Laboratory.pptx

Laboratory Diagnostics in Hepatitis
‫و‬ ‫هپاتیت‬ ‫انواع‬
‫تست‬ ‫بالینی‬ ‫تفسیر‬
‫ها‬
‫حاجوی‬ ‫جعفر‬
‫دکتری‬
‫پزشکی‬ ‫شناسی‬ ‫ایمنی‬ ‫تخصصی‬

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

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
Clinical Terms

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

• 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

Source of
virus
feces blood/
blood-derived
body fluids
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

ssRNA virus, Non-enveloped
Only one 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

Hepatitis A Diagnosis
Clinical manifestations
Viral antibodies
– Immunoelectron microscopy
– RIA
– ELISA
– Immune adherence hemagglutination (IAHA)
(old method)

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

Diagnosis of hepatitis A
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)

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

DNA virus from the
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

Parenterally ie via blood, 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

There are 10x more 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

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

Course of HBV Infection
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

Laboratory Tests for HBV
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

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:

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:

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:

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

HBsAg
 First specific marker
 Detectable during incubation, peaks in acute stage
 Declines upon recovery
 Elevated in carriers

HBeAg
 Appears shortly after HBsAg and parallels HBsAg
 Present during active replication of virus (most
infectious phase)

Anti-HBc
 First detectable antibody
 IgM present in the interval between disappearance of
HBsAg and appearance of Anti-HBs (core window)
 IgG produced during convalescense and persists for life

Anti-HBe
 Appears after disappearance of HBeAg
 Indicates resolution

Anti-HBs
 Appears during recovery and lasts for years
 Indicates immunity (also produced as a result of
vaccination)

Acute HBV Infection with Recovery
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

Interpretation of Laboratory Tests 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

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

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

Diagnosis, treatment and monitoring of
chronic HBV infection

strategies for diagnosis of chronic HBV
infection

Progression to Chronic Hepatitis B Virus
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

Disease Phases in Chronic 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

Virological and Biochemical Course
of Chronic Hepatitis B

Chronic Hepatitis B Serologic Patterns
HBs
Ag
Anti-
HBs
Anti-
HBc
HBeAg Anti-
HBe
Interpretation
+ - IgG + - Chronic Hepatitis B,
High infectivity
+ - IgG - + Chronic Hepatitis B,
Low infectivity

Summary of laboratory Investigation of HBV

Enveloped RNA virus
Not possible 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

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

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

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)

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)

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

confirms infection,
may just be 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

HCV core antigen testing
 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

GENOTYPING
• HCV Genotype
 11 genotypes, >50 subtypes
 clinical importance: counseling and treatment
 epidemiology
LIVER FUNCTION TEST
• ALT
• SGPT

very variable in HCV infection
Weak association between ALT levels
and severity of histopathology
Resolution of high levels is good indicator
of response to therapy
ALT

HCV infection with Self-resolving HCV infection
 Spontaneous
clearance can
occur in 14%

Serologic Pattern of Acute 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

HCV infection with Chronic HCV
infection

strategy for detection of HCV
antibody, irrespective of prevalence

Diagnosis, treatment and monitoring of
chronic HCV infection

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

Hepatitis D infection
Hepatitis D 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.

 Percutanous exposures
injecting drug use
 Permucosal exposures
sex contact
Hepatitis D Virus Modes
of Transmission

Hepatitis D
Diagnosis
Clinical manifestations
Delta antigen
Immunofluorescence
RIA
ELISA
Anti delta antigen
same as above

Hepatitis D tests
HDV Ag
– 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

anti-HBs
Symptoms
ALT Elevated
Total anti-HDV
IgM anti-HDV
HDV RNA
HBsAg
HBV - HDV Coinfection
Time after Exposure
Titre

Jaundice
Symptoms
ALT
Total anti-HDV
IgM anti-HDV
HDV RNA
HBsAg
HBV - HDV Superinfection
Time after Exposure
Titre

Hepatitis E infection
Single stranded 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

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)

Symptoms
ALT IgG anti-HEV
IgM anti-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
Titer

Hepatitis G virus
Hepatitis G 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

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

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

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%

HBV DNA Quantification Assays
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

Quantifying HBV: HBV DNA 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

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:
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

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

HCV RNA Detection Assays
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)

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)

Laboratory Diagnosis of Resistance
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.)

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
Resistance/Genotyping requires amplification first
– Increasing role in making treatment decisions as more drugs
become available for HBV

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