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)

HCV

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

HIV

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

HIV-NAT and HIV-Combo assayPreseroconversion (antiHIV1/2 negative) follow-ups• Quantitative NAT (IU/ml)• Architect HIV Ag/Ab Combo (Abbott) (s/co)

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

1,00E+06

1,00E+07

0,1 1 10

sample/cut-off

IU H

IV1-

RN

A/m

l

NAT minimal sensitivity (D)