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 HassanProf. of Immunology

Med Res Inst, Alex UnivE-mail: [email protected]

Objectives

Define some clinical terms of hepatitisknow different types of viral hepatitisReview the serologic diagnosis of viral hepatitisReview the methods available for molecular testing

Hepatitis: inflammation of liverAcute Viral Hepatitis: symptoms last less than 6 monthsChronic 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

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) antibody2. Hepatitis A IgM3. Hepatitis A IgGFirst tests available since 1978No antigen testAntibody response is similar following vaccination or infectionIncubation time is 7 to 28 days

Diagnosis of hepatitis AIgM 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

FecalHAV

Symptoms

Liver Enzymes

IgM anti-HAV

Total anti-HAV

Months after Exposure

Tite

r

Typical Serologic Course

0 1 2 3 4 5 6 12

24

Hepatitis A Virus Infection

Hepatitis B virus infectionTransmission– 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

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

Serologic markers:

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• No antigen test

Serologic markers (cont.):

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 (cont.):

Acute Hepatitis B Virus Infection with RecoveryTypical Serologic Course

Weeks after Exposure

Titer

Symptoms

HBeAg anti-HBe

Total anti-HBc

IgM anti-HBc anti-HBsHBsAg

0 4 8 12 16 20 24 28 32 36 52 100

Hepatitis B – Laboratory Tests

Serologic markers – caveats:Persistent HBsAg for >6 mos = chronic infectionHBsAg 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 infectionEnveloped RNA virusParenteral infection60-85% get chronic infectionTreatment with interferon+ribavirin cures virus in only 25-40%

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

Laboratory Tests for HCV

Serology:Detection of anti-HCV antibodiesSerologic test available since 1990

Molecular:HCV RNA detectionDetermination 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

Symptoms +/-

Time after Exposure

Tite

ranti-HCV

Liver Enzymes

Normal

0 1 2 3 4 5 6 1 2 3 4YearsMonths

HCV RNA

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 testsAvailable 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 infectionRNA 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 virusHepatitis 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-termNo 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 AssaysAssay Method LLD*

(IU/ml)a

Versant Qualitative (Siemens) 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

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 amplificationaConversion 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)

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.

محمد – – – دنيا القادر عبد خلود الله عبد يمنى على وليدعطية– دينا

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