Download Hepatitis A

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