HBV Genotype Panel AREVIR-GenaFor-Meeting Bonn, 23. – 24. April 2009 Michael Chudy Section of Molecular Virolgy Division of Virology E-Mail: chumi@pei.de 1
Background � During the ‘WHO Consultation on Global Measurement Standards and their use in the in vitro Biological Diagnostic Field’ in June 2004 concern was raised that HBsAg and HBV NAT test kits might be less sensitive for some HBV genotypes other than A2, which is represented by the in the WHO reference preparation (2 nd International Standard) � During the ECBS meeting in October 2005 the PEI proposed a project to establish WHO International Reference Panels representing different HBV genotypes/HBsAg subtypes � This project was assigned as high priority by ECBS 2
HBV genotype reference panels � HBV genotype panel (NAT tests) � HBV genotype panel (HBsAg tests) 3
HBV - genotypes/subtypes, frequency and geographical distribution Genotype Subgenotype HBsAg subtype Frequency Main Geographical Distribution A A1 high Africa adw2, ayw1 A2 adw2 high Europe, North America, Australia A3 Cameroon, Democratic Republic of Congo, Gabon B B1 adw2 high Far East (Japan, China, Taiwan) B2 high/low Far East (China, Japan, Viet Nam/Thailand) adw2/adw3, B3 adw2 high Far East (Indonesia, Sumatra, Sulawesi) B4 ayw1 high Viet Nam C C1 adr/ayr/adw2 high/high/low Far East (Japan, China) C2 high/high Thailand, China/Viet Nam adr/ayr; ad C3 adrq-/adw2 high/low Pacific (New Zealand to Polynesia), Micronesia/Far East C4 ayw3 low Northeast Australia D D1 ayw2 high Mediterranean, Middle East, India D2 high/low worldwide/USA ayw3/ayw4 D3 ayw2/ayw3 high/high South Africa, Alaska/Europe, Costa Rica D4 ayw2, high Oceania, Somalia, not identified adw3 low Eastern Europe Spain E - high Africa ayw4 F F1 adw4q-/ayw4 high/low Cental America, Argentina, Spain, Alaska/Nicaragua F2/F3/F4… adw4q-/ayw4 high/low South America, Polynesia, France/Venezuela G - adw2 low USA, Mexico, Europe H - low Central America (Nicaragua, Mexico), California adw4 Recombinant Strains A/D adw2 India C/D ayw2 Tibet 4 C/? Viet Nam adw2
HBV genotype reference panels � Efforts to collect plasma units worldwide - HBV DNA/HBsAg high titre plasma samples with sufficient volume - NAT testing, HBsAg-testing, and HBV genotyping of about 215 potential candidate members � HBV genotypes A – H - One genotype H sample received recently - This sample could not be considered for the NAT panel � HBV genotype panel (NAT tests): 15 panel members � HBV genotype panel (HBsAg tests): 15 panel members � 12 samples are member in both panels � Project in close cooperation with Prof Gerlich (Institute of Virology, University Giessen) 5
HBV genotype panel (NAT tests) � Final characterization of 15 panel members - Sequencing of entire S ORF - Genosubtype, HBsAg subtype, escape mutations � Characterization of negative plasma � Dilution � Filling and lyophilisation � Collaborative study � Report to ECBS in 2009 6
HBV Genotype Panel (NAT Tests) Characterization of the panel members HBV HBV HIV1/HCV HBV DNA HBsAg anti-HBc HBeAg anti-HBe Sample HBsAg Genotype Genotype RNA Origin No Subtype INNO-LiPA Sequencing (IU/mL) ARCHITECT* ARCHITECT Elecsys ARCHITECT Procleix 1 South Africa A A1 6,08E+08 131,9 pos pos neg neg adw2 2 Brazil adw2 A A1 6,53E+08 94,0 pos pos neg neg 3 Germany adw2 A A2 6,87E+08 74,3 pos pos neg neg 4 Japan adw2 B B1 1,48E+08 51,4 pos pos neg neg 5 Japan B B2 2,84E+08 95,3 pos pos neg neg adw2 6 Viet Nam ayw1 B B4 6,29E+06 4,6 pos pos neg neg 7 Japan adr C C2_Ce 3,99E+08 70,2 pos pos neg neg 8 Japan adr C C2_Ce 1,25E+08 47,0 neg pos neg neg 9 Russia C C2_Ce 2,92E+08 54,4 neg pos neg neg adr 10 Germany ayw2 D D1 1,17E+09 130,4 pos pos neg neg 11 South Africa ayw2 D D3 1,04E+08 63,8 pos pos neg neg 12 Iran ayw2 D D1 1,00E+08 17,7 pos pos neg neg 13 West Africa E E 9,45E+08 82,6 pos pos neg neg ayw4 14 Brazil adw4 F F3 1,10E+07 32,2 pos neg neg neg 15 Germany adw2 G G 1,40E+07 0,9 pos neg neg neg 7
HBV Genotype Panel (NAT Tests) Characterization of the panel members HBV HBV HIV1/HCV HBV DNA HBsAg anti-HBc HBeAg anti-HBe Sample HBsAg Genotype Genotype RNA (IU/mL) (KIU/mL) Origin No Subtype INNO-LiPA Sequencing qNATs ARCHITECT ARCHITECT Elecsys ARCHITECT Procleix 1 South Africa A A1 6,08E+08 131,9 pos pos neg neg adw2 2 Brazil adw2 A A1 6,53E+08 94,0 pos pos neg neg 3 Germany adw2 A A2 6,87E+08 74,3 pos pos neg neg 4 Japan adw2 B B1 1,48E+08 51,4 pos pos neg neg 5 Japan B B2 2,84E+08 95,3 pos pos neg neg adw2 6 Viet Nam ayw1 B B4 6,29E+06 4,6 pos pos neg neg 7 Japan adr C C2_Ce 3,99E+08 70,2 pos pos neg neg 8 Japan adr C C2_Ce 1,25E+08 47,0 pos pos neg neg 9 Russia C C2_Ce 2,92E+08 54,4 pos pos neg neg adr 10 Germany ayw2 D D1 1,17E+09 130,4 pos pos neg neg 11 South Africa ayw2 D D3 1,04E+08 63,8 pos pos neg neg 12 Iran ayw2 D D1 1,00E+08 17,7 pos pos neg neg 13 West Africa E E 9,45E+08 82,6 pos pos neg neg ayw4 14 Brazil adw4 F F3 1,10E+07 32,2 pos neg pos neg 15 Germany adw2 G G 1,40E+07 0,9 pos neg neg neg 8
HBV Genotype Panel (NAT Tests) Characterization of the panel members - Genotyping INNO-LiPA HBV Genotyping Assay 9
HBV Genotype Panel (NAT Tests) Characterization of the panel members – qNAT testing 1,000E+10 1,000E+09 HBV DNA (IU/mL CAP/CTM Abbott RealTime 1,000E+08 artus HBV LC PCR Kit CA HBV Monitor Mean 1,000E+07 1,000E+06 1/A1 2/A1 3/A2 4/B1 5/B2 6/B4 7/C2 8/C2 9/C2 10/D1 11/D3 12/D1 13/E 14/F3 15/G HBV Genotype Panel - Candidate Panel Members 10
HBV genotype panel (NAT tests) – Dilution of panel members � Dilution matrix for panel members: Negative plasma pool - Testing of 117 negative pre-screened plasma units (CSL Behring) • HBV serology: anti-HBs; anti-HBc • HIV/HCV NAT: cobas Taqscreen MPX Test; Procleix Ultrio Assay - Pooling of negative plasma units � If possible dilution to a HBV DNA concentration of about 10 6 IU/ml � Dilution to a volume of 1.2 litre per sample 11
HBV genotype panel (NAT tests) – Filling and lyophilisation � Certified company in Switzerland � 4-ml screw-cap glass vials with rubber stopper � Filling volume 0.5 ml per sample � 15 x 2,000 vials � Add 144 vials filled with 0.5 ml of negative plasma pool (for determination of residual moisture content) � Instrument CHRIST Epsilon 2-25 D (LPC-16/NT process documentation) � Five lyo batches, no mixing of genotypes per tray Characterization of the final product: � Pre-study (PEI): Control of HBV DNA concentration before and after lyo; no significant loss of HBV DNA concentration � Collaborative study � Programme of stability testing � Residual moisture content: 0.82%; SD ± 0.03% (method acc. EP) 12
HBV genotype panel (NAT tests) – Collaborative study � Study purpose: evaluation of the HBV genotype panel using different NAT assays; parallel testing of the 2 nd HBV DNA IS (97/750) � Invited 23 labs � Reply from 19 labs � 20 participants (incl. PEI) � 5 NCLs: NIBSC, CBER, ISS, PEI, NIID Japan � 6 special labs (special diagnostic expertise in viral hepatology): Brazil, Argentina, South Africa, Spain, USA, Germany � 9 kit manufacturers: Korea, Taiwan, USA (4), Sweden, Switzerland, Germany � HBV NAT quant: 15 labs (18 tests) � HBV NAT qual: 5 labs � HBV NAT sequencing whole genome (data to GenBank) and genotyping: 1 lab � Report to ECBS in 2009 13
HBV Mutation Assays 14
HBV Mutation Assays - G1896A (Pre-C) Detection with Real-Time PCR and Melting Curve Analysis (LightCycler) 3‘ 5‘ Forward Primer WILD TYPE Anchor Probe Sensor Probe Reverse Primer 3‘ 5‘ Red 640 Fluorescein Mutation in the sensor sequence (preferential on the 3’) 15
HBV Pre-C 1896: Mixture of Wild-Type and Mutation Samples 1 3 2 4 5 NC sample Wild-Type (N2810) 4*10 3 copies/reaction 1 Mutation (N2875) 7*10 3 copies/reaction 2 3 Wild-Type/Mutation 75:25 4 Wild-Type/Mutation 50:50 5 Wild-Type/Mutation 25:75 16
HBV Pre-C 1896: Analysis of Clinical HBV Samples Mutation Wild-Type N2810 HBV plasmid N2811 N3595 N3596 N3761 N2875 N2876 NC - N2876: mixture of mutation and wild-type - N3761, N2811: show a hint for an additional mutation 17
Acknowledgements - Prof Wolfram Gerlich, Institute of Virology, University Giessen - Dr Michael Schmidt, German Red Cross Frankfurt/Main Dr Christoph Jochum, Biotest, Dreieich - - Dr Eugene Zhiburt, Federal Blood Center, Moscow, Russia - Prof Majid Cheraghali, Iranian Blood Transfusion Organization, Tehran, Iran - Prof Hiroshi Yoshizawa, Hiroshima University, Japan David J. Watts, South African National Blood Service - - Dr Márcia Otani, Fundação Pró-Sangue Homocentro de São Paulo, Brazil - Dr Ana Padilla, WHO, Geneva People from PEI - - Section of Molecular Virology and IVD-Section - Administrative staff 18
19
Recommend
More recommend
