1. ICBS Master Panels for Kit Evaluation: HCV, HBV, and HIV (WWW.ICBS-WEB.ORG) Howard A. Fields, Ph.D. Susan Diaz, MPH Division of Viral Hepatitis Centers for Disease Control and Prevention

2. Objective of Master Panels  Identify, validate, and make available high- quality affordable tests for HBV, HCV, and HIV because cost is one of the major impediments to universal screening.  Use as a model for BTEP project to produce Master Panels with Dr. Alexander Kanev, Vector currently in year 3.

3. HCV Master Panels positive and negative

4. HCV Master Panel: Diversity Country of origin Country of origin Genotype Genotype Subtype Subtype RIBA 3.0 banding patterns RIBA 3.0 banding patterns

5. Location of International HCV Collaborators N=14 for HCV some collaborators different for HBV and HIV *

6. Methods to Characterize Panel Members Screening Tests Screening Tests  Anti-HCV by Ortho 3.0; positives repeated in duplicate Confirmatory Tests Confirmatory Tests  RIBA 3.0 for HCV by Chiron Quantitative PCR Quantitative PCR  Roche Amplicor for HCV RNA Genotyping / Subtyping by direct DNA Sequencing Genotyping / Subtyping by direct DNA Sequencing  CDC: ABI Prism 3100 Capillary Sequencer (NS5b default to 5’-UTR); phylogenetic analysis  Visible Genetics, Inc. Clip Sequencing Reaction for HCV (5’UTR/NS5b) percent homology

7. Collections HCV: 1007 HCV: 1007 HBV: 450 HBV: 450 HIV: 200 HIV: 200 COINFECTION: 62 COINFECTION: 62 NEGATIVES: 450 NEGATIVES: 450  Total Units Received: 2169

8. HCV Positive Units Description of Selectable Units HCV Positive Units Description of Selectable Units Serologically confirmed as positive (exclude indeterminate specimens) Serologically confirmed as positive (exclude indeterminate specimens) Concordant genotype by direct DNA sequencing from two laboratories Concordant genotype by direct DNA sequencing from two laboratories Minimum volume 100 ml Minimum volume 100 ml Additional selection criteria based upon diversity Additional selection criteria based upon diversity  By geographic regions  By genotype/subtype  By RIBA banding patterns

9. Diversity by Genotype among Selected Units Among 1007 HCV IR units received and characterized, 539 units met the definition of selectable (54%) Among 1007 HCV IR units received and characterized, 539 units met the definition of selectable (54%)  Genotype 1 (n=296)  Genotype 2 (n=26)  Genotype 3 (n=42)  Genotype 4 (n=129)  Genotype 5 (n=3)  Genotype 6 (n=43)

10. 66233633240 Diversity by subtype RIBA 3.0 banding patterns Diversity by Country and Genotype among Official HCV Panel Members

11. geographic diversity Negative Panel geographic diversity Manufacturing completed June 18, 2002 Manufacturing completed June 18, 2002 First panel to be completed First panel to be completed 200 Members from both the developed and developing world 200 Members from both the developed and developing world  181 domestic members utilized from ARC  19 members pooled by region: Egypt, Indonesia, and Ivory Coast

12. Origin of Kits Sent to PEI China 13 Russia9 India7 South Korea5 EU4 Costa Rica1 Indonesia1 Uzbekistan1 Brazil1 Argentina1 USA (not FDA approved) 1 Total = 44

13. Russian Kits Sent to PEI for Evaluation Biotechnologitzeskaja Kompanija Bioservis (No. 23, Hepascreen) IMBIO (No. 24, Anti-HCV ELISA) Medizinische Biologische Union (No.27, HCV-DCM) Union–Diagnostisches-System (No.25, Anti-HCV ELISA) Vector-Best (No. 29, RecombiBest anti-HCV-Strip, OPD) Received, but Not Evaluated as of August 23, 2004  Institut Pastera Sankt-Peterburg (Anti-HCV ELISA-G)  NPP Akwapast (Immunoenzyme Test-System for Detection of Anti-HCV)  Radiopreparat (Anti-HCV)  Vector-Best (RocombiBest anti-HCV-Strip, TMB)

14. Specimens ICBS HCV Master Panel (n=200) ICBS negative Master Panel (n=181 and 200) Seroconversion Panel (commercial)  core only, genotype 2b  NS3 only  Core & NS3, genotype 1a  Mean day delay

15. * Significant reduction in specificity (n = 181 vs. 200) * * * * * ^^ ^ ^ ^ ^ Russian kits

16. * * * * * * Significant reduction in specificity (n = 181 vs. 200) ^ ^ ^ ^ ^ ^ Russian kits

17. PI of Russian Kits Evaluated Union–Diagnostisches-System 235.28 232.83 Biotech Kompanija Bioservis 235.00 231.00 IMBIO 232.83 232.33 Vector-Best 231.78 229.83 Medizinische Biolog Union 231.28 229.30 PI N=181 N=200 * * * Statistical significant reduction in specificity (n = 181 vs. 200)

18. Conclusions (1) ICBS HCV Negative Master Panel manufactured June, 2002.   N = 181 US specimens   N = 19 specimens from the developing world yielded weakly screening test positive when tested with some assays licensed in the EU. Some specimens also tested positive/indeterminate with some of the EU license supplemental tests.   N = 200 specimens confirmed as true negative at CDC (Ortho 3.0, Chiron RIBA 3.0, and Roche HCV Amplicor)

19. Conclusions (2) ICBS HCV Positive Master Panel manufactured September, 2002   Diversity based on geographic origin, genotype, subtype (concordance by DNA sequencing), and RIBA 3.0 banding patterns   All confirmed as true positive by screening (Ortho 3.0), supplemental testing (RIBA 3.0), and positive PCR (Roche HCV Amplicor)

20. Conclusion (3) Russian kits evaluated by PEI 2003 – 2004   PI less than kits licensed in the US and EU and several other kits manufactured in resource-challenged regions of the world  probably equal to the kit manufactured by  Highest PI manufactured by Union–Diagnostisches-System probably equal to the kit manufactured by Biotech Kompanija Bioservis based on 181 US specimens  Union–Diagnostisches-System Biotechnologitzeskaja Kompanija Bioservis  When 19 specimens from the developing world was added (n=200), the specificity of all 5 Russian kits decreased; however, only kits Union–Diagnostisches-System and Biotechnologitzeskaja Kompanija Bioservis decreased to a statistical significant level.  continued to have the highest PI value among the Russian kits evaluated.  Despite this decrease in specificity, the kit manufactured by Union–Diagnostisches-System continued to have the highest PI value among the Russian kits evaluated.