1. False negative HBsAg detectionFalse negative HBsAg detection n Is rare in patients with signs of chronic hepatitis B. n May be an issue in low-risk populations, such as: –blood donors, –organ, tissue and cell donors. It may result of:It may result of: n Undetectable, low-level HBsAg. n Substitutions in HBsAg “Major Hydrophilic Region”: –located at positions 99 to 160, –encompasses the “a” determinant, a major HBV epitope. False-Negative HBsAg Detection in Chronic Hepatitis B

2. Mutations in “a” Determinant 107 138 139 137 149 147 A R D 144 G 145 210 S-S S-S --S-S- 99 121 124 S-S Loop 1 of “a” determinant Loop 2 of “a” determinant HBs1 HBs2 HBs3 HBs4 HBs5 141 K T 126 N Q 129 H M 133 L E

3. Aims of the Study To determine the prevalence of falsely negative HBsAg detection in a large population of organ, tissue and cell donors.To determine the prevalence of falsely negative HBsAg detection in a large population of organ, tissue and cell donors. To understand the role of HBsAg mutants in the lack of HBsAg detection in HBV DNA positive donors.To understand the role of HBsAg mutants in the lack of HBsAg detection in HBV DNA positive donors.

4. Patients 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti- HBc Ab, anti-HBs Ab) between May 2000 and May 2004: 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti- HBc Ab, anti-HBs Ab) between May 2000 and May 2004:  First group: - 626 donors (5.6%), - with at least one of the three HBV serological markers, - excluding vaccination profiles (anti-HBs Ab alone).

5. 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti- HBc Ab, anti-HBs Ab) between May 2000 and May 2004: 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti- HBc Ab, anti-HBs Ab) between May 2000 and May 2004:  First group: - 626 donors (5.6%), -Brain-dead, heart-beating organ donors -Living organ donors -Tissue donors -Stem cell donors -Cord blood donors -Cornea donors 199131657556118 Patients

6. 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti- HBc Ab, anti-HBs Ab) between May 2000 and May 2004: 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti- HBc Ab, anti-HBs Ab) between May 2000 and May 2004:  First group : - 626 donors (5.6%), - with at least one of the three HBV serological markers, - excluding vaccination profiles (anti-HBs Ab alone).  Second group: - 1433 brain-dead organ donors, - seronegative for HBV or with anti-HBs Ab only. Patients

7. Methods (1) HBsAg was assessed with 3 different assays:HBsAg was assessed with 3 different assays: n Vitros Eci TM (Ortho-Clinical Diagnostics), n Architect TM (Abbott), n Vidas TM (BioMérieux). HBV DNA was systematically sought by means of a real-time PCR assay:HBV DNA was systematically sought by means of a real-time PCR assay: n Cobas TaqMan HBV (Roche Molecular Systems), n Lower limit of detection: 6 IU/ml, n Dynamic range of quantification: 30-10 8 IU/ml.

8. Full-length preS1-preS2-HBsAg sequence was determined in all HBV DNA-positive donors:Full-length preS1-preS2-HBsAg sequence was determined in all HBV DNA-positive donors: n Nested PCR using previously described primers a n Direct sequencing, n Alignment of sequences with prototype strains of HBV genotypes A to H. a Stuyver et al., J Gen Virol 2000 Methods (2)

9. HBV DNA in HBsAg-positive Donors 3.3 log IU/ml 2.5 log IU/ml 5.4 log IU/ml 2.0 log IU/ml 2.7 log IU/ml Brain-dead organ donors (n = 199) Living organ donors (n = 13) TissueDonors (n = 165) Cord blood Donors (n = 56) Stem cell Donors (n = 75) CorneaDonors (n = 118) 17/20 (85%) 2/2 (100%) 3/5 (60%) 2/2 (100%) 1/8 (12.5%) 20% 40% 60% 80% 100% 0%

10. HBV DNA in Donors with Isolated Anti-HBc Ab 1.6 log IU/ml 3.6 log IU/ml 20% 40% 60% 80% 100% 0% 2/53 (3.8%) 2/21 (9.5%) Brain-dead organ donors (n = 199) Living organ donors (n = 13) TissueDonors (n = 165) Cord blood Donors (n = 56) Stem cell Donors (n = 75) CorneaDonors (n = 118)

11. 2.4 log IU/ml 20% 40% 60% 80% 100% 0% 3/62 (4.8%) HBV DNA in Donors with Both Anti-HBc and Anti-HBs Ab Brain-dead organ donors (n = 199) Living organ donors (n = 13) TissueDonors (n = 165) Cord blood Donors (n = 56) Stem cell Donors (n = 75) CorneaDonors (n = 118)

12. 3.5 log IU/ml 3.6 log IU/ml 20% 40% 60% 80% 100% 0% 2/5 (40%) 1/27 (3.7%) HBV DNA in Donors with Three Markers Brain-dead organ donors (n = 199) Living organ donors (n = 13) TissueDonors (n = 165) Cord blood Donors (n = 56) Stem cell Donors (n = 75) CorneaDonors (n = 118)

13. HBsAg Gen A: MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH HBsAg Gen B:............................................................ HBsAg Gen C:....A..L............S..K.....L.............ET..........QI.S. HBsAg Gen D:............................................TT.............. HBsAg Gen E:..S....................K....................A............... HBsAg Gen F:.......L....R....VC....K....................L.R.P........... HBsAg Gen G:.......L....R....VC....K....................L.R.P........... HBsAg Gen H:.......L.R.......VC....K...................VP.G.P.......I... HBsAg-9 :..S....................K....................A............... HBsAg-10 :....A..L...............K....................T..........QI.S. HBsAg-11 :...T....................................... E G A.P.....L....... HBsAg-12 :............................................................ HBsAg-13 :...........................................ENT.............. HBsAg-14 :....... L F.... L F..........................S.... A S............... HBsAg-16 :............. A V...... S L................ S F.S...EA.........C..... HBsAg-18 :...T........................................A.K.P...L....... HBsAg-19 :..ST........................................A.T.P........... HBsAg-20 :............................................TT.............. HBsAg-21 :.......L.R...................K..................R........... HBsAg-23 :..K....S..................................TT................ HBsAg-24 :..S....................K....................A............... HBsAg-27 :............................................TT.............. HBsAg-147 :............................. K Q..............TT.............. HBsAg-148 :..............................N.............NT.............. HBsAg-149 :............. A V...... S L..... T I T I. Q R........... P L. E G A..... Q L......... HBsAg-150 :....A..L...............K....................TH.........QI.S. HBsAg-153 :..S....................K....................A.............S. HBsAg-154 :............................................TT.............. HBsAg-29 : -----------------------------------------------------------. HBsAg-112 :............................................V...P.L......... HBsAg-143 :............................................TT.............. HBsAg-144 :....A..L...............K....................T..........Q..S. HBsAg-145 :............................................TT.............. HBsAg Sequence Analysis Group 1 HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers 45 Reference prototype strains of A to H genotypes

14. HBsAg Gen A: MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH HBsAg Gen B:............................................................ HBsAg Gen C:....A..L............S..K.....L.............ET..........QI.S. HBsAg Gen D:............................................TT.............. HBsAg Gen E:..S....................K....................A............... HBsAg Gen F:.......L....R....VC....K....................L.R.P........... HBsAg Gen G:.......L....R....VC....K....................L.R.P........... HBsAg Gen H:.......L.R.......VC....K...................VP.G.P.......I... HBsAg-9 :..S....................K....................A............... HBsAg-10 :....A..L...............K....................T..........QI.S. HBsAg-11 :...T....................................... E G A.P.....L....... HBsAg-12 :............................................................ HBsAg-13 :...........................................ENT.............. HBsAg-14 :....... L F.... L F..........................S.... A S............... HBsAg-16 :............. A V...... S L................ S F.S...EA.........C..... HBsAg-18 :...T........................................A.K.P...L....... HBsAg-19 :..ST........................................A.T.P........... HBsAg-20 :............................................TT.............. HBsAg-21 :.......L.R...................K..................R........... HBsAg-23 :..K....S..................................TT................ HBsAg-24 :..S....................K....................A............... HBsAg-27 :............................................TT.............. HBsAg-147 :............................. K Q..............TT.............. HBsAg-148 :..............................N.............NT.............. HBsAg-149 :............. A V...... S L..... T I T I. Q R........... P L. E G A..... Q L......... HBsAg-150 :....A..L...............K....................TH.........QI.S. HBsAg-153 :..S....................K....................A.............S. HBsAg-154 :............................................TT.............. HBsAg-29 : -----------------------------------------------------------. HBsAg-112 :............................................V...P.L......... HBsAg-143 :............................................TT.............. HBsAg-144 :....A..L...............K....................T..........Q..S. HBsAg-145 :............................................TT.............. HBsAg Sequence Analysis Group 1 45 HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers Reference prototype strains of A to H genotypes

15. HBsAg Gen A: MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH HBsAg Gen B:............................................................ HBsAg Gen C:....A..L............S..K.....L.............ET..........QI.S. HBsAg Gen D:............................................TT.............. HBsAg Gen E:..S....................K....................A............... HBsAg Gen F:.......L....R....VC....K....................L.R.P........... HBsAg Gen G:.......L....R....VC....K....................L.R.P........... HBsAg Gen H:.......L.R.......VC....K...................VP.G.P.......I... HBsAg-9 :..S....................K....................A............... HBsAg-10 :....A..L...............K....................T..........QI.S. HBsAg-11 :...T....................................... E G A.P.....L....... HBsAg-12 :............................................................ HBsAg-13 :...........................................ENT.............. HBsAg-14 :....... L F.... L F..........................S.... A S............... HBsAg-16 :............. A V...... S L................ S F.S...EA.........C..... HBsAg-18 :...T........................................A.K.P...L....... HBsAg-19 :..ST........................................A.T.P........... HBsAg-20 :............................................TT.............. HBsAg-21 :.......L.R...................K..................R........... HBsAg-23 :..K....S..................................TT................ HBsAg-24 :..S....................K....................A............... HBsAg-27 :............................................TT.............. HBsAg-147 :............................. K Q..............TT.............. HBsAg-148 :..............................N.............NT.............. HBsAg-149 :............. A V...... S L..... T I T I. Q R........... P L. E G A..... Q L......... HBsAg-150 :....A..L...............K....................TH.........QI.S. HBsAg-153 :..S....................K....................A.............S. HBsAg-154 :............................................TT.............. HBsAg-29 : -----------------------------------------------------------. HBsAg-112 :............................................V...P.L......... HBsAg-143 :............................................TT.............. HBsAg-144 :....A..L...............K....................T..........Q..S. HBsAg-145 :............................................TT.............. 45 HBsAg Sequence Analysis Group 1 HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers Reference prototype strains of A to H genotypes

16. HBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC HBsAg Gen B:............................ HBsAg Gen C:.........T.................. HBsAg Gen D: R....T...T..Y........S...... HBsAg Gen E: R..M.L...T........S..S...... HBsAg Gen F:....AL...T........S..S...... HBsAg Gen G:....AL...T........S..S...... HBsAg Gen H:.....L...T...........S...... HBsAg-9 : R....L...T........S..S...... HBsAg-10 :.........T.................. HBsAg-11 :. M R T I......T...........S...... HBsAg-12 :............ RL............... HBsAg-13 : R........T..Y........S...... HBsAg-14 :............................ HBsAg-16 :............................ HBsAg-18 :....I....T...........S...... HBsAg-19 :....I....T........ T S..S...... HBsAg-20 : R........T..Y........S...... HBsAg-21 :...........T................ HBsAg-23 : R........T..Y........S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 :............................ HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S...... HBsAg-29 : R........................... HBsAg-112 :............Y........S...... HBsAg-143 : R........A..Y........S...... HBsAg-144 :.........T.................. HBsAg-145 : R..M.T...T..Y........S...... “a” Determinant Sequence Analysis Group 1 Reference prototype strains of A to H gentotypes HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers

17. HBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC HBsAg Gen B:............................ HBsAg Gen C:.........T.................. HBsAg Gen D: R....T...T..Y........S...... HBsAg Gen E: R..M.L...T........S..S...... HBsAg Gen F:....AL...T........S..S...... HBsAg Gen G:....AL...T........S..S...... HBsAg Gen H:.....L...T...........S...... HBsAg-9 : R....L...T........S..S...... HBsAg-10 :.........T.................. HBsAg-11 :. M R T I......T...........S...... HBsAg-12 :............ RL............... HBsAg-13 : R........T..Y........S...... HBsAg-14 :............................ HBsAg-16 :............................ HBsAg-18 :....I....T...........S...... HBsAg-19 :....I....T........ T S..S...... HBsAg-20 : R........T..Y........S...... HBsAg-21 :...........T................ HBsAg-23 : R........T..Y........S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 :............................ HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S...... HBsAg-29 : R........................... HBsAg-112 :............Y........S...... HBsAg-143 : R........A..Y........S...... HBsAg-144 :.........T.................. HBsAg-145 : R..M.T...T..Y........S...... Reference prototype strains of A to H gentotypes HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers “a” Determinant Sequence Analysis Group 1

18. HBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC HBsAg Gen B:............................ HBsAg Gen C:.........T.................. HBsAg Gen D: R....T...T..Y........S...... HBsAg Gen E: R..M.L...T........S..S...... HBsAg Gen F:....AL...T........S..S...... HBsAg Gen G:....AL...T........S..S...... HBsAg Gen H:.....L...T...........S...... HBsAg-9 : R....L...T........S..S...... HBsAg-10 :.........T.................. HBsAg-11 :. M R T I......T...........S...... HBsAg-12 :............ RL............... HBsAg-13 : R........T..Y........S...... HBsAg-14 :............................ HBsAg-16 :............................ HBsAg-18 :....I....T...........S...... HBsAg-19 :....I....T........ T S..S...... HBsAg-20 : R........T..Y........S...... HBsAg-21 :...........T................ HBsAg-23 : R........T..Y........S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 :............................ HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S...... HBsAg-29 : R........................... HBsAg-112 :............Y........S...... HBsAg-143 : R........A..Y........S...... HBsAg-144 :.........T.................. HBsAg-145 : R..M.T...T..Y........S...... Reference prototype strains of A to H gentotypes HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers “a” Determinant Sequence Analysis Group 1

19. HBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC HBsAg Gen B:............................ HBsAg Gen C:.........T.................. HBsAg Gen D: R....T...T..Y........S...... HBsAg Gen E: R..M.L...T........S..S...... HBsAg Gen F:....AL...T........S..S...... HBsAg Gen G:....AL...T........S..S...... HBsAg Gen H:.....L...T...........S...... HBsAg-9 : R....L...T........S..S...... HBsAg-10 :.........T.................. HBsAg-11 :. M R T I......T...........S...... HBsAg-12 :............ RL............... HBsAg-13 : R........T..Y........S...... HBsAg-14 :............................ HBsAg-16 :............................ HBsAg-18 :....I....T...........S...... HBsAg-19 :....I....T........ T S..S...... HBsAg-20 : R........T..Y........S...... HBsAg-21 :...........T................ HBsAg-23 : R........T..Y........S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 :............................ HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S...... HBsAg-29 : R........................... HBsAg-112 :............Y........S...... HBsAg-143 : R........A..Y........S...... HBsAg-144 :.........T.................. HBsAg-145 : R..M.T...T..Y........S...... Reference prototype strains of A to H gentotypes HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers “a” Determinant Sequence Analysis Group 1

20. Reference prototype strains of A to H gentotypes HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers “a” Determinant Sequence Analysis Group 1 HBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC HBsAg Gen B:............................ HBsAg Gen C:.........T.................. HBsAg Gen D: R....T...T..Y........S...... HBsAg Gen E: R..M.L...T........S..S...... HBsAg Gen F:....AL...T........S..S...... HBsAg Gen G:....AL...T........S..S...... HBsAg Gen H:.....L...T...........S...... HBsAg-9 : R....L...T........S..S...... HBsAg-10 :.........T.................. HBsAg-11 :. M R T I......T...........S...... HBsAg-12 :............ RL............... HBsAg-13 : R........T..Y........S...... HBsAg-14 :............................ HBsAg-16 :............................ HBsAg-18 :....I....T...........S...... HBsAg-19 :....I....T........ T S..S...... HBsAg-20 : R........T..Y........S...... HBsAg-21 :...........T................ HBsAg-23 : R........T..Y........S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 :............................ HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S...... HBsAg-29 : R........................... HBsAg-112 :............Y........S...... HBsAg-143 : R........A..Y........S...... HBsAg-144 :.........T.................. HBsAg-145 : R..M.T...T..Y........S......

21. PreS1-PreS2 Sequence Analysis Group 1 No specific features were observed in the preS1 and preS2 regions in the donors with no detectable HBsAg compared to HBsAg positive donors.No specific features were observed in the preS1 and preS2 regions in the donors with no detectable HBsAg compared to HBsAg positive donors.

22. Frequency of HBV DNA Detection in the Second Study Group Brain-dead organ donors Seronegative Anti-HBs alone TOTALn9125211433 HBV DNA + confirmed 011

23. HBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC HBsAg Gen B:............................ HBsAg Gen C:.........T.................. HBsAg Gen D: R....T...T..Y........S...... HBsAg Gen E: R..M.L...T........S..S...... HBsAg Gen F:....AL...T........S..S...... HBsAg Gen G:....AL...T........S..S...... HBsAg Gen H:.....L...T...........S...... HBsAg-122 : R....L.PST..Y........S...... “a” Determinant Sequence Analysis Group 2 Reference prototype strains of A to H genotypes Vaccinated donors Position 129

24. PreS1-preS2 Sequence Analysis Group 2 No additional preS1 or preS2 changes were observed that could explain the lack of HBsAg detection in this donor.No additional preS1 or preS2 changes were observed that could explain the lack of HBsAg detection in this donor.

25. HBV DNA:HBV DNA: n is detected in most HBsAg-positive organ, tissue and cell donors, n can be detected in HBsAg-negative donors, with or without anti-HBc Ab. In HBV DNA-positive donors, whatever their serological profile, the level of HBV replication is substantially lower than in patients with chronic hepatitis B.In HBV DNA-positive donors, whatever their serological profile, the level of HBV replication is substantially lower than in patients with chronic hepatitis B. Conclusions (I)

26. The lack of HBsAg detection in our study could not be explained by HBsAg or preS1-preS2 amino acid substitutions (except in one donor without any HBV serological marker).The lack of HBsAg detection in our study could not be explained by HBsAg or preS1-preS2 amino acid substitutions (except in one donor without any HBV serological marker). The lack of HBsAg detection was most likely related to a lack of sensitivity of enzyme immunoassays for low-level HBsAg in individuals with low-level viral replication.The lack of HBsAg detection was most likely related to a lack of sensitivity of enzyme immunoassays for low-level HBsAg in individuals with low-level viral replication. Conclusions (II)

27. Organ, tissue and cell transplantation safety may greatly benefit from:Organ, tissue and cell transplantation safety may greatly benefit from: n Implementation of highly-sensitive HBsAg assays. n Implementation of highly-sensitive HBV DNA detection assays. Perspectives

28. French National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, INSERM U 635 Hôpital H. Mondor, Université Paris XII, Créteil Dominique CHALLINE Stéphane CHEVALIEZ Rozenn BRILLET Jean-Michel PAWLOTSKY