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

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

4. 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

5. 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 Cirrhosis: Replacement of liver tissue  fibrosis, scar tissue Fulminant Hepatitis: severe impairment of liver functions Clinical Terms

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

7. 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

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

9. Hepatitis A - Diagnosis Three serologic markers available: 1. 1.Hepatitis A Total (IgG and IgM) antibody 2. 2.Hepatitis A IgM 3. 3.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

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

11. Fecal HAV Symptoms Liver Enzymes IgM anti-HAV Total anti-HAV Months after Exposure Titer Typical Serologic Course 0123 4561212 2424 Hepatitis A Virus Infection

12. 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.

13. 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

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

15. Hepatitis B – Laboratory Tests 3) Anti - HB c (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 Serologic markers (cont.):

16. Hepatitis B – Laboratory Tests 4) HB e Ag (Hepatitis B e antigen): indicates person is highly infectious Selecting patients for therapy 5) Anti-HB e (Antibody to HBV e antigen): prognostic for resolution of infection; less infectious; Serologic markers (cont.):

17. Acute Hepatitis B Virus Infection with Recovery Typical Serologic Course Weeks after Exposure Titer Symptoms HBeAg anti-HBe Total anti-HBc IgM anti-HBc anti-HBs HBsAg 0481216 20 242832 36 52100

18. 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

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

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

21. 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

22. Laboratory Tests for HCV Serology: Screening: – –3 rd 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

23. Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection Symptoms +/- Time after Exposure Titer anti- HCV Liver Enzymes Normal 012345 61234 Years Months HCV RNA

24. 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

25. 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

26. 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

27. 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.

28. 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.

29. Acute Hepatitis Infection IgM-HAVHBsAg IgM anti-HBc Anti-HCV

30. Molecular Tests for Hepatitis

31. 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 4 o C for several days or -70 o C for long-term No licensed tests for diagnostic purposes   all tests are for monitoring or donor screening

32. 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

33. HCV RNA Detection Assays AssayMethodLLD* (IU/ml) a Versant Qualitative (Siemens)TMA*5 - 10 Amplicor Qualitative v2.0 (Roche)RT-PCR50 Ampliscreen (Roche)RT-PCR50 Amplicor Monitor v2.0 (Roche)RT-PCR600 Cobas Taqman (Roche)RT-PCR15 Abbott RealTime (Abbott)RT-PCR12 - 30 Versant Quantitative v3.0 (Siemens) bDNA615 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 *LLD = Lower Limit of Detection; *TAM= Transcription-mediated amplification a Conversion 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)

34. 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

35. Molecular Laboratory Tests for HCV Used for treatment monitoring (and in some circumstances for confirmation of positive or indeterminate serology) HCV RNA is detectable 2 to 14 days after an exposure

36. Study Questions: Write a short note on: Serologic markers of HBV laboratory tests.

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

38. THANK YOU