Viral markers

Overview of Viral Markers

1. VIRAL MARKERS Presented by: Dr Deepak Mishra Moderated by: Dr Nishant Sharma

2. Hepatitis: Inflammation of the liver, characterized by the presence of inflammatory cells in the tissue of the organ VIRAL HEPATITIS : Infection of liver caused by hepatotropic viruses Hepatitis viruses A, B, C, D, and E Cytomegalovirus, Epstein–Barr virus , Yellow fever virus

3. Hepatitis: Global burden of disease About 1 million people die each year from causes related to viral hepatitis ( 2.7% of all deaths) An estimated 57% of liver cirrhosis and 78% of primary liver cancer are due to HBV or HCV infection HBV related disease: About 2 billion people have been infected About 600 000 people die each year HCV related disease: About 150 million people are chronically infected with HCV (about 10 times higher than HIV estimates) More than 350 000 people die each year WHO (2013). Media Centre. Hepatitis C Fact Sheet No 164. Updated July 2013; WHO (2013) Media Centre. Hepatitis B Fact sheet No 204. Updated July 2013 . WHO (2013). Immunization, Vaccines and Biologicals. Hepatitis.

4. Hepatitis B and C : WHO European Region Hepatitis B and C each is estimated to affect up to 2% of the population in the Region 13.3 million people living with chronic hepatitis B, 15 million with chronic hepatitis C T ogether , they cause over 120 000 deaths per year Two-thirds of infected persons live in eastern Europe and central Asia Co-infection of HCV and HIV is common, especially among people who inject drugs Only 1 in 5 persons exposed to the hepatitis B and C virus develops acute symptoms, but chronic infection is common Hope VD et al. (2013). Prevalence and estimation of hepatitis B and C infections in the WHO European Region: a review of data focusing on the countries outside the European Union and the European Free Trade Association WHO Regional Office for Europe. Hepatitis. Hope VD et al. (2013). Prevalence and estimation of hepatitis B and C infections in the WHO European Region: a review of data focusing on the countries outside the European Union and the European Free Trade Association WHO Regional Office for Europe. Hepatitis . . www.euro.who.int/hepatitis .

5. Self limiting disease No evidence of chronic disease Average incubation period of 28 days ( 15 – 50 days) Nonspecific constitutional signs, symptoms( usually last 2 months) No evidence of chronic liver disease or persistent infection Rare cause of Acute liver failure 6.8% of all ALF cases(US)

6. HAV: Structure Virus: Genus – Hepatovirus , Picornaviridae family Nonenveloped ,single stranded RNA virus 27- 32 nm diameter,Icosahedral symmetry Unlike other members of the family Requires a long adaptation period to grow in cell culture:Replicates slowly Rarely produces a cytopathic effect More resistant to heating and chlorine inactivation Seven genotypes with unique geographical distribution Only a single serotype of HAV exists

7. HAV Viral markers: IgM anti- HAV H as been used as the primary marker of acute infection Comprised mainly of antibodies against capsid proteins Positive at the time of onset of symptoms Usually accompany the first rise in the alanine aminotransferase (ALT) level

8. HAV Viral markers: IgM anti- HAV Methods used: Radioimmunoassay Immunochemical staining Enzyme-linked immunosorbent assay Immunoblotting Dot blot immunogold filtration Remain positive for 3-6 months after the primary infection and for as long as 12 months in 25% of patients

9. HAV Viral markers: IgG anti- HAV Appears soon after IgM and generally persists for many years The presence of anti-HAV IgG in the absence of IgM indicates past infection or vaccination rather than acute infection IgG provides protective immunity Antibodies to structural proteins are produced following immunization with hepatitis A vaccine A small proportion ( 8to 20%) of vaccinated persons have a transient IgM anti-HAV response IgG anti-HAV is produced by all successfully immunized persons Commercially available tests for total anti-HAV are not sensitive enough to detect antibody concentrations in a significant proportion of immunized persons

10. HAV Viral markers IgG and IgA anti-HAV are also detected in saliva , urine , and and feces Saliva tests have been reported as an alternative to conventional serum testing as a screening tool in outbreak investigations and epidemiological studies However , the sensitivity of detecting anti-HAV in saliva is 1 to 3 log 10 units lower than that with serum

11. HAV Viral markers: Molecular diagnosis Viremia occurs within 1 to 2 weeks after HAV exposure and persists through the period of liver enzyme elevation HAV RNA detection is the most sensitive technique for screening clinical specimens( serum, plasma, saliva, fecal suspension and environmental samples ) Can be detected in blood earlier than antibodies Viremia may be present for a much longer period during the convalescent phase Serum: 10 2  to 10 5   copies/ml Stool: High viral load( 2 to 3 log10 units higher than serum),detected until 81  and 90 days Saliva: 1 to 3 log10 units lower than that found in serum

12. HAV Viral markers: Molecular diagnosis Patients with severe infection: Higher initial viral load than patients with less severe infection The molecular diagnosis of hepatitis A is not used in clinical laboratories and blood banks, as is currently done for HIV, HBV and HCV infections

13. Belongs to Hepadnavirus family : Dane particle Outer lipid envelope : Embedded proteins involved in viral binding Nucleocapsid : Encloses the viral DNA 8 genotypes, 4 serotypes

14. Genotype A,D : USA, Europe B,C : Asia A,E,G: Africa Genotype B : less severe disease,less HCC than Genotype C

15. Serology for HBV: Antigen Antibodies Hepatitis B Surface Antigen (HBsAg) Antibody to Hepatitis B Surface Antigen (Anti HBs) Hepatitis B Core Antigen (HBcAg) Antibody to Hepatitis B Core Antigen (Anti HBc IgM & Anti HBc) Hepatitis B ‘e’ Antigen (HBeAg) Antibody to Hepatitis B ‘e’ Antigen (Anti HBe)

16. 1 st virological marker detectable in serum usually between 8 th and 12 th weeks of infection It preceeds elevation of aminotransferase activity and clinical symptoms by 2-6 weeks It remains elevated during the entire icteric or symptomatic phase of the disease. Typically, it disappeares 1-2 months after the onset of jaundice and rarely persists beyond 6 months. Hepatitis B Surface antigen ( HBsAg )

17. AASLD: Chronic Hepatitis B is defined as HBsAg positivity for more than six months Chronic HBV Infection

18. Strategies for prevention of HBV is based on providing susceptible persons with anti HBs The protective a n tibo d y Anti HBs HBsAg prepared by r ec o mbina n t DNA technology A lmost indefinite protection B ooster required after 5 y r s Hepatitis B surface antibody (Anti HBs)

19. Hepatitis B core antigen Expressed on the surface of the nucleocapsid core Not secreted and remains within hepatocytes Anti HBc IgM First antibody to appear Indicates acute HBV infection May be the only marker in core window Anti HBc IgG : Persists along with A nti HBs in patients who recover from acute infection HBsAg in patients who progress to chronic HBV infection

20. HBeAg Secreted into the circulation Not essential for replication in vivo Acces s ory protein of HBV An index of viral replication, infectivity, severity of disease, and response to treatment High probability of progression to a chronic carrier state when HBeAg persists longer than 12 weeks Pregnant women withHBeAg positive have a risk of transmission of virus to fetus is > 90%. Hepatitis B e antigen

21. Hepatitis B e antibody (Anti Hbe ) Detectable when HBeAg disappears (12 - 16 wks ) HBeAg is a marker of replicative infection Seroconversion to Anti HBe indicates resolution of infection( switch from active state to inactive carrier state) HBeAg seroconversion is an important therapeutic milestone and goal

22. HBV DNA Direct product & hallmark of infection Measure of virus replication in the liver and infectivity Liver disease progression intrinsically linked to extent of viral replication Clinical application: M anagement of HBV carriers Identify of disease progression Select candidates for antiviral therapy Guide treatment with nucleoside/nucleotide analogues Loss of detectable HBV DNA is an earlier indicator of response to antiviral therapy than loss of HBeAg

23. Immune T olerant C learance C ontrol E scape status (healthy carrier)

24. About 180 million people are infected with hepatitis C virus 3 percent of global population 3-4 million people become infected with HCV annually About 25 million people are infected in Europe HCV prevalence 5 times exceeds HIV prevalence

25. Infection remains chronic in 70-85% of infected individuals. In 20-40% cases ,C hronic hepatitis C infection leads to end stage liver diseases: Cirrhoses , hepatocel lular carcinoma and liver failure after 20-30 years of i nfection

26. Anti HCV Anti HCV HCV RNA qualitative and quantitative HCV RNA qualitative and quantitative Serologic and virologic markers of H C V infection

27. Major laboratory methods for HCV diagnosis: 1984- ELISA 1985 -Western blot 1995 -Qualitative and quantitative PCR 2003 -HCV genotyping 2007- HCV quantit . test ( using Real Time PCR) 2010 -HCV NS5B and 5’UTR/Core region sequencing

28. HCV : Antibody testing Indicated for standard testing/ screening A negative test rules out chronic infection Techniques EIA : Most sensitive Rapid Immunoassay tests: PoC testing Recombinant Immunoblot assay No further testing indicated

29. HCV : RNA assays Indicated for confirming presence or absence of infection, monitor response to treatment Not a measure of severity of disease Techniques PCR based methods: Detect RNA at 25 IU/ml TMA based methos : Detect RNA at 10 IU/ml Signal amplification technology: Technically easier, less sensitive ( Detect RNA at 615 IU/ml)

30. Quantitative tests : Used before treatment to measure baseline viral load and to assess on treatment response to therapy Qualitative tests: Capable of detecting low levels of HCV RNA Indicated for confirming the diagnosis

31. A L T

32. 85%

33. Interpretation of HCV assays Anti HCV HCV RNA interpretation + + Acute or chronic HCV depending upon clinical context + -- Resolution of HCV -- + Early acute HCV, chronic HCV in immunosuppressed states -- -- Absence of HCV

34. VIRION : D efective virus which shows similarities with the viroids in plants Spherical, C onsists of a particle 35 nm in diameter consisting of the delta antigen surrounded by an outer coat of HBsAg HBV capsid HDV nucleoprotein NUCLEIC ACID: (-) ss RNA, circular Satellite virus : replicates only in the presence of HBV

35. 63 Consequences of hepatitis B and delta virus infection L ow risk of chronic infection Severe acute disease Usually develop chronic infection High risk of severe CLD May present as an acute hepatitis

36. Presence of HBsAg is necessary for diagnosis of HDV infection Additional presence of IgM anti H Bc is necessary for diagnosis of acute HBV/HDV coinfection

37. Anti HDV antibody Total anti HDV antibody Appears after four weeks of acute infection Repeated testing required May be the only way to diagnose acute HDV infecton IgG anti HDV High titre present in chronic infection Correlates well with ongoing HDV replicationAdditional presence of IgM anti H Bc is necessary for diagnosis of acute HBV/HDV coinfection IgG anti HDV Rarely used ,not approved for clinical use

38. HDVAg Acute Infection: Appears early Detection by EIA is short lived Chronic Infection: Present in high titres Serum HDV RNA Can be detected by RT-PCR based assays May detect viral loads of as low as 10 genomes/ml Primary end point of treatment: Suppression of HDV RNA

39. Calicivirus -like viruses U nenveloped RNA virus, 32-34nm in diameter + ve stranded RNA genome, 7.6 kb in size V ery labile and sensitive

40. Anti HEV IgG Appears shortly after IgM response Titre increases throughout the acute phase into convalescent phase Discordance between assays present Anti HEV IgM Appears during the early phase of clinical illness Disappears rapidly over four to five months Low sensitivity

41. HEV RNA assay Stool: Detected approximately one week before the onset of illness Can persist as long as two weeks thereafter Serum: Detected two to six weeks after infection Can persist for two to four weeks in those who resolve acute infection Primary end point of treatment: Disappearance of RNA

42. A L T Ig G a n t i - H E V Ig M a n t i - H E V V i r u s i n s t o o l Typical Serologic Course Symptoms T i t e r 0 1 2 3 4 5 6 7 8 9 10 11 1 2 13 Weeks after Exposure

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