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 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

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

Virological and Biochemical Course of Chronic Hepatitis B

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

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 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% Liquid Hybridization (Abbott) 1.6 5 x 105 5 x 105 - 1 x 1010 12 – 22% Cobas Amplicor Monitor (Roche) 0.001 2 x 102 2 x 102 - 2 x 105 14 – 44% Cobas Taqman (Roche) 35 (Manual) 70 (Automated) 2 x 102 - 1 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

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 anti-HCV Symptoms +/- HCV RNA Titer ALT Normal 1 2 3 4 5 6 1 2 3 4 Months Years Time after Exposure

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

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):92-106.

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 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) 600 600-800,000 Cobas Taqman V2.0 (Roche) 10 25 – 3 x 108 Abbott RealTime (Abbott) 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) 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

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 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) 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 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

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/-- http://iasusa.org/sites/default/files/uploads/2016hivsny_naggie.pdf

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