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, andE
Cytomegalovirus, Epstein–Barr
virus, Yellow fever virus

5. 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. UpdatedJuly 2013;
WHO (2013) Media Centre. Hepatitis B Fact sheet No 204. Updated July 2013.
WHO (2013). Immunization, Vaccines and Biologicals.Hepatitis.

6. 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
 Together, 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 FreeTrade 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 FreeTrade Association
WHO Regional Office for Europe. Hepatitis. . www.euro.who.int/hepatitis
.

7. 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)

8. 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

9. HAV Viral markers: IgM anti- HAV
 Has 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

10. 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

11. 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

12. 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 log10 units lower than that with serum

13. 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: 102 to 105 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

14. 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

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

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

18. 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)

20. 1st virological marker
detectable in serum usually
between 8th and
12th 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)

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

22. Strategies for
prevention of HBV is
based on providing
susceptible persons
with anti HBs
The
protective
antibody
Anti HBs
HBsAg prepared
by recombinant
DNA technology
 Almost indefinite protection
 Booster required after 5 yrs
Hepatitis B surface antibody (Anti HBs)

24. 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
Anti HBs in patients who recover from acute infection
HBsAg in patients who progress to chronic HBV infection

26. HBeAg
 Secreted into
the circulation
 Not essential for
replication in vivo
 Accessory
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

27. 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

29. HBV DNA
 Directproduct & hallmarkof infection Measure of virus
replication in the liver and infectivity
 Liverdiseaseprogression intrinsically linked to extent of viral replication
 Clinicalapplication:
 Management of HBVcarriers
 Identify of diseaseprogression
 Selectcandidatesfor antiviraltherapy
 Guidetreatment with nucleoside/nucleotideanalogues
 Loss of detectable HBV DNA is an earlier indicator of response to
antiviral therapy than loss of HBeAg

32. Immune Tolerant Clearance Control Escape
status (healthy carrier)

35.  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

36.  Infection remains chronic in 70-85% of infected individuals.
 In 20-40% cases ,Chronic hepatitis C infection leads to end stage
liver diseases: Cirrhoses, hepatocellular carcinoma and liver failure
after 20-30 years of infection

37. Anti HCVAnti HCV HCV RNA
qualitative and quantitative
HCV RNA
qualitative and quantitative
Serologic and virologic markers of HCV infection

38.  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

39. 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

40. 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)

41.  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

42. ALT

45. 85%

48. Interpretation ofHCV
assays
Anti HCV HCVRNA interpretation
+ + Acute or chronic HCVdepending
upon clinical context
+ -- Resolution of HCV
-- + Early acute HCV,chronic HCVin
immunosuppressed states
-- -- Absenceof HCV

50.  VIRION: Defective virus which shows
similarities with the viroids in plants
 Spherical, Consists 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

51. 63
Consequences of hepatitis B and delta virus infection
 Low risk of chronic infection
 Severe acute disease
 Usually develop chronic infection
 High risk of severe CLD
 May present as an acute hepatitis

52. Presence of HBsAg is necessary for diagnosis
of HDV infection
Additional presence of IgM anti HBc is
necessary for diagnosis of acute HBV/HDV
coinfection

53. 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 HBc is necessary for diagnosis of acute HBV/HDV
coinfection
IgG anti HDV
 Rarely used ,not approved for clinical use

54. 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

58.  Calicivirus-like viruses
 Unenveloped RNA virus, 32-34nm in diameter
 +ve stranded RNA genome, 7.6 kb in size
 Very labile and sensitive

61. 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

62. 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

63. A LT IgG anti-
H E V
Ig M a n ti-
H E V
V ir u s in
s t o o l
Typical Serologic Course
Symptoms
Titer
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Weeks after Exposure