Ergebnisse der Abfrage zu NAT-only positiven Befunden beim Blutspendescreening (NAT yield) Micha Nübling, PEI

Ergebnisse der Abfrage zu NAT-only positiven Befunden beim Blutspendescreening (NAT yield) Micha Nübling, PEI

NAT for cellular blood components in Germany 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)

Evolution of NAT systems in-house NATs Cobas Amplicor HCV Cobas Amplicor HCV v2.0 Procleix HCV/HIV Cobas AmpliScreen HCV, HIV, HBV Procleix Ultrio Assay Procleix TIGRIS System with the Ultrio Cobas S201 with TaqScreen

Procleix with the Ultrio

Cobas S201 with TaqScreen configuration 1/2/1 Pooling Manager Workstation AmpliLink Workstation Hamilton Microlab STAR Pipettor Data Manager Workstation COBAS AmpliPrep Instrument COBAS TaqMan Analyzer Reporting of Results

European Countries Leading Pool size by country Pool size Pool of 1 ( Chiron ) Pool < 10 ( Roche ) Pool > 10 or unknown No NAT in routine Other countries: South Africa Israël Russia Egypt

NAT for cellular blood components in Germany 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) NAT yield versus breakthrough transmissions??

Viremia during the "diagnostic window" HCV HCV-RNA (copies / ml) 1,0E+10 1,0E+09 1,0E+08 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 1,0E+01 HCV-RNA doubling time 10,8 h 5.000 IU / ml 1,0E+00 0 10 20 30 40 50 60 70 80 90 Days after infection HCV-infection antihcv pos antihcv negative phase

NAT onlies: HCV 92 HCV-NAT onlies = 1 / 440 000 donations -2007) 16 15 14 13 13 12 11 11 11 10 8 6 4 4 6 8 2 0 1999 2000 2001 2002 2003 2004 2005 2006 2007 Nübling et al. (2009) Transfusion 49, 1850-58

92 HCV-NAT onlies = 1 / 440.000 donations NAT onlies 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

Viral Loads in HCV NAT Yield Cases 40 yield cases with viral load data 39 (99%) >> 5.000 IU/ml 1,00E+08 1,00E+07 Viral Load (IU/mL) 1,00E+06 1,00E+05 1,00E+04 1,00E+03 1,00E+02 1,00E+01 1,00E+00 5.000 IU / ml Nübling et al. (2009) Transfusion 49, 1850-58

HCV genotypes in NAT Yield Cases 26 yield cases with HCV genotype data HCV gt 1 50% HCV gt 3 38% HCV gt 2 12%

Transfusion-associated HCV transmissions Cases reported to PEI 18 16 14 Introduction of NAT HCV Transmissions 12 10 8 6 4 2 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 Year missed by AmpliScreen HCV / 24 2000 2001 2002 2003 2004 2005 2006 2007

HCV transmission 01-2005 repeat donor: seroconversion (antihcv, HCV- HCV genotype 2b look-back 10-2004 previous donation antihcv (Ortho 3.0) neg HCV-RNA (AmpliScreen/24) neg recipient (erythrocytes) HCV-pos HCV genotype 2b plasma still available replicate testing (n=5) in all available NAT systems

HCV transmission HCV window phase, genotype 2b 95% LOD (WHO IS, gt1) pos results (ID) Cobas Amplicor 43 IU/ml 1 / 5 Cobas AmpliScreen HCV Test v 2.0 29 IU/ml 1 / 5 HPS Cobas TaqMan HCV Test 15 IU/ml 3 / 5 CAP Cobas Amplicor HCV Test v 2.0 11 IU/ml 3 / 5 CAP Cobas TaqMan HCV Test 13 IU/ml 3 / 5 CAP Cobas TaqScreen 11 IU/ml 5 / 5 Procleix HIV-1/HCV Assay 2 IU/ml 0 / 5 Procleix Ultrio Assay 3 IU/ml 2 / 5 Versant HCV RNA Qual 10 IU/ml 0 / 5 Abbott RealTime HCV 11 IU/ml 4 / 5 Kretzschmar et al. (2007) Vox Sanguinis 92, 297-301

Viremia during the "diagnostic window" HIV-1 HIV-RNA (copies / ml) 1,0E+10 1,0E+09 1,0E+08 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 1,0E+01 1,0E+00 HIV1-RNA doubling time 21,5 h p24-positive p24-negative 0 3 6 9 12 15 18 21 24 27 30 33 36 Days after infection 10.000 IU / ml HIV-infection antihiv-negative phase antihiv pos

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) 16 14 12 10 8 6 4 2 1 4 1 3 2 4 2 0 1999 2000 2001 2002 2003 2004 2005 2006 2007 Nübling et al. (2009) Transfusion 49, 1850-58

11 yield cases with viral load data 10 > 10 000 IU/ml 1,00E+08 1,00E+07 1,00E+06 Viral Load (IU/mL) 1,00E+05 1,00E+04 1,00E+03 1,00E+02 10,000 IU/ml 1,00E+01 1,00E+00 Nübling et al. (2009) Transfusion 49, 1850-58

Transfusion-associated HIV transmissions Cases reported to PEI 16 14 12 10 8 6 4 2 0 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Introduction of HIV NAT

HIV transmission 04-2007 repeat donor: seroconversion (antihiv1/2) HIV1 subtype B look-back 01-2007 previous donation antihiv1/2 neg HIV1- neg recipient (erythrocytes) HIV1-seroconversion HIV1 subtype B, same variant as in donor -up sample 147 IU/ml ID CAP CTM HIV Ag/Ab test negative (Abbott)

01-2007 back-up sample HIV transmission 147 IU/ml ID CAP CTM HIV HIV Ag/Ab test negative (Abbott) recovered plasma into manufacturing pool: HIV1-RNA neg 04-2007 repeat donor: seroconversion (antihiv1/2) quantitative HIV-NATs screening HIV-NATs CAP CTM HIV (IU/ml) HPS CTM HIV (IU/ml) CAM HIV (IU/ml) Abbott RealTime HIV-1 (IU/ml) Procleix Ultrio Cobas AmpliScreen HIV (MP) Cobas TaqScreen neg neg <LLQ 924 pos () pos (2/2) pos

HIV-1 subtype B Transmission ) 100 fold underdetection of virus variant 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 24 Schmidt et al. (2009) Transfusion 49, 1836-44

HBV : early infection phase IU/ml 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 DNA t x2 =48 h HBsAg Pool NAT ID NAT antihbc 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 weeks

HBV : early infection + low level carrrier phase IU/ml 10 9 introduction of antihbc (Oct 1, 2006) 2% antihbc pos 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 DNA HBsAg antihbc 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 weeks years

HBV-DNA detection in antihbc (+) HBV-DNA (+) Donor # 1 2 3 4 5 6 7 8 9 10 % DNApos 95% LOD Screening HBV-NATs CAP / Cobas TaqScreen 3/5 62% 4 IU/ml (30) AmpliScreen HBV (MP) 3/5 3/5 44% 5 IU/ml (22/50) Procleix Ultrio 3/5 3/5 26% 7 IU/ml (13/50) Procleix Ultrio Plus 2/5 2/5 2/5 52% 1,9 IU/ml (26/50)

Screening HBV-NATs HBV-DNA detection in antihbc (+) HBV-DNA (+) Donor # 1 2 3 4 5 6 7 8 9 10 % DNApos 95% LOD CAP / Cobas TaqScreen 3/5 62% 4 IU/ml (30) AmpliScreen HBV (MP) 3/5 3/5 44% (22/50) 5 IU/ml Procleix Ultrio Assay 3/5 3/5 26% 7 IU/ml (13/50) Procleix Ultrio Plus 2/5 2/5 2/5 52% 1,9 IU/ml (26/50) Diagnostic qt NATs artus HBV TaqMan PCR 2/5 3/5 42% (20) 4 IU/ml HPS / Cobas TaqMan 3/5 64% 5 IU/ml (32/50) Abbott RealTime 3/5 66% 6 IU/ml (33/50) artus HBV LC PCR 3/5 3/5 50% (20) 6 IU/ml CAP / Cobas TaqMan 2/5 2/5 3/5 42% 12 IU/ml (20)

Zusammenfassung S NAT: Hohe Virussicherheit bei Plasma- und Blutprodukten S ären Blutkomponenten unvermeidlich low level viremia mismatch S Weiterer Handlungsbedarf bei HBV?

VIELEN DANK an... S S Dr Evelyne Kretzschmar, Duisburg S PD Dr Michael Schmidt, Frankfurt FG 2/4 Molekulare Virologie S Ines Amberg S Dr Michael Chudy S Christine Hanker-Dusel S Claudia König S Magdalene Köstermenke S Dr Julia Kress S Christine Pfannkuch