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Laboratory.pptx
- 1. Laboratory Diagnostics inHepatitis و هپاتیت انواع تست بالینی تفسیر ها حاجوی جعفر دکتری پزشکی شناسی ایمنی تخصصی
- 2. Objectives Define some clinicalterms of hepatitis know different types of viral hepatitis Review the serologic diagnosis of viral hepatitis Review the methods available for molecular testing
- 3. Hepatitis: inflammation ofliver 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 Clinical Terms
- 4. 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
- 5. • Fever, Fatigue,Loss of appetite, Nausea,… • Swelling and tenderness of liver • Jaundice -yellow tinge in the skin and eyes • dark urine • transaminase, alkaline phosphatase levels increased Hepatitis symptoms
- 6. Source of virus feces blood/ blood-derived bodyfluids blood/ blood-derived body fluids blood/ blood-derived body fluids feces Route of transmission fecal-oral percutaneous permucosal percutaneous permucosal percutaneous permucosal fecal-oral Chronic infection no yes yes yes no Prevention pre/post- exposure Immunization vaccine available pre/post- exposure Immunization vaccine available blood donor screening; risk behavior modification pre/post- exposure immunization; risk behavior modification ensure safe drinking water Type of Viral Hepatitis A B C D E
- 7.
- 8. ssRNA virus, Non-enveloped Onlyone serotype(Picornavirus) Fecal-oral transmission Usually self-limited illness low mortality No carrier state In rare cases, fulminant hepatic necrosis Hepatitis A Structure and Clinical manifestations
- 9. Hepatitis A Diagnosis Clinicalmanifestations Viral antibodies – Immunoelectron microscopy – RIA – ELISA – Immune adherence hemagglutination (IAHA) (old method)
- 10. 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 Incubation time is 7 to 28 days Antibody response is similar following vaccination or infection
- 11. Diagnosis of hepatitisA IgM anti-HAV: appears 4 wks after exposure and disappears by 3 -6 months(40-60 days) Indicates acute infection IgG anti-HAV: after IgM peaks 60-80 days(during convalescence) persists for life. Indicates exposure (Past Infection) or immunity(vaccination)
- 12. Fecal HAV Symptoms Liver Enzymes IgM anti-HAV Totalanti-HAV Months after Exposure Titer Typical Serologic Course 0 1 2 3 4 5 6 1 2 2 4 Hepatitis A Virus Infection
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- 16. DNA virus fromthe Hepadnaviridae family. Enveloped and encapsulated. Human is only known host. Infectivity retain for 7 days in room T C. Important proteins : An surface protein called surface antigen – HBsAg A structural nucleocapsid core protein – HBcAg A soluble nucleocapsid protein – HBeAg HEPATITIS B VIRUS
- 17. Parenterally ie viablood, saliva, menstrual and vaginal discharges, semen and breast milk Infected blood and blood products Sexual contact Prenatally from mother to child Incubation period of 1-6 mo. ̶ 25% acute hepatitis, 1% fulminant hepatic necrosis ̶ 10% chronic carriers Hepatitis B Epidemiology
- 18. There are 10xmore people with chronic HBV than HIV/AIDS worldwide (WHO) HBV is 50-100x more infectious than HIV (WHO) 2 billion people worldwide (1/3 world population) infected with HBV (WHO) 350 million people worldwide have chronic (lifelong) HBV (CDC) 1 million people die each year from liver disease and liver cancer (CDC) Every 30–45 seconds, one person dies from the vaccine-preventable HBV HBV Worldwide
- 19. Cell mediated Immunity –important for recover in acute phase Humoral Immunity – not always protective – HBsAg for Vaccines Interferon(Innate immunity) – not detected during infection – exogenous application effective Hepatitis B Host Defenses
- 20. Course of HBVInfection Adult Infant 90-95% Acute Infection Full Recovery Chronic Hepatitis B 5-10% Virus Persists 70-90% Virus Persists Chronic Hepatitis B 10-30% Acute Infection Full Recovery Course of Hepatitis B virus infection
- 21. Laboratory Tests forHBV Serology: – Many tests available – most common tests are Enzyme Immunoassays (EIAs, MEIAs) – First tests available in 1972 – No single test tells you everything Molecular: – HBV DNA (quantitative) – HBV genotyping – HBV resistance testing
- 22. Hepatitis B –Laboratory Tests 1) HBsAg (Hepatitis B surface antigen): • First serological marker that appear • 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 Serologic markers:
- 23. Hepatitis B –Laboratory Tests 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 • IgG - past or chronic infection • No antigen test Serologic markers:
- 24. Hepatitis B –Laboratory Tests 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 Serologic markers:
- 25. HBV Viral GenomeOrganization HBsAg HBcAg HBeAg 3200 Base Pair Genome Hepatocyte receptor bindng site HBV DNA Polymerase Protein that transactivates transcriptional promotors
- 26. Hepatitis B serologyprofile
- 27. HBsAg First specificmarker Detectable during incubation, peaks in acute stage Declines upon recovery Elevated in carriers
- 28. HBeAg Appears shortlyafter HBsAg and parallels HBsAg Present during active replication of virus (most infectious phase)
- 29. Anti-HBc First detectableantibody IgM present in the interval between disappearance of HBsAg and appearance of Anti-HBs (core window) IgG produced during convalescense and persists for life
- 30. Anti-HBe Appears afterdisappearance of HBeAg Indicates resolution
- 31. Anti-HBs Appears duringrecovery and lasts for years Indicates immunity (also produced as a result of vaccination)
- 32. Acute HBV Infectionwith Recovery Typical Serologic Course Weeks after Exposure Titer Symptoms HBeAg anti-HBe Total anti-HBc IgM anti-HBc anti-HBs HBsAg 0 4 8 12 16 20 24 28 32 36 52 100
- 33. Interpretation of LaboratoryTests in Hepatitis B Test Acute Hepatitis B Immunity through Infection Immunity through Vaccination Immune tolerance Immune clearance Chronic active Infection with Precore Mutant Inactive Carrier HBsAg + - - + + + + HBsAb - + + - - - - HBeAg + - - + + - - HBeAb - +/- - - - + + HBcAb IgM + - - - - - - HBV DNA + - - >20,000 IU/mL >20,000 IU/mL >20,000 IU/mL <2,000 IU/mL ALT Elevated Normal Normal Normal Elevated Elevated Normal
- 34. 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%; chronically infected individuals may have low titres which rise during acute flares
- 35. Hepatitis B –Laboratory Tests Serologic markers – caveats: Isolated HBcAb may be due to: – Co-infection with HCV HBeAg negative mutants: – Due to mutation in precore – More difficult to treat; greater risk of cirrhosis Co-infection with HCV may suppress both HBeAg and HBsAg
- 36. Diagnosis, treatment andmonitoring of chronic HBV infection
- 39. strategies for diagnosisof chronic HBV infection
- 40. Progression to ChronicHepatitis B Virus Typical Serologic Course Weeks after Exposure Titer IgM anti-HBc Total anti- HBc HBsAg Acute (6 months) HBeAg Chronic (Years) anti-HBe 0 4 8 12 16 20 24 28 32 36 52 Years
- 41. Disease Phases inChronic HBV Infection Phase HBsAg HBeAg Anti- HBe ALT HBV DNA range Immune Tolerant + + - Normal >8 log IU/mL Immune Clearance + + - Normal or elevated 3-8 log IU/mL Inactive Disease + - + Normal <3 log IU/mL HBeAg- negative Chronic HBV + - + Normal or elevated 3-8 log IU/mL
- 42. Virological and BiochemicalCourse of Chronic Hepatitis B
- 43. Chronic Hepatitis BSerologic Patterns HBs Ag Anti- HBs Anti- HBc HBeAg Anti- HBe Interpretation + - IgG + - Chronic Hepatitis B, High infectivity + - IgG - + Chronic Hepatitis B, Low infectivity
- 44. Summary of laboratoryInvestigation of HBV
- 46.
- 47. Enveloped RNA virus Notpossible to grow virus in culture 60-85% get chronic infection Highly heterogeneous - • 11 genotypes (1-11) • many subtypes (a,b,c, etc.) • 100 different strains (1,2,3,etc.) A 2011 study estimated that as many as 5.2 million persons are living with HCV in the US. 30 - 40% of HIV+ people in US also infected with HCV. Hepatitis C infection
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- 49. Laboratory Tests forHCV Serology: Detection of anti-HCV antibodies Serologic test available since 1990 Molecular: HCV RNA detection Determination of HCV genotype Viral load determination
- 50. SEROLOGIC TESTS Enzyme Immunoassay(EIA) for Anti-HCV Positive: past or current infection Verification of Anti-HCV (+) screening test 1. Reflex supplemental testing*: follow-up with more specific serologic test, e.g. HCV RIBA or NAT HCV RIBA* (Recombinant Immunoblot Assay) Detects antibodies to individual HCV antigens and increased specificity compared to EIA-2 Some RIBA-positive patients are HCV RNA-negative Laboratory Tests for HCV
- 51. Third generation EIA: –sensitivity > 99%, specificity = 99%, in immunocompetent patients – No need for confirmatory test in pts with clinical liver disease False positives: autoimmune disorders – No need for further testing in case of negative EIA in immune-competent patients False negatives: hemodialysis, immune-deficiencies Enzyme Immunoassay (EIA)
- 52. No liver symptoms,+ve EIA – negative test implies false +ve EIA +ve EIA, but -ve HCV RNA Problem: Presence of antibody does not indicate if the virus is replicating Advantage? Can be ordered on the same sample as the original EIA Recombinant Immunoblot Assay(RIBA)
- 53. VIRAL LOAD TESTS -Used to confirm positive EIA - measure HCV RNA (genetic material). - Specificity >98% A.Qualitative test • Nucleic Acid Test (NAT)* for HCV RNA using RT-PCR Detects HCV RNA in the blood, very sensitive B. Quantitative test Branched-chain DNA (bDNA) Measures viral loads greater than 50 IU/ml Transcription-mediated Amplification (TMA) Measure viral loads as few as 5-10 IU/ml Laboratory Tests for HCV
- 54. confirms infection, may justbe below the level of detection. Single +ve: -ve: Indications – Acute HCV, before antibodies made (+in 1 - 3 wks) – Chronic hepatitis with indeterminate serology – Chronic hepatitis and autoantibodies, with false positive serology – Persistent HCV replication after liver transplantation, when antibodies persist HCV RNA test-qualitative
- 55. HCV core antigentesting Precede antibody detection Indicates viraemia Less sensitive than RNA Levels correlate with RNA levels Specificity: Good in HCV antibody negative samples, but false positives in HCV antibody pos RNA negative samples! Potential role: Confirming HCV positive serology Diagnosis of incident infections
- 56. GENOTYPING • HCV Genotype 11 genotypes, >50 subtypes clinical importance: counseling and treatment epidemiology LIVER FUNCTION TEST • ALT • SGPT Laboratory Tests for HCV
- 57. very variable inHCV infection Weak association between ALT levels and severity of histopathology Resolution of high levels is good indicator of response to therapy ALT
- 58.
- 59. HCV infection withSelf-resolving HCV infection Spontaneous clearance can occur in 14%
- 60. Serologic Pattern ofAcute HCV Infection with Progression to Chronic Infection Symptoms +/- Time after Exposure Titer anti- HCV ALT Normal 0 1 2 3 4 5 6 1 2 3 4 Years Months HCV RNA
- 61. HCV infection withChronic HCV infection
- 62. strategy for detectionof HCV antibody, irrespective of prevalence
- 63. Diagnosis, treatment andmonitoring of chronic HCV infection
- 65. Anti-HCV HCV RNA(PCR) Interpretation Negative Negative • No infection Positive Positive • HCV present (acute or chronic infection) Negative Positive • Chronic infection in immunosuppressed patient • Early infection Positive Negative • Resolved infection • Treated infection, HCV below detectable levels (verify with qualitative HCV RNA PCR) • False-positive anti-HCV test (<1%) Interpreting Hepatitis C Test
- 66.
- 67. Hepatitis D infection HepatitisD virus is an incomplete small RNA virus that needs HBV to survive Only occurs in the presence of HBV Incubation time – similar to Hepatitis B Can occur initially as a co-infection, where it runs the same course as hepatitis B – severe acute disease. – low risk of chronic infection.
- 68. Percutanous exposures injectingdrug use Permucosal exposures sex contact Hepatitis D Virus Modes of Transmission
- 69. Hepatitis D Diagnosis Clinical manifestations Deltaantigen Immunofluorescence RIA ELISA Anti delta antigen same as above
- 70. Hepatitis D tests HDVAg – Present only during prodrome, not tested – Anti-HDV Total (IgG & IgM) available 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
- 71. anti-HBs Symptoms ALT Elevated Total anti-HDV IgManti-HDV HDV RNA HBsAg HBV - HDV Coinfection Typical Serologic Course Time after Exposure Titre
- 72. Jaundice Symptoms ALT Total anti-HDV IgM anti-HDV HDVRNA HBsAg HBV - HDV Superinfection Typical Serologic Course Time after Exposure Titre
- 73.
- 74. Hepatitis E infection Singlestranded RNA virus Present in animals without causing disease Human HEV infection rare in the US. Endemic in many countries. Fecal oral transmission Disease is most common in ages 15-40 Causes self limiting disease in adults and there is no chronic state
- 75. Hepatitis E Virus- Diagnosis • Both IgG and IgM antibody tests are available • Incubation period – 7 to 28 days Anti-HEV IgM - Arise with the onset of symptoms Anti-HEV IgG- will develop after symptoms resolve (2-4 weeks)
- 76. Symptoms ALT IgG anti-HEV IgManti-HEV Virus in stool 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 Hepatitis E Virus Infection Typical Serologic Course Titer Weeks after Exposure
- 77. Hepatitis G virus HepatitisG virus or GBV-C is closely related to HCV Common in HCV infected patients Mode of transmission: ?parenteral ?sexual Role in human disease is controversial. Usually mild acute or chronic hepatitis. May delay progression of HIV disease
- 78. Molecular Tests forHepatitis
- 79. 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. 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
- 80. Hepatitis Virus –Molecular Tests Lower limit of Detection (LLD) does not equal dynamic (linear) range of quantitative assays. Results of different assays may (HBV) or may not (HCV) be interchangeable
- 81. Nucleic Acid AmplificationTests (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%
- 82. HBV DNA QuantificationAssays Assay Sensitivity (pg/ml)* LLD (copies/ml)* Linearity (copies/ml) Coefficient of Variation Versant bDNA v3.0 (Siemens) 2.1 2 x 103 2 x 103 to 1 x 108 15 - 37% Hybrid Capture II (Digene) 0.02 to 0.5 5 x 103 5 x 103 to 6 x 107 10 – 15% Liquid Hybridization (Abbott) 1.6 6 x 105 5 x 105 to 1 x 1010 12 – 22% Cobas Amplicor Monitor (Roche) 0.001 2 x 102 2 x 102 to 2 x 105 14 – 44% Cobas Taqman (Roche) 35 (Manual) 70 (Automated) 2 x 102 to 1 x 1010 16 – 54% RealArt HBV PCR (artus/Qiagen) 10 1 to 4 x 108 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
- 83. Quantifying HBV: HBVDNA level or HBsAg? cDNA best marker of viral burden Serum HBV DNA level Realtime PCR: accumulation of PCR product Fluorescent probes Standard curve sensitive Dynamic range: 10-108 ge/ml 0 10 20 30 40 50 1 2 3 4 5 6 Concentration log 10 Threshold Cycle
- 84. HBV DNA inClinical 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
- 85. Laboratory Tests forHCV 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
- 86. VIRAL LOAD TESTS -Used to confirm positive EIA - measure HCV RNA (genetic material), types: - Specificity >98% A.Qualitative test • Nucleic Acid Test (NAT)* for HCV RNA using RT-PCR Detects HCV RNA in the blood, very sensitive B. Quantitative test Branched-chain DNA (bDNA) Measures viral loads greater than 50 IU/ml Transcription-mediated Amplification (TMA) Measure viral loads as few as 5-10 IU/ml Laboratory Tests for HCV
- 87. confirms infection, may justbe below the level of detection. Single +ve: -ve: Indications – Acute HCV, before antibodies made (+in 1 - 3 wks) – Chronic hepatitis with indeterminate serology – Chronic hepatitis and autoantibodies, with false positive serology – Persistent HCV replication after liver transplantation, when antibodies persist HCV RNA test-qualitative
- 88. HCV RNA DetectionAssays Assay Method LLD* (IU/ml)a Linearity (IU/ml) Versant Qualitative (Siemens) TMA 5 - 10 NA Amplicor Qualitative v2.0 (Roche) RT-PCR 50 NA Ampliscreen (Roche) RT-PCR 50 NA Amplicor Monitor v2.0 (Roche) RT-PCR 600 600-800,000 Cobas Taqman (Roche) RT-PCR 15 15 – 1 x 108 Abbott RealTime (Abbott) RT-PCR 12 - 30 10 – 1 x 107 Versant Quantitative v3.0 (Siemens) bDNA 615 615 - 7,700,000 *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)
- 89. Genotyping Used for: – Detectionof 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)
- 90. Laboratory Diagnosis ofResistance Pros Cons Sequencing Discovers Labor-intensive new mutations Low sensitivity (15-20% pop.) Line Probe High throughput Detects known High sensitivity mutations only (5-10% pop.)
- 91. Diagnostics in ViralHepatitis: 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 Resistance/Genotyping requires amplification first – Increasing role in making treatment decisions as more drugs become available for HBV