2013:

HCV GenomeHCV Genome

-HCV genome is about 9400 nucleotides long, it is ssRNA and positive sense

-the 10 viral proteins are first made as a large polyprotein

-individual proteins are released from polyprotein by cellular and viral proteases

-core, E1 and E2 are the structural proteins which form the virus particle

-remaining proteins are nonstructural and have roles in viral replication

HCV protein and its functionHCV protein and its function

HCV VariabilityHCV Variabilityyy

Marked genetic heterogenity due to hyper variable g g y ypregion

RNA virus, RDRP lacks proof-reading functionMutations arise during replication are not corrected

– Genotypesi ll di HCV i l h bgenetically divergent HCV isolates that can be

grouped phylogenetically

HCV Genotypes and QuasispeciesHCV Genotypes and Quasispeciesyp pyp p

Term Definition Nucleotide Similarity

GenotypeHeterogeneity among different viruses

66% – 69%

SubtypeClosely related viruses within each genotype

77% – 80%genotype

QuasispeciesComplex of genetic variants within individual viruses

91% – 99%individual viruses

Hepatitis C GenotypesHepatitis C Genotypes

HCV genotypes are substantially divergent in sequence from each other and fall into 6 phylogenetic clades designated aseach other and fall into 6 phylogenetic clades, designated as genotypes

• More than 70 subtype are identified (21 unclassified)Genotypeyp ( ) yp

• dictates length of therapy and predicts therapeutic responseGenotype 1 requires longer therapy and has lower responseGenotype 1 requires longer therapy and has lower response

GENOTYPESGENOTYPES1 and 2: global distribution1a: west europe1a: west europe1b: USA and japan, blood transfusion3a: asia, infects injecting drug users3a: asia, infects injecting drug users4: middle east and africa5: south africa6: east asia

In Iran :1a: 49%3a: 34%1b: 13%4: 0.4%

Clinical symptomsClinical symptoms

In acute phaseJust about 15% of HCV cases are acute infectionClinical features of acute hepatitis divide into four stages:1. incubation period: 15-160(50) days, RNA are detected after 1-2 w

asymptomatic, anti HCV negative1. Pre- icteric period: elevate LFT level, non specific symptoms,

1. Icteric period: urine darkness and jaundice occurs, in 80% of cases Bili level at least 3mg/dl and ALT elevate 600IU, HCV RNA are positive gbut anti-HCV exist only in 50-70% , 4-6 weeks

2. Convalescent period: recovery of symptom, normalized LFT,

Natural HistoryNatural HistoryNatural HistoryNatural HistoryAcute Infection

Symptoms– SymptomsAre uncommonOn average, appear 6 to 7 weeks after infection.g pp

– Testing6 t 8 k A ti tib di b6 to 8 weeks: Average time antibodies can be detected.1 to 3 weeks: Average time virus can be detected. 4 to 12 weeks: Often elevation in ALTs

15 to 25 percent of people resolve acute infection– 15 to 25 percent of people resolve acute infection.

Serologic Pattern of Acute HCV Infection i h Rwith Recovery

Symptoms +/

anti-HCV

Symptoms +/-

HCV RNA

Tite

r

ALT

Normal

0 1 2 3 4 5 6 1 2 3 4

Time after ExposureYearsMonths

chronic phasec o c p ase85% of cases, patients not able clear virus and after 6 mounts cause viremia and persistent infection with highest ALT level

d HCV RNA itiand HCV-RNA positive Factors that cause chronically infections aren't distinct to know but: virus quasispecies susceptibility of E gene to rapidknow but: virus quasispecies, susceptibility of E gene to rapid mutation and immune system dodging

Gradually progressing disease and absence of symptoms first two decades are critical characteristicsTiredness and lethargy are most commonTiredness and lethargy are most commonCirrhosis (20-30% o patients)Severity indicators of disease are ambiguousSeverity indicators of disease are ambiguous

Natural HistoryNatural HistoryNatural HistoryNatural HistoryChronic Infection

– Diagnosed by the detection of HCV RNA in the blood for at least six months.

– 60 to 70 percent of people will have persistent or fluctuating ALT elevations.

– Chronic liver disease usually progresses at a slow rate without symptoms.rate without symptoms.

– Estimated that 10 to 20 percent of people will develop cirrhosis 20 to 30 years after infection.

– The rate of progression is highly variable.

Serologic Pattern of Acute HCV Infection with P i t Ch i I f tiProgression to Chronic Infection

Symptoms +/

anti-HCV

Symptoms +/-

HCV RNA

Tite

r

ALT

Normal

0 1 2 3 4 5 6 1 2 3 4

Time after ExposureYearsMonths

Natural HistoryNatural HistoryNatural HistoryNatural History

Factors that Influence Progressiong– Greater than age 40 at time of infection– Male gender– Alcohol use– Co-infection with HIV or HBV

C– Co-morbid conditions such as obesity

Factors that Don’t Influence ProgressionFactors that Don t Influence Progression– Viral load– GenotypeGenotype

FULMINANT VIRAL HEPATITISFULMINANT VIRAL HEPATITIS

R d i l th 1% f i t i h titiRare and occurs in less than 1% of icteric hepatitis infectionsSevere progressive manifestation of vial hepatitisSevere, progressive manifestation of vial hepatitisCauses extensive liver necrosisLi dd l b llLiver may suddenly become smaller

Marked ↑ in prothrombine time (PT), that does not improve with vitamin Kimprove with vitamin K

F t lit h 80%Fatality approaches 80%

TransmissionTransmissionTransmissionTransmission

How is hepatitis C o s epat t s Cbeing transmitted?

Routes of TransmissionRoutes of Transmission

S l 15%Injecting drug use 60% Sexual 15%Injecting drug use 60%

Transfusion 10%(before screening)

Occupational 4%

Other 1%*

Unknown 10%Unknown 10%

* Nosocomial; iatrogenic; perinatal Nosocomial; iatrogenic; perinatal

Source: Centers for Disease Control and Prevention

HCV TestingHCV TestingHCV TestingHCV Testing

Who should o s ou dbe tested?

CDC Testing RecommendationsCDC Testing RecommendationsCDC Testing RecommendationsCDC Testing RecommendationsTesting Routinely Recommended Based on Risk of Infection

Person who ever injected illegal drugs

Persons with selected medical conditions– Persons who received clotting factor concentrates – Persons who were ever on long-term hemodialysis– Persons with persistently abnormal alanine

aminotransferace levels (persons with chronic liver disease)

Prior recipients of transfusions or solid organsPersons who were notified that they received blood from a– Persons who were notified that they received blood from a donor who later tested positive for HCV infection

CDC Testing RecommendationsCDC Testing RecommendationsCDC Testing RecommendationsCDC Testing Recommendations

Testing Routinely Recommended Based on Need for g yExposure

– Health care, emergency medical, and public safety workers after needlesticks, sharps, or mucosal exposures to HCV positive bloodmucosal exposures to HCV positive blood

Child b t HCV iti– Children born to HCV positive women

HCV TestingHCV Testing

Diagnostic methods of HCV infectionDiagnostic methods of HCV infectiongg

Specific and nonSpecific and non--specific tests are available:specific tests are available:

specific Non-specific

serologic Liver enzyme

virologic

enzyme

Liver virologic

G t

biopsy

Liver Genotype determination radiograp

hy

The role of testing in The role of testing in di i d t t tdi i d t t tdiagnosis and treatmentdiagnosis and treatment

Method Screen Confirm Duration of therapy

Predicting sustained response

Assessing treatment response

HCV antibody test (EIA) X

PCR: HCV genotype X

PCR: HCV RNA qualitative assay X X

PCR: HCV RNA quantitative assay X

HCV TestingHCV TestingHCV TestingHCV Testing

Initial Screening – Negative Resultg g

– A negative test most likely means that a person is not infected.

– False negatives are uncommon.May occur if a person has been recently infected.May occur in individuals who are immuno-suppressed or on long-term hemodialysis.

HCV TestingHCV TestingHCV TestingHCV TestingInitial Screening - Positive Result

F l iti– False positives are uncommon.Most likely to occur in individuals at low-risk for infection.for infection.May occur in individuals with autoimmune liver disease.

– A positive test, especially in a person with known risk factors most likely means that they haverisk factors, most likely means that they have been exposed to the virus.

– Screening test results can be verified with a supplemental or confirmatory test.

HCV TestingHCV TestingHCV TestingHCV TestingConfirmatory Testing

– To ensure that a positive screening test result is a true positivea true positive.

To distinguish between a resolved and an active– To distinguish between a resolved and an active infection.

– They can be used alone or more than one test can be used.

VirologicVirologicgg

Detection of HCV-RNA are most useful particularly when Ab is not producedPCRG ld t d d f HCV iti i RT PCRGold standard of HCV recognition is RT-PCRThere are two qualitative and quantitative RT-PCR for HCV recognitionfor HCV recognitionQualitative: Amplicor HCV test and Cobas Amplicor HCV testHCV testQuantitative: RT-PCR

Testing Asymptomatic PeopleTesting Asymptomatic PeopleEIA (-) No HCV

(+)

RT-PCR ( )(-)

RIBAActive

(+)

(-)

RIBAActive HCV

Infection (+)

False Positive EIANo Exposure

(Probable) Prior HCV Infection with Recovery py

Liver BiopsyLiver BiopsyLiver BiopsyLiver BiopsyLiver Biopsy– Most sensitive measure of disease severity– Most sensitive measure of disease severity.

– Used to determine stage of fibrosis.g

– Can be used to help predict natural history of disease.

– Often used to determine the need for treatment.

– Can also be used to predict response to treatment.

Tests asessing liver damageTests asessing liver damageg gg g

Noninvasive tests of fibrosis and activityNoninvasive tests of fibrosis and activity– Panel of biochemical markers, e.g.

FibroTest– Ultrasonography, e.g. FibroScan

Liver biopsy– Gold standard for grading inflammation– Gold standard for grading inflammation

and disease stage

Staging of Fibrosis on Liver BiopsyStaging of Fibrosis on Liver Biopsyg g p yg g p y

S tage H is to logy

0 N o fib ro s is0 N o fib ro s is

1 M in im al fib ro s is

2 M o d era te fib ro s is

3 M o d era te to S evere fib ros is

4 C irrh o s is

HistologicHistologic Progression of HCVProgression of HCVHistologicHistologic Progression of HCVProgression of HCV

NormalNormal Mild Chronic HepatitisMild Chronic Hepatitis

Moderate Chronic HepatitisModerate Chronic Hepatitis CirrhosisCirrhosis

Role of liver biopsy in HCV infectionRole of liver biopsy in HCV infectionConfirm clinical 

diagnosisAssess severity of  fibrosis and 

necroinflammation1

p yp y

diagnosis

Role of liver biopsy in 

HCV infection

Can aid decision making

Associated risks:bleeding <1% 

death 0.1–0.01%2 HCV infection

Evaluate possible concomitant disease processes (e.g., alcoholic liver 

disease)

Assess therapeutic intervention1

TreatmentTreatmentTreatmentTreatment

Who should o s ou dbe treated?

Contraindications to treatmentContraindications to treatmentContraindications to treatmentContraindications to treatment

Patients in whom HCV therapy is currentlyPatients in whom HCV therapy is currently contraindicated include:– Pregnant patients– Patients with severe uncontrolled systemic

diseasesP ti t ith k h iti it t d d– Patients with known hypersensitivity to drugs used to treat HCV

TreatmentTreatmentTreatmentTreatmentTreatment Goal

– To prevent complications of infection; principally achieved by eradication of the virus.

– HCV is considered to be eradicated when there is a Sustained Viral Response (SVR).

– An SRV is defined as the absence of detectable HCV RNA (virus) six months after treatment ends.HCV RNA (virus) six months after treatment ends.

– A qualitative viral detection test is used for this purpose.

Pegylated interferon and ribavirinPegylated interferon and ribaviringygy

Peginterferon alfa-2a (40KD)PEGASYS

Ribavirin

S b t i j ti B thSubcutaneous injection, once weekly

By mouth, twice daily

Evolution of hepatitis C treatmentEvolution of hepatitis C treatment

Discovery of HCV genomeDiscovery of HCV genome

Treatment with IFN alfa for 24 or 48 weeks – 3x weekly dosing – Poor outcomes

Addition of RBV to IFN alfa improved outcomes

Peg‐IFN mono – once‐weekly dosing

Peg‐IFN alfa plus RBV becomes gold standard

Response‐guided therapy emerging

New antivirals enter development

20101989

Evolution of hepatitis C treatmentEvolution of hepatitis C treatment

NS3 protease inhibitor classes include:p- linear covalent(boceprevir and telaprevir),- linear noncovalent(asunaprevir)- macrocyclic inhibitors (vaniprevir)

f Sinhibitor of NS5A: DaclatasvirDaclatasvir is a potent inhibitor of NS5A that was used in combination with asunaprevirused, in combination with asunaprevir

Treatment Side EffectsTreatment Side EffectsCommon Side Effects of Peginterferon

Occurring in more than 10 percent of patientspatientsFatigueMuscle achesHeadachesNausea and vomitingSkin irritation on injection siteLow-grade feverWeight lossWeight lossDepressionMild bone marrow suppressionMild bone marrow suppressionHair loss

Treatment Side EffectsTreatment Side EffectsCommon Side Effects of Ribavirin– Occurring in more than 20 percent of patientsg p p

AnemiaFatigue and irritabilityg yItchingRashNasal stuffiness, sinusitis, and cough

Ribiviran can cause birth defectsMust use strict contraceptive methods during treatment and for six months after.

In January 2004, the role of occult hepatitis C virus (HCV) i f ti i h i li di f k ti l iinfection in chronic liver disease of unknown etiology in approximately 10% of cases with abnormal LFTs was first d ib d b C till t ldescribed by Castillo et al.

. This cases was termed cryptogenic liver hepatitis

This occult infection can be present in two f pdifferent clinical situations:

I. in antianti--HCV negative:HCV negative: serum HCV-RNA negative patients with abnormal liver function tests

II. and in antianti--HCV positiveHCV positive subjects with normal values of liver enzymes and without serum HCV RNA

I 57 f 100 ti t h ti f ti HCV tib di dI 57 f 100 ti t h ti f ti HCV tib di dIn 57 of 100 patients who were negative for anti-HCV antibodies and for serum HCV RNA and who had occult HCV infection, as demonstrated by the detection of HCV RNA in liver-biopsy specimens

In 57 of 100 patients who were negative for anti-HCV antibodies and for serum HCV RNA and who had occult HCV infection, as demonstrated by the detection of HCV RNA in liver-biopsy specimensdemonstrated by the detection of HCV RNA in liver biopsy specimens by RT-PCR demonstrated by the detection of HCV RNA in liver biopsy specimens by RT-PCR

HCV in extrahepatic region was reported like PBMCs, BM, CSF, spleen, pancreas,… HCV in extrahepatic region was reported like PBMCs, BM, CSF, spleen, pancreas,… p pp p

Therefore PBMCs are so beneficial, although gold standard is liver biopsyTherefore PBMCs are so beneficial, although gold standard is liver biopsyp yp y

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