Yield obtained by mandatory HCV- and HIV1-NAT in Germany
and break through infections
–
1999 - 2007
Micha Nübling
Paul-Ehrlich-Institut
NAT Background•All blood collection centers were contacted in regard to their testing experience 1999-2007 (questionnaire, 2008)
•4,5 mio RBCs / year
German Red Cross 7 centers 75%Hospital assoc. 75 centers 20%Private 5 centers 5%
•NAT-assays •CE marked screening NATs•CE marked diagnostic NATs validated for sensitivity etc•Validated in-house NATs
NAT Background
•Mandatory NATs for all blood / plasma donations
•Definition of minimal NAT sensitivity limit (ID)
01.04.1999 HCV-NAT 5.000 IU / ml ( = 20.000 cps / ml)
01.05.2004 HIV1-NAT 10.000 IU / ml ( = 5.000 cps / ml)
Viremia during the "diagnostic window"HCV
antiHCV pos
5.000 IU / ml
HCV-infectionantiHCV negative phase
HCV-RNA doubling time10,8 h
1,0E+00
1,0E+01
1,0E+02
1,0E+03
1,0E+04
1,0E+05
1,0E+06
1,0E+07
1,0E+08
1,0E+09
1,0E+10
0 10 20 30 40 50 60 70 80 90
Days after infection
HC
V-R
NA
(c
op
ies
/ ml)
NAT onlies: HCV
92 HCV-NAT onlies among 40.8 million donations (76 centers; 1999-2007)
= 1 / 440 000 donations
4
6
11
13
11 11
13
8
15
0
2
4
6
8
10
12
14
16
1999 2000 2001 2002 2003 2004 2005 2006 2007
Nübling et al. (2009) Transfusion (in press)
NAT onlies: HCV
92 HCV-NAT onlies= 1 / 440.000 donations
„Higher incidence“ centers
#47 6 / 202.435 donations = 1 / 33.700#15 16 / 587.000 donations = 1 / 36.700#08 9 / 427.000 donations = 1 / 47.400
„Lower incidence“ centers
#30 4 / 4.415.033 donations = 1 / 1.100.000#52 6 / 6.713.709 donations = 1 / 1.120.000#23 0 / 903.948 donations = 0 / 903.948
4
6
11
13
11 11
13
8
15
0
2
4
6
8
10
12
14
16
1999 2000 2001 2002 2003 2004 2005 2006 2007
HCV genotypes in NAT Yield Cases
26 yield cases with HCV genotype data
HCV gt 338%
HCV gt 150%
HCV gt 212%
Viral Loads in HCV NAT Yield Cases
40 yield cases with viral load data39 (99%) >> 5.000 IU/ml
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
Vir
al L
oad
(IU
/mL
)
5.000 IU / ml
Nübling et al. (2009) Transfusion (in press)
Transfusion-associated HCV transmissionsCases reported to PEI
0
2
4
6
8
10
12
14
16
18
Year
HC
V T
ran
smis
sio
ns
Introduction of NAT
missed by AmpliScreen HCV / 24
10-2004 previous donationantiHCV (Ortho 3.0) neg HCV-RNA (AmpliScreen/24) neg
HCV transmission
01-2005 repeat donor: seroconversion (antiHCV, HCV-RNA)HCV genotype 2b
look-back
recipient (erythrocytes) HCV-pos
HCV genotype 2b
plasma still availablereplicate testing (n=5) in all available
NAT systems
HCV window phase, genotype 2b
95% LOD (WHO IS, gt1) pos results (ID)Screening CE-NATs
Cobas AmpliScreen HCV Test v 2.0 29 IU/ml 1 / 5CAP Cobas TaqScreen 11 IU/ml 5 / 5
Procleix HIV-1/HCV Assay 2 IU/ml 0 / 5Procleix Ultrio Assay 3 IU/ml 2 / 5
Diagnostic CE-NATsCobas Amplicor HCV Test v 2.0 43 IU/ml 1 / 5HPS Cobas TaqMan HCV Test 15 IU/ml 3 / 5CAP Cobas Amplicor HCV Test v 2.0 11 IU/ml 3 / 5
CAP Cobas TaqMan HCV Test 13 IU/ml 3 / 5
Versant HCV RNA Qual 10 IU/ml 0 / 5
Abbott RealTime HCV 11 IU/ml 4 / 5
HCV transmission
Viremia during the "diagnostic window"HIV-1
p24-positive
p24-negative
antiHIV-negative phase
HIV1-RNA doubling time 21,5 h
1,0E+00
1,0E+01
1,0E+02
1,0E+03
1,0E+04
1,0E+05
1,0E+06
1,0E+07
1,0E+08
1,0E+09
1,0E+10
0 3 6 9 12 15 18 21 24 27 30 33 36
Days after infection
HIV
-RN
A (
cop
ies
/ ml)
10.000 IU / ml
antiHIV posHIV-infection
1
4
1
32
4
2
0
2
4
6
8
10
12
14
16
1999 2000 2001 2002 2003 2004 2005 2006 2007
NAT onlies: HIV
11 HIV-NAT onlies among 19 million donations (2004-2007)= 1 / 1 700 000 donations
(17 HIV-NAT onlies from 1999 to 2007= 1 / 2 400 000 donations)
Introduction of HIV NAT
Nübling et al. (2009) Transfusion (in press)
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
Vir
al L
oad
(IU
/mL
)
10,000 IU/ml
Viral Loads in HIV NAT Yield Cases
11 yield cases with viral load data10 > 10 000 IU/ml
Nübling et al. (2009) Transfusion (in press)
Transfusion-associated HIV transmissionsCases reported to PEI
0
2
4
6
8
10
12
14
16
Introduction of HIV NAT
01-2007 previous donationantiHIV1/2 neg HIV1-RNA (CAP CTM/96) neg
HIV transmission
04 - 2007 repeat donor: seroconversion (antiHIV1/2)HIV1 subtype B
look-back
recipient (erythrocytes) HIV1-seroconversion
HIV1 subtype B, variant as in donor
back-up sample147 IU/ml ID CAP CTMHIV Ag/Ab test negative (Abbott)
HIV transmission
04 - 2007 repeat donor: seroconversion (antiHIV1/2)
pos
CobasTaqScreen
pos (5/5)
ProcleixUltrio
pos (2/2)
CobasAmpliScreen
HIV (MP)
924<LLQnegneg
Abbott RealTime
HIV-1 (IU/ml)
CAM HIV(IU/ml)
HPS CTMHIV
(IU/ml)
CAP CTMHIV
(IU/ml)
• quantitative HIV-NATs• screening HIV-NATs
01 - 2007 back-up sample147 IU/ml ID (CAP CTM HIV)
HIV Ag/Ab test negative (Abbott)
recovered plasma into manufacturing pool: HIV1-RNA neg
HIV-1 subtype B Transmission≈ 100 fold underdetection in routine NAT
Sense primer CAP CTM HIV-1 Test (nt 1788 – nt 1819)
CTM Primer: 5´ AGT GGG GGG ACA TCA AGC AGC CAT GCA AA 3´Donor: 5´ AGT GGG GGG ACA TCA AGC AGC CAT GCA AA 3´
Probe CAP CTM HIV-1 Test (nt 1821 – nt 1856)
CTM Probe: 5´ TCT GCA GCT TCC TCA TTG ATG GT A TCT TTT AAC 3´ Donor: 5´ TCT GCA GCT TCC TCA TTG ATG GT T TCT TTT AAC 3´
Antisense primer CAP CTM HIV-1 Test (nt 1921 – nt 1950)
CTM Primer: 5´ G G T ACT AGT AGT TCC TGC TAT GTC ACT T CC 3´ Donor: 5´ G T T ACT AGT AGT TCC TGC TAT GTC ACT A CC 3´
Schmidt et al. (2009) Transfusion (in press)Nübling et al. (2009) Transfusion (in press)
Challenges for Pathogen Testing
Virus safety with routine measures high safety margins achieved
testing experiencelimitations low virus level may be NAT negvulnerability mismatches unpredictable
sensitivity levels (HCV, HIV) appear as reasonable compromise for low incidence population
Conclusions