Ergebnisse der Abfrage zu NAT-only positiven Befunden beim Blutspendescreening (NAT yield) Micha Nübling, PEI
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1 Ergebnisse der Abfrage zu NAT-only positiven Befunden beim Blutspendescreening (NAT yield) Micha Nübling, PEI
2 NAT for cellular blood components in Germany Definition of minimal NAT sensitivity limit (ID) HCV-NAT IU / ml ( = cps / ml) HIV1-NAT IU / ml ( = cps / ml)
3 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
4 Procleix with the Ultrio
5 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
6 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
7 NAT for cellular blood components in Germany Mandatory NATs for all blood / plasma donations Definition of minimal NAT sensitivity limit (ID) HCV-NAT IU / ml ( = cps / ml) HIV1-NAT IU / ml ( = cps / ml) NAT yield versus breakthrough transmissions??
8
9 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 IU / ml 1,0E Days after infection HCV-infection antihcv pos antihcv negative phase
10 NAT onlies: HCV 92 HCV-NAT onlies = 1 / donations -2007) Nübling et al. (2009) Transfusion 49,
11 92 HCV-NAT onlies = 1 / donations NAT onlies incidence centers #47 6 / donations = 1 / #15 16 / donations = 1 / #08 9 / donations = 1 / Lower incidence centers #30 4 / donations = 1 / #52 6 / donations = 1 / #23 0 / donations = 0 /
12 Viral Loads in HCV NAT Yield Cases 40 yield cases with viral load data 39 (99%) >> 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 IU / ml Nübling et al. (2009) Transfusion 49,
13 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%
14 Transfusion-associated HCV transmissions Cases reported to PEI Introduction of NAT HCV Transmissions Year missed by AmpliScreen HCV /
15 HCV transmission repeat donor: seroconversion (antihcv, HCV- HCV genotype 2b look-back 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
16 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 IU/ml 1 / 5 HPS Cobas TaqMan HCV Test 15 IU/ml 3 / 5 CAP Cobas Amplicor HCV Test v 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,
17
18 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 Days after infection IU / ml HIV-infection antihiv-negative phase antihiv pos
19 NAT onlies: HIV 11 HIV-NAT onlies among 19 million donations ( ) = 1 / donations (17 HIV-NAT onlies from 1999 to 2007 = 1 / donations) Nübling et al. (2009) Transfusion 49,
20 11 yield cases with viral load data 10 > 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,
21 Transfusion-associated HIV transmissions Cases reported to PEI Introduction of HIV NAT
22 HIV transmission repeat donor: seroconversion (antihiv1/2) HIV1 subtype B look-back 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)
23 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 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
24 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,
25
26 HBV : early infection phase IU/ml DNA t x2 =48 h HBsAg Pool NAT ID NAT antihbc weeks
27
28 HBV : early infection + low level carrrier phase IU/ml 10 9 introduction of antihbc (Oct 1, 2006) 2% antihbc pos DNA HBsAg antihbc weeks years
29 HBV-DNA detection in antihbc (+) HBV-DNA (+) Donor # % 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)
30 Screening HBV-NATs HBV-DNA detection in antihbc (+) HBV-DNA (+) Donor # % 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)
31 Zusammenfassung S NAT: Hohe Virussicherheit bei Plasma- und Blutprodukten S ären Blutkomponenten unvermeidlich low level viremia mismatch S Weiterer Handlungsbedarf bei HBV?
32 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
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