Laboratory Diagnostics in Hepatitis T. Mazzulli, MD, FRCPC Department of Microbiology UHN/Mount Sinai Hospital

Objectives Review the serologic diagnosis of viral hepatitis Review the methodologies available for molecular testing and describe some of the advantages and disadvantages Discuss the currently available commercial assays that are available and those which are in use in Toronto Discuss the use of molecular methods for genotyping and resistance testing

Hepatitis A - Diagnosis Three serologic markers available: Hepatitis A Total (IgG and IgM) antibody Hepatitis A IgM Hepatitis A IgG First tests available since 1978 No antigen test Antibody response is similar following vaccination or wild type infection Incubation time is 7 to 28 days

Hepatitis A Virus Infection Typical Serologic Course Symptoms Total anti-HAV ALT Titer Fecal HAV IgM anti-HAV 1 2 3 4 5 6 12 24 Months after Exposure

Laboratory Tests for HBV Serology: Many tests available – most common tests are Enzyme Immunoassays (EIAs, MEIAs) First tests available in 1972 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: 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: 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; spontaneous seroconversion in 10 to 20% of healthy adults per year

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

Disease Phases in Chronic HBV Infection 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 <3 log IU/mL HBeAg-negative Chronic HBV

Interpretation of Serologic Tests in Hepatitis B

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; likely due to mutations in the “a” determinant of the S gene Surface antigen escape mutants described in infants infected with HBV after HBIG + vaccination and in Liver transplants after prolonged HBIG Anti-HBc IgM may persist for up to 2 years in 20%; chronically infected individuals may have low titres which rise during acute flares

Hepatitis B – Laboratory Tests Serologic markers – caveats: Precore or HBeAg negative mutants: Due to mutation in precore (abolishes HBeAg production) or core promoter region (down-regulates HBeAg production) No effect on viral replication (may be enhanced) More difficult to treat; greater risk of cirrhosis Co-infection with HCV may suppress both HBeAg and HBsAg

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

Hepatitis B – Laboratory Tests Serologic markers – caveats: Isolated HBcAb may be due to: Remote infection (immune or chronic carrier) “Window” period between HBsAg and HBsAb Co-infection with HCV False positive test result – HBcAb is marker most prone to false positives HBV DNA may help sort this out

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 The diagnosis of HCV infection is based on the detection of specific antibodies and the viral RNA genome.

Laboratory Tests for HCV Serology: Screening: 3rd generation EIAs measure antibodies directed against recombinant peptides NS4, core, NS3, and NS5 proteins Sensitivity = 97% Detects antibodies within 6 to 8 weeks No HCV IgM test available Confirmatory/supplementary: RIBA, LiPA, Second EIA, HCV RNA The diagnosis of HCV infection is based on the detection of specific antibodies and the viral RNA genome.

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

Rational Use of HCV Diagnostic Tests TREATMENT Diagnosis Serological assays Qual HCV RNA As mentioned, diagnostic tests can be used at different time points during chronic hepatitis C. After having diagnosed HCV infection, using serological and molecular assays, the determination of ALT values, viral load levels and liver histology helps in defining prognosis, indications to therapy and treatment protocols as well as the probability of patients’ response to therapy. Once treatment begins, it is important to monitor ALT and viral load for an early assessment of treatment outcomes and the need to suspend or modify the initial protocols. Prognosis Liver histology Decision to treat ALT Liver histology Qual HCV RNA Treatment duration Genotyping Viral load Response and resistance assessment Qual HCV RNA Viral load

Hepatitis D Virus - Diagnosis Anti-HDV Total (IgG & IgM) available Incubation time – similar to Hepatitis B High titres of HDV antibodies indicate ongoing chronic infection Available only at National Microbiology Lab in Winnipeg

Hepatitis E Virus - Diagnosis Both IgG and IgM antibody tests are available Incubation period – 7 to 28 days No domestically acquired cases in Canada Available only at the National Microbiology Lab in Winnipeg

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 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 Determined by PROBIT analysis to determine the value that is consistently detected 95% of the time 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 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

Measuring HBV DNA Gish and Locarnini, Clin Gastro Hep 2006

Comparison of Quantitative HBV DNA Assays Versant 3.0 vs. Versant 1.0: R2 = 0.9001 Versant vs. Cobas: R2 = 0.7711 Versant vs. Digene: R2 = 0.9849 Yao J et al. J Clin Microbiol 2004:42(2)

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 (Roche) 15 15 – 1 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

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

Laboratory Tests for HCV 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 The diagnosis of HCV infection is based on the detection of specific antibodies and the viral RNA genome.

Genotyping Used for: Methods include: 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.)

InnoLiPA Principle Chromogen (NBT/BCIP) Purple precipitate Marker line Conj.cont. Amp.cont. L180 M180 M204 V204 I204 V207 L207 M207 I207 Alkaline Phosphatase Streptavidin Biotin Amplified target DNA-probe Nitrocellulose strip

InnoLIPA HBV Drug Resistance

HBV Resistance Testing

HBV Resistance Testing

InnoLiPA vs. Sequencing Hussein et al, J Clin Micro 2006

Drug Resistance Report LAMIVUDINE Present Mixed Absent L80V    X V173L X L180M M204V M204I M204S X     Present Mixed Absent A181V    X A181T X   N236T ADEFOVIR

Interpreting HBV DR Reports   Resistance Mutation Lamivudine Resistance L180M +M204V Adefovir Resistance N236T A181V/T Sensitive Adefovir Tenofovir Lamivudine Entecavir Telbivudine Emtricitabine Resistant or Reduced Susceptibility Clevudine · L180M + M204V/I are the key lamivudine-resistant mutations · A181V/T leads to 4-fold increase in IC50 ·  N236T leads to 7-fold increase in IC50

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 Sensitivity and dynamic range varies between assays Standardization allows (to some degree) interchangeability of the results with different assays Resistance/Genotyping requires amplification first Increasing role in making treatment decisions as more drugs become available for HBV