Diagnose van hepatitis B en hepatitis C

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Diagnose van hepatitis B en hepatitis C

• Prof. Patrick Goubau
• UCL
• Antwerpen 28/02/2005

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Blood of chronic hepatitis B carrier in
electronic microscopy
HBs Ag
Dane particles
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Replication of Hepatitis B virus
circular partly double stranded viral DNA
particle
Covalently closed circular DNA
cell
Pregenomic RNA
Reverse Transcriptase
particle
circular partly double stranded viral DNA
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HBV genomic structure
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Amino acids specifying determinants of HBsAg
Magius Norder, Intervirol. 19953824-34
Position
Amino acid
Specificity
122
Lys
d
Arg
y
127
Pro
w1/w2
Thr
w3
Leu/Ile
w4
160
Lys
w
Arg
r
w1 also requires Arg122, Phe134 and/or Ala 159
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Geographical distributionof genotypes and
serotypes
Magius Norder, Intervirol. 19953824-34
Genotype
A
Northwestern Europe, Central Africa
B
Indonesia, China, Vietnam
C
Korea, China, Japan, Polynesia,Vietnam
D
Mediterranean area, India
E
West Africa
F
American Natives,Polynesia
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F
Genotypes of HBV in Belgian children, 1999
D
DA
AI
GM
WS
100
KrMe
VC
DeAy
98.4
CM
72.6
99.7
DL
VA
RR
BM
PI
100
TA
LP
A
100
KM
DV
E
C
LS
B
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Chronic hepatitis B
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HBV core et antigène e
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HBV precore and promotor mutants
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HBV transmission

• Mother gt child essentially if HBeAg positive
• Horizontal
• child gt child
• Institutions for mentally handicaped
• Sexual
• Parenteral
• Transfusion, transplantation
• Nosocomial and iatrogenic
• Shared siringes among drug addicts

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Geographic Distribution of Chronic HBV Infection
Prévalence AgHBs
?8 - High
2-7 - Medium
lt2 - Low
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Transmission patterns of HBV
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Evolution of HBV infection

• Acute infection mortality 0,5 1
• Chronic carriership
• Favourable evolution negative for Ag HBe
• Unfavourable evolution AgHbe pos or precore
  mutant
• Chronic hepatitis and fibrosis
• Cirrhosis
• Primary hepatocarcinoma (possible without
  cirrhosis)
• Cure often persistance of hepatic DNA
• Cave liver transplants!

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Outcome of Hepatitis B Virus Infection by Age at
Infection
100
100
80
80
60
60
Chronic Infection
Chronic Infection ()
Symptomatic Infection ()
40
40
20
20
Symptomatic Infection
0
0
1-6 months
7-12 months
Older Children and Adults
Birth
1-4 years
Age at Infection
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Age at Aquisition of Acute and Chronic HBV
Infection United States, 1989 Estimates
(4 ) Perinatal (24) (4) Children
(12) (1-10 yrs) (8) Adolescent (6)
Adult (59)
Adult (83)
Acute HBV Infections
Chronic HBV Infections
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Treatment of chronic hepatitis B with interferon
aMeta-analysis of 15 studies (837 patients) Wong
et al. 1993
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Treatment options for HBV

• Interferon and pegylated interferon
• Lamivudine (3TC) frequent resistance (YMDD)
• Adefovir remains active on strains resistant to
  lamivudine

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LAMIVUDINE
Incidence of detectable serum variant HBV
after 1 yr 24 2 yr 42 3 yr 53
Lai NEJM 1998 Liaw Gastroenterology 2000 Leung
Hepatology 2001
ADEFOVIR
Incidence of detectable serum variant HBV
after 48 weeks 0 96 weeks lt2
Angus Gastroenterology 2003
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Peg IFN a-2b with/without lamivudineJanssen et
al. Lancet, 365123-129, 2005

• 52 weeks of treatment
• 26 weeks post-treatment follow-up
• Pegylated interferon a-2b with / without
  lamivudine (3TC)
• Combined therapy n130
• Monotherapy n136

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Peg IFN a-2b with/without lamivudineJanssen et
al. Lancet, 365123-129, 2005
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Peg IFN a-2b with/without lamivudineJanssen et
al. Lancet, 365123-129, 2005
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Hepatitis B diagnostics
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Isolated anti-HBc antibody

• Post-acute window phase
• Years later loss of anti-HBs antibody
• False positive result
• Check hepatitis C!!

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Post-immunization control
Vaccine schedule
Month 0, 1, 6-12 Month 0, 1, 2, 12
serological control 1-2 months later
Anti-HBs 10 IU/l
5 non-responders in adults
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Hepatitis B diagnostics
Chronic hepatitis B
High viral replication
Low viral replication or precore mutant
gttest DNA
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HBV quantitative DNA test

• Indications
• institution of therapy in chronic HBsAg
  patients,
• follow-up of chronic hepatitis B treatment-
  acute rebound in chronic hepatitis B
• Conditions
• - abnormal liver tests, particularly ALT- HBsAg
  positivity- maximum twice a year per patient

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HBV quantitative DNA test

• Limits
• Maximal sensitivity few responders
• Sensitivity 103 to 105 IU/ml (IU X 6 copies)
  most useful for follow-up of treatment
• Is it sufficient for pre-core mutants?

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HBV qualitative DNA test
Prior telephone contact with the laboratory is
necessary. The test may be useful if infection is
doubtful e.g. if- isolated positive
anti-HBcore- seronegative chronic hepatitis
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Real time PCRwith sybr green fluorescence
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Real time PCR with use of probes
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Real time PCR advantages

• No contamination
• Rapid results
• Standardized procedures
• Automation possible
• Quantitative results and wide dynamic range

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Protein S
832
178
336
680
TP
Reverse Transcriptase
RNAse H
Spacer
1
F A
B
C
Domains
D
E
SNLSWLSLDVSAAFYHI
SLGIHLNPNKTT
LNFMGYVIGSW
VLGFRKIPMGVGLSPFLLAQFTSAICS
AFSYMDDVVLG
75
163
200
230
247
91
189
210
241
257
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Virus de lhépatite C (HCV)
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HCV genome and processing
C
E1
E2
NS2
NS3
NS4
NS5
5UTR
3UTR
signal peptidase
serine protease
p21
gp31
gp70
p7
p23
p70
p8
p27
p58
p68
capsid
envelope
protease
protease helicase NTPase
RNA polymerase
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Genotypes of HCV
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HCV Clinical évolution
Infection
80
20
Resolution
Chronic infection
1/3
1/3
Subclinical
1/3
Moderate disease
Severe disease fibrosis cirrhosis liver failure
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Risk history in HCV infected
Mother to child 1
Sexual 5
Unknown 34
Parenteral 60
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Prevalence of HCV infection

• Weak 0.5
• Scandinavia, Australia, Canada, Switzerland
• Intermediate 1
• Belgium, USA, western Europe
• High gt2
• Third world, Asia
• Very high gt10
• Selected areas Nile delta, southern Cameroon

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Antibody negative window in acute hepatitis C

• Second generation EIA 82 days
• Third generation EIA 52 days
• Kupek E.J., J Vir Hepat 2001 8 78-82.

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Retreatment of hepatitis CVirological clearance
Davis et al. 1998
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Treatment of chronic hepatitis CVirological
clearance
McHutchinson et al. 1998 Poynard et al. 1998
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Innolipa genotyping of HCV
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Detection de lARN de lHCV
ROCHE
Amplicor HCV
Amplicor HCV Monitor
HCV Taqman
Bayer
Versant
TMA
bDNA
Abbott
LCX
IU/ml
0
10
101
102
103
104
105
106
107
108
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HCV qualitative assaysother indications

• Determination of a vertical transmission
  infection of a baby of a HCV-RNA positive mother,
  test to be performed on blood of the baby at 6
  months of age.
• Detection of HCV-RNA in a HCV positive patient
  with chronic hepatitis with at least 2 x elevated
  transaminases at 6 months interval.
• Confirmation of a HCV infection, in the absence
  of anti-HCV, in immunosuppressed patients (renal
  dialysis, HIV, treatment,...

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HCV antigen test compared to PCR
Aoyagi K. et al. J Clin Microbiol 1999371802-8
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HCV antigen test (Ortho) performance in
seroconverting patients

• 83 (20/24) positive together with RT-PCR
• 87 of RT-PCR positives contained Ag
• Mean antigen positivity one day later than RT-PCR

Peterson J. et al. Vox Sang 2000 78 80-5