HAV HBV

HCV

HDV
HEV
SEROLOGICAL
MARKERS FOR
VIRAL
HEPATITIS
 HEPATITIS A

• IgM Antibody to Hepatitis A (Anti HAV IgM)

– Positive result indicates recent acute HAV infection.
– Present for 3-6 months after onset.
– In patients with relapses can persist for more than 12
months.
– Detected by RIA and ELISA methods


• Total antibody to Hepatitis A (Anti HAV Ab)

– Past infection and immunity to HAV.
– Individuals given Serum Ig for HAV prophylaxis may test
positive for 6 months.

 HEPATITIS A

• HAV Antigen detection

– Detected in stool
– Expensive, No routine clinical
application
– Detected by IEM and EIA methods


• HAV RNA
– Detected in Blood and Stool
specimens
– No routine clinical application
– Detected by RT – PCR.
HEPATITIS A
 HEPATITIS B

SEROLOGICAL MARKERS FOR HBV INFECTION

ANTIGENS 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)

Hepatitis B ‘x’ Antigen (HBxAg) Antibody to Hepatitis B ‘x’ Antigen (Anti HBx)
 HEPATITIS B

• Hepatitis B Surface Antigen (HBsAg)

– Used to diagnose acute or chronic infection.
– First marker to appear in an acute infection. Detected
approx. 6 weeks after infection.
– Disappearance indicates recovery from infection.
– Persistence for > 6 months indicates chronic infection
(Carrier state)
– Individuals tested within 72 hours after administration of
vaccine may test as positive.
 HEPATITIS B

• Antibody to Hepatitis B Surface antigen (Anti HBs)

– Only test to assess presence of protective immunity after
immunization with Hepatitis B vaccine.
– Levels of > 10 mIU / ml are protective.
– Positive result in individuals with recent acute HBV infection
indicates convalescence.
– Some cases of Chronic HBV infection may have both
HBsAgand Anti HBs. These antibodies are heterotypic
and likely not protective.
 HEPATITIS B

• IgM antibody to Hepatitis B core antigen (Anti HBcIgM)

– First antibody to appear.
– Indicates acute HBV infection.
– Detectable for 3 – 12 months.
– May be the only marker in “CORE WINDOW”.
– May also become detectable during an exacerbation of
Chronic hepatitis.
– Not helpful to differentiate between acute HBV infection
and reactivation of chronic HBV infection.
 HEPATITIS B

• Total antibody to Hepatitis B core antigen

(Anti HBc)
– Indicates past infection with HBV.
– Persists for life.
– Absent in individuals who are immune solely as a
result of vaccination.
– Up to 10% false positive rates has been described
in individuals with no documented infection to
HBV.

 HEPATITIS B

• Hepatitis B ‘e’ antigen (HBeAg)

– Marker of active HBV replication. Indicates
high infectivity.
– Develops 1 week after HBsAg is detectable.
– Correlates well with the level of infectivity,
the quantity of virus present and the
presence of viral DNA polymerase in the
serum.
 HEPATITIS B

• Hepatitis B ‘e’ antigen (HBeAg)

– High probability of progression to a chronic carrier
state when HBeAg persists longer than 12 weeks.
– If a pregnant woman is HBeAg positive than the risk of
transmission of virus to fetus is > 90%.
– Can be used to monitor therapy of patients with
chronic HBV infection.
– Mutant strains replicate without producing HBeAg.
 HEPATITIS B

• Antibody to Hepatitis B ‘e’ antigen (Anti-HBe)

– Detectable when HBeAgdisappears (12 – 16 wks).
– Seroconversion to Anti HBe indicates resolution of infection.
– In chronic HBV infection, indicates resolving or minimal liver
disease and low contagiousness.
– Individuals who are HBsAg positive and have Anti HBe must
still be considered infectious.
– Does not imply immunity to infection.
 HEPATITIS B

• Hepatitis B Core antigen (HBcAg)

– Not detectable in the blood stream.
– HBcAg peptides are expressed on the surface of
hepatocytes and can be demonstrated by IF.
– Marker of the infectious viral material and it is the most
accurate index of viral replication.


• HBV DNA Polymerase

– Detectable with in 1 week of initial infection.
– Mutations are associated with treatment failure.
 HEPATITIS B

• HBV DNA
– Detectable as soon as 1 week after infection.
– Used to detect mutants.
– HBV DNA in circulating blood is a measure of virus
replication in the liver and infectivity.
– Helpful in monitoring treatment in patients with chronic
HBV infection.
– Loss of detectable HBV DNA is an earlier indicator of
response to antiviral therapy than loss of HBeAg.
– Detection methods
• Signal amplification: Hybrid capture & bDNA
• Target amplification: PCR & TMA.
 HEPATITIS B
SEROLOGY OF ACUTE HEPATITIS B
Symptoms

Increased ALT
Total Anti-HBc

Relative Value

HBsAg

IgM Anti-HBs
anti-HBc

Detection
Limit

0 1 2 3 4 5 6 12 18 24

Months since exposure

HEPATITIS B
 HEPATITIS B

• Discordant or Unusual profiles

– HBsAg – Pos / Anti HBc – Neg
– HBsAg – Pos / Anti HBs – Pos / Anti HBc – Pos
– Anti HBc – Positive only
– HBeAg – Pos / Anti HBe – Pos
– HBeAg – Pos / HBsAg – Neg
– Anti HBs – Positive only in a non immunized person
 HEPATITIS C

• Anti HCV antibody

– Screening tests
• Fourth generation ELISA (Antigens: NS3 + NS4 +
Core Protein + NS5)
• Sensitivity – 100% in Immunocompetent and 50 –
95% in Immunosuppressed and Hemodialysis
• Specificity – 99%
– Supplementary tests
• RIBA and LIA: Modification of W.Blot.
• More specific and Less sensitive
• Anti HCV will not differentiate between acute and
chronic HCV infection.
•
 HEPATITIS C

• Anti HCV IgM

– Positive in 50 – 93% of acute HCV and in 50
– 70% of chronic HCV infection.
– So cannot be relied upon for acute HCV.
 HEPATITIS C

• HCV RNA – QUALITATIVE

– Hybridization methods
• Not useful
• HCV replicates at relatively low levels and viral genomes may
be present only in small amounts.
• So amplification is necessary.
– Amplification methods
• Polymerase chain reaction (RT-PCR)
• Transcription mediated amplification (TMA)
– Single Qualitative Positive test – Active viral replication
– Single Qualitative negative test – Reflects only a viral
load below the detection limit of the assay – Repeat
test.
 HEPATITIS C
• HCV RNA – QUANTITATIVE
– Target amplification methods – RT-PCR
• Cut-off: 1000 copies / ml
– Signal amplification methods – bDNA assay
• Cut-off: 200,000 copies / ml
– Real-Time PCR – Best method
– Quantification is done only for genotypes 1, 4, 5
and 6. For genotypes 2 and 3 only Qualitative
analysis is done.
 HEPATITIS C

• HCV Genotyping
– 6 genotypes and numerous subtypes
– Genotyping – Direct sequencing of the NS5B or E1
region.
– Methods – RFLP, Nested PCR, Reverse Hybridization,
Direct sequencing INNO-LIPA, Serological
detection of antibodies to genotype specific HCV
epitopes.
– Sub typing is not necessary for treatment purposes.
 HEPATITIS C

• HCV Core antigen detection

– Useful for screening in window period.
– Detected 1 – 2 days after HCV RNA positivity.
– Low sensitivity.
– Recently Immune complex dissociation EIA is done
• Detection limit of antigen – 2 pg / ml
• 1 pg / ml of HCV Core antigen = 8000 HCV RNA IU / ml
– Total HCV Core antigen quantification can be used as an
alternate to HCV RNA PCR for treatment purposes.
 HEPATITIS C
SEROLOGY OF ACUTE HCV

Symptoms

HCV RNA
Relative Value

ALT Anti-HCV

Detection
Limit

0 1 2 3 4 5 6 12 18 24

Months since exposure

 HEPATITIS D

• Anti HDV IgM and IgG antibodies

– Acute HBV-HDV Co infection
• HBsAg – Pos, Anti HBc IgM – Pos
• Anti HDV IgM – Positive
• Anti HDV IgG antibodies develop late in acute
phase and usually decline after infection to
sub detectable levels.
• Both IgM and IgG disappear with in months to
years after recovery.

•
 HEPATITIS D


– HBV-HDV Super infection

• HBsAg – Pos, Anti HBc IgM – Neg
• Results in persistent HDV infection.
• High titres of IgM and IgG anti HDV are
detectable in acute phase, persisting
indefinitely.
• Progression to chronicity is associated
with persisting high levels of anti HDV
IgM and anti HDV IgG.

 HEPATITIS D

• HDV Antigen (Delta antigen)

– Present during the late incubation
period of acute infection and lasts
for 21 – 45 days.


• HDV RNA
– Detected in Serum
– By RT PCR.
 HEPATITIS E

• IgM Antibody to HEV (Anti HEV IgM)

– Acute HEV infection. Indicates recent exposure to HEV.
– Titres decline rapidly during early convalescence.
• IgG Antibody to HEV (Anti HEV IgG)
– Indicates immunity and Old infection.
– Persists for long periods of time. Sometimes up to > 14
years.
 HEPATITIS E

• HEV Antigen detection

– Detected in Serum and Liver by IFA
technique.
– No routine clinical application.
• HEV RNA
– Detected in Serum and Stool
– By RT PCR
– Detected in acute phase faeces in
approximately 50% of cases.