false-negative hbsag detection in chronic hepatitis b

• False negative HBsAg detectionFalse negative HBsAg detection Is rare in patients with signs of chronic hepatitis B.Is rare in patients with signs of chronic hepatitis B. May be an issue in low-risk populations, such as:May be an issue in low-risk populations, such as:

– blood donors,blood donors,

– organ, tissue and cell donors.organ, tissue and cell donors.

• It may result of:It may result of: Undetectable, low-level HBsAg.Undetectable, low-level HBsAg. Substitutions in HBsAg “Major Hydrophilic Region”:Substitutions in HBsAg “Major Hydrophilic Region”:

– located at positions 99 to 160,located at positions 99 to 160,

– encompasses the “a” determinant, a major HBV epitope.encompasses the “a” determinant, a major HBV epitope.

False-Negative HBsAg False-Negative HBsAg Detection in Chronic Detection in Chronic

Hepatitis BHepatitis B

Mutations in “a” DeterminantMutations in “a” Determinant

107107 138138 139139

137137 149149

147147

AA

RRD D 144144

G G 145145

210210

S-SS-SS-SS-S

--S-S---S-S-

9999

121121 124124S-SS-SLoopLoop 1 of “a” determinant 1 of “a” determinant

LoopLoop 2 of “a” determinant 2 of “a” determinant

HBs1HBs1

HBs2HBs2

HBs3HBs3

HBs4HBs4

HBs5HBs5

141 141 KK

TT 126126

NN

QQ 129129

HH

MM 133133

LL

EE

Aims of the StudyAims of the Study

• To determine the prevalence of falsely negative To determine the prevalence of falsely negative

HBsAg detection in a large population of organ, HBsAg detection in a large population of organ,

tissue and cell donors.tissue and cell donors.

• To understand the role of HBsAg mutants in the To understand the role of HBsAg mutants in the

lack of HBsAg detection in HBV DNA positive lack of HBsAg detection in HBV DNA positive

donors.donors.

PatientsPatients

• 11,155 organ, tissue and cell donors were systematically 11,155 organ, tissue and cell donors were systematically tested for the presence of HBV markers (e.g. HBsAg, anti-tested for the presence of HBV markers (e.g. HBsAg, anti-HBc Ab, anti-HBs Ab) between May 2000 and May 2004:HBc Ab, anti-HBs Ab) between May 2000 and May 2004:

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


First groupFirst group::- 626 donors (5.6%),626 donors (5.6%),

- Brain-dead, heart-beating organ donorsBrain-dead, heart-beating organ donors

- Living organ donorsLiving organ donors

- Tissue donorsTissue donors

- Stem cell donorsStem cell donors

- Cord blood donorsCord blood donors

- Cornea donorsCornea donors

199199

1313

165165

7575

5656

118 118

PatientsPatients


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

Second group:Second group:- 1433 brain-dead organ donors,1433 brain-dead organ donors,- seronegative for HBV or with anti-HBs Ab only.seronegative for HBV or with anti-HBs Ab only.

PatientsPatients

Methods (1)Methods (1)

• HBsAg HBsAg was assessed with 3 different assays:was assessed with 3 different assays:

Vitros EciVitros EciTM TM (Ortho-Clinical Diagnostics), (Ortho-Clinical Diagnostics), ArchitectArchitectTMTM (Abbott), (Abbott), VidasVidasTMTM (BioMérieux). (BioMérieux).

• HBV DNAHBV DNA was systematically sought by means of a was systematically sought by means of a real-time PCR assay:real-time PCR assay:

Cobas TaqMan HBV (Roche Molecular Systems),Cobas TaqMan HBV (Roche Molecular Systems), Lower limit of detection: 6 IU/ml,Lower limit of detection: 6 IU/ml, Dynamic range of quantification: 30-10Dynamic range of quantification: 30-1088 IU/ml. IU/ml.

• Full-length Full-length preS1-preS2-HBsAg sequencepreS1-preS2-HBsAg sequence was was determined in all HBV DNA-positive donors:determined in all HBV DNA-positive donors:

Nested PCR using previously described primersNested PCR using previously described primersa a

Direct sequencing,Direct sequencing,

Alignment of sequences with prototype strains of HBV Alignment of sequences with prototype strains of HBV genotypes A to H.genotypes A to H.

aaStuyver et al., J Gen Virol 2000 Stuyver et al., J Gen Virol 2000

Methods (2)Methods (2)

HBV DNA in HBsAg-positive DonorsHBV DNA in HBsAg-positive Donors

3.3 log3.3 logIU/mlIU/ml





Brain-deadBrain-deadorgan donorsorgan donors

(n = 199)(n = 199)

LivingLivingorgan donorsorgan donors

(n = 13)(n = 13)

TissueTissueDonorsDonors

(n = 165)(n = 165)

Cord bloodCord bloodDonorsDonors(n = 56)(n = 56)

Stem cellStem cellDonorsDonors(n = 75)(n = 75)

CorneaCorneaDonorsDonors

(n = 118)(n = 118)

17/20 (85%)17/20 (85%)

2/2 (100%)2/2 (100%)

3/5 (60%)3/5 (60%)

2/2 (100%)2/2 (100%)

1/8 (12.5%)1/8 (12.5%)20%20%

40%40%

60%60%

80%80%

100%100%

0%0%

HBV DNA in Donors with HBV DNA in Donors with Isolated Anti-HBc AbIsolated Anti-HBc Ab



20%20%

40%40%

60%60%

80%80%

100%100%

0%0%

2/53 (3.8%)2/53 (3.8%)2/21 (9.5%)2/21 (9.5%)


(n = 199)(n = 199)


(n = 13)(n = 13)


(n = 165)(n = 165)




(n = 118)(n = 118)


20%20%

40%40%

60%60%

80%80%

100%100%

0%0%

3/62 (4.8%)3/62 (4.8%)

HBV DNA in Donors with Both HBV DNA in Donors with Both Anti-HBc and Anti-HBs AbAnti-HBc and Anti-HBs Ab


(n = 199)(n = 199)


(n = 13)(n = 13)


(n = 165)(n = 165)




(n = 118)(n = 118)



20%20%

40%40%

60%60%

80%80%

100%100%

0%0%

2/5 (40%)2/5 (40%)

1/27 (3.7%)1/27 (3.7%)

HBV DNA in Donors with HBV DNA in Donors with Three MarkersThree Markers


(n = 199)(n = 199)


(n = 13)(n = 13)


(n = 165)(n = 165)




(n = 118)(n = 118)

HBsAg Gen AHBsAg Gen A: MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH: MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHHBsAg Gen BHBsAg Gen B: ............................................................ : ............................................................ HBsAg Gen CHBsAg Gen C: ....A..L............S..K.....L.............ET..........QI.S. : ....A..L............S..K.....L.............ET..........QI.S. HBsAg Gen DHBsAg Gen D: ............................................TT.............. : ............................................TT.............. HBsAg Gen EHBsAg Gen E: ..S....................K....................A............... : ..S....................K....................A............... HBsAg Gen FHBsAg Gen F: .......L....R....VC....K....................L.R.P........... : .......L....R....VC....K....................L.R.P........... HBsAg Gen GHBsAg Gen G: .......L....R....VC....K....................L.R.P........... : .......L....R....VC....K....................L.R.P........... HBsAg Gen HHBsAg Gen H: .......L.R.......VC....K...................VP.G.P.......I...: .......L.R.......VC....K...................VP.G.P.......I... HBsAg-9 : ..S....................K....................A............... HBsAg-9 : ..S....................K....................A............... HBsAg-10 : ....A..L...............K....................T..........QI.S. HBsAg-10 : ....A..L...............K....................T..........QI.S. HBsAg-11 : ...T.......................................HBsAg-11 : ...T.......................................EE

GGA.P.....L....... A.P.....L....... HBsAg-12 : ............................................................ HBsAg-12 : ............................................................ HBsAg-13 : ...........................................ENT.............. HBsAg-13 : ...........................................ENT.............. HBsAg-14 : .......HBsAg-14 : .......LL

FF........LLFF..........................S..............................S....AA

SS............... ............... HBsAg-16 : .............HBsAg-16 : .............AA

VV............SSLL................................SS

FF.S...EA.........C..... .S...EA.........C..... HBsAg-18 : ...T........................................A.K.P...L....... HBsAg-18 : ...T........................................A.K.P...L....... HBsAg-19 : ..ST........................................A.T.P........... HBsAg-19 : ..ST........................................A.T.P........... HBsAg-20 : ............................................TT.............. HBsAg-20 : ............................................TT.............. HBsAg-21 : .......L.R...................K..................R........... HBsAg-21 : .......L.R...................K..................R........... HBsAg-23 : ..K....S..................................TT................ HBsAg-23 : ..K....S..................................TT................ HBsAg-24 : ..S....................K....................A............... HBsAg-24 : ..S....................K....................A............... HBsAg-27 : ............................................TT.............. HBsAg-27 : ............................................TT.............. HBsAg-147 : .............................HBsAg-147 : .............................KK

QQ..............TT.............. ..............TT.............. HBsAg-148 : ..............................N.............NT.............. HBsAg-148 : ..............................N.............NT.............. HBsAg-149 : .............HBsAg-149 : .............AA

VV............SSLL..........TT

IITTII..QQ

RR......................PPLL..EE

GGA.....A.....QQLL......... .........

HBsAg-150 : ....A..L...............K....................TH.........QI.S. HBsAg-150 : ....A..L...............K....................TH.........QI.S. HBsAg-153 : ..S....................K....................A.............S. HBsAg-153 : ..S....................K....................A.............S. HBsAg-154 : ............................................TT.............. HBsAg-154 : ............................................TT..............

HBsAg-29 : -----------------------------------------------------------. HBsAg-29 : -----------------------------------------------------------. HBsAg-112 : ............................................V...P.L.........HBsAg-112 : ............................................V...P.L.........

HBsAg-143 : ............................................TT.............. HBsAg-144 : ....A..L...............K....................T..........Q..S. HBsAg-145 : ............................................TT..............

HBsAg Sequence Analysis HBsAg Sequence Analysis Group 1Group 1

HBsAg-positive donorsHBsAg-positive donors

Isolated anti-HBc Ab donorsIsolated anti-HBc Ab donors

Donors with three Donors with three serological markersserological markers

45

Reference prototype strains Reference prototype strains of A to H genotypesof A to H genotypes


GGA.P.....L....... A.P.....L....... HBsAg-12 : ............................................................ HBsAg-12 : ............................................................ HBsAg-13 HBsAg-13 : ...........................................ENT.............. : ...........................................ENT.............. HBsAg-14 : .......HBsAg-14 : .......LL

FF........LLFF..........................S..............................S....AA

SS............... ............... HBsAg-16 : .............HBsAg-16 : .............AA

VV............SSLL................................SS


QQ..............TT.............. ..............TT.............. HBsAg-148HBsAg-148 : ..............................N.............NT.............. : ..............................N.............NT.............. HBsAg-149 : .............HBsAg-149 : .............AA

VV............SSLL..........TT

IITTII..QQ

RR......................PPLL..EE

GGA.....A.....QQLL......... .........


HBsAg-29 : -----------------------------------------------------------. HBsAg-29 : -----------------------------------------------------------. HBsAg-112 : ............................................V...P.L.........HBsAg-112 : ............................................V...P.L.........



45






GGA.P.....L....... A.P.....L....... HBsAg-12 : ............................................................ HBsAg-12 : ............................................................ HBsAg-13 HBsAg-13 : ...........................................ENT.............. : ...........................................ENT.............. HBsAg-14 : .......HBsAg-14 : .......LL

FF........LLFF..........................S..............................S....AA

SS............... ............... HBsAg-16 : .............HBsAg-16 : .............AA

VV............SSLL................................SS


QQ..............TT.............. ..............TT.............. HBsAg-148HBsAg-148 : ..............................N.............NT.............. : ..............................N.............NT.............. HBsAg-149 : .............HBsAg-149 : .............AA

VV............SSLL..........TT

IITTII..QQ

RR......................PPLL..EE

GGA.....A.....QQLL......... .........


HBsAg-29 : -----------------------------------------------------------. HBsAg-29 : -----------------------------------------------------------. HBsAg-112 HBsAg-112 : ............................................V...P.L......... : ............................................V...P.L.........


45






HBsAg Gen AHBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC: KTCTTPAQGNSMFPSCCCTKPTDGNCTCHBsAg Gen BHBsAg Gen B: ............................ : ............................ HBsAg Gen CHBsAg Gen C: .........T.................. : .........T.................. HBsAg Gen DHBsAg Gen D: R....T...T..Y........S...... : R....T...T..Y........S...... HBsAg Gen EHBsAg Gen E: R..M.L...T........S..S...... : R..M.L...T........S..S...... HBsAg Gen FHBsAg Gen F: ....AL...T........S..S...... : ....AL...T........S..S...... HBsAg Gen GHBsAg Gen G: ....AL...T........S..S......: ....AL...T........S..S......HBsAg Gen HHBsAg Gen H: .....L...T...........S......: .....L...T...........S...... HBsAg-9 : R....L...T........S..S......HBsAg-9 : R....L...T........S..S......HBsAg-10 : .........T..................HBsAg-10 : .........T..................HBsAg-11 : .HBsAg-11 : .MM

RRTTII......T...........S...... ......T...........S......

HBsAg-12 : ............HBsAg-12 : ............RLRL..............................HBsAg-13 : R........T..Y........S......HBsAg-13 : R........T..Y........S......HBsAg-14 : ............................ HBsAg-14 : ............................ HBsAg-16 : ............................ HBsAg-16 : ............................ HBsAg-18 : ....I....T...........S......HBsAg-18 : ....I....T...........S......HBsAg-19 : ....I....T........HBsAg-19 : ....I....T........TT

SS..S........S......HBsAg-20 : R........T..Y........S......HBsAg-20 : R........T..Y........S......HBsAg-21 : ...........T................HBsAg-21 : ...........T................HBsAg-23 : R........T..Y........S......HBsAg-23 : R........T..Y........S......HBsAg-24 : R....L...T........S..S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 : ............................ HBsAg-149 : ............................ HBsAg-150 : R......H.T.................. HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S......HBsAg-154 : R........T..Y........S......

HBsAg-29 : R........................... HBsAg-29 : R........................... HBsAg-112 : ............Y........S...... HBsAg-112 : ............Y........S...... HBsAg-143 : R........A..Y........S...... HBsAg-144 : .........T.................. HBsAg-145 : R..M.T...T..Y........S......

““a” Determinant Sequence Analysisa” Determinant Sequence AnalysisGroup 1Group 1

Reference prototype strains Reference prototype strains of A to H gentotypesof A to H gentotypes



Donors with three serological Donors with three serological markersmarkers


RRTTII......T...........S...... ......T...........S......

HBsAg-12 : ............HBsAg-12 : ............RLRL..............................HBsAg-13 : R........T..Y........S......HBsAg-13 : R........T..Y........S......HBsAg-14 : ............................ HBsAg-14 : ............................ HBsAg-16 : ............................ HBsAg-16 : ............................ HBsAg-18HBsAg-18 : ....I....T...........S...... : ....I....T...........S......HBsAg-19HBsAg-19 : ....I....T........ : ....I....T........TT

SS..S........S......HBsAg-20 : R........T..Y........S......HBsAg-20 : R........T..Y........S......HBsAg-21 : ...........T................HBsAg-21 : ...........T................HBsAg-23 : R........T..Y........S......HBsAg-23 : R........T..Y........S......HBsAg-24 : R....L...T........S..S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 : ............................ HBsAg-149 : ............................ HBsAg-150 : R......H.T.................. HBsAg-150 : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S......HBsAg-154 : R........T..Y........S......








RRTTII......T...........S...... ......T...........S......


SS..S........S......HBsAg-20 : R........T..Y........S......HBsAg-20 : R........T..Y........S......HBsAg-21 : ...........T................HBsAg-21 : ...........T................HBsAg-23 : R........T..Y........S......HBsAg-23 : R........T..Y........S......HBsAg-24 : R....L...T........S..S...... HBsAg-24 : R....L...T........S..S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-27 : R..M.T...T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-147 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-148 : R........T..Y........S...... HBsAg-149 : ............................ HBsAg-149 : ............................ HBsAg-150HBsAg-150 : R......H.T.................. : R......H.T.................. HBsAg-153 : R....L...T........S..S...... HBsAg-153 : R....L...T........S..S...... HBsAg-154 : R........T..Y........S......HBsAg-154 : R........T..Y........S......







HBsAg Gen AHBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC: KTCTTPAQGNSMFPSCCCTKPTDGNCTCHBsAg Gen BHBsAg Gen B: ............................ : ............................ HBsAg Gen CHBsAg Gen C: .........T.................. : .........T.................. HBsAg Gen DHBsAg Gen D: R....T...T..Y........S...... : R....T...T..Y........S...... HBsAg Gen EHBsAg Gen E: R..M.L...T........S..S...... : R..M.L...T........S..S...... HBsAg Gen FHBsAg Gen F: ....AL...T........S..S...... : ....AL...T........S..S...... HBsAg Gen GHBsAg Gen G: ....AL...T........S..S......: ....AL...T........S..S......HBsAg Gen HHBsAg Gen H: .....L...T...........S......: .....L...T...........S...... HBsAg-9 : R....L...T........S..S......HBsAg-9 : R....L...T........S..S......HBsAg-10 : .........T..................HBsAg-10 : .........T..................HBsAg-11 HBsAg-11 : . : .MM

RRTTII......T...........S...... ......T...........S......









PreS1-PreS2 Sequence PreS1-PreS2 Sequence AnalysisAnalysisGroup 1Group 1

• No specific features were observed in the No specific features were observed in the preS1 and preS2 regions in the donors with preS1 and preS2 regions in the donors with no detectable HBsAg compared to HBsAg no detectable HBsAg compared to HBsAg positive donors.positive donors.

Frequency of HBV DNA Detection Frequency of HBV DNA Detection in the Second Study Groupin the Second Study Group


Seronegative Seronegative

Anti-HBs alone Anti-HBs alone

TOTALTOTAL

nn

912 912

521521

14331433

HBV DNA +HBV DNA +confirmedconfirmed

0 0

11

11

HBsAg Gen AHBsAg Gen A: KTCTTPAQGNSMFPSCCCTKPTDGNCTC: KTCTTPAQGNSMFPSCCCTKPTDGNCTCHBsAg Gen BHBsAg Gen B: ............................ : ............................ HBsAg Gen CHBsAg Gen C: .........T.................. : .........T.................. HBsAg Gen DHBsAg Gen D: R....T...T..Y........S......: R....T...T..Y........S......HBsAg Gen EHBsAg Gen E: R..M.L...T........S..S...... : R..M.L...T........S..S...... HBsAg Gen FHBsAg Gen F: ....AL...T........S..S...... : ....AL...T........S..S...... HBsAg Gen GHBsAg Gen G: ....AL...T........S..S...... : ....AL...T........S..S...... HBsAg Gen HHBsAg Gen H: .....L...T...........S......: .....L...T...........S...... HBsAg-122 : R....L.PST..Y........S...... HBsAg-122 : R....L.PST..Y........S......

““a” Determinant Sequence a” Determinant Sequence AnalysisAnalysis

Group 2Group 2


Vaccinated donorsVaccinated donors

Position 129Position 129

PreS1-preS2 Sequence AnalysisPreS1-preS2 Sequence AnalysisGroup 2Group 2

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

• HBV DNA:HBV DNA:

is detected in most HBsAg-positive organ, tissue and cell is detected in most HBsAg-positive organ, tissue and cell donors,donors,

can be detected in HBsAg-negative donors, with or without can be detected in HBsAg-negative donors, with or without anti-HBc Ab.anti-HBc Ab.

• In HBV DNA-positive donors, whatever their serological In HBV DNA-positive donors, whatever their serological profile, the level of HBV replication is substantially profile, the level of HBV replication is substantially lower than in patients with chronic hepatitis B.lower than in patients with chronic hepatitis B.

Conclusions (I)Conclusions (I)

• The lack of HBsAg detection in our study could not be The lack of HBsAg detection in our study could not be explained by HBsAg or preS1-preS2 amino acid explained by HBsAg or preS1-preS2 amino acid substitutions (except in one donor without any HBV substitutions (except in one donor without any HBV serological marker).serological marker).

• The lack of HBsAg detection was most likely related The lack of HBsAg detection was most likely related to a lack of sensitivity of enzyme immunoassays for to a lack of sensitivity of enzyme immunoassays for low-level HBsAg in individuals with low-level viral low-level HBsAg in individuals with low-level viral replication.replication.

Conclusions (II)Conclusions (II)

• Organ, tissue and cell transplantation safety may Organ, tissue and cell transplantation safety may greatly benefit from:greatly benefit from:

Implementation of highly-sensitive HBsAg assays.Implementation of highly-sensitive HBsAg assays.

Implementation of highly-sensitive HBV DNA Implementation of highly-sensitive HBV DNA detection assays.detection assays.

PerspectivesPerspectives

French National Reference Center for Viral French National Reference Center for Viral Hepatitis B, C and Delta,Hepatitis B, C and Delta,

Department of Virology, INSERM UDepartment of Virology, INSERM U635635

HHôpital H. Mondorôpital H. Mondor, Université Paris XII, Créteil, Université Paris XII, Créteil

Dominique CHALLINE Dominique CHALLINE Stéphane CHEVALIEZStéphane CHEVALIEZ

Rozenn BRILLETRozenn BRILLETJean-Michel PAWLOTSKYJean-Michel PAWLOTSKY

false-negative hbsag detection in chronic hepatitis b

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