1. HBV-DNA detection by hybridization assay in the serum of anti-HBe positive HBV carriers HBV-DNA + Chronic Hepatitis 11/19 58% 11/14 79% Bonino 1980 7/13 54% - Scotto 1983 16/32 50% - Liebermann 1983 14/24 58% 14/18 78% Hadziyannis 1983 28/106 26% 28/46 65% Bonino 1984 8/30 27% 8/21 38% Lok 1984 10/153 7% 1/5 20% Tur Kaspa 1984 14/78 18% 10/38 26% Wu 1986 116/455 25% 72/139 52%

2. Chronic anti-HBe positive hepatitis B was born in 1980 in the Mediterranean area < 2% - Low2-7% - Intermediate> 8% - High

3. Chronic anti-HBe positive hepatitis B Features Virological : mean viremia levels lower than those observed in HBeAg positive patientsClinical: progressive liver disease with low rate of spontaneous resolutions, in spite of temporary remissions high relapse rate after the end of IFN, in spite of the response obtained during treatment Brunetto MR, Hepatology 1989

4. HBeAg positive infection Anti-HBe positive infection

5. Identification of 1896 G to A switch as causative agent of anti-HBe positive chronic hepatitis B Brunetto MR et al, Ital. J Gastro 1989 Carman A et al, Lancet 1989 Santantonio T et al, Virology 1991

6. NUCLEOCAPSID PROTEINS: HBeAg PRE-CCORE 183 aa E.R. 29 aa 149 (154) aa 10 aa 17 kDa non particulate, secreted HBeAg Substitution of 7cys restores particle formation and HBcAg antigenicity

8. Hepatitis B Virus Li J-S et al, J Virol 1993 Sequence of pre-core codon 15 Genotype Size of gene (nucleotides) core Pre-S CCC CCU CCC/U CCU CCC/U ABCDEFABCDEF 555 549 519 486 Bestmatchingwith codon 28

9. Genotype D is the prevailing HBV genotype in the Mediterranean area where Chronic anti-HBe positive hepatitis B was originally described < 2% - Low2-7% - Intermediate> 8% - High

10. Reports from South Europe on the various features, the outcome and response to IFN of HBeAg negative/anti-HBe positive CHB, mainly concern 1896 GtoA,genotype D CHB and do not necessarily apply to other geographical areas where other virologic form of HBeAg-negative CHB may prevail Hadziyannis SJ, J Hep 2002

11. Modulation of HBeAg expression Transcriptional Translational Basic core promoter mutants HDV super/co-infections Pre-core mutants: 1896 G to A Stop at codon 28 1817 C to T Stop at codon 2 1897 G to A Stop at codon 28 1816 G to T Elimination of 1815 T to C start codon 1

12. Anti-HBe positive chronic hepatitis B: 1896 G to A switch by oligohybridization assay All patients were of Italian origin 52 (31.7%) 99 (60.4%) 13 (7.9 %) 161 / 164 (98.2%) Brunetto MR,1999

13. Anti-HBe positive chronic hepatitis B: pre-core heterogeneity by direct sequencing in 18 patients from Tuscany 1762 1764 1896 1899 A - T G - A G - A G - A 12/18 12/18 11/18 6/18 66.6% 66.6% 61.1% 33.3% + + - - in 5 patients - - - - in 2 patients Moscato G, 2001

14. Gaeta et al. Year of analisys:1997 Chronic HBsAg Hepatitis in Italy 834 cases in 14 centres

15. HBeAg pos.ve HBeAg pos.ve anti-HBe pos.ve anti-HBe pos.ve Cases = 538 Cases = 718 Giusti et al. 1975-85 Gaeta et al. 1997 Change of HBeAg and anti-HBe Distribution in positive HBsAg Carriers in Italy

16. Chronic anti-HBe positive hepatitis B Reasons for the increase of its prevalence in Italy: - reduction of HBeAg positive cases - long lasting unresolving disease inducing a progressive accumulation of cases in the population - increase in the diagnostic accuracy (more sensitive assays, more diagnostic accuracy by the clinicians)

17. Geographic Distribution of HBsAg Carriers < 2% - Low2-7% - Intermediate> 8% - High

18. Chronic anti-HBe positive hepatitis B Reasons for the increase of its prevalence in Italy: - reduction of HBeAg positive cases - long lasting unresolving disease inducing a progressive accumulation of cases in the population progressive accumulation of cases in the population - increase in the diagnostic accuracy (more sensitive assays, more diagnostic accuracy by the clinicians)

19. Outcome of Chronic Anti-HBe Positive Hepatitis B 010203040506070 80 INFECTION 36 - 40 y. Age (years) 45 y. CIRRHOSIS asymptomatic HBeAg minus selection High intrafamiliar infection (43.9%) Low parenteral exposure (13.4%) Small number of patients with history of acute hepatitis (4.8%) or HBeAg pos. (2.4%) High rate of asymptomatic carriers among relatives High rate of acute hepatitis in sexual partners DIAGNOSIS

20. Outcome of chronic anti-HBe positive hepatitis B 4 Signs of portal hypertension 2 Died 1 OLT 2nd Biopsy 63/87 72.4% 102 patients (mean age 36 y) with chronic hepatitis at histology Median follow-up 6y. (mean 5.9y.; range 2-12y.) Biochemical and Clinical Outcome 85.3% 7.8% 6.9% 38.1% 49.2% 12.7%

21. Gaeta et al. 2000 20 years old 50 years old Prevalence of Chronic anti-HBe positive Hepatitis B by Age % %

22. Chronic anti-HBe positive hepatitis B Reasons for the increase of its prevalence in Italy: - reduction of HBeAg positive cases - long lasting unresolving disease inducing a progressive accumulation of cases in the population - increase in the diagnostic accuracy (more sensitive assays, more diagnostic (more sensitive assays, more diagnostic accuracy by the clinicians) accuracy by the clinicians)

23. Outcome of Chronic Anti-HBe Positive Hepatitis B Biochemical patterns in 164 untreated patients after 23 months (range 12-36) monthly monitoring 01224 months With flares and normalization Without flares With flares and without normalization 73 pts ( 44.5% ) 59 pts ( 36.0% ) 32 pts ( 19.5% ) Asymptomatic flare-up: 90% of cases ALTALT Flare-up yearly frequency: once 57.1% twice 20% < once 22.8%

24. Viremia profile persistently > 10 6 gen/ml 15 ( 9.1%) occasionally < 10 6 gen/ml 52 (31.7%) frequently < 10 6 gen/ml 87 (53.1%) persistently < 10 6 gen/ml 10 ( 6.3%) IgM anti-HBc profile persistently > 7 PEI I.U. 110 (67.1%) 57.9% 82.2% fluctuations < 7 PEI I.U. 54 (32.9%) 42.1% 17.8% fluctuations < 4 PEI I.U. 5 ( 3.0%) ALL Chronic Hepatitis Cirrhosis  2 9.332, p = 0.002 Anti-HBe positive chronic hepatitis B: virological patterns in 164 patients 23 months (range 12-36) monthly monitoring Brunetto MR,1999

25. A favoring condition due to the changed HBV epidemiology in Italy A better understanding of what we are looking for and how to do it Are the major factors responsible for the increased prevalence of anti-HBe positive CHB in Italy

26. AKNOWLEDGEMENT Anna Maria Maina Giovanna Moscato Filippo Oliveri Barbara Coco Piero Colombatto Ferruccio Bonino