Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)
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Transcript of 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
THANK YOU