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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
2
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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,
3
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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
4
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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
5
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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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6
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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
7
)������ # ��������
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
8
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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
9
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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
10
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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
�����7
• 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
11
8��������� ���9����#��������:������'�������������� �����������
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
12
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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)
13
-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
14
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15
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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
16
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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
17
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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)
18
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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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19
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
8��#/?$#αααα3��>2�@���*������> ������������
20
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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
����#*����������������������������������������5��)6�
a simulation for Belgium
21
����#*����������∆∆∆∆���3����'��!�(3���������� ��������
• 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%
����#*����������∆∆∆∆���3����'��!�(3
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%)
22
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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
23
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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