal immunity - natural - vaccine-induced protective level 10-30 IU/L • HAV detection in blood and blood products in faeces, saliva, … in shelfish, food products in water,sewage,

7. HAV vaccines • Children (2-15) Havrix Junior (720 U/dose), 0.5 ml ml/d scheme : 0 and 6-12 mo (2 doses, IM) • Adults (>15) Havrix 1440 (1440 U/dose, 1 ml/d scheme : 0 and 6-12 mo (2 doses, IM)

8. Epidemiology of HEVepidemische en sporadische gevallen Rare cases in western countries after recent travel in endemic area

9. Hepatitis E virus Serology anti-HEV IgM anti-HEV IgG HEV-RNA

10. Hepatitis B virus Diagnostic tools and interpretation

11. HBV: genome and gene products • 3200 baseparen • 4 open reading frames • 7 proteins • large (preS1-preS2-S) • Middle (preS2-S) • Small or HBsAg • nucleocapsid or HBcAg • secreted HBe-Ag • Polymerase • X protein

12. Inflammation and liver cell damage Transaminases ALT/AST en other biochemical markers Antigens Antibodies HBsAganti-HBs HBcAg anti-HBc-Tot anti-HBc-IgM HBeAg anti-HBe • Detection/quantific. • of HBV DNA • Commercial assays • Branched DNA (Bayer) • PCR (Roche Amplicor) • In-house nested PCR • In-house real-time PCR Diagnostic markers of HBV infection

14. Usefulnessof HBV-DNA quantification • Diagnosis • Acute HBV infection : HBV DNA is not useful • Chronic HBV infection – Is HBV replicating ? • HBeAgpos : not useful • HBeAgneg/anti-HBepos : useful, “threshold value” ? • Prognosis • Therapy • Decision to treat : ALT, biopsy, HBeAgpos • Selection treatment • Monitoring : HBV DNA, ALT, HBeAg, anti-HBe,

15. Evolution of an acute, self-limited HBV infection

16. Serologic profile of a chronic HBV infection, with a late seroconversion to anti-HBe

17. Case 1 • 41 years old Afghan male • political refugee • In training for assistant-cook • Medical screening exam for ‘hepatitis’ • No symptoms • No history of hepatitis

18. Results - blood chemistry

19. Question 1 Can HBsAg and anti-HBs occur concomitantly ? Results - serology

20. [HBsAg-anti-HBs] immune complexes • Are present during the clearance phase of an acute HBV infection in the “window phase” and in some chronic HBV patients • Routine tests for HBsAg/anti-HBs do not detect immune complexes (IC) • IC dissociatie (ICD) by treatment of serum (100µl) with HCl (50 µl, 0.5 N, 1h at 37°C) and neutralisation with NaOH (50 µl, 0.5N)(Rabenau et al. 1996) • Research tests can detect IC’s

21. HAV serostatus • anti-HAV-IgM antibodies are only present in the acute phase of HAV infections • ALT/AST activities are normal • Anti-HAV status (IgG antibodies) would have been useful to see whether this person still needs HAV vaccination • Food handling • Chronic HBV infection

22. Question 2 Is this person infectious ??? Question 3 Does he need treatment ? Results – additional HBV tests

23. Infectivity is LOW • Spouse and daughter show no signs or markers of HBV infection (HBsAlneg) • Vaccination of household (sexual contact) ! • Twinrix is an alternative for Engerix-B • Therapy is not indicated • Normal transaminases, low DNA • Follow-up : annually ?

24. Case 2 • Man, born in 1947 • 1986 – Ulcerative colitis • 1992 - liver enzymes slightly elevated, no further investigations • 1998 – abnormal liver tests, alcohol consumption, • June 1998 – exacerbation of colitis • Nov 2001 - exacerbation of colitis

25. Evolution of laboratory tests

26. Spontaneous seroconversions in chronic HBV • HBeAg to anti-HBe seroconversion • Inflammation (ALT/AST) = 8-15% per year • Normal ALT <2% in children < 3 years 4-5% in patients > 3 years • HBsAg to anti-HBs seroconversion • Active HBeAg- hepatitis 0.5% /jaar • Asymptomatic HBeAg- carrier • In a western population 1-2% /jaar • Post perinatal infection 0.05-0.8% /jaar

27. HBeAg/anti-HBe seroconversion • Transition to ‘inactive carrier’ • Normalisation of transaminases • Low viral replication and HBV DNA (<105 gEq/ml) • Active hepatitis with HBeAg-/anti-HBe+ • Elevated transaminases • High(er) HBV DNA (> 105 gEq/ml) • Precore mutation (G1896A: stop codon) • Core/precore promotor mutations

28. -29 aa 183 aa 1 HBeAg precursor P G1896A aa -10 aa 1 aa 149 AUG AUG mature HBeAg aa 183 aa 1 HBcAg Hepatitis B core and e antigen

29. Interpretation of the serology and its evolution in this patient (case 2) • Spontaneous seroconversion of HBsAg to anti-HBs • No detectable [HBsAg-anti-HBs] IC’s • HBV DNA detection, quantification and sequence analysis are needed for a correct diagnosis and prognosis

30. Case 3 • Man, 45 years • Traffic accident => brain death • Possible organ donor (liver ?) • Serology : • HBsAgneg, anti-HBsneg,anti-HBcpos, anti-HCVneg,anti-HIVneg, CMVneg • Biochemistry : no abnormalities

31. Prevalence of “anti-HBc alone” in low seroprevalence populations

32. Prevalence of “anti-HBc alone” in high seroprevalence populations

33. 1 2 1 = window phase 2 = late immunity, only anti-HBc persists 3 3 = chronic infection with low replication/production of HBV or with HBsAg mutant Possible causes of “Anti-HBc alone” serology

34. “Occult hepatitis” • Typical serology HBsAgneg, anti-HBsneg, anti-HBcpos • HBV DNA < detection limit of routine PCR test (e.g. < 200 gEq/mL) • HBV DNA in the liver

35. Influence of HCV infection on HBV replication • HCV core protein suppresses HBV replication with a factor 2 to 4 • HCV infection reduces the expression of HBsAg in the liver • Treatment of HCV with IFNaalsohas an effect on HBV

36. “anti-HBc alone” can be : • Window phase • Late immunity – only sign of past infection • Chronic infection – “occult infection” • HBsAg mutant • “False” positive test result • really “false positive” – low signal, 2nd EIA • Core-binding antibodies (IgM vs IgG)

37. HBV Replication Host Imm Resp Liver Disease ALT = Hi Hi = to Hi = to Hi = HBsAg ++ ++ ++ + - + Anti-HBc + + + + + + HBeAg + + - - - - Anti-HBe - - + + + or - + or - HBV DNA Hi PCR Hi PCR Hi PCR Lo PCR Nested PCR - Active Hepatitis Tolerance HBeAg+ HBeAg- Occult Inactive carrier mutant serocon

38. Lab Tools IgM anti-HDV HDV-Ag anti-HDV HDV-RNA

41. Hepatitis C virus Diagnostic tools and interpretation

42. The HCV genome and expressed polyprotein

43. Markers of HCV infection • Indirect markers • anti-HCV • ELISA 3-4th generation • Confirmation tests RIBA, LIA • Direct markers = HCV genome • RT-PCR qualitative and quantitative • Branched-DNA (quantitative) • Genotyping • HCV core antigen

44. Anti-HCV assays: generations 1 to 4 1989 1993 2000 Generation 1 2 3 4 Antigens NS3/4 C,NS3,NS4 C,NS3,NS4A/B,NS5A Sensitivity 95-98% >99% Specificity <95% 99.8% Lag time (wks) 16-24 4-12 4-8

45. Confirmation of anti-HCV+ • Repeat ELISA on the same sample • Another ELISA on the same sample • Same ELISA on a new sample • Confirmation assays • RIBA (recombinant immunoblot assay) • LIA (Line immuno assay) • Confirmation by PCR

46. Molecular tests for HCV • Molecular detection – qualitative • Molecular quantification • Genotyping

47. HCV-RNA detection – qualitative

48. Qualitative HCV-RNA tests • Confirms diagnosis of HCV infection • Useful for the early diagnosis of acute hepatitis C • Demonstrates the presence of active infection • « Gold standard » for documenting response to therapy

49. Interpretation of combined anti-HCV and HCV-RNA results

50. Treatment of HCV • Clinical studies evaluating the efficacy of different treatment protocols : drugs, doses, duration, … have revealed the importance of 1) HCV-RNA quantification 2) genotyping