1 HBV Genotype Panel Michael Chudy Section of Molecular Virolgy Division of Virology SoGAT XXI Meeting Brussels, 28 - 29 May 2009.

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Presentation transcript:

1 HBV Genotype Panel Michael Chudy Section of Molecular Virolgy Division of Virology SoGAT XXI Meeting Brussels, May 2009

2 GenotypeSubgenotypeHBsAg subtypeFrequencyMain Geographical Distribution AA1 adw2, ayw1 highAfrica A2 adw2 highEurope, North America, Australia A3 Cameroon, Democratic Republic of Congo, Gabon BB1 adw2 highFar East (Japan, China, Taiwan) B2 adw2/adw3, high/lowFar East (China, Japan, Viet Nam/Thailand) B3 adw2 highFar East (Indonesia, Sumatra, Sulawesi) B4 ayw1 highViet Nam CC1 adr/ayr/adw2 high/high/lowFar East (Japan, China) C2 adr/ayr; ad high/highThailand, China/Viet Nam C3 adrq-/adw2 high/lowPacific (New Zealand to Polynesia), Micronesia/Far East C4 ayw3 lowNortheast Australia DD1 ayw2 highMediterranean, Middle East, India D2 ayw3/ayw4 high/lowworldwide/USA D3 ayw2/ayw3 high/highSouth Africa, Alaska/Europe, Costa Rica D4 ayw2, highOceania, Somalia, not identified adw3 lowEastern Europe Spain E - ayw4 highAfrica FF1 adw4q-/ayw4 high/lowCental America, Argentina, Spain, Alaska/Nicaragua F2/F3/F4… adw4q-/ayw4 high/lowSouth America, Polynesia, France/Venezuela G - adw2 lowUSA, Mexico, Europe H - adw4 lowCentral America (Nicaragua, Mexico), California Recombinant Strains A/D adw2 India C/D ayw2 Tibet C/? adw2 Viet Nam HBV - genotypes/subtypes, frequency and geographical distribution

3 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 in the current WHO International Standard preparations  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 Expert Committee on Biological Standardization

4 International reference panels for HBV genotypes  HBV genotype panel (NAT tests)  HBV genotype panel (HBsAg tests)

5 HBV genotype panels  Efforts to collect plasma units worldwide - HBV DNA/HBsAg high titre plasma samples with sufficient volume  Characterization of 215 potential candidate materials - quant HBV DNA, quant HBsAg - Sequencing of entire S ORF, - genotyping, escape mutations, HBsAg subtyping  HBV genotypes A – G (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): 16 panel members  12 samples are member in both panels  Project in close cooperation with Prof Gerlich (Institute of Virology, University Giessen)

6 HBV genotype panel (NAT tests) Final characterization of the panel members Sample No Origin HBsAg Subtype HBV Genotype HBV Genotype HBV DNAHBsAganti-HBcHBeAganti-HBe HIV1/HCV RNA INNO-LiPASequencing(IU/mL)ARCHITECT*ARCHITECTElecsysARCHITECTProcleix 1South Africaadw2AA16,08E+08131,9pos neg 2Braziladw2AA16,53E+0894,0pos neg 3Germanyadw2AA26,87E+0874,3pos neg 4Japanadw2BB11,48E+0851,4pos neg 5Japanadw2BB22,84E+0895,3pos neg 6Viet Namayw1BB46,29E+064,6pos neg 7JapanadrCC2_Ce3,99E+0870,2pos neg 8JapanadrCC2_Ce1,25E+0847,0negposneg 9RussiaadrCC2_Ce2,92E+0854,4negposneg 10Germanyayw2DD11,17E+09130,4pos neg 11South Africaayw2DD31,04E+0863,8pos neg 12Iranayw2DD11,00E+0817,7pos neg 13West Africaayw4EE9,45E+0882,6pos neg 14Braziladw4FF31,10E+0732,2posneg 15Germanyadw2GG1,40E+070,9posneg

7 HBV genotype panel (NAT tests) Final characterization of the panel members Sample No Origin HBsAg Subtype HBV Genotype HBV Genotype HBV DNA* (IU/mL) HBsAg (KIU/mL) anti-HBcHBeAganti-HBe HIV1/HCV RNA INNO-LiPASequencingqNATsARCHITECT ElecsysARCHITECTProcleix 1South Africaadw2AA16,08E+08131,9pos neg 2Braziladw2AA16,53E+0894,0pos neg 3Germanyadw2AA26,87E+0874,3pos neg 4Japanadw2BB11,48E+0851,4pos neg 5Japanadw2BB22,84E+0895,3pos neg 6Viet Namayw1BB46,29E+064,6pos neg 7JapanadrCC2_Ce3,99E+0870,2pos neg 8JapanadrCC2_Ce1,25E+0847,0pos neg 9RussiaadrCC2_Ce2,92E+0854,4pos neg 10Germanyayw2DD11,17E+09130,4pos neg 11South Africaayw2DD31,04E+0863,8pos neg 12Iranayw2DD11,00E+0817,7pos neg 13West Africaayw4EE9,45E+0882,6pos neg 14Braziladw4FF2 or F31,10E+0732,2posnegposneg 15Germanyadw2GG1,40E+070,9posneg *Concentration based on quantification by four different assays

8 HBV genotype panel (NAT tests) Dilution of panel members, filling and lyophilisation  Dilution matrix for panel members: Negative plasma pool - Testing of 117 negative pre-screened plasma units HBV serology: anti-HBs; anti-HBc HBV/HCVHIV 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 (12 / 15 samples)  Dilution to a volume of 1.2 litre per sample  15 x 2,000 vials  Filling volume 0.5 ml per sample  Filling and lyo by a certified Swiss company

9 HBV genotype panel (NAT tests) Characterization of the final product  Pre-study (PEI): Control of HBV DNA concentration before and after lyo - no loss of HBV DNA and HBsAg concentration  Stability testing programme  Residual moisture content: 0.82% (SD ± 0.03%) (method acc. EP)  Collaborative study

10 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 18 labs  19 participants (incl. PEI) - 5 NCLs: CBER, ISS, NIBSC, NIID Japan, PEI, - 6 special labs (special diagnostic expertise in viral hepatology): Argentina, Brazil, Germany, South Africa, Spain, USA - 8 kit manufacturers: Germany, Korea, Sweden, Switzerland, Taiwan, USA (3)  HBV NAT: quant 15 labs (17 tests), qual 3 labs  HBV DNA sequencing and genotyping: 1 lab  Statistical analysis  Draft report for circulating  Report to ECBS in (16), 2

11 HBV genotype panel (NAT tests) – collaborative study Preliminary results Arithmetic mean values from 3 independent runs (replicate testing) Mean 6.01 ± 0.17 log 10 IU/ml

12 HBV genotype panel (NAT tests) – collaborative study Preliminary results Arithmetic mean values from 3 independent runs (replicate testing)

13 Blast HBV genotyping tool – sequence comparison WHO IS (GenBank AJ012207) vs. reference sequences pre-S2 S C P X pre-S1 P

14 HBV genotype panel (HBsAg tests) Prof. Gerlich, Univ. Giessen  Cloning and sequencing of entire S ORF - Genosubtype, HBsAg subtype, escape mutations  Determination of HBs antigen concentration by - Laurell immune electrophoresis (PEI-units) - qCLIA (Architect, IU)  Determination of HBsAg protein by - UV photometry after purification (ng)  Removal of virions from the HBsAg subviral particles by ultracentrifugation over sucrose cushion (decrease of infectivity about 99%)  Determination of HBsAg content in the supernatant by qCLIA (IU/ml) PEI  Residual HBV-DNA by quant NATs  Dilution in negative plasma pool (HBsAg concentration about 30 IU/ml)  Pilot study to investigate the effects of lyophilisation on the consistency of HBsAg detection  Filling and lyophilisation  Collaborative study  Report for establishment to ECBS 2010     ()() 

15 International reference panels for HBV genotypes Conclusion  Two panels (NAT tests and HBsAg tests)  Well characterized panels - serological and molecular characterization, protein characterization - NAT:16 different assays (13 quant; 3 qual), overall good correlation in detecting most of the genotypes, no unitage of panel members - HBsAg:results from pilot studies provide hints for different detection efficiency  Intended use - Assay validation - Assay comparison - BV testing (NCLs, manufacturers)  Establishment by ECBS - NAT test panel probably HBsAg panel 2010  Global availability

16 Acknowledgements - Prof Gerlich, Institute of Virology, University Giessen - Prof Yoshizawa & Prof Tanaka, Hiroshima University, Japan - Biotest AG, Dreieich - Federal Blood Center, Moscow, Russia - Fundação Pró-Sangue Homocentro de São Paulo, Brazil - German Red Cross Frankfurt/Main - Iranian Blood Transfusion Organization, Tehran, Iran - South African National Blood Service - WHO Collaborative Study Group - Dr Ana Padilla, WHO, HSS / EMP / QSM, Geneva - NIBSC - People from PEI - Section of Molecular Virology and IVD-Section - Administrative staff

17 Thank you for your attention!