Laboratory Diagnostics
in Viral Hepatitis
Heidar Sharafi, PhDc
Baqiyatallah Research Center for
Gastroenterology and Liver Disease (BRCGL)
Laboratory Diagnosis of Hepatitis B
Laboratory Tests for HBV
Serology:
– Many tests available – most common tests are
Enzyme Immunoassays (EIAs, MEIAs)
– No single test tells you everything
Molecular:
– HBV DNA (quantitative)
– HBV genotyping and resistance testing
Hepatitis B – Laboratory Tests
Serologic markers:
1) HBsAg (Hepatitis B surface antigen):
• if positive, person is infectious
2) HBsAb (Antibody to HBV surface antigen):
• indicates immunity to HBV and protection from disease
3) HBcAb (Antibody to HBV core antigen):
• Total - indicates past or active infection
• IgM - early indicator of acute infection
4) HBeAg (Hepatitis B e antigen):
• Selecting patients for therapy
5) Anti-HBe (Antibody to HBV e antigen):
• prognostic for resolution of infection
Acute Hepatitis B Virus Infection with Recovery
Typical Serologic Course
Symptoms
anti-HBe
HBeAg
Total anti-HBc
Titer
0
4
anti-HBs
IgM anti-HBc
HBsAg
8
12
16
20 24
28
32
Weeks after Exposure
36
52
100
Progression to Chronic Hepatitis B Virus
Typical Serologic Course
Acute
(6 months)
Chronic
(Years)
HBeAg
anti-HBe
HBsAg
Total antiHBc
Titer
IgM anti-HBc
0 4 8 12 16 20 24 28 32 36
Weeks after Exposure
52
Years
Virological and Biochemical Course
of Chronic Hepatitis B
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
Elevated
Normal
Normal
Normal
Elevated
Elevated
Normal
ALT
Viral Hepatitis – Molecular Tests
Molecular assays available as follows:
– Commercial and In-house assays for HBV DNA
and HCV 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
Nucleic Acid Tests (NAT) 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 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 1 x 108
15 - 37%
Hybrid Capture II
(Digene)
0.02 to 0.5
5 x 103
5 x 103 6 x 107
10 – 15%
1.6
5 x 105
5 x 105 1 x 1010
12 – 22%
0.001
2 x 102
2 x 102 2 x 105
14 – 44%
35 (Manual)
70 (Automated)
2 x 102 1 x 1010
16 – 54%
10
1 - 4 x 108
Liquid Hybridization
(Abbott)
Cobas Amplicor
Monitor (Roche)
Cobas Taqman
(Roche)
RealArt HBV PCR
(artus/Qiagen)
*283,000 copies/pg; 5.26 copies/IU
A. Lok et al. Hepatology 2001;34; J. Servoss et al. Infect Dis
Clin N Am 2006;20; B. Weber. Future Drugs 2005
HBV DNA Level in Clinical Practice
Routine monitoring on therapy to assess
response to treatment
– Every 3 months on oral agents
– Every 1 month on PEG/IFN
Routine monitoring off therapy to estimate
prognosis and to evaluate need for treatment
– Every 6-12 months normally
Laboratory Diagnosis of Hepatitis C
Laboratory Tests for HCV
Serology:
Detection of anti-HCV antibodies
Molecular:
HCV RNA detection
Determination of HCV genotype
HCV RNA level determination
HCV resistance testing
Laboratory Tests for HCV
Serology:
Screening:
– Many tests available – most common tests are Enzyme
Immunoassays
– Sensitivity > 97%
– Detects antibodies within 6 to 8 weeks
Confirmatory/supplementary:
– HCV RNA RT-PCR
Serologic Pattern of Acute HCV Infection with
Progression to Chronic Infection
antiHCV
Symptoms +/-
Titer
HCV RNA
ALT
Normal
0
1
2
3
4
Months
5
6
1
2
3
Years
Time after Exposure
4
Virological Markers for Hepatitis C
Assessment
HCVAb HCV RNA
Positive
Positive
Interpretation
Acute or chronic HCV depending on the clinical
context
Positive
Negative
Resolution of HCV; Acute HCV during period of
low-level viremia
Negative Positive
Early acute HCV infection; chronic HCV in
setting of immunosuppressed state; false positive
HCV RNA test
Negative Negative
Absence of HCV infection
Protease Inhibitors
Simeprevir (SMV)
Paritaprevir (PTV)
Grazoprevir (GZR)
Bertino G. World Journal of Hepatology. 2016;8(2):92-106.
4A
5’UTR
P7
Approved HCV Direct-acting
Antiviral (DAA) Agents
3’UTR
NS5B NUC Inhibitors NS5B non-NUC
NS5A Inhibitors
Sofosbuvir (SOF)
Inhibitors
Ledipasvir (LDV)
Dasabuvir (DSV)
Daclatasvir (DCV)
Ombitasvir (OMV)
Elbasvir (EBR)
Velpatasvir (VEL)
HCV RNA Quantification
HCV RNA detection/quantification should
be tested before initiation of treatment, at
the end of treatment and 12 or 24 weeks
after treatment completion.
HCV RNA quantification should be made
by a reliable sensitive assay (LLD <15
IU/ml) and HCV RNA levels should be
expressed in IU/ml.
HCV RNA Detection Assays
Assay
Method
LLD*
(IU/ml)a
Linearity
(IU/ml)
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 V2.0 (Roche)
RT-PCR
10
25 – 3 x 108
Abbott RealTime (Abbott)
RT-PCR
12 - 30
10 – 1 x 107
bDNA
615
615 -7,700,000
Versant Qualitative (Siemens)
Versant Quantitative v3.0 (Siemens)
*LLD = Lower Limit of Detection;
aConversion
S. Chevaliez et al. World J Gastro 2007;13; J
factor IU/ml to copies/ml varies with each
Scott et al. JAMA 2007;297; A. Caliendo et al. J
assay (e.g. PCR: 1 IU/ml = 2.4 copies/ml; bDNA: 1IU/ml
Clin Microbiol 2006;44
= 5.2 copies/ml)
DAAs and Virologic Responses
Sustained virologic response (SVR)
Undetectable HCV RNA 12 w (SVR12) or
24 w (SVR24) after the end of therapy
by a sensitive molecular method with a
LLD <15 IU/ml
Both SVR12 and SVR24 have been accepted
as endpoints of therapy given that their
concordance is 99%.
DAAs and Virologic Responses
Non-response
Detectable HCV RNA at the end of
treatment
Relapse
Reappearance of HCV RNA in serum
after therapy is discontinued
No RVR, cEVR and pEVR
HCV Genotyping
Strains of HCV are classified into seven genotypes
(1–7) and a large number of subtypes.
The HCV genotype, including genotype 1 subtype,
should also be assessed prior to treatment initiation.
Genotyping/subtyping should be performed with an assay
that accurately discriminates subtype 1a from 1b.
Methods:
–
–
–
–
DNA Sequencing and phylogenetic analysis (Reference method)
Line probe assay
Primer specific amplification
RFLP
Mechanism of Action of DAAs
Mechanism of action:
Binding and blocking the active
site of the protein (NS3, NS5A &
NS5B)
Resistance:
Mutations close to active site
reduce affinity to drug
HCV displays a high genetic diversity
• Production of 1012 virions daily
• ~1 error per 10,000 bases for RNA polymerase
• Within one patient HCV exists as a population of
genetically distinct but closely related variants (quasispecies)
• RASs: Resistance-associated Substitutions
Barriers to Genetic Resistance by
DAAs Approved (GT1)
Protease
Inhibitors
NS5A Inhibitors
NS5B NUC
Inhibitors
NS5B non-NUC
Inhibitors
DAAs in class
Simeprevir
Paritaprevir
Grazoprevir
Ledipasvir
Daclatasvir
Ombitasvir
Elbasvir
Sofosbuvir
Dasabuvir
Barrier to
resistance
Low (1a < 1b)
Low (1a < 1b)
High (1a = 1b) Very Low (1a < 1b)
• RAVs to one DAA are generally cross-resistant to other
DAAs within a class, although this is not always the case
Bertino G. World Journal of Hepatology. 2016;8(2):92-106.
NS5A Resistance Overview
• Baseline polymorphisms associated with resistance are relatively
prevalent (15-30%)
• Currently available NS5A inhibitors suffer from broad crossresistance at key positions
– Q30R, L31M/V, Y93H/N
• 75% of patients harbor RAVs after treatment failure with
SOF/LDV
• NS5A variants persist for prolonged periods
• Selected NS5A RASs impact re-treatment responses
Broad Cross-resistance with NS5A inhibitors
Fold-change in resistance
GT1a
GT1b
M28T
Q30R
L31M/V
Y93H/N
LDV
20x
>100x
>100x/ >1,000x
>1,000x/
>10,000x
OMV
>1000x
>100x
<3x
>1,000x/
>10,000x
<10x
20x/50x
>100x
L31V
Y93H/N
>100x/--
DCV
>100x
>1000x
>100x/ >1,000x
>1,000x/
>10,000x
<10x
20x/50x
EBR
20x
>100x
>10x
<10x
>100x/--
>100x
>1,000x/
>10,000x
20x/50x
>100x/ >1,000x
VEL
<10x
<3x
http://iasusa.org/sites/default/files/uploads/2016hivsny_naggie.pdf
<3x/--
Consideration for NS5A Resistance Testing in
DAA-Naïve Patients – G1a Only
• SOF/LDV (or DCV)
– Apparent role in treatment-naïve patients
• Cirrhosis
– Could baseline testing be used to “optimize” therapy in
TE patients, particularly those with cirrhosis?
• 24 weeks + RBV for all TE cirrhosis with baseline NS5A RAVs?
Diagnostics in Viral Hepatitis: Summary
Serology remains the cornerstone for diagnosis and
screening
NAT is critical to patient management
Of the many NAT tests available, PCR, bDNA and
TMA remain most popular
– Sensitivity and dynamic range varies between
assays
– Standardization allows (to some degree)
interchangeability of the results with different
assays