Werkkopie van Hepatitis A-Z-Kortijk-04-09-30 (FINAL)an acute HBV infection in the “window phase”...

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1 Geert Leroux-Roels Laboratorium voor Klinische Biologie UZ Gent Virus Discovered Genome Envelope Classification HAV 1975 ss-RNA Picorna HBV 1970 ds-DNA HBsAg HepaDNA HCV 1989 ss-RNA E1-E2 Flaviviridae HDV 1978 ds-RNA HBsAg viroid HEV 1990 ss-RNA Caliciviridae HGV 1995 ss-RNA E1-E2 Flaviviridae TTV 1997 ss-DNA Circoviridae Virus Serology Antibodies Antigens Molecular det/quant Advanced molec anal HAV IgM, IgG HBV HBsAl HBeAl HBcAl HBsAg HBeAg det/quant genotype resistance HCV IgG det/quant genotype HDV Anti-delta Delta-Ag HEV IgM, IgG HGV IgG TTV IgG det

Transcript of Werkkopie van Hepatitis A-Z-Kortijk-04-09-30 (FINAL)an acute HBV infection in the “window phase”...

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Geert Leroux-RoelsLaboratorium voor Klinische Biologie

UZ Gent

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Virus Discovered Genome Envelope Classification

HAV 1975 ss-RNA Picorna

HBV 1970 ds-DNA HBsAg HepaDNA

HCV 1989 ss-RNA E1-E2 Flaviviridae

HDV 1978 ds-RNA HBsAg viroid

HEV 1990 ss-RNA Caliciviridae

HGV 1995 ss-RNA E1-E2 Flaviviridae

TTV 1997 ss-DNA Circoviridae

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Virus Serology Antibodies Antigens

Molecular det/quant

Advanced molec anal

HAV IgM, IgG

HBV HBsAl HBeAl HBcAl

HBsAg HBeAg

det/quant genotype resistance

HCV IgG det/quant genotype

HDV Anti-delta Delta-Ag

HEV IgM, IgG

HGV IgG

TTV IgG det

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• Serology– Anti-HAV IgM acute HAV infection– Anti-HAV Totaal immunity

- natural- vaccine-inducedprotective level 10-30 IU/L

• HAV detection in blood and blood productsin faeces, saliva, …in shelfish, food productsin water,sewage,

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• Children (2-15)Havrix Junior (720 U/dose), 0.5 ml ml/dscheme : 0 and 6-12 mo (2 doses, IM)

• Adults (>15)Havrix 1440 (1440 U/dose, 1 ml/dscheme : 0 and 6-12 mo (2 doses, IM)

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Rare cases in western countries after recent travel in endemic area

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Serologyanti-HEV IgManti-HEV IgG

HEV-RNA

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3200 baseparen4 open reading frames7 proteins• large (preS1-preS2-S)• Middle (preS2-S)• Small or HBsAg• nucleocapsid or HBcAg• secreted HBe-Ag• Polymerase• X protein

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Antigens Antibodies

HBsAg anti-HBs

HBcAg anti-HBc-Totanti-HBc-IgM

HBeAg anti-HBe

Inflammation and liver cell damageTransaminases ALT/ASTen other biochemicalmarkers

Detection/quantific. of HBV DNACommercial assays- Branched DNA (Bayer)- PCR (Roche Amplicor)In-house nested PCRIn-house real-time PCR

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• Diagnosis– Acute HBV infection : HBV DNA is not useful– Chronic HBV infection – Is HBV replicating ?

• HBeAgpos : not useful• HBeAgneg/anti-HBepos : useful, “threshold value” ?

• Prognosis• Therapy

– Decision to treat : ALT, biopsy, HBeAgpos

– Selection treatment– Monitoring : HBV DNA, ALT, HBeAg, anti-HBe,

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• 41 years old Afghan male• political refugee• In training for assistant-cook• Medical screening exam for ‘hepatitis’• No symptoms• No history of hepatitis

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8-61U/L20γ−GT0-128U/L106Alk Fosf0-480U/L351LDH0-38U/L21AST0-41U/L16ALT0.20-0.80mg/dL0.55Bili Ind0.00-0.20mg/dL0.10Bili Dir0.00-1.00mg/dL0.66Bili TotRef. intervalUnitResultTest

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NegativeAnti-HCV

NegativeAnti-HAV-IgM

PositiveAnti-HBc-Tot

46 IU/LAnti-HBs

PositiveHBsAg

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Question 1Can HBsAg and anti-HBsoccur concomitantly ?

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• Are present during the clearance phase of an acute HBV infection in the “window phase” and in some chronic HBV patients

• Routine tests for HBsAg/anti-HBs do not detect immune complexes (IC)

• IC dissociatie (ICD) by treatment of serum (100µl) with HCl (50 µl, 0.5 N, 1h at 37°C) and neutralisation with NaOH (50 µl, 0.5N)(Rabenau et al. 1996)

• Research tests can detect IC’s

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• anti-HAV-IgM antibodies are only present in the acute phase of HAV infections

• ALT/AST activities are normal• Anti-HAV status (IgG antibodies) would

have been useful to see whether this person still needs HAV vaccination– Food handling– Chronic HBV infection

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8 400 gEq/mlHBV DNA

PositiveAnti-HBe

NegativeHBeAg

PositiefAnti-HBc-Tot

46 IU/LAnti-HBs

PositiveHBsAg

ResultaatTest Question 2Is this personinfectious ???

Question 3Does he needtreatment ?

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– Spouse and daughter show no signs or markers of HBV infection (HBsAlneg)

– Vaccination of household (sexual contact) !– Twinrix is an alternative for Engerix-B

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– Normal transaminases, low DNA– Follow-up : annually ?

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• Man, born in 1947• 1986 – Ulcerative colitis • 1992 - liver enzymes slightly elevated,

no further investigations • 1998 – abnormal liver tests, alcohol

consumption,• June 1998 – exacerbation of colitis• Nov 2001 - exacerbation of colitis

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<0.7 mEq/mlHBV DNAPos PosAnti-HBeNegNegHBeAg

PosPosPosPosAnti-HBcNegNegNegNegAnti-HBsNegPos PosPosHBsAg582856120γ-GT3317 3094AST422435129ALT

Sep 2003June 1999June 1998May 1998Test

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• HBeAg to anti-HBe seroconversion

– Inflammation (ALT/AST) = 8-15% per year– Normal ALT <2% in children < 3 years

4-5% in patients > 3 years

• HBsAg to anti-HBs seroconversion– Active HBeAg- hepatitis 0.5% /jaar– Asymptomatic HBeAg- carrier

• In a western population 1-2% /jaar• Post perinatal infection 0.05-0.8% /jaar

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• Transition to ‘inactive carrier’– Normalisation of transaminases– Low viral replication and HBV DNA (<105 gEq/ml)

• Active hepatitis with HBeAg-/anti-HBe+

– Elevated transaminases– High(er) HBV DNA (> 105 gEq/ml)– Precore mutation (G1896A: stop codon)– Core/precore promotor mutations

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-29 aa 183aa 1

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aa 1aa 183

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aa -10 aa 149aa 1

AU

G

AU

GPG

1896

A

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• Spontaneous seroconversion of HBsAg to anti-HBs

• No detectable [HBsAg-anti-HBs] IC’s• HBV DNA detection, quantification and

sequence analysis are needed for a correct diagnosis and prognosis

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• Man, 45 years• Traffic accident => brain death• Possible organ donor (liver ?)• Serology :• HBsAgneg, anti-HBsneg,anti-HBcpos, anti-

HCVneg,anti-HIVneg, CMVneg

• Biochemistry : no abnormalities

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2/51 (3.9)51 (0.5)9751Blooddonors

Switserland1999

2/27 (7.4)27 (0.2)15,000Blooddonors

Germany1998

0/104 (0)104 (1.2)9000Pregnant Switserland1996

94 (25)377IVD usersGermany1997

5/65 (7.7)65 (1.4)5300Unselected18-70 yrs

Germany1998

HBV DNA+

number (%)Prevalencenumber(%)

Populationstudied

Studygroup

Country (year)

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1.3404001Chinese Hong-Kong(1992)

11.9681801ChineseHong-Kong(1988)

20.888.73033Africans Senegal (1987)

5.018.8685Prisoninmates

USA (1984)

3.355.21461Male homosexuals

USA (1984)

Anti-HBc alone (%)

Total HBV(%)

Populationstudied

Studygroup

Country (year)

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1

1 = window phase

2

2 = late immunity, only anti-HBc persists

3 = chronic infection with lowreplication/production of HBV or with HBsAg mutant

3

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• Typical serology

HBsAgneg, anti-HBsneg, anti-HBcpos

• HBV DNA < detection limit of routine PCR test (e.g. < 200 gEq/mL)

• HBV DNA in the liver

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• HCV core protein suppresses HBV replication with a factor 2 to 4

• HCV infection reduces the expression of HBsAg in the liver

• Treatment of HCV with IFNα also has an effect on HBV

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1. Window phase2. Late immunity – only sign of past infection3. Chronic infection – “occult infection”4. HBsAg mutant5. “False” positive test result

• really “false positive” – low signal, 2nd EIA

• Core-binding antibodies (IgM vs IgG)

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-Nested PCR

Lo PCRHi PCRHi PCRHi PCRHBV DNA

+ or -+ or -++--Anti-HBe

----++HBeAg

++++++Anti-HBc

+-+++++++HBsAg

== to Hi= to HiHiHi=ALT

HBVReplication

HostImm Resp

LiverDisease

Tolerance

Active Hepatitis

HBeAg+ HBeAg-

mutant serocon

Occult Inactivecarrier

Lab Tools IgM anti-HDV HDV-Aganti-HDV HDV-RNA

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� ����#HCV• ELISA 3-4th generation• Confirmation tests RIBA, LIA

� ���������!����;�����������• RT-PCR qualitative and quantitative• Branched-DNA (quantitative)• Genotyping• HCV core antigen

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1989 1993 2000

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Antigens NS3/4 C,NS3,NS4 C,NS3,NS4A/B,NS5A

Sensitivity 95-98% >99%

Specificity <95% 99.8%

Lag time (wks) 16-24 4-12 4-8

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• Repeat ELISA on the same sample• Another ELISA on the same sample• Same ELISA on a new sample• Confirmation assays

– RIBA (recombinant immunoblot assay)– LIA (Line immuno assay)

• Confirmation by PCR

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• Molecular detection – qualitative

• Molecular quantification

• Genotyping

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10 IU/mlManual qualitTMA

Bayer Corpor, DiagnosticsDiv

Versant HCV RNA qualitativeassay

50 IU/mlSemi-automated, qual PCR

Roche MolecSystems

Cobas AmplicorHCVV2.0

50 IU/mlManual, qualitPCR

Roche MolecSystems

Amplicor HCV v2.0

Lower limit of detection

MethodManufacturerAssay

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• Confirms diagnosis of HCV infection• Useful for the early diagnosis of acute

hepatitis C• Demonstrates the presence of active

infection• « Gold standard » for documenting

response to therapy

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Acute HCV infection with Ab’sChronic HCV infection

PosPos

Past, recovered infectionNegPos

Acute infection (no Ab’s yet)Chronic in immune deficient p.

PosNeg

No infectionEarly post exposure (<1 week)

NegNeg

InterpretationHCV-RNAAnti-HCV

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• Clinical studies evaluating theefficacy of different treatmentprotocols : drugs, doses, duration, … have revealed the importance of1) HCV-RNA quantification 2) genotyping

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• Generally less sensitive thanqualitative HCV-RNA test => Taqman !

• Positive in >95% of untreated patients with chronic hepatitis C

• Useful in predicting response to therapy and determination of earlyvirological response (EVR)

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Amplicor Monitor v2.0(PCR)

VersantHCV RNA v3.0(b-DNA)

LCx 25HCV RNA

SuperQuant

20 200 2,000 20,000 200,000 2,000,000 IU/ml

600

615

500,000

7,700,000

2,630,000

30 1,470,000

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• Direct Sequencing– ‘Home-made’ methods: NS5B-, E1-based– 5’-noncoding: Trugene - Visible Genetics

• ‘Reverse Hybridization’– Inno LiPA (Innogenetics)

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Treatment of HCV

• Interferon-αααα

• Interferon-αααα + Ribavirine

• Pegylated IFN-αααα + Ribavirine

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• 1997 Consensus conference– IFNα monotherapy : stop therapy when

HCV-RNA (sensitive qualitative test)remains POSITIVE after 12 weeks

• 1998 McHutchison – NEJM 339:1485

– IFNα + ribavirin : stop therapy when HCV-RNA (sensitive qualitative test)remains POSITIVE after 24 weeks

n = 390 n = 390 (86%)(86%)

n = 63n = 63(14%)(14%)

2 log reduction or 2 log reduction or HCV RNA (HCV RNA (--) )

YESYES

NONO

Week 12 (N = 453)Week 12 (N = 453)n = 253n = 253(65%)(65%)SVRSVR

n = 137 n = 137 (35%)(35%)No SVRNo SVR

n = 2n = 2(3%)(3%)

n = 61 n = 61 (97%)(97%)FriedFried et al. NEJM 2002;347:975et al. NEJM 2002;347:975--982982

SVRSVR

No SVRNo SVR

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• in patients treated with PEG-IFN + ribavirin• undetectable HCV-RNA or log 2 drop at

week 12, is predictive for sustained response (>60%)

• absence of 2 log drop is extremely (>99%) predictive for non-sustained response

• should lead to early stop of treatment• leads to significant cost reduction • avoids inconvenience and side effects

GENOTYPE 2 OR 3GENOTYPE 2 OR 3 GENOTYPE 1 (and 4, 5 or 6)GENOTYPE 1 (and 4, 5 or 6)==

PEGPEG--IFNIFN--αααααααα + 800 mg + 800 mg ribavirinribavirin24 weeks24 weeks

EndEnd--ofof--treatment treatment virologicalvirological responseresponseSustained Sustained virologicalvirological responseresponse

> 2 log decrease> 2 log decreaseor HCV RNA (or HCV RNA (--))

at week 12at week 12

<2 log decrease<2 log decreaseat week 12at week 12

EndEnd--ofof--treatment treatment virologicalvirological responseresponseSustained Sustained virologicalvirological responseresponse

==Stop treatmentStop treatment

or continue in order or continue in order to slow evolutionto slow evolutionof liver diseaseof liver disease

HCV Genotype determinationHCV Genotype determination

Viral load quantification Viral load quantification at baseline and week 12at baseline and week 12

HCV RNA detection HCV RNA detection (sensitive qualitative assay) (sensitive qualitative assay)

at the end of treatment and 24 weeks laterat the end of treatment and 24 weeks later

Liver biopsyLiver biopsy

Bad prognosisBad prognosis==

PEGPEG--IFNIFN--αααααααα + + 10001000--1200 1200 mgribavirinmgribavirin

48 weeks48 weeks

Good prognosisGood prognosis==

No treatmentNo treatment

CHRONIC HEPATITIS CCHRONIC HEPATITIS C

HCV RNA detection HCV RNA detection (sensitive qualitative assay) (sensitive qualitative assay)

at the end of treatment and 24 weeks laterat the end of treatment and 24 weeks later

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a simulation for Belgium

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• Number of new cases per year = 1000• Number of treated cases = 600• Fraction of HCV genotype 1 = 70 %• Cost of quantitative HCV-RNA = 150 Euro per

test, x 2 (w0 and w12) = 300 Euro• Cost of PEG-IFN + Riba = 400 Euro per week• Diagnostic sensitivity (detection of NR) = 33%• NPV of EVR = 100%

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1000 new HCV

600 treated

180 non-gt1 420 gt1

350 EVR

HCV-RNA at w12 63000

HCV-RNA at w0 63000

Cost Benefit

210 SR 140 NSR

70 No EVR

&+18 126000 1008000

w0

w12

W48 End of treatment Net savings AA3BBB

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• Correct diagnosis• Selection of treatment and duration• Decision to “Stop treatment”

– Reduce costs (medication, doctor visits, ..)– Reduce the discomfort and suffering of patients– Reduce the loss of labour time

– The impact on costs (direct, indirect) will increase if the diagnostic sensitivity of the EVR algorythm can be improved (>33%)

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• 12% o post-transfusion hepatitis unrelated to A-E

• 18% of acute hepatitis unrelated to A-E• Up to 40% of fulminant hepatitis no

etiology is present• Cases of acute hepatitis followed by

aplastic anemia

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• HGV• TTV• TLMV• TTV-like minivirus• Sanban• Yonban• Sen virus

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• Related to HCV - Flaviviridae• Parenteral transmission• Replicates in lymphocytes and not in

hepatocytes• HGV infection prolongs survival in HIV• Does not cause hepatitis and not even

co-morbidity in (frequent) association with HBV or HCV

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8.3Fulminant hep

50.5Chronic HCV

5.6Chronic HBV

14.217Haemodialysis

25.714.8Clotting disord

61.8Blood donors

Anti-E2 Ab (%)HGV-RNA (%)

TT virus infection in acute and chronic liver diseases and in patients regularly receiving blood products in BelgiumAli S, van Pelt JF, Verslype C, Nevens F, Fevery J, Yap SH – Acta Gastroenterol Belg 2004,67:161

TTV-DNA was present in • 49% of patients with chronic HCV• 54% in patients with chronic HBV• 47% in patients receiving clotting factors• 64% in patients in chronic haemodialysis• 29.7% in (340) healthy blood donors

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Dr. Robert Vranckx HEV-Al

Wetenschappelijk Instituut Volksgezondheid HDV-Ag ?

Juliette Wytmansstraat 14 HGV-RNA ?1050 Brussel

Prof. Dr. Patrick Goubau HDV-Al

AIDS Reference Laboratory, UCLAvenue Hippocrate 54/92B-1200 Brussels