Presentazione standard di PowerPointNovel diagnostic tools Molecular HBV genotype cccDNA Assessment...
Transcript of Presentazione standard di PowerPointNovel diagnostic tools Molecular HBV genotype cccDNA Assessment...
HBsAg quantitativo: impiego nella diagnosi dello
stato di portatore inattivo e nelle stopping rules in
corso di terapia con PEGIFN
G Missale
UO Malattie Infettive ed Epatologia
Azienda Ospedaliero-Universitaria di Parma
Conventional HBV Markers
Direct markers
- HBs antigen - HBe antigen
POL
HBcAg
LHBs
MHBs
SHBs
Partially double- stranded DNA
Indirect markers
- Anti-HBs
- Anti-HBc - Anti-HBc IgM - Anti-HBe
Molecular marker
HBV DNA
Novel diagnostic tools
Molecular HBV genotype cccDNA Assessment of virus mutations -Precore and core promoter
mutations -HBV envelope mutations -Antiviral resistance mutations
Serological
• QUANTITATIVE HBsAg
• QUANTITATIVE HBeAg
HBV ccc DNA
RC primer
CCC primer
RC
primer
CCC primer
Commercial tests
Elecsys II – Roche
Architect – Abbott
ADVIA Centaur HBsAg Assay – Bayer
Hepanostika HBsAg – Biomerieux
In-house assays widely used in some countries
HBsAg measuring
Dynamic Range: 0 – 250.0 IU/ml (WHO) Specimens with values exceeding 250 IU/ml are flagged and may be diluted with the Manual Dilution Procedure. Operator enters the dilution factor in the Patient or Control order screen. System uses this dilution factor to automatically calculate the concentration of the sample before dilution and report the result. Dual epitope capture format, detects all known HBsAg mutants
Quantitative HBsAg measurement with some assays after dilution
Architect HBsAg (QT)
Qualitative screening assay for HBsAg universally available
nucleus cytoplasm
repair
mRNA
transcription
Core particle plus
strand synthesis
recycling
mRNA
transport
translation Ribosome
Transport to cell
membrane
HBV
preCore
HBX
Secreted HBeAg
ER
Spheres and Filaments
containing HBsAg
Small HBs Medium HBs
Large HBs
Envelope
Subgenomic and
Genomic RNA
Pre-genomic RNA Core
Pol
cccDNA
Golgi
complex
Entry of HBV
into cell and
uncoating
Core particle minus
strand synthesis
Core assembly and
RNA packaging
HBV Life Cycle
Raimondo et al, J Hepatol 2007
Hepatic HBV cccDNA Levels in Different Patient Populations
Werle-Lapostolle, Petersen, Locarnini, Zoulim Gastroenterology 2004
HBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV DNA (147- fold) levels compared to HBeAg- patients (p<0.001, Wilcoxon tests)
Tota
l HB
V D
NA
(c
op
ies/
cell)
10 -3
10 -2
10 -1
10 0
10 1
10 2
10 3
10 4
nucleus cytoplasm
repair
mRNA
transcription
Core particle plus
strand synthesis
recycling
mRNA
transport
translation Ribosome
Transport to cell
membrane
HBV
preCore
HBX
Secreted HBeAg
ER
Spheres and Filaments
containing HBsAg
Small HBs Medium HBs
Large HBs
Envelope
Subgenomic and
Genomic RNA
Pre-genomic RNA Core
Pol
cccDNA
Golgi
complex
Entry of HBV
into cell and
uncoating
Core particle minus
strand synthesis
Core assembly and
RNA packaging
HBV Life Cycle
Raimondo et al, J Hepatol 2007
NUCs
Changes in Serum HBsAg Levels are Positively Correlated with Changes in cccDNA Levels
p<0.01
-3 -2 -1 0 1
-3
-2
-1
0 ADV+PEG
Change in Serum HBsAg (log 10 ng/mL)
Ch
ange
in c
ccD
NA
(l
og
10
co
pie
s/ce
ll)
Petersen J et al Hepatology 2006
HBsAgsl decline predicts SVR of Peginterferon treated HBeAg pos CH-B patients
Sonneveld MJ et al, Hepatology 2010;52:1251-57
HBsAgsl <1500 UI/ml at week 12 is associated with SVR of Peginterferon treated HBeAg pos CH-B patients
Piratvisuth T et al, Hepatol Int 2011
NPV 84%
HBsAg levels week 12 HBsAg levels week 24
Neptune study confirms prediction of response at week 12 for HBsAgsl decline <1500 UI/ml
Gane E et al, J Hepatol 2011 Abst 69
0.5 Log10 IU/ml HBsAg decline at week 12 has high predictive value of SVR in Peginterferon treated HBeAg-neg patients
Moucari R, et al. Hepatology 2009; 49:1151-57
Stopping rule for in Peginterferon treatment of HBeAg-neg patients by combining on-treatment HBsAg and HBV-DNA
Rijckborst V, et al. Hepatology 2010; 52:454-61
PARC trial
Validation of topping rule for in Peginterferon treatment of HBeAg-neg patients combining HBsAg and HBV-DNA
Rijckborst V, et al. J Hepatol 2012; 56:1006-11
PreS/S mutations impact on HBsAg and cccDNA levels
Pollicino T et al, Hepatology 2012; 56:434-43
Conclusions: HBeAg-pos patients
• In HBeAg-positive CHB, no decline of HBsAg levels at 12 weeks is associated with a very low probability of subsequent anti-HBe seroconversion
• In HBeAg-positive CHB, decline of HBsAg levels below 1500 IU/ ml at 12 weeks is a strong predictor of anti-HBe seroconversion, while HBsAg levels >20,000 UI/ml are associated with a very low probability of anti-HBe seroconversion
Conclusions: HBeAg-neg patients
• In HBeAg-negative CHB, a combination of no HBsAg decline and <2 log10 IU/ml decline of HBV DNA seems to be a predictor of nonresponse in European HBeAg-negative patients with genotype D
Kinetics of HBsAg during 3 years of Telbivudine
in HBeAg-pos patients
Wursthorn K, Hepatology 2010; 52:1611-20
Prediction of treatment response to ETV by
HBsAg serum levels
Lee JM, Hepatology 2011; 53:1486-93
86.8% sensitivity 78.9% specificity
Conclusions: NUC treated patients
• A decline of HBsAg during NA treatment in HBeAg-positive patients may identify cases with subsequent HBeAg or HBsAg loss
HBsAgsl helps to distinguish Active from Inactive HBsAg Gen D carriers
Brunetto MR et al, Gastroenterology 2010;139:483-90
>
HBsAgsl helps to distinguish Active from Inactive HBsAg Gen D carriers
Brunetto MR et al, Gastroenterology 2010;139:483-90
HBsAgsl helps to distinguish Active from Inactive HBsAg Gen D carriers
Brunetto MR et al, Gastroenterology 2010;139:483-90
>
at baselineHBsAgsl: 1709 vs 62.12 IU/ML
Conclusions
Inactive carriers may be identified by single determination of serum HBsAg levels <1000 IU/ml and HBV-DNA <2000 UI/ml but such levels may occasionally be detected in CHB patients as well