HEV Assay Development Update

Transcription

1 HEV Assay Development Update Jeffrey M. Linnen, Ph.D. Senior Director, Research & Development Hologic Gen-Probe, San Diego, California USA IPFA/PEI 20th International Workshop on Surveillance and Screening of Blood Borne Pathogens - Helsinki, Finland 24 April 2013

2 Disclaimers The Procleix Panther System and Ultrio Elite Assay are CE-marked but are not available for commercial sale in the U.S. The performance characteristics of these products in development have not been evaluated by the FDA. The Procleix Dengue and HEV Assays are currently under development and are not available for commercial sale. The performance characteristics (under the IVD Directive 98/79/EC) have not been established. The Procleix Parvo/HAV Assay is an in-process test that is CE-marked and available for commercial sale in the U.S. The Master File is on file with the FDA. PROCLEIX and ULTRIO are trademarks of Novartis Vaccines and Diagnostics, Inc.; ULTRIO PLUS and ULTRIO ELITE are trademarks of Novartis AG. PANTHER and TIGRIS are trademarks of Gen-Probe Incorporated. All other trademarks are the property of their respective owners. 2

3 Research & Development Activities Update Procleix Ultrio Elite Assay on the Panther System CE marked in July 2012 TMA Assay for screening for HIV-1, HIV-2, HCV and HBV Prospective testing using the Procleix Dengue Virus Assay initiated in the US in August 2012 under an investigative protocol (IND application) Two US sites: Charlotte, NC (American Red Cross) and Tampa, FL (Creative Testing Solutions) testing donations from Puerto Rico Procleix WNV Assay on the Panther System CE marked in March 2013 Feasibility of using the Procleix Parvo/HAV Assay on the Panther System completed Development of the Procleix HEV Assay on the Panther System is underway 3

4 Procleix HEV Assay on the Panther System Under Development Qualitative Transcription Mediated Amplification (TMA) assay to detect HEV RNA on the fully automated Procleix Panther System Design will be also compatible with the TIGRIS System Assay design is anticipated to allow detection of all clinically relevant genetic variants of HEV: Genotypes 1 and 2 (restricted to humans) Genotypes 3 and 4 (known to infect humans, pigs, and other species) Assay design allows detection of sequences that are provisionally designated as belonging to genotype 6 Preliminary assay performance data: Analytical sensitivity, genotype detection, specificity 4

5 Preliminary HEV Analytical Sensitivity Comparison of WHO Standard to in-vitro synthesized transcript (IVT) 5 Detection Probability by Probit Analysis HEV WHO Standard, IU/mL (95% Fiducial Limits) ~ 1.6 copies of transcript per 1 IU of WHO Standard (virus) IVT, Copies/mL (95% Fiducial Limits) 95% 8.4 ( ) 13.7 ( ) 50% 1.7 ( ) 2.5 ( ) Data on File, Hologic Gen-Probe, Feb

6 Preliminary HEV Analytical Sensitivity Comparison of Procleix Panther and TIGRIS Systems Panther: n = TIGRIS: n = 68 Error bars = 95% Confidence Interval HEV WHO Standard, IU/mL (PEI code 6329/10) Detection Probability by Probit Analysis Panther System TIGRIS System (95% Fiducial Limits) (95% Fiducial Limits) 95% 12.3 ( ) 12.6 ( ) 50% 2.4 ( ) 2.5 ( ) p-value Very similar performance on the two platforms Data on File, Hologic Gen-Probe, May

7 Confirmation of Analytical Sensitivity Results Positive Plasma Donor Specimens from Sanquin (The Netherlands) Detection Probability by Probit Analysis c Sanquin Specimen 1 a (95% Fiducial Limits) Sanquin Specimen 2 b (95% Fiducial Limits) 95% 6.1 ( ) 12.3 ( ) 50% 2.2 ( ) 2.8 ( ) a Original viral load is 255,000 IU/mL using Sanquin HEV RT-PCR assay b Original viral load is 270,000 IU/mL using Sanquin HEV RT-PCR assay c SAS 9.2 Probit analysis using normal model Data on File, Hologic Gen-Probe, Mar

8 Preliminary HEV Analytical Sensitivity Comparison of HEV genotype in vitro synthesized transcripts (IVT) Copies/mL (n = 81) Percent Reactivity of Genotype 1 IVTs 1 2 3a 3b 3f 4c Detection Probability by Probit Analysis 2 95% LOD (95% fiducial limits) 50% LOD (95% fiducial limits) 12.7 ( ) 2.8 ( ) 13.0 ( ) 2.4 ( ) Limit of Detection in Copies/mL 13.7 ( ) 2.5 ( ) 14.6 ( ) 2.9 ( ) 10.2 ( ) 2.2 ( ) 1 Based on GenBank accession numbers NC (1), M74506 (2), AB (3a), AB (3b), FJ (3f), AB (4c) 2 Using SAS 9.2 PROBIT normal model 18.9 ( ) 2.9 ( ) Data on File, Hologic Gen-Probe, Mar

9 Preliminary HEV Assay Specificity Results from Testing Source Plasma and Volunteer Donors 2,000 unlinked, frozen normal plasma specimens collected in the US 418 plasma samples from Source Plasma 1582 plasma samples from volunteer whole blood donors 3 Procleix Panther instruments used in this study n % Specimens Tested 2, Reactive Repeatedly Reactive Initial Specificity (95% CI) ( ) Final Specificity (95% CI) 100 ( ) CI: confidence interval using SCORE method Reactive Specimen ID Initial S/CO Repeat S/CO Data on File, Hologic Gen-Probe, May 2012 R

10 Confirmation of HEV TMA Repeatedly Reactive Donation PCR and Sequencing TMA Repeatedly reactive specimen (ID: R149449) was found to be non-reactive for both HEV IgG and IgM (Focus Diagnostics, Cypress, CA)* Positive by in-house PCR assay; yielded a unique sequence matching most closely to swine HEV Presence of swine HEV sequence suggests a zoonotic infection in the donor Based on this result, preliminary assay specificity was 100% (n = 2000); prevalence of HEV RNA was 1/2000 (or 0.05%) Summary of Testing 150 bp 100 bp M = 50-bp DNA ladder Lane 1 = HEV WHO Standard Lane 2 = HEV RR (R149449) Lane 3 = Neg Control Specimen ID TMA S/CO PCR Genotype IgG IgM R (initial), 4.5 (repeat) Positive 3a Non-Reactive Non-Reactive *Focus Diagnostics HEV IgG and IgM tests have not been cleared or approved by the FDA Data on File, Hologic Gen-Probe, May

11 Confirmation of HEV TMA Repeatedly Reactive Donation Phylogenetic Analysis 3f Based on ORF2 region sequence Software: Geneious Pro Tree building method: Neighbor Joining Substitution Model: Tamara-Nei 3e 3a 3b 11 11

12 Ongoing or Planned Studies HEV RNA Prevalence in Various Geographic Regions HEV RNA Prevalence in US Blood Donors (in collaboration with Susan Stramer, ARC) Minimum of 20,000 donations from the ARC will be tested as individual donations on the Panther System in the Gen-Probe R&D Laboratory in San Diego; matched specimens (serum, plasma) of any reactive donations will be available for additional testing (testing started April 2013) Supplemental testing will include alternative NAT (for confirmation) and sequencing (for determining genotype) US Geographic Location Target Matched Sets Northeast 3,000 Southeast 3,000 South Central 3,000 Pacific Northwest 3,000 Midwest (3 regions) 8,000 Total 20,000 Additional studies are planned for testing donations from The Netherlands and Spain 12

13 Summary Procleix HEV Assay Under Development Preliminary results indicated that the HEV TMA Assay under development is sensitive and specific All 4 HEV genotypes were detected with 95% detection levels between 10 to 20 copies/ml Assay design is compatible for use on both the TIGRIS and Panther Systems Study to investigate RNA prevalence in US donors is underway; world-wide studies are planned 13

14 Thank You!