Diagnosis of Hepatitis B

1. Hepatitis b infectiondiagnosis and interpretation Dr.T.V.Rao MD Dr.T.V.Rao MD

2. 2 billion people have been infected (1 out of 3 people). 400 million people are chronically infected. 10-30 million will become infected each year. An estimated 1 million people die each year from hepatitis B and its complications. Approximately 2 people die each minute from hepatitis B. Hepatitis B In the World Dr.T.V.Rao MD

3. Prevalence of Hepatitis B carriers .Worldwide prevalence of hepatitis B carriers and primary hepatocellular carcinoma. (Courtesy Centers for Disease Control and Prevention, Atlanta.) From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 66, published by Mosby Philadelphia,,

4. Amember of the hepadnavirus group Circular partiallydouble-stranded DNA viruses Replication involves a reverse transcriptase. Endemic in the human population and hyper endemic in many parts of the world. Anumber of variants It has not yet been possible to propagate the virus in cell culture 1、Properties of HBV Dr.T.V.Rao MD

5. Hepatitis B • Hepadnaviridae family • DNA virus • Double-shelled particles • Outer lipoprotein envelope (surface Ag) • Inner viral nucleocapsid (core) • seven genotypes • four major subtypes. • All HBV subtypes share one common antigenic determinant - "a.“ • Thus, antibodies to the "a" determinant confer protection to all HBV subtypes Diagrammatic representation of the hepatitis B virion and the surface antigen components EM of Hepatitis B viron

6. Hepatitis b virus a major cause of hepatitis Dr.T.V.Rao MD

7. HEPATITIS B DNA virus – hepadnavirus 3200 bp Compact - uses overlapping genes Complicated replication – has a ssDNA component to RNA to DNA Difficult to grow Liver damage may be due to host immunity Dr.T.V.Rao MD

8. Structure of hepatitis b virus Dr.T.V.Rao MD

9. HBV Structure & Antigens Dane particle HBsAg = surface (coat) protein ( 4 phenotypes : adw, adr, ayw and ayr) HBcAg = inner core protein (a single serotype) HBeAg = secreted protein; function unknown Dr.T.V.Rao MD

10. HBsAg-containing particles are released into the serum of infected people and outnumber the actual virions. Spherical or filamentous They are immunogenic and were processed into the first commercial vaccine against HBV. Surface particles Dr.T.V.Rao MD

11. Structure and major antigens: 22 nm – most abundant – extra viral envelope protein - spheres and tubes 42 nm double-shelled – intact virus Both covered by HBsAg – see in blood Disrupt 42 nm with mild detergent – get 27nm core particle – covered by HBcAg – never see in blood HBeAg – soluble – binds to the smooth ER and gets exported into the circulation – see in blood HEPATITIS B Dr.T.V.Rao MD

12. Open Reading Frames • There are 4 open reading frames derived from the same strand (the incomplete + strand) • S - the 3 polypeptides of the surface antigen (preS1, preS2 and S - produced from alternative translation start sites. • C - the core protein • P- the polymerase • X - a transactivator of viral transcription (and cellular genes?). HBx is conserved in all mammalian (but not avian) hepadnavirus. Though not essential in transfected cells, it is required for infection in vivo. Dr.T.V.Rao MD

13. Acute infection HBsAg positive and anti-HBcAg IGM Rarely, IgM anti-HBc only marker Usually seen in acute fulminate Hep B Chronic infection HBsAg positive and anti-HBcAg Previous Infection HBsAg negative anti-HBs positive IgG anti-HBc positive Hbv – serology interpretation

14. HBcAg—anti-HBc system HBcAg can be found in the nuclei of liver cells, no free HBcAg in serum HBcAg is the marker of replication of HBV The stage called window phase Anti-HBc IgM is a marker of acute infection and acute attack of chronic infection of HBV. Anti-HBc IgG is the marker of past infection, high titer means low level replication of HBV Etiology Dr.T.V.Rao MD

15. Secretory protein that is processed from the precore protein Elevated early in infection and usually coverts to antibody early on. Traditionally used as a marker for viral load as viral load was undetectable with early assays when Ag was absent. However, certain variants of the Hep B virus do not create the HBeAg as it has no known function. When present, it does correlate with elevated viral load and seroconversion the antibody usually correlates with a decrease in viral load by a magnitude of 4-5. Hepatitis B virus – e antigen

16. Three antigen-antibody system • Include HBsAg, anti-HBs, pre-s1,s2 antigen and anti-pres1, s2 • HBsAg appears 1-2 weeks (late to 11-12 weeks) after exposure, persists for 1-6 weeks( even 5 months) in acute hepatitis B. In chronic patients or carrier, HBsAg persist many years • HBsAg antigencity but no infectivity Dr.T.V.Rao MD

17. Clinical outcomes of Hepatitis B infections .Clinical outcomes of acute hepatitis B infection. (Redrawn from White DO, Fenner F: Medical virology, ed 3, New York, 1986, Academic Press From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 62, published by Mosby Philadelphia,,

18. Determinants or acute and chronic HBV infection From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 66, published by Mosby Philadelphia,, Figure 66-7

19. Who should be screened Persons born in hyper endemic areas Men who have sex with men Injection drug users Patients on dialysis HIV infected patients Pregnant women Family and household contacts and sexual contacts of HBV-infected persons. Testing should be performed by obtaining an HBsAg and anti-HBs. Screening – Who?

20. Serological markers Dr.T.V.Rao MD

21. HBsAg:Present in acute or chronic infection. HBsAb:Present in recovery or immunization. Anti -HB Core:May be “Total” (IgG&IgM) or IgM. Lifelong marker of past and active infection in either acute or chronic. HBeAg:Acute infection, and extremely infectious. Anti-Hbe: Usually prognostic for resolution. HEPATITIS B MARKERS: Dr.T.V.Rao MD

22. HBcAg—anti-HBc system HBcAg can be found in the nuclei of liver cells, no free HBcAg in serum HBcAg is the marker of replication of HBV The stage called window phase Anti-HBc IgM is a marker of acute infection and acute attack of chronic infection of HBV. Anti-HBc IgG is the marker of past infection, high titer means low level replication of HBV Etiology Dr.T.V.Rao MD

23. Acute Hepatitis B Virus Infection withRecovery Typical Serologic Course Symptoms anti-HBe HBeAg Total anti-HBc Titre anti-HBs IgM anti-HBc HBsAg 0 4 8 12 16 24 28 32 52 100 20 36 Weeks after Exposure Dr.T.V.Rao MD

24. Acute (6 months) Chronic (Years) HBeAg Anti-HBe HBsAg Total anti-HBc Titer IgM anti-HBc 0 4 8 12 16 20 24 28 32 36 52 Years Weeks after Exposure Acute HBV Infection with Progression to Chronic Infection: Typical Serologic Course Dr.T.V.Rao MD

25. HEPATITIS B Serologic Markers During Acute HBV Hepatitis and Recovery Anti-HBcAb Titer HBsAg Anti-HBsAb “Window” Anti-HBeAb HBeAg Months after exposure Incubation Jaundice Recovery IMMUNE Elevated ALT Dr.T.V.Rao MD

26. HBcAg—anti-HBc system HBcAg can be found in the nuclei of liver cells, no free HBcAg in serum HBcAg is the marker of replication of HBV The stage called window phase Anti-HBc IgM is a marker of acute infection and acute attack of chronic infection of HBV. Anti-HBc IgG is the marker of past infection, high titer means low level replication of HBV Etiology Dr.T.V.Rao MD

27. Serology: - acute: HBsAg+ (HBsAb-) = acute infection or chronic carrier HBeAg+ = highly infectious HBsAb = immune – naturally or vaccine Window = HBsAg- and HBsAb- will be HBcAb+ (IgM – acute) HEPATITIS B Dr.T.V.Rao MD

28. HBeAg—anti-HBe system HBeAg is a soluble antigen HBeAg is a reliable indicator of active replication of HBV Anti-HBe is a marker of reduced infectivity. If exist long may be a marker of integration of HBV into liver cell Etiology Dr.T.V.Rao MD

29. Acute (6 months) Chronic (Years) HBeAg anti-HBe HBsAg Total anti-HBc Titer IgM anti-HBc 0 4 8 16 20 24 28 36 12 32 52 Weeks after Exposure Progression to Chronic Hepatitis B Virus Infection Typical Serologic Course Dr.T.V.Rao MD

30. The marker of molecular biology of HBV HBV-DNA The direct indicator of HBV infection Can integrate into the genome of hepatocytes HBV DNA polymerase Possesses the ability of reverse transcriptase and the indicator of the ability of replication of HBV Etiology Dr.T.V.Rao MD

31. Possible Outcomes of HBV Infection Acute hepatitis B infection 3-5% of adult-acquiredinfections 95% of infant-acquired infections Chronic HBV infection Chronic hepatitis 12-25% in 5 years Cirrhosis 20-23% in 5 years 6-15% in 5 years Hepatocellularcarcinoma Liver failure Liver transplant Death Dr.T.V.Rao MD Death

32. HBsAg N. HBcAB (TOTAL) N. HBsAB N. HAV-IGM N. HCV N. NO evidence of viral hepatitis viruses. PRACTICE!!!!!!!!!!!!!!! Dr.T.V.Rao MD

33. Solving the problems • HBsAG N. • HBcAB (TOTAL) P. • HBsAB P. • HAV-IGM N. • HCV N. • PAST INFECTION. Dr.T.V.Rao MD

34. Immunized for HBV infection • HBsAg N. • HBcAB (total) N. • HBsAB P. • HAV-IGM N. • HCV N. Dr.T.V.Rao MD

35. Practice…….. • HBsAg P • HBcAB (Total) P • HBsAB N • HAV-IGM N • HCV N • MAY BE ACUTE OR CHRONIC. • Order Hep. B Core IgM to clarify. • The IgM will be positive , If Acute. Dr.T.V.Rao MD

36. Co infection with HBV, HAV, and HCV • HBsAg P • HBcAB (TOTAL) P • HBsAB N • HAV-IGM P HCV P Dr.T.V.Rao MD

37. Past infection with recovery, and then re-infection that has become chronic, this is very rare but does happen. • HBsAG P • HBcAB (total) P • HBsAB P • HAV-IGM N • HCV N • . Dr.T.V.Rao MD

38. Hepatitis B persistent infection • Persistent viral load that declines over time • HBeAg declines overtime, converting eventually to anti-HBe antibody • Seroconversion correlates with rise in LFTs and 5 order of magnitude decline in viral load. • Classically, to Anti-HBe antibody = no viral DNA circulating, which is incorrect • 0.5% clear HBsAg annually

39. Molecular Advances in Diagnosis of HBV Infection • Recent diagnostic developments including HBV genotyping and precore/core promoter assays that could well play important future roles in HBV patient management

40. Emerging tools in diagnosis of HBV infection • Current HBV DNA assays make use of differing technologies and can generally be divided into (i) signal amplification assays (liquid phase hybridization, antibody capture approach, branched DNA) and (ii) DNA amplification tests based on the polymerase chain reaction (PCR) . Signal amplification assays have sensitivities approaching 1 pg of DNA (105-106 genome copies) or even to 103 genome copies. Alternatively, HBV DNA detection based on a nested PCR approach can detect as few as 102-103 genome copies. Dr.T.V.Rao MD

41. Programme created by Dr.T.V.Rao MD for Health care workers in the Developing world • Email • doctortvrao@gmail.com Dr.T.V.Rao MD