Transcript Hepatitis C een verborgen epidemie

Hepatitis A, B, C, D, E, G,…
diagnostic tools and their use
Geert Leroux-Roels
Laboratorium voor Klinische Biologie
UZ Gent
Human Hepatitis Viruses
Discovery and characteristics
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
Human Hepatitis Viruses
Diagnostic tools
Virus
Serology
Molecular Advanced
Antibodies Antigens det/quant molec anal
HAV IgM, IgG
HBV HBsAl
HBeAl
HBcAl
HBsAg
HBeAg
HCV IgG
HDV Anti-delta
det/quant
genotype
resistance
det/quant
genotype
Delta-Ag
HEV IgM, IgG
HGV IgG
TTV
IgG
det
Epidemiology of HAV
Hepatitis A virus – diagnostic tools
• Serology
– Anti-HAV IgM
acute HAV infection
– Anti-HAV Totaal immunity
- natural
- vaccine-induced
protective level 10-30 IU/L
• HAV detection
in blood and blood products
in faeces, saliva, …
in shelfish, food products
in water,sewage,
HAV vaccines
• Children (2-15)
Havrix Junior (720 U/dose), 0.5 ml ml/d
scheme : 0 and 6-12 mo (2 doses, IM)
• Adults (>15)
Havrix 1440 (1440 U/dose, 1 ml/d
scheme : 0 and 6-12 mo (2 doses, IM)
Epidemiology of HEV
epidemische en sporadische gevallen
Rare cases in western countries
after recent travel in endemic area
Hepatitis E virus
Serology
anti-HEV IgM
anti-HEV IgG
HEV-RNA
Hepatitis B virus
Diagnostic tools and interpretation
HBV: genome and gene products
3200 baseparen
4 open reading frames
7 proteins
• large (preS1-preS2-S)
• Middle (preS2-S)
• Small or HBsAg
• nucleocapsid or HBcAg
• secreted HBe-Ag
• Polymerase
• X protein
Diagnostic markers of HBV infection
Inflammation and
liver cell damage
Transaminases ALT/AST
en other biochemical
markers
Antigens
Antibodies
HBsAg
anti-HBs
HBcAg
anti-HBc-Tot
anti-HBc-IgM
HBeAg
anti-HBe
Detection/quantific.
of HBV DNA
Commercial assays
- Branched DNA (Bayer)
- PCR (Roche Amplicor)
In-house nested PCR
In-house real-time PCR
Usefulness
of HBV-DNA quantification
• 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,
Evolution of an acute,
self-limited HBV infection
Serologic profile of a chronic HBV infection,
with a late seroconversion to anti-HBe
Case 1
•
•
•
•
•
•
41 years old Afghan male
political refugee
In training for assistant-cook
Medical screening exam for ‘hepatitis’
No symptoms
No history of hepatitis
Results - blood chemistry
Test
Bili Tot
Bili Dir
Bili Ind
ALT
AST
LDH
Alk Fosf
g-GT
Result
0.66
0.10
0.55
16
21
351
106
20
Unit
mg/dL
mg/dL
mg/dL
U/L
U/L
U/L
U/L
U/L
Ref. interval
0.00-1.00
0.00-0.20
0.20-0.80
0-41
0-38
0-480
0-128
8-61
Results - serology
Test
Result
HBsAg
Positive
Question 1
Anti-HBs
46 IU/L
Can HBsAg and anti-HBs
occur concomitantly ?
Anti-HBc-Tot
Positive
Anti-HAV-IgM
Negative
Anti-HCV
Negative
[HBsAg-anti-HBs] immune complexes
• 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
HAV serostatus
• 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
Results – additional HBV tests
Test
Resultaat
HBsAg
Positive
Anti-HBs
46 IU/L
Anti-HBc-Tot
Positief
HBeAg
Negative
Anti-HBe
Positive
HBV DNA
8 400 gEq/ml
Question 2
Is this person
infectious ???
Question 3
Does he need
treatment ?
• Infectivity is LOW
– Spouse and daughter show no signs or
markers of HBV infection (HBsAlneg)
– Vaccination of household (sexual contact) !
– Twinrix is an alternative for Engerix-B
• Therapy is not indicated
– Normal transaminases, low DNA
– Follow-up : annually ?
Case 2
• 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
Evolution of laboratory tests
Test
May 1998 June 1998
June 1999 Sep 2003
ALT
AST
129
94
35
30
24
17
42
33
g-GT
HBsAg
Anti-HBs
Anti-HBc
120
Pos
Neg
Pos
56
Pos
Neg
Pos
28
Pos
Neg
Pos
58
Neg
Neg
Pos
HBeAg
Neg
Neg
Anti-HBe
HBV DNA
Pos
<0.7 mEq/ml
Pos
Spontaneous seroconversions
in chronic HBV
• 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
HBeAg/anti-HBe seroconversion
• 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
-29
AUG
G1896A
P
AUG
Hepatitis B core and e antigen
aa 183
aa 1
HBeAg precursor
aa -10
aa 1
aa 149
mature HBeAg
aa 1
HBcAg
aa 183
Interpretation of the serology and
its evolution in this patient (case 2)
• 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
Case 3
•
•
•
•
•
Man, 45 years
Traffic accident => brain death
Possible organ donor (liver ?)
Serology :
HBsAgneg, anti-HBsneg,anti-HBcpos, antiHCVneg,anti-HIVneg, CMVneg
• Biochemistry : no abnormalities
Prevalence of “anti-HBc alone” in low
seroprevalence populations
Country
(year)
Study
group
Population
studied
Prevalence
number
(%)
HBV DNA+
number (%)
Germany
1998
Unselected
18-70 yrs
5300
65 (1.4)
5/65 (7.7)
Germany
1997
IVD users
377
94 (25)
Switserland Pregnant
1996
9000
104 (1.2)
0/104 (0)
Germany
1998
15,000
27 (0.2)
2/27 (7.4)
9751
51 (0.5)
2/51 (3.9)
Blood
donors
Switserland Blood
1999
donors
Prevalence of “anti-HBc alone”
in high seroprevalence populations
Country
(year)
Study
group
Population Total HBV
studied
(%)
Anti-HBc
alone (%)
USA (1984)
Male
homosexuals
1461
55.2
3.3
USA (1984)
Prison
inmates
685
18.8
5.0
Senegal
(1987)
Africans
3033
88.7
20.8
Hong-Kong Chinese
(1988)
1801
68
11.9
Hong-Kong Chinese
(1992)
4001
40
1.3
Possible causes of “Anti-HBc alone” serology
1 = window phase
1
2
2 = late immunity, only
anti-HBc persists
3
3 = chronic infection with low
replication/production of HBV
or with HBsAg mutant
“Occult hepatitis”
• 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
Influence of HCV infection
on HBV replication
• 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 IFNa also has an
effect on HBV
“anti-HBc alone” can be :
1.
2.
3.
4.
5.
Window phase
Late immunity – only sign of past infection
Chronic infection – “occult infection”
HBsAg mutant
“False” positive test result
•
•
really “false positive” – low signal, 2nd EIA
Core-binding antibodies (IgM vs IgG)
Active Hepatitis
HBeAg-
Tolerance HBeAg+
HBV
Replication
mutant
serocon
Occult
Inactive
carrier
Host
Imm Resp
Liver
Disease
ALT
=
Hi
Hi
= to Hi
= to Hi
=
HBsAg
++
++
++
+
-
+
Anti-HBc
+
+
+
+
+
+
HBeAg
+
+
-
-
-
-
Anti-HBe
-
-
+
+
+ or -
+ or -
HBV DNA
Hi PCR
Hi PCR
Hi PCR
Lo PCR
Nested
PCR
-
Lab Tools
IgM anti-HDV
anti-HDV
HDV-Ag
HDV-RNA
Hepatitis C virus
Diagnostic tools and interpretation
The HCV genome and expressed polyprotein
Markers of HCV infection
• Indirect markers
– anti-HCV
• ELISA
• Confirmation tests
3-4th generation
RIBA, LIA
• Direct markers = HCV genome
• RT-PCR qualitative and quantitative
• Branched-DNA (quantitative)
• Genotyping
• HCV core antigen
Anti-HCV assays: generations 1 to 4
1989
Generation 1
1993
2
2000
3
4
Antigens
NS3/4
Sensitivity
95-98%
>99%
Specificity
<95%
99.8%
Lag time
(wks)
16-24
C,NS3,NS4
4-12
C,NS3,NS4A/B,NS5A
4-8
+
Confirmation of anti-HCV
•
•
•
•
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
Molecular tests for HCV
• Molecular detection – qualitative
• Molecular quantification
• Genotyping
HCV-RNA detection – qualitative
Assay
Manufacturer
Method
Lower limit of
detection
Amplicor HCV
v2.0
Roche Molec
Systems
Manual, qualit 50 IU/ml
PCR
Cobas Amplicor
HCV
V2.0
Roche Molec
Systems
Semiautomated,
qual PCR
50 IU/ml
Versant HCV
RNA qualitative
assay
Bayer Corpor,
Diagnostics
Div
Manual qualit
TMA
10 IU/ml
Qualitative HCV-RNA tests
• 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
Interpretation of combined
anti-HCV and HCV-RNA results
Anti-HCV
HCV-RNA Interpretation
Neg
Neg
No infection
Early post exposure (<1 week)
Neg
Pos
Acute infection (no Ab’s yet)
Chronic in immune deficient p.
Pos
Neg
Past, recovered infection
Pos
Pos
Acute HCV infection with Ab’s
Chronic HCV infection
Treatment of HCV
• Clinical studies evaluating the
efficacy of different treatment
protocols : drugs, doses, duration, …
have revealed the importance of
1) HCV-RNA quantification
2) genotyping
Quantitative HCV RNA tests
• Generally less sensitive than
qualitative HCV-RNA test => Taqman !
• Positive in >95% of untreated patients
with chronic hepatitis C
• Useful in predicting response to
therapy and determination of early
virological response (EVR)
Range of HCV-RNA assays
Amplicor
Monitor v2.0
(PCR)
600
Versant
HCV RNA v3.0
(b-DNA)
LCx
HCV RNA
SuperQuant
500,000
7,700,000
615
2,630,000
25
30
20
1,470,000
200
2,000
20,000
200,000
2,000,000
IU/ml
Molecular Genotyping
• Direct Sequencing
– ‘Home-made’ methods: NS5B-, E1-based
– 5’-noncoding: Trugene - Visible Genetics
• ‘Reverse Hybridization’
– Inno LiPA (Innogenetics)
Analysis of the viral genome sequence
Reverse hybridisation of PCR amplicons
Treatment of HCV
• Interferon-a
• Interferon-a + Ribavirine
• Pegylated IFN-a + Ribavirine
Early stopping rules
• 1997 Consensus conference
– IFNa monotherapy : stop therapy when
HCV-RNA (sensitive qualitative test)
remains POSITIVE after 12 weeks
• 1998 McHutchison – NEJM 339:1485
– IFNa + ribavirin : stop therapy when
HCV-RNA (sensitive qualitative test)
remains POSITIVE after 24 weeks
PEG-IFN-a2a QW + ribavirin QD
All genotypes
Week 12 (N = 453)
YES
SVR
n = 253
(65%)
No SVR
n = 137
(35%)
SVR
n=2
(3%)
No SVR
n = 61
(97%)
n = 390
(86%)
2 log reduction or
HCV RNA (-)
NO
n = 63
(14%)
Fried et al. NEJM 2002;347:975-982
2 log drop of HCV-RNA at week 12
• 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
CHRONIC HEPATITIS C
HCV Genotype determination
GENOTYPE 2 OR 3
=
PEG-IFN-a + 800 mg ribavirin
24 weeks
HCV RNA detection
(sensitive qualitative assay)
at the end of treatment and 24 weeks later
End-of-treatment virological response
Sustained virological response
GENOTYPE 1 (and 4, 5 or 6)
Liver biopsy
Good prognosis
Bad prognosis
=
=
No treatment
PEG-IFN-a +
1000-1200 mgribavirin
48 weeks
Viral load quantification
at baseline and week 12
> 2 log decrease
or HCV RNA (-)
at week 12
HCV RNA detection
(sensitive qualitative assay)
at the end of treatment and 24 weeks later
End-of-treatment virological response
Sustained virological response
<2 log decrease
at week 12
=
Stop treatment
or continue in order
to slow evolution
of liver disease
Cost-benefit of monitoring
early viral response (EVR)
a simulation for Belgium
Cost-benefit of Dlog2 at week 12
Assumptions and data
•
•
•
•
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%
Cost-benefit of Dlog2 at week 12
1000 new HCV
Cost
600 treated
w0
HCV-RNA at w0
180 non-gt1
63000
420 gt1
w12
HCV-RNA at w12 63000
350 EVR
70 No EVR
STOP
210 SR
W48
Benefit
126000
1008000
Net savings
882000
140 NSR
End of treatment
Molecular diagnostic tools for detection
and monitoring of HCV infections
• 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%)
Additional Hepatitis Agents
• 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
New viruses not proven to
cause human hepatitis
•
•
•
•
•
•
•
HGV
TTV
TLMV
TTV-like minivirus
Sanban
Yonban
Sen virus
HGV – GBV-C
• 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
Prevalence in Belgium
Sheng - Doctoral Thesis - KUL 1998
HGV-RNA (%) Anti-E2 Ab (%)
Blood donors
1.8
6
Clotting disord
14.8
25.7
Haemodialysis
17
14.2
Chronic HBV
5.6
Chronic HCV
50.5
Fulminant hep
8.3
TT virus infection in acute and chronic
liver diseases and in patients regularly
receiving blood products in Belgium
Ali 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
Analysis of samples for less
common forms of hepatitis
Dr. Robert Vranckx
Wetenschappelijk Instituut Volksgezondheid
Juliette Wytmansstraat 14
1050 Brussel
Prof. Dr. Patrick Goubau
AIDS Reference Laboratory, UCL
Avenue Hippocrate 54/92
B-1200 Brussels
HEV-Al
HDV-Ag ?
HGV-RNA ?
HDV-Al