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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: [email protected] 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 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 Viral Hepatitis Hepatotropic viruses – Hepatitis A, B, C, D, E and G viruses Generalized infection plus infection of liver – EBV, CMV and HSV 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 Hepatitis A infection RNA virus Fecal-oral transmission Usually self-limited illness No carrier state In rare cases, fulminant hepatic necrosis Hepatitis A - Diagnosis Three serologic markers available: 1. Hepatitis A Total (IgG and IgM) antibody 2. Hepatitis A IgM 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 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 Hepatitis A Virus Infection Typical Serologic Course Symptoms Total anti-HAV Titer Liver Enzymes Fecal HAV 0 1 IgM anti-HAV 2 4 5 6 3 Months after Exposure 1 2 2 4 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. 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 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 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 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; Acute Hepatitis B Virus Infection with Recovery Typical Serologic Course Symptoms HBeAg anti-HBe Total anti-HBc Titer HBsAg 0 4 8 anti-HBs IgM anti-HBc 12 16 20 24 28 32 36 Weeks after Exposure 52 100 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 Hepatitis C infection Enveloped RNA virus Parenteral infection 60-85% get chronic infection Treatment with interferon+ribavirin cures virus in only 25-40% Sources of infection for persons with newly-diagnosed Hepatitis C Injection drug use 60% Sexual 15% Transfusion 10% (before screening) * Nosocomial Other* 5% Unknown 10% Health-care work Perinatal CDC 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 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 Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection antiHCV Symptoms +/- Titer HCV RNA Liver Enzymes Normal 0 1 2 3 4 Months 5 6 1 2 3 Years Time after Exposure 4 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 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 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 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. 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. Acute Hepatitis Infection IgM-HAV HBsAg IgM anti-HBc Anti-HCV Molecular Tests for Hepatitis 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 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 HCV RNA Detection Assays Assay Method LLD* (IU/ml)a TMA* 5 - 10 Amplicor Qualitative v2.0 (Roche) RT-PCR 50 Ampliscreen (Roche) RT-PCR 50 Amplicor Monitor v2.0 (Roche) RT-PCR 600 Cobas Taqman (Roche) RT-PCR 15 Abbott RealTime (Abbott) RT-PCR 12 - 30 Versant Quantitative v3.0 (Siemens) bDNA 615 Versant Qualitative (Siemens) *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 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 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 Study Questions: Write a short note on: Serologic markers of HBV laboratory tests. Assignment Write shortly on serological diagnosis of HBV. وليد على – يمنى عبد هللا – خلود عبد القادر – دنيا محمد – دينا عطية