Lab diagnosis of hepatitis

Lab diagnosis of hepatitis

1. Lab diagnosis of hepatitis b Dr Geethunath KIMS Hubbli DR CV Hima Bindu JNMC Belagavi

2. Contents Assessment and staging of HBV chronic infection Diagnosis and Management Rapid diagnostic tests ELISA/EIA Newer Techniques Whom to treat When to stop the treatment Monitoring Special situations Pregnancy Other co-morbidities

3. Assessment and Staging of HBV Chronic infection 1. Serological markers of HBV infection 2. Measurement of HBV DNA levels and 3. Assessing severity of liver disease by a. Liver enzymes b. Non-invasive tests (NITs) such as aspartate aminotransferase (AST)-to-platelet ratio index(APRI), FIB-4, transient elastography ( FibroScan ). c. Liver biopsy, if available

4. Serological markers of HBV infection   Antigen markers: HBsAg, HBeAg and HBcAg HBsAg: First marker to appear; elevated in acute, chronic or in carriers HBeAg : Indicates active viral replication and high infectivity HBcAg : Not detectable in serum; can be detected in hepatocytes by immunofluorescence test

5. Antibody markers: Anti-HBs, Anti- HBe and Anti-HBc Anti-HBc Ab: IgM indicates acute hepatitis and IgG appears in chronic hepatitis or carriers or after recovery Anti-HBs Ab: Marker of recovery or vaccination Anti- HBe Ab: Indicates low viral replication and low infectivity

6. Molecular markers: HBV DNA detection Indicates active viral replication and high infectivity Viral DNA load helps in monitoring treatment response Non-specific markers: Elevated liver enzymes and serum bilirubin; indicates clinically active infection (increase in AST and ALT values upto 1000-2000IU/L) ALT>AST The prothrombin time is the best indicator of prognosis. In patients who recover, the normalization of serum aminotransferases usually occurs within one to four months. A persistent elevation of serum ALT for more than six months indicates a progression to chronic hepatitis.

7. Serological markers of hepatitis B virus in various stages of hepatitis B virus infection Lau KCK, Burak KW, Coffin CS. Impact of Hepatitis B Virus Genetic Variation, Integration, and Lymphotropism in Antiviral Treatment and Oncogenesis. Microorganisms [Internet]. 2020;8:1470. Available from: http://dx.doi.org/10.3390/microorganisms8101470

8. Serological markers of hepatitis B virus in various stages of hepatitis B virus infection The serologic and clinical patterns observed during acute infection [4]. Hollinger FB. Hepatitis B virus infection and transfusion medicine: science and the occult. Transfusion 2008

9. Santantonio T, Fasano M,Current concepts on management of chronic hepatitis B. http//dx.doi.org/10.5772/54759.

10. Immune tolerance In the case of transmission perinatally, high levels of HBV replication is present with high levels of HBeAg and viral DNA but no evidence of liver dsease . This phase lasts for 10-30 years, during which there is slow clearance of HbeAg . Immune clearance It is usually seen in 3 rd and 4 th decades Spontaneous clearance of HBeAg is seen at rate of 10-20% annually.   Immune Control Phase This phase is characterized by HBeAg seroconversion to anti- HBe , low to undetectable HBV DNA, and normal ALT level. However, about 10–30% of HBeAg seroconversion patients still have elevated levels of ALT and HBV DNA; consequently, they are classified as HBeAg -negative CHB patients and mostly have a mutation in the core promoter or pre-core region (antibody HBe , only core) Immune Reactivation Phase Recurrence of replication of HBV DNA is seen in around 10–20% of inactive carriers after years of quiescence. Most of them have a mutation in the core promoter or pre-core region and show necroinflammation and fibrosis in liver histology

11. Modified from: Hepatitis B Virus Infection, Yun-Fan Liaw , Chia-Ming Chu, Lancet 2009; 373 : 582-92

12. HBsag Anti-HBs Anti- HBc HBeag anti-HBe Symptoms Liver enzymes DNA Interpretation + – – – – Absent Normal + Early acute hepatitis (incubating) + – IgM ++ – Present Elevated ++ Acute hepatitis B, high infectivity + – IgG ++ – Present Elevated ++ Chronic hepatitis B, high infectivity (Immunoreactive chronic hepatitis) + – IgG – + Present Elevated + Chronic hepatitis B, low infectivity (Chronic inactive hepatitis) + – IgG + – Absent Normal ++ Immunotolerant chronic HBV infection (previously called as super carriers) + – IgG – +/– Absent Normal + Chronic inactive HBV infection (previously called as simple carriers)

13. Rapid diagnostic test Rapid diagnostic tests (RDTs) are single-use disposable assays and are read visually and can give a simple qualitative result ≤ 30 minutes. They are based on immunologic principles like particle agglutination, lateral flow immunoassay, immune-filtration etc.

14. The capture antibody and control antibody will be lined on the membrane pad, while the detection antibody will be immobilized on the conjugation pad. The sample with the target analyte will be applied on the sample pad and will flow under capillary force to bind with the detection antibody on the membrane pad. In the sandwich format, the color appearing on both lines of the membrane pad will indicate a positive result and if the color only appears on the control line it indicates a negative result. As in a competitive format, the analyte-carrier molecule and secondary antibody will be lined at the test line and control lines on the membrane pad, respectively. The sample with the target analyte will flow to the membrane pad, the presence of color on the control line will indicate a positive result while the presence of color on both lines indicates a negative result Abu, N.; Mohd Bakhori , N.; Shueb , R.H. Lateral Flow Assay for Hepatitis B Detection: A Review of Current and New Assays.  Micromachines   2023 ,  14 , 1239

15. ELISA/EIA It is a solid phase enzyme immune assay based on sandwich principle. The solid phase of the microtiter plate is made of polystyrene wells coated with mouse monoclonal antibodies specific for HBsAg; whereas guinea pig polyclonal antibody purified by affinity chromatography is used to prepare the anti-HBs• peroxidase (horseradish) conjugate in the liquid-phase. When a serum or plasma specimen containing HBsAg is added to the anti-HBs antibody-coated wells together with the peroxidase conjugated anti-HBs antibody and incubated, an antibody-HBsAg-antibody-peroxidase complex will form on the wells.

16. After washing the microtiter plate to remove unbound material, a solution of TMB substrate is added to the wells and incubated. A color develops in proportion to the amount of HBsAg bound to Anti-HBs. The peroxidase-TMB reaction is stopped by addition of sulfuric acid. The optical density of developed color is read with a suitable photometer at 450 nm with a selected reference wavelength within 620 nm to 690 nm.

17. CLIA  In this technique, an enzyme converts a substrate into a chemiluminescent signal that is emitted as photons of light in the visible or near visible range. CLIA analysers are useful for the detection of serological markers of hepatitis viruses B virus (HBV) CLIA has advantages of being more reliable, precise, technically simple, shorter execution times (30–40 minutes) and high-speed throughput.

18. Nucleic acid testing Nucleic acid testing (NAT) are highly sensitive and specific in detecting the presence of viral nucleic acid It is used to identify individuals with high viral loads, to monitor disease progression and the efficacy of antiviral therapies, to detect drug resistant mutants, and to identify relapse after the discontinuation of an antiviral therapy.

19. Algorithm

20. Organisation of lab services for viral hepatitis

21. Whom to treat? The persistence of HBsAg beyond 6 months defines the person to have a chronic hepatitis B. T he people who need treatment rely upon the presence of cirrhosis, fibrosis, levels of liver enzymes and platelet count. It is recommended to use the noninvasive techniques (NIT) like APRI and FIB-4 for assessing the extent of fibrosis.

22. What to treat with Treatment for CHB in adults and their doses Tenofovir disproxil fumarate (TDF) :300mg once daily Entecavir (compensated liver disease) :0.5mg once daily Entecavir (decompensated liver disease) :1mg once daily Tenofovir alafenamide fumarate :25mg once daily

23. Monitoring the treatment 1. Monitoring for disease progression and treatment response in persons with CHB prior to, during and posttreatment 2. Monitoring for tenofovir or entecavir side-effects 3. Monitoring for hepatocellular carcinoma

24. When to stop The discontinuation of treatment of Hepatitis B is usually not recommended and should be done at specialized centers under expertise

25. Hepatitis B infection and Pregnancy All pregnant women with HBV should be evaluated for the need of treatment for hepatitis B and any associated liver disease, and given advice about prevention of transmission. Only a proportion of those with hepatitis B virus infection (pregnant or otherwise) need treatment. In the absence of prophylaxis, a large proportion of viraemic mothers, especially those who are seropositive for HBeAg , transmit the infection to their infants at the time of, or shortly after birth. The risk of developing chronic infection is 90% following perinatal infection (up to 6 months of age) but decreases to 20–60% between the ages of 6 months and 5 years.

26. Care for the baby The newborn baby should be administered a timely first dose (the ‘birth dose’) of hepatitis B vaccine (monovalent) as soon as possible after birth, ideally within 24 hours. This birth dose must be followed by timely administration of 3-doses of hepatitis B-containing vaccine [e.g. monovalent hepatitis B vaccine, tetravalent combination vaccine with DPT (DPT-Hep B) or a pentavalent vaccine ( DPT+HepB+Hib ). The hepatitis B vaccine birth dose followed by these three doses is the most effective method for prevention of mother-to child transmission of hepatitis B.

27. References The serologic and clinical patterns observed during acute infection [4]. Hollinger FB. Hepatitis B virus infection and transfusion medicine: science and the occult. Transfusion 2008 Santantonio T, Fasano M,Current concepts on management of chronic hepatitis B. Modified from: Hepatitis B Virus Infection, Yun-Fan Liaw , Chia-Ming Chu, Lancet 2009; 373 : 582-92 Abu, N.; Mohd Bakhori , N.; Shueb , R.H. Lateral Flow Assay for Hepatitis B Detection: A Review of Current and New Assays.  Micromachines   2023 ,  14 , 1239 National laboratory guidelines for testing viral hepatitis WHO guidelines on testing hepatitis b and c Technical guidelines for testing and management of hepatitis b National guidelines for testing and management of hepatitis b

28. Thank you