1. Laboratory Diagnostics in Viral Hepatitis
Heidar Sharafi, PhDc
Baqiyatallah Research Center for Gastroenterology and Liver Disease (BRCGL)

2. Laboratory Diagnosis of Hepatitis B

3. 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

4. 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

5. Acute Hepatitis B Virus Infection with Recovery
Typical Serologic Course
Symptoms
HBeAg
anti-HBe
Total anti-HBc
Titer
anti-HBs
HBsAg
IgM anti-HBc 4 8 12 16 20 24 28 32 36 52
100
Weeks after Exposure

6. Progression to Chronic Hepatitis B Virus Typical Serologic Course
Acute
(6 months)
Chronic
(Years)
HBeAg
anti-HBe
HBsAg
Total anti-HBc
Titer
IgM anti-HBc 4 8 12 16 20 24 28 32 36 52
Years
Weeks after Exposure

7. Virological and Biochemical Course of Chronic Hepatitis B

8. Interpretation of Laboratory Tests in Hepatitis B
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
<2,000 IU/mL
ALT
Elevated
Normal

9. 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

10. 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

11. HBV DNA Quantification Assays
Sensitivity (pg/ml)*
LLD (copies/ml)*
Linearity (copies/ml)
Coefficient of Variation
Versant bDNA v3.0 (Siemens)
2.1
2 x 103
2 x x 108 %
Hybrid Capture II (Digene)
0.02 to 0.5
5 x 103
5 x x 107
10 – 15%
Liquid Hybridization (Abbott)
1.6
5 x 105
5 x x 1010
12 – 22%
Cobas Amplicor Monitor (Roche)
0.001
2 x 102
2 x x 105
14 – 44%
Cobas Taqman (Roche)
35 (Manual)
70 (Automated)
2 x x 1010
16 – 54%
RealArt HBV PCR (artus/Qiagen) 10
1 - 4 x 108
*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

12. 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

13. Laboratory Diagnosis of Hepatitis C

14. 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

15. 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

16. Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection
anti-HCV
Symptoms +/-
HCV RNA
Titer
ALT
Normal 1 2 3 4 5 6 1 2 3 4
Months
Years
Time after Exposure

17. Virological Markers for Hepatitis C Assessment
HCVAb
HCV RNA
Interpretation
Positive
Acute or chronic HCV depending on the clinical context
Negative
Resolution of HCV; Acute HCV during period of low-level viremia
Early acute HCV infection; chronic HCV in setting of immunosuppressed state; false positive HCV RNA test
Absence of HCV infection 17

18. Approved HCV Direct-acting Antiviral (DAA) Agents
Core E1 E2 P7
NS2
NS3 4A
NS4B
NS5A
NS5B
5’UTR
3’UTR
Protease Inhibitors
Simeprevir (SMV)
Paritaprevir (PTV)
Grazoprevir (GZR)
NS5A Inhibitors
Ledipasvir (LDV)
Daclatasvir (DCV)
Ombitasvir (OMV)
Elbasvir (EBR)
Velpatasvir (VEL)
NS5B NUC Inhibitors
Sofosbuvir (SOF)
NS5B non-NUC Inhibitors
Dasabuvir (DSV)
Bertino G. World Journal of Hepatology. 2016;8(2):

19. 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.

20. HCV RNA Detection Assays
Method
LLD* (IU/ml)a
Linearity (IU/ml)
Versant Qualitative (Siemens)
TMA
5 - 10 NA
Amplicor Qualitative v2.0 (Roche)
RT-PCR 50
Ampliscreen (Roche)
Amplicor Monitor v2.0 (Roche)
600
,000
Cobas Taqman V2.0 (Roche) 10
25 – 3 x 108
Abbott RealTime (Abbott)
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)
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

21. 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%.

22. 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

23. 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

24. Mechanism of Action of DAAs
Binding and blocking the active site of the protein (NS3, NS5A & NS5B)
Resistance:
Mutations close to active site reduce affinity to drug

25. 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

26. 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)
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):

27. NS5A Resistance Overview
Baseline polymorphisms associated with resistance are relatively prevalent (15-30%)
Currently available NS5A inhibitors suffer from broad cross-resistance 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

28. Broad Cross-resistance with NS5A inhibitors
Fold-change in resistance
GT1a
GT1b
M28T
Q30R
L31M/V
Y93H/N
L31V
LDV
20x
>100x
>100x/ >1,000x
>1,000x/ >10,000x
>100x/--
OMV
>1000x
<3x
<10x
20x/50x
DCV
EBR
>10x
VEL
<3x/--

29. 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?

30. 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