Assays to Address Emerging Threats to Blood Safety

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1 Assays to Address Emerging Threats to Blood Safety Jeffrey M. Linnen, Ph.D. Director, Product Development Gen-Probe Incorporated San Diego, CA The IPFA/PEI 17th Workshop on Surveillance and Screening of Blood Borne Pathogens 27 May 2010, Zagreb, Croatia 1

2 Blood Screening R&D Activities New platform development: PANTHER Instrument Totally integrated, fully automated NAT platform Parvovirus B19/HAV early stage development Xenotropic murine leukemia virus-related virus (XMRV) Research Alternative sample types for blood screening Arboviral blood safety research Studies planned to investigate dengue transfusion transmission in Brazil Prototype multiplexed arboviral assay (WNV, chikungunya, and dengue virus) 2

3 PANTHER Instrument* Totally integrated NAT platform: fully automated from sample to result Will extend advantages of TIGRIS platform s full automation to low- and midvolume laboratories PANTHER Instrument is intended to run key current Gen-Probe assays and future assays: Dual Format instrument Chemiluminescent detection assays (e.g. PROCLEIX ULTRIO Plus Assay) Quantitative and qualitative real time fluorescent detection assays (4 colors upgradeable to 6) New blood screening assay will be introduced on PANTHER Instrument *currently under development PROCLEIX and ULTRIO are trademarks of Novartis Vaccines and Diagnostic, Inc 3

4 Duplex Parvovirus B19/ Hepatitis A Virus Assay: Quantitative Parvovirus B19/Qualitative HAV Assay on TIGRIS System* Quantitative Parvovirus B19: End-point quantitation based on chemiluminescent signal ~2.4 log dynamic range (e.g ,000 IU/mL) Allows variable pool sizes: ~1:16-1:500 Equivalent quantitation of genotypes 1, 2, and 3 Qualitative HAV: Qualitative detection Design goal of 95% detection at copies/ml (as sensitive as possible) Internal Control added during specimen processing; control for specimen processing, amplification and detection Determines the validity of each individual reaction Initial result will allow discrimination between Parvovirus and HAV (no need for discriminatory assays) TIGRIS is a trademark of Gen-Probe Incorporated *Under development 4

5 Linearity of Quantitative TMA Assay Allows parvovirus B19 quantification over a range of ~2.5 log copies Suitable for screening pools from 16 to ~500 donations 5

6 Detection of Parvovirus B19 Genotypes GP Assay was re-designed after participation in WHO international genotype collaborative study in October 2008 GP in-house standards for parvovirus B19 genotypes 1, 2, and 3 were serially diluted and tested with the prototype assay 10 replicates at each copy level 50% detection (by probit analysis) for 3 genotypes ranged from 7-11 copies/ml 6

7 Qualitative HAV TMA Assay Previous prototype assay was redesigned due to new variant sequences identified Evaluation of analytical sensitivity of prototype assay Gen-Probe in vitro synthesized transcript (IVT) WHO International Standard for HAV RNA NAT assays, NIBSC Code 00/560 IVT Copies/ ml N # Reactive % Reactive WHO Standard IU/mL N # Reactive % Reactive % % % % % % % % % % % % % Target Detection Probability 50% (95% CI) 95% (95% CI) IVT (copies/ml) 2.67 ( ) ( ) HAV WHO Std (IU/mL) 0.88 ( ) 3.17 ( ) 7

8 Research TMA Assay for XMRV Xenotropic murine leukemia virus-related virus (XMRV): gammaretrovirus discovered in human prostate tumor tissue in 2006 Recent studies have produced contradictory evidence for association of XMRV with prostate cancer and/or chronic fatigue syndrome (CFS) Both disease association and transmission routes for XMRV are unclear Prototype Transcription-Mediated Amplification (TMA) assay for the detection of XMRV RNA and DNA Runs on the fully automated TIGRIS System (capable of generating 1000 test results in 14 hours) Assay may be useful for large scale studies to investigate XMRV transmission and disease association Initial evaluations with research assay: Analytical sensitivity (IVT and virus from tissue culture) Detected in normal blood donors? 8

9 Analytical Sensitivity of XMRV TMA Assay Evaluation of analytical sensitivity of prototype assay Gen-Probe in vitro synthesized transcript (IVT) Virus obtained from Graham Simmons and Mike Busch (BSRI, San Francisco, CA) IVT Copies/ ml N # Reactive % Reactive Virus Copies/mL* N # Reactive % Reactive % % % % % % % % % % % % % % *copies/ml based on BSRI estimate Target Detection Probability Copies/mL 50% (95% CI) 95% (95% CI) IVT 2.4 ( ) 17.4 ( ) Virus 0.6 ( ) 2.5 ( ) 9

10 XMRV in Plasma Samples from Normal Blood Donors* PPT tubes (plasma samples) from American Red Cross (Charlotte, NC) were screened on the TIGRIS System with the research XMRV Assay Testing at Gen-Probe (San Diego, CA) N Average Analyte S/ CO # Reactive % Reactive Specificity (95% CI) % 100% ( ) No evidence of XMRV nucleic acid in 1435 plasma samples screened from normal US blood donors *Work done in collaboration with Susan Stramer and Roger Dodd (American Red Cross) 10

11 Future XMRV Investigations Blood Safety/Chronic Fatigue Syndrome Future studies will be expanded to include additional sample types Whole blood vs. plasma Investigate possible transfusion transmission using linked blood donor/recipient samples Association with prostate cancer RNA/DNA from prostate tumor tissue RNA/DNA from urine from prostate cancer patients Association with other diseases? High throughput TIGRIS system will allow large scale studies 11

12 Alternative Specimens for Blood Screening Can use of whole blood increase sensitivity for detection of HIV-1 in blood donors? 50 HIV-1 positive blood donors from US* HIV antibody/16-donation pool NAT reactive HIV treatment-naïve Equal volumes (50 µl) of matched whole blood or plasma were tested in quantitative HIV-1 TMA Assay on TIGRIS System Average of 2 replicates of viral load measurements for each sample type used for comparison *Work done in collaboration with Mike Busch, Lani Montalvo; and Tzong-Hae Lee (Blood Systems Research Institute, San Francisco, CA) and Val Winkelman (Blood Systems Laboratories/Creative Testing Solutions, Tempe, AZ) 12

13 Comparison of HIV-1 Viral Loads in Plasma and Whole Blood Specimens US Blood Donor Population 13

14 Regression Analysis of HIV-1 Viral Loads in Plasma vs. Whole Blood The viral load results from paired plasma and whole blood specimens were analyzed by the Passing-Bablok linear regression method Slope: (95% CI to 1.043); intercept: log copies/ml (95% CI to 0.399) Good correlation between whole blood and plasma viral loads in this population over the range of viral loads measured 14

15 Comparison of HIV-1 Viral Loads in Plasma and Whole Blood Higher HIV-1 viral loads (on average) seen in plasma Study suggests that there may be no benefit gained by screening for HIV-1 in whole blood rather than plasma More detailed investigation needed for infrequent (but reproducible) differences observed Results may not necessarily be extrapolated to very low level viremic donations (early WP donations, donations by elite controllers, or HAARTsuppressed patients) 15

16 Summary New platform and assay development is ongoing Early stage development of duplex parvovirus B19/HAV Assay is underway Research TMA assay for XMRV on TIGRIS System has been developed Assay system suitable for large scale studies Comparison of HIV-1 levels in plasma and whole blood thus far suggests no benefit to screening for HIV-1 in whole blood 16