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Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201). Laboratory Diagnostics in Hepatitis. Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst , Alex Univ E-mail: elgreatlyem@hotmail.com. Objectives. Define some clinical terms of hepatitis

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Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

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  1. Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

  2. Laboratory Diagnostics in Hepatitis Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com

  3. Objectives • Define some clinical terms of hepatitis • know different types of viral hepatitis • Review the serologic diagnosis of viral hepatitis • Review the methods available for molecular testing

  4. Clinical Terms • Hepatitis: inflammation of liver • Acute Viral Hepatitis: symptoms last less than 6 months • Chronic Hepatitis: Inflammation of liver for at least 6 months • Cirrhosis: Replacement of liver tissue fibrosis, scar tissue • Fulminant Hepatitis: severe impairment of liver functions

  5. Viral Hepatitis • Hepatotropic viruses • Hepatitis A, B, C, D, E and G viruses • Generalized infection plus infection of liver • EBV, CMV and HSV

  6. Some basic serology… • Presence of Viral Proteins/Nucleic acids (mostly called ‘antigens’) indicates: • Virus is present • Virus might be replicating • Presence of antibodies to Viral proteins indicares • Virus may be currently present (or not) • Could indicate either immunity or ongoing infection

  7. Hepatitis A infection • RNA virus • Fecal-oral transmission • Usually self-limited illness • No carrier state • In rare cases, fulminant hepatic necrosis

  8. Hepatitis A - Diagnosis • Three serologic markers available: • Hepatitis A Total (IgG and IgM) antibody • Hepatitis A IgM • Hepatitis A IgG • First tests available since 1978 • No antigen test • Antibody response is similar following vaccination or infection • Incubation time is 7 to 28 days

  9. Diagnosis of hepatitis A • IgM anti-HAV: • appears 4 wks after exposure and disappears by 3 -6 months. • Indicates acuteinfection • IgG anti-HAV: • peaks during convalescence and persists for life. • Indicates exposure or immunity

  10. Hepatitis A Virus Infection Typical Serologic Course Symptoms Total anti-HAV Liver Enzymes Titer Fecal HAV IgM anti-HAV 4 5 6 12 24 0 1 2 3 Months after Exposure

  11. Hepatitis B virus infection • Transmission • Parenteral (injections, blood transfusion ….) • Clinical: • 25% acute hepatitis, 1% fulminant hepatic necrosis • 10% chronic carriers • Incubation period of 1-6 mo.

  12. Laboratory Tests for HBV • Serology: • Many tests available [most common tests are Enzyme Immunoassays (ELISA)] • For every rule, there is an exception/caveat • No single test tells you everything • Molecular: • HBV DNA (quantitative) • HBV genotyping • HBV resistance testing

  13. Hepatitis B – Laboratory Tests Serologic markers: • 1) HBsAg (Hepatitis B surface antigen): • if positive, person is infectious • Sensitivity = 0.15 ng/ml • Specificity = 99.5% • 2) Anti-HBs (Antibody to HBV surface antigen): • indicates immunity to HBV and protection from disease • Protective level is >10 IU/ml

  14. Hepatitis B – Laboratory Tests Serologic markers (cont.): • 3)Anti - HBc (Antibody to HBV core antigen): • Total - indicates past or active infection; present whether person is immune or chronic carrier • Specificity = 99.8% to 99.9% • IgM - early indicator of acute infection • No antigen test

  15. Hepatitis B – Laboratory Tests Serologic markers (cont.): • 4) HBeAg (Hepatitis Be antigen): • indicates person is highly infectious • Selecting patients for therapy • 5) Anti-HBe (Antibody to HBVe antigen): • prognostic for resolution of infection; • less infectious;

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

  17. Hepatitis B – Laboratory Tests Serologic markers – caveats: • Persistent HBsAg for >6 mos = chronic infection • HBsAg and anti-HBs may co-exist in up to 24% of chronically infected individuals; • Anti-HBc IgM may persist for up to 2 years in 20% of chronically infected individuals

  18. Hepatitis C infection • Enveloped RNA virus • Parenteral infection • 60-85% get chronic infection • Treatment with interferon+ribavirin cures virus in only 25-40%

  19. Sources of infection for persons with newly-diagnosed Hepatitis C Injection drug use 60% Sexual 15% Transfusion 10% (before screening) * Nosocomial Health-care work Perinatal Other* 5% Unknown 10% CDC

  20. Laboratory Tests for HCV Serology: • Detection of anti-HCV antibodies • Serologic test available since 1990 Molecular: • HCV RNA detection • Determination of HCV genotype • Viral load determination

  21. Laboratory Tests for HCV Serology: • Screening: • 3rd generation ELISA measure antibodies directed against virus peptides • Sensitivity = 97% • Detects antibodies within 6 to 8 weeks • No HCV IgM test available • Confirmatory/supplementary: • RIBA, Line Probe Assay (LiPA), Second EIA, HCV RNA

  22. Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection anti-HCV Symptoms +/- HCV RNA Titer Liver Enzymes Normal 6 1 2 3 4 0 1 2 3 4 5 Years Months Time after Exposure

  23. Hepatitis D infection • small RNA virus that needs HBV to survive • Only occurs in the presence of HBV • Test for D if suspicion that it might be a cause of disease exacerbation in chronic hepatitis B • Incubation time – similar to Hepatitis B

  24. Hepatitis D tests • Available only at National Microbiology Labs. • HDV Ag • Present only during prodrome, not tested for • Anti-HDV IgM • Acute infection • Anti-HDV IgG • Appear during convalescence • But remain elevated in carriers • High titres of HDV antibodies indicate ongoing chronic infection

  25. Hepatitis E infection • RNA virus • Present in animals without causing disease • Fulminant hepatic necrosis in pregnant women (case fatality rate is 10-50%) • Incubation period – 7 to 28 days

  26. Hepatitis E Virus - Diagnosis • IgM antibodies to HEV, • HEV RNA assay • Both IgG and IgM antibody tests are available • Available only at the National Microbiology Labs.

  27. Hepatitis G virus • Hepatitis G virus is closely related to HCV • Common in HCV infected patients • Mode of transmission: ?parenteral • Role in human disease is controversial. Usually mild acute or chronic hepatitis.

  28. Acute Hepatitis Infection • IgM-HAV • HBsAg • IgM anti-HBc • Anti-HCV

  29. Molecular Tests for Hepatitis

  30. Hepatitis Virus – Molecular Tests • Molecular assays available as follows: • Commercial assays for HBV DNA and HCV RNA • In-house assays for HAV RNA & HDV RNA • No molecular assay for HEV RNA • HCV RNA & HBV DNA, • plasma or serum must be separated from cells within 6 hrs and plasma can be stored at 4oC for several days or -70oC for long-term • No licensed tests for diagnostic purposes • all tests are for monitoring or donor screening

  31. Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNA • Quantitation of RNA or DNA may be reported as copies/ml or IU/ml • Conversion factor for copies/ml to IU/ml is not the same for different assays measuring the same target or different targets • HBV DNA: 5.82 copies/IU • HCV RNA: PCR - 2.4 copies/IU; bDNA: 5.2 copies/IU

  32. HCV RNA Detection Assays *LLD = Lower Limit of Detection; *TAM= Transcription-mediated amplification aConversion factor IU/ml to copies/ml varies with each assay (e.g. PCR: 1 IU/ml = 2.4 copies/ml; bDNA: 1IU/ml = 5.2 copies/ml) S. Chevaliez et al. World J Gastro 2007;13; J Scott et al. JAMA 2007;297; A. Caliendo et al. J Clin Microbiol 2006;44

  33. HBV DNA in Clinical Practice • Routine monitoring on therapy to assess response to treatment • Every 3 months X years on oral agents • Every 1 month X 6-12 on PEG/IFN • Routine monitoring off therapy to estimate prognosis and to evaluate need for treatment • Every 6 –12 months normally • Diagnosis of occult HBV infection

  34. MolecularLaboratory Tests for HCV • Used for treatment monitoring (and in somecircumstances for confirmation of positive or indeterminateserology) • HCV RNA isdetectable 2 to 14 daysafter an exposure

  35. Study Questions: • Write a short note on: Serologic markers of HBVlaboratory tests.

  36. Assignment • Write shortly on serological diagnosis of HBV. وليد على – يمنى عبد الله – خلود عبد القادر – دنيا محمد – دينا عطية

  37. THANK YOU

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