Slide 1: HAV HBV HCV HDV HEV SEROLOGICAL
HEPATITIS Dr.H.R.V.Rajkumar HEPATITIS A : 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 : HEPATITIS A HAV Antigen detection
Detected in stool
Expensive, No routine clinical application
Detected by IEM and EIA methods
Detected in Blood and Stool specimens
No routine clinical application
Detected by RT – PCR. HEPATITIS A : HEPATITIS A HEPATITIS B : HEPATITIS B HEPATITIS B : 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 : 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 HBsAg and Anti HBs. These antibodies are heterotypic and likely not protective. HEPATITIS B : 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 : 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 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.
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 : HEPATITIS B Antibody to Hepatitis B ‘e’ antigen (Anti HBe)
Detectable when HBeAg disappears (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 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 : 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.
Signal amplification: Hybrid capture & bDNA
Target amplification: PCR & TMA. HEPATITIS B : HEPATITIS B Detection Limit Relative Value Months since exposure 0 1 2 3 4 5 6 12 18 24 Symptoms IgM anti- HBc Total Anti- HBc HBsAg Increased ALT Anti- HBs SEROLOGY OF ACUTE HEPATITIS B HEPATITIS B : HEPATITIS B 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 : HEPATITIS C Anti HCV antibody
Fourth generation ELISA (Antigens: NS3 + NS4 + Core Protein + NS5)
Sensitivity – 100% in Immunocompetent and 50 – 95% in Immunosuppressed and Hemodialysis
Specificity – 99%
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 : 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 : HEPATITIS C HCV RNA – QUALITATIVE
HCV replicates at relatively low levels and viral genomes may be present only in small amounts.
So amplification is necessary.
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 : 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 : 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 : HEPATITIS C HCV Core antigen detection
Useful for screening in window period.
Detected 1 – 2 days after HCV RNA positivity.
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 : HEPATITIS C SEROLOGY OF ACUTE HCV HEPATITIS D : 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 : 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 : HEPATITIS D HDV Antigen (Delta antigen)
Present during the late incubation period of acute infection and lasts for 21 – 45 days.
Detected in Serum
By RT PCR. HEPATITIS E : 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 : HEPATITIS E HEV Antigen detection
Detected in Serum and Liver by IFA technique.
No routine clinical application.
Detected in Serum and Stool
By RT PCR
Detected in acute phase faeces in approximately 50% of cases. Slide 29: Thank You