Diagnosis of Hepatitis B Infection

Hepatitis b infection diagnosis and interpretation: 

Dr.T.V.Rao MD Hepatitis b infection diagnosis and interpretation Dr.T.V.Rao MD 1

Hepatitis B In the World : 

Hepatitis B In the World 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. Dr.T.V.Rao MD 2

Prevalence of Hepatitis B carriers: 

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 5 th edition, 2005, Chapter 66, published by Mosby Philadelphia,,

1、Properties of HBV: 

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

Hepatitis B: 

Hepatitis B Diagrammatic representation of the hepatitis B virion and the surface antigen components EM of Hepatitis B viron 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

Hepatitis b virus a major cause of hepatitis: 

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

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 HEPATITIS B Dr.T.V.Rao MD 7

Structure of hepatitis b virus: 

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

HBV Structure & Antigens : 

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 9

Surface particles: 

Surface particles 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. Dr.T.V.Rao MD 10

PowerPoint Presentation: 

HEPATITIS B 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 Dr.T.V.Rao MD 11

PowerPoint Presentation: 

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. Open Reading Frames Dr.T.V.Rao MD 12

Hbv – serology interpretation: 

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

Etiology: 

Etiology 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 Dr.T.V.Rao MD 14

Hepatitis B virus – e antigen: 

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

Three antigen-antibody system : 

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 16

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 Clinical outcomes of Hepatitis B infections From Murray et. al., Medical Microbiology 5 th edition, 2005, Chapter 62, published by Mosby Philadelphia,,

Determinants or acute and chronic HBV infection : 

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

Screening – Who?: 

Screening – Who? 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.

Serological markers : 

Serological markers Dr.T.V.Rao MD 20

HEPATITIS B MARKERS:: 

HEPATITIS B MARKERS: 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. Dr.T.V.Rao MD 21

Etiology: 

Etiology 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 Dr.T.V.Rao MD 22

PowerPoint Presentation: 

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

PowerPoint Presentation: 

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

PowerPoint Presentation: 

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

Etiology: 

Etiology 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 Dr.T.V.Rao MD 26

PowerPoint Presentation: 

HEPATITIS B 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) Dr.T.V.Rao MD 27

Etiology: 

Etiology 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 Dr.T.V.Rao MD 28

PowerPoint Presentation: 

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

Etiology: 

Etiology 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 Dr.T.V.Rao MD 30

PowerPoint Presentation: 

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

PRACTICE!!!!!!!!!!!!!!!: 

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

Solving the problems: 

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

Immunized for HBV infection : 

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

Practice……..: 

Practice…….. Dr.T.V.Rao MD 35 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.

Co infection with HBV, HAV, and HCV : 

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

Past infection with recovery, and then re-infection that has become chronic, this is very rare but does happen. : 

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 37

Hepatitis B persistent infection: 

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

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 Molecular Advances in Diagnosis of HBV Infection 39

Emerging tools in diagnosis of HBV infection: 

Emerging tools in diagnosis of HBV infection Dr.T.V.Rao MD 40 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 (10 5 -10 6 genome copies) or even to 10 3 genome copies. Alternatively, HBV DNA detection based on a nested PCR approach can detect as few as 10 2 -10 3 genome copies.

PowerPoint Presentation: 

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 41