Hepatitis B and Hepatitits C virus lab diagnosis and managementa

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RECENT ADVANCES IN DIAGNOSIS AND MANAGEMENT OF HEPATITIS B & C VIRAL INFECTION. GAURAV DOGRA

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DNA Virus, Member of Hepadnaviridae family. Humans are the only host. Numerous antigenic properties. May retain the infectivity for more than 7 days at RT. INTRODUCTION: Hepatitis B Virus

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HBV GENOME: Virion is 42 nm in diameter. Genome is Partially double standard. 3020-3320bp or 1700-2800bp long. Contain 48%GC rich.

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Genes Region Gene Product S S Major Protein (S) S+ Pre-S2 Middle Protein (M) S+ PreS1& S2 Large Protein (L) C C HBcAg P DNA Polymerase X HBxAg HBsAg 8 major genotypes (A-H). Sequence diversity b/w genotypes 8 to 14% CONT….

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4 million infected in India Hepatitis B Virus: Epidemiology More than 350 million chronically infected world wide Established cause of CH, cirrhosis and HCC. Approximately 30% of world population ~ 2 billion India has intermediate endemicity of HBV Infection. Prevalence b/w 2% to 10% among general population. CDC: 2005

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Geographical distribution of HBV infection CDC, 2005

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loss of appetite, weakness, nausea, vomiting, abdominal pain, jaundice (yellow skin or eyes), dark urine and joint pain. Incubation period: 3 to 4 months. Clinical Symptoms? Persons with chronic HBV infection often do not feel sick for decades after infection Acute HBV: Chronic HBV : The patients with HBsAg (+) for more than SIX months chronic HBV infection

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Sexual~15% Perinatal~45% IVDA~20% Health Workers~ 6% Blood transfusion~7% Modes of Transmission Unknown~5% Shahid Jameel et. al Dassarathy et. al S.K. Acharya et. al

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CLINICAL OUTCOME OF HBV Acute hepatitis Resolution 90% Fulminant hepatitis 1% HBsAg >6 months 9% Resolution Asymp. carrier Chronic Hepatitis Cirrhosis HCC ~2-7%

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Serology ELISA HBsAg HBcIgM HBcIgG HBeAg Anti HBs Ab Anti HBe Ab RIA Pathological Assays Liver Biopsy wherever feasible. Molecular Assays PCR RT- PCR Genotyping Biochem Raised ALT Raised AST Raised S. Bil Deranged PT HBV Diagnostic Assays Radiological examination: Endoscopy ,Ultra sonography , CT etc.

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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 Acute Hepatitis B Virus Infection: Serological course Weeks after Exposure Titer

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Acute (6 months) HBeAg Chronic (Years) anti-HBe 0 4 8 12 16 20 24 28 32 36 52 Years Progression to CHB Infection – Serol. course Titer IgM anti-HBc Weeks after Exposure Total anti-HBc HBsAg

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HBV MARKER IN DIFFERENT STAGES OF INFECTION

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Commercial system for serological testing of HBV:

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CONT…....

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Radioimmunoassay: widely-used in research because of its sensitivity Detect a few picograms (10−12 g) of antigen

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Molecular Methods to Detect HBV DNA Target amplification assays : PCR, LCR (ligase chain reaction ) Hybridization assays: Dot blot hybridization assays Signal amplification assays: b DNA assays Direct Sequencing

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Polymerase chain reaction: Target Sequence: Surface Region Size: 321bp Initial Denaturation - 94˚C for 3min. Final extension- 72˚C for 10min. Extension- 72˚C for 50 sec Cycle Denaturation- 94˚C for 30 sec. Annealing- 58˚C for 50 sec. 34 cycles Result: 2% Agarose gel electrophoresis.

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Dot blot hybridization assays: Sample: Serum or tissue Detection limit: 0.1 top 1.0pg (28,000 to 280,000 genomic) Time: 5 days Method: Autoradiography Lower sensitivity Long turnaround time

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b- DNA assays: Detection by chemiluminescence emission Detect 2x103 HBV copies/ ml & up to 5x109 copies/ ml Bayer Quantiplex /Versant HBV DNA ASSAY

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Molecular assay used to detect HBV nucleic acid

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Genotyping assays: 3. Restriction fragment length Polymorphism. Line probe HBV genotyping assays. 2. Sequence analysis: A product of 806 base pairs of pre-S and a part of the S gene were amplified with the primers. Genotyping is not of much significance as the therapy is not based on the genotypes

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Occult HBV Infection: Detectable HBV DNA in blood or liver in the absence of HBsAg Testing for occult hepatitis B recommended in: In cryptogenic liver disease. Organ transplant donors who are anti-core Ab. +ve. Molecular assays with high sensitivity are required. Diagnosis of occult HBV Infection

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Hepatitis B vaccine Plasma derived vaccine produced form 22nm HBsAg particles. Licensed in USA in 1981. Safe effective but not well accepted. Recombinant hepatitis B vaccine was licensed in united states in July 1986 This vaccine is produced by inserting a plasmid containing the gene for HBsAg in to saccharomyces cerevisiae Vaccine is produced by two manufactures Merck (Recombivax HB) and GlaxoSmithKline Pharmaceuticals (Engerix-B) Prophylaxis

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Recommended doses of licensed hepatitis B Vaccine Recombinant hepatitis B surface antigen protein dose.

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Interferon Alfa- first Drug approved in 1992 for chronic Hepatitis B in USA & Europe. Bind to specific cell receptor & produces immunomodulatory antiviral and antifibrotic effect. Dose: 10 MU three times a week for 4 months Factor associated with sustained response to interferon AGE High transminase Low serum HBV DNA

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Treatment: Goal: Suppress viral infection. Halt the Progression of liver damage before it leads into cirrhosis &HCC. Only three approved therapies are available: The Alpha Interferon Lamivudine Adefovir

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Peg IFN:- Peg IFN alfa2a Greater decline in HBV DNA Higher HbsAg seroconversion rate than standard interferon. Peg IFN Alfa 2b leads to sustained response in 44% of patients. Liver biopsy : 6 months after completion of treatment (wherever indicated & feasible). Alpha Interferon

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Lamivudine: Enantiomer of 2’3’-dideoxy-3’-thiacytidine It is phosphorylated to the triphosphate, which compete with other triphosphate for incorporation into DNA causing chain termination Dose/Duration : 100mg per day /52 weeks Factor associated with beneficial response to Lamivudine Wild type (HBeAg- Positive) Virus High transminase activity Low serum HBV DNA

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Drawback of lamivudine monotherapy : Emergence of resistant HBV with mutation at Tyrosine Methionine Aspartate Aspartate (YMDD) motif at catalytic domain of the viral reverse transcriptase/ DNA polymerase YMDD- 15-30% in first year 7% in First five years STOP Lamivudine: When there is HBeAg seroconversion with detectable HBeAb undetectable HBV DNA by PCR

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Determination of drug resistance: lamivudine resistance mutation, due to changes in the YMDD motif of the RNA dependent DNA polymerase in P ORF regions Substitutions of either valine or isoleucine for methionine in the highly conserved motif Tyr-Met-Asp-Asp of the C domain . YMDD

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Adefovir Cyclic analog of Deoxyadenosine monoposphate (dAMP) Inhibit the covalently closed circular DNA in HBV infected hepatocyte Dose/ Duration : 10mg Daily/ 48 weeks Adefovir dipivoxil is also active against lamivudine resistant YMDD mutants Adverse effect: Headache, Pharyngitis, abdominal pain and flu like syndrome

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First line treatment of chronic hepatitis B: Adefovir, Lamivudine or interferon?

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New Drugs in development Tenofovir disoproxil fumarate :Similar to adefovir & approved for HIV is also effective in suppressing the replication of YMDD mutants. (CLINICAL TRIAL) Entecavir: Guanosine analog, has a strong inhibitory effect on the priming of HBV polymerase by guanosine tri phosphate Dose: 0.1-0.5mg & 1mg against YMDD mutants

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Telbuvidine (β-L-2’-deoxythymidine,LdT) is one of three L-nucleoside with specific HBV inhibitory activity. LdT almost without side effect DOSE: 400-60mg daily / 6 months of therapy

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Hepatitis C Virus

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HCV CHARACTERISTIC FAMILY Flaviviridae Enveloped Positive sense single standard RNA (9.6kb) 3300 aminoacid polyprotein. No. RNA proof reading ability~ Quasispeicies Half life ~2.7 hours Daily production 1012 virions

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Risk factor for hepatitis C virus infection

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92% 81% 55% 65% 57% 70% 65% 66% 26% Genomic organization and homology between the isolates of HCV

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Genotype/ Subtypes: Genome heterogeneity among different HCV isolates. Varies from 31% to 35% of bases Six major genotypes and 120 subtypes Quasispecies: Population of closely related yet heterogeneous sequences within the same individual. The quasispecies vary from each other by 1% to 9% of bases.

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Chronic hepatitis Clinical Basis: Anorexia, weight loss, jaundice, ascites, biochemical features SGOT, SGPT more than normal for more than 6 months duration. Morphological changes of periportal hepatitis, presence of variable inflammation and hepatocyte regeneration of the lobule

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Cirrhosis: Ascites, Splenomegaly, Shrunken Liver. Endoscopy shows esophageal varices Biochemical features of hepatocellular failure, characterized by low serum albumin level (<2.5 gm %) Hepatocellular Carcinoma (HCC): The diagnosis of HCC will be based on cytology and relevant histopathological changes in liver biopsy. The serum alpha- fetoprotein > 1000mg/ml.

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HCV infection: Worldwide Distribution of Genotypes 6-GENOTYPES/ 120 SUB TYPES/QUASISPECIES

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Natural course of HCV infection HCV Infection < 20% self-limiting > 80% chronic 15-40% Cirrhosis 10-30% HCC Immune response Hepatitis Re-infection

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Serology ELISA Anti HCV Ab Core Ag RIBA Pathological Assays Liver biopsy FNAC Molecular Assays PCR RT- PCR b-DNA TMA Genotyping Biochem Raised SGOT Raised SGPT Raised S. Bil Deranged PT HCV Diagnostic Assays Radiological examination: Endoscopy , Ultra sonography , CT.

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Serologic Pattern of Acute HCV 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 200 400 600 800 1000

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Serologic Pattern of Chronic Infection Symptoms +/- Time after Exposure Titer anti-HCV ALT Normal 0 2 4 6 1 2 3 4 Years Months HCV RNA 200 400 600 800 1000

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HCV Polyprotein

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Recombinant immunoblot assay (RIBA) Chiron corporation develop a strip immunoassay to resolve true positive from false positive EIA result FDA approved second generation RIBA in 1993 followed by third generation 1999. EIA Antigen(NS3+NS5) + Super oxide Dismutase (hSOD) Recombinant hSOD included on RIBA strip to detect nonspecific antibodies

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Guidelines for HCV RNA Testing CDC

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Cont..

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HCV RNA Detection: HCV RNA in plasma defines active infection HCV RNA can be detected 1 to 3 weeks post exposure. Nucleic acid test can be classified in to: Qualitative: RT-PCR, TMA Quantitative: bDNA, Real Time PCR

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Transcription mediated amplification (TMA) Involves reaction with T7 RNA Polymerase and RT under isothermal condition The TMA based VERSANT HCV RNA qualitative assay and procleix HIV/HCV assay was approved by FDA Detection limit: as low as 5 copies/ml Highly Sensitive: 96%

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Real Time PCR Syber Green TaqMan (Primer Probe) Detection

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Advantages and Disadvantages of both the chemistry

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Commercial available HCV RNA detection assay

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Different Methods of Genotyping

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Fast, Easy Highly specific DNA hybridization test Rapid diagnosis of HCV genotypes. Expansive The INNO-LiPA HCV II:

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Restriction Fragment Length Polymorphism: HCV genotyping typically involves RT-PCR amplification of the 5’UTR. Restriction Endonuclease Digestion of he PCR Product 3 to 5 different enzymes are used All six genotypes can be detected Used in routine lab practice. Sensitivity 96%

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Occult HCV Infection:- HCV RNA undetectable in serum or plasma by current assays, but found in serum, PBMC and/or liver by enhanced molecular tests Very high sensitivity molecular assay like nested RT-PCR followed by nucleic acid hybridization technique required Diagnosis of occult HCV Infection

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Response of therapy Disease severity Geographical distribution Design of HCV vaccine and therapeutic agents. Significance of genotyping

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Management protocol in chronic hepatitis C Considering the natural history of hepatitis c, there are different goal for treatment. To prevent the occurrence of cirrhosis and its complication To reduce the extra hepatic manifestation To prevent the contamination of other people (i.e. surgeon or drug user)

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IFN- is glycoprotein that have both direct antiviral and immunomodulatory activities. The activity of IFN-  against a wide range of DNA and RNA viruses prompted its use among the patients with chronic HCV. INTERFERON+ Ribavirin Sustained virological response Peginterferon-  2a [Pegasys] Peginterferon-  2b [PEG- intron] 56% 54% Genotype 2,3 response rate : 76-82% Genotype1 response rate: 42-46%

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Candidate for HCV therapy with no contradictions HCV Genotyping Genotype 1,4,5 &6 Genotype 2 & 3 Liver biopsy and viral load measurement to determine benefits of therapy PEG-IFN+ Ribavirin Week12: measure viral load PEG-IFN+ Ribavirin Week24: stop therapy & measure viral load <2 log drop ≥ 2log drop but still detectable by qualitative HCV RNA Assay ≥ 2log drop & undetectable Therapy failure End –of- treatment response; stop therapy Non response ; Stop therapy Continue therapy Continue therapy Week 24: Qualitative HCV RNA + - Non response; stop therapy Continue therapy Week 48: Qualitative HCV RNA + - Non response; stop therapy End –of- treatment response; stop therapy Week 72: Qualitative HCV RNA + - Relapse Sustain Virological response Week 48: Qualitative HCV RNA + - Relapse Sustain Virological response SUMMARY

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New drugs in development: These are small molecules, inhibitors of the HCV virus replication, protease, helicase and polymerase An Oral HCV serine protease inhibitor (BILN 2061 KG pharma Germany) was administered to 31 patients of genotype1. 25 patients viral load return to the base line with in 7 days. 5 patients does not respond to therapy {CLINICAL TRIAL} Other drugs include new enzyme modulators, new ribavirin analogs deprive of hematologic toxicity, ribozymes etc. The Development of an effective HCV vaccine has so far been disappointing

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THANKS GAURAV DOGRA

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Acute hepatitis patients, immunocompromised patients, infants born to HCV-infected mothers Symptomatic patients, recipients of blood or blood products prior to 1991, hemophiliacs, injection drug users, organ, tissue, or blood donors Qualitative HCV RNA HCV EIA-3 Stop† Stop† Quantitative HCV RNA Liver biopsy/treatment decision‡ HCV genotyping‡ Ty 2 or 3 Ty 1, 4, or 5 12 mo of IFN monotherapy (ribavirin contraindicated) 6 mo of IFN-ribavirin Qualitative HCV RNA: 3 mo (discontinue if positive) Qualitative HCV RNA: 3 mo Qualitative HCV RNA:12mo Quantitative HCV RNA: 6 mo after treatment to confirm a sustained response Positive Negative Negative Positive

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http://faculty.vassar.edu/lowry/newcs.html http://faculty.vassar.edu/lowry/fisher2x3.html http://www.quantitativeskills.com/sisa/

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cirrhosis

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Use of Mol Tech in initiation & monitoring of Rx. In CHB ? HBV DNA >20,000 IU/mL in HBeAg +ve suggest repli. Persistence of ALT 3-6 months Demonstration of histological changes. Baseline viral load req. to predict Rx response. HBeAg +ve Rx 4-6 m & HBeAg –ve Rx 6-12 m. Endpoint Rx: anti-HBe +ve & DNA <20,000 IU/mL. Follow up: 3 months for a year.

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Chromatograph Showing Precore gene mutation (Valine to Phenylalanine)