False negative HBsAg detection

1
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.

2
Mutations in a Determinant
HBs2
121
124
S-S
Loop 1 of a determinant
T 126
N
HBs1
Q 129
HBs3
H
Loop 2 of a determinant
L
99
M 133
HBs5
149
--S-S-
137
107
S-S
138
139
147
S-S
141 K
R
G 145
D 144
E
A
210
HBs4
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 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
• First group
• 626 donors (5.6),
• with at least one of the three HBV serological
  markers,
• excluding vaccination profiles (anti-HBs Ab
  alone).

5
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
6
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.

7
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.

8
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
9
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)
Cord blood Donors (n 56)
Stem cell Donors (n 75)
Cornea Donors (n 118)
10
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)
Cord blood Donors (n 56)
Stem cell Donors (n 75)
Cornea Donors (n 118)
11
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)
Cord blood Donors (n 56)
Stem cell Donors (n 75)
Cornea Donors (n 118)
12
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)
Cord blood Donors (n 56)
Stem cell Donors (n 75)
Cornea Donors (n 118)
13
HBsAg Sequence Analysis Group 1
45
HBsAg Gen A MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWW
TSLNFLGGSPVCLGQNSQSPTSNH 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
14
HBsAg Sequence Analysis Group 1
45
HBsAg Gen A MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWW
TSLNFLGGSPVCLGQNSQSPTSNH 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
15
HBsAg Sequence Analysis Group 1
45
HBsAg Gen A MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWW
TSLNFLGGSPVCLGQNSQSPTSNH 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
16
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
17
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
18
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
19
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
20
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
21
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.

22
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
23
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
24
PreS1-preS2 Sequence AnalysisGroup 2

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

25
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.

26
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.

27
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.

28
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