False-Negative HBsAg Detection in Chronic Hepatitis B

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

Mutations in “a” Determinant HBs2 121 124 S-S Loop 1 of “a” determinant T126 N HBs1 Q129 HBs3 H Loop 2 of “a” determinant L 99 M133 HBs5 149 --S-S- 137 107 S-S 138 139 147 S-S 141 K R G 145 D 144 E A 210 HBs4

Aims of the Study • 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.

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: • First group: • 626 donors (5.6%), • with at least one of the three HBV serological markers, • excluding vaccination profiles (anti-HBs Ab alone).

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: • 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 199 13 165 75 56 118

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: • 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.

Methods (1) • HBsAg was assessed with 3 different assays: • Vitros EciTM (Ortho-Clinical Diagnostics), • ArchitectTM (Abbott), • VidasTM (BioMérieux). • HBV DNA was systematically sought by means of a real-time PCR assay: • Cobas TaqMan HBV (Roche Molecular Systems), • Lower limit of detection: 6 IU/ml, • Dynamic range of quantification: 30-108 IU/ml.

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

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

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

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

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

HBsAg Sequence Analysis Group 1 45 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.......................................EGA.P.....L....... HBsAg-12 : ............................................................ HBsAg-13 : ...........................................ENT.............. HBsAg-14 : .......LF....LF..........................S....AS............... HBsAg-16 : .............AV......SL................SF.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 : .............................KQ..............TT.............. HBsAg-148 : ..............................N.............NT.............. HBsAg-149 : .............AV......SL.....TITI.QR...........PL.EGA.....QL......... 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.............. Reference prototype strains of A to H genotypes HBsAg-positive donors Isolated anti-HBc Ab donors Donors with three serological markers

“a” Determinant Sequence AnalysisGroup 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 : .MRTI......T...........S...... HBsAg-12 : ............RL............... HBsAg-13 : R........T..Y........S...... HBsAg-14 : ............................ HBsAg-16 : ............................ HBsAg-18 : ....I....T...........S...... HBsAg-19 : ....I....T........TS..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

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

Frequency of HBV DNA Detection in the Second Study Group Brain-deadorgan donors Seronegative Anti-HBs alone TOTAL n 912 521 1433 HBV DNA +confirmed 0 1 1

“a” Determinant Sequence AnalysisGroup 2 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...... Reference prototype strains of A to H genotypes Vaccinated donors Position 129

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

Conclusions (I) • HBV DNA: • is detected in most HBsAg-positive organ, tissue and cell donors, • 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.

Conclusions (II) • 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.

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

French National Reference Center for Viral Hepatitis B, C and Delta,Department of Virology, INSERM U635Hôpital H. Mondor, Université Paris XII, Créteil Dominique CHALLINE Stéphane CHEVALIEZ Rozenn BRILLET Jean-Michel PAWLOTSKY

False-Negative HBsAg Detection in Chronic Hepatitis B