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Laboratory Diagnostics in Hepatitis

Laboratory Diagnostics in Hepatitis. T. Mazzulli, MD, FRCPC Department of Microbiology UHN/Mount Sinai Hospital. Objectives. Review the serologic diagnosis of viral hepatitis Review the methodologies available for molecular testing and describe some of the advantages and disadvantages

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Laboratory Diagnostics in Hepatitis

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  1. Laboratory Diagnostics in Hepatitis T. Mazzulli, MD, FRCPC Department of Microbiology UHN/Mount Sinai Hospital

  2. Objectives • Review the serologic diagnosis of viral hepatitis • Review the methodologies available for molecular testing and describe some of the advantages and disadvantages • Discuss the currently available commercial assays that are available and those which are in use in Toronto • Discuss the use of molecular methods for genotyping and resistance testing

  3. 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 wild type infection • Incubation time is 7 to 28 days

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

  5. Laboratory Tests for HBV • Serology: • Many tests available – most common tests are Enzyme Immunoassays (EIAs, MEIAs) • First tests available in 1972 • For every rule, there is an exception/caveat • No single test tells you everything • Molecular: • HBV DNA (quantitative) • HBV genotyping • HBV resistance testing

  6. 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

  7. Hepatitis B – Laboratory Tests Serologic markers: • 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

  8. Hepatitis B – Laboratory Tests Serologic markers: • 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; spontaneous seroconversion in 10 to 20% of healthy adults per year

  9. 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

  10. Progression to Chronic Hepatitis B Virus Typical Serologic Course Acute (6 months) Chronic (Years) HBeAg anti-HBe HBsAg Total anti-HBc Titer IgM anti-HBc Years 0 4 8 16 20 24 28 36 12 32 52 Weeks after Exposure

  11. Virological and Biochemical Course of Chronic Hepatitis B

  12. Disease Phases in Chronic HBV Infection

  13. Interpretation of Serologic Tests in Hepatitis B

  14. 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; likely due to mutations in the “a” determinant of the S gene • Surface antigen escape mutants described in infants infected with HBV after HBIG + vaccination and in Liver transplants after prolonged HBIG • Anti-HBc IgM may persist for up to 2 years in 20%; chronically infected individuals may have low titres which rise during acute flares

  15. Hepatitis B – Laboratory Tests Serologic markers – caveats: • Precore or HBeAg negative mutants: • Due to mutation in precore (abolishes HBeAg production) or core promoter region (down-regulates HBeAg production) • No effect on viral replication (may be enhanced) • More difficult to treat; greater risk of cirrhosis • Co-infection with HCV may suppress both HBeAg and HBsAg

  16. HBV Viral Genome Organization HBcAg Hepatocyte receptor bindng site HBeAg Protein that transactivates transcriptional promotors HBsAg 3200 Base Pair Genome HBV DNA Polymerase

  17. Hepatitis B – Laboratory Tests Serologic markers – caveats: • Isolated HBcAb may be due to: • Remote infection (immune or chronic carrier) • “Window” period between HBsAg and HBsAb • Co-infection with HCV • False positive test result – HBcAb is marker most prone to false positives • HBV DNA may help sort this out

  18. 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

  19. Laboratory Tests for HCV Serology: • Screening: • 3rd generation EIAs measure antibodies directed against recombinant peptides NS4, core, NS3, and NS5 proteins • Sensitivity = 97% • Detects antibodies within 6 to 8 weeks • No HCV IgM test available • Confirmatory/supplementary: • RIBA, LiPA, Second EIA, HCV RNA

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

  21. Rational Use of HCV Diagnostic Tests TREATMENT Diagnosis Serological assays Qual HCV RNA Prognosis Liverhistology Decision to treat ALT Liver histology Qual HCV RNA Treatment duration Genotyping Viral load Response and resistance assessment Qual HCV RNA Viral load

  22. Hepatitis D Virus - Diagnosis • Anti-HDV Total (IgG & IgM) available • Incubation time – similar to Hepatitis B • High titres of HDV antibodies indicate ongoing chronic infection • Available only at National Microbiology Lab in Winnipeg

  23. Hepatitis E Virus - Diagnosis • Both IgG and IgM antibody tests are available • Incubation period – 7 to 28 days • No domestically acquired cases in Canada • Available only at the National Microbiology Lab in Winnipeg

  24. Molecular Tests for Hepatitis

  25. 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 • HCV RNA will be done in HIV or other immunocompromised patients if requested

  26. Hepatitis Virus – Molecular Tests • Lower limit of Detection (LLD) does not equal dynamic (linear) range of quantitative assays • Determined by PROBIT analysis to determine the value that is consistently detected 95% of the time • Results of different assays may (HBV) or may not (HCV) be interchangeable

  27. 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 • Coefficient of variation (COV) may range from 15 to 50%

  28. HBV DNA Quantification Assays A. Lok et al. Hepatology 2001;34; J. Servoss et al. Infect Dis Clin N Am 2006;20; B. Weber. Future Drugs 2005 *283,000 copies/pg; 5.26 copies/IU

  29. Measuring HBV DNA Gish and Locarnini, Clin Gastro Hep 2006

  30. Comparison of Quantitative HBV DNA Assays Versant 3.0 vs. Versant 1.0: R2 = 0.9001 Versant vs. Cobas: R2 = 0.7711 Versant vs. Digene: R2 = 0.9849 Yao J et al. J Clin Microbiol 2004:42(2)

  31. HCV RNA Detection Assays *LLD = Lower Limit of Detection; 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

  32. 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

  33. Laboratory Tests for HCV Molecular: • Both qualitative and quantitative HCV RNA assays available • 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

  34. Genotyping • Used for: • Detection of mutations that confer resistance to antiviral agents • Genotyping of isolates for epidemiological purposes; categorizes patient isolates into 8 different HBV genotypes (A to H) and 6 different HCV genotypes (1 to 6 with 24 subtypes) • Methods include: • Sequencing • Hybridization (Line Probe Assay, Trugene Assay)

  35. Laboratory Diagnosis of Resistance ProsCons SequencingDiscovers Labor-intensive new mutations Low sensitivity (15-20% pop.) Line ProbeHigh throughput Detects known High sensitivity mutations only (5-10% pop.)

  36. Conj.cont. Amp.cont. Marker line L180 M180 M204 V204 I204 V207 L207 M207 I207 InnoLiPA Principle Chromogen(NBT/BCIP) Purple precipitate Alkaline Phosphatase Streptavidin Biotin Amplified target DNA-probe Nitrocellulose strip

  37. InnoLIPA HBV Drug Resistance

  38. HBV Resistance Testing

  39. HBV Resistance Testing

  40. InnoLiPA vs. Sequencing Hussein et al, J Clin Micro 2006

  41. LAMIVUDINE Present Present Mixed Mixed Absent Absent L80V A181V X X V173L A181T X X N236T L180M X X M204V X M204I X M204S X Drug Resistance Report ADEFOVIR

  42. Resistance Mutation Lamivudine Resistance Sensitive Adefovir Tenofovir Lamivudine Entecavir Telbivudine Emtricitabine Lamivudine Tenofovir Entecavir Emtricitabine L180M +M204V Adefovir Resistance Resistant or Reduced Susceptibility Lamivudine Entecavir Telbivudine Emtricitabine Clevudine Adefovir Tenofovir Clevudine Adefovir N236T Adefovir Resistance A181V/T Interpreting HBV DR Reports ·L180M + M204V/I are the key lamivudine-resistant mutations ·A181V/T leads to 4-fold increase in IC50 ·N236T leads to 7-fold increase in IC50

  43. Diagnostics in Viral Hepatitis: Summary • Serology remains the cornerstone for diagnosis and screening • NAAT is critical to patient management • Of the many NAAT tests available, PCR, bDNA and TMA remain most popular • Sensitivity and dynamic range varies between assays • Standardization allows (to some degree) interchangeability of the results with different assays • Resistance/Genotyping requires amplification first • Increasing role in making treatment decisions as more drugs become available for HBV

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