CBER Chagas Serological Lot Release Panel Development Hira L. Nakhasi, Ph.D. Director, Division of Emerging and Transfusion Transmitted Diseases, OBRR/CBER/FDA

Challenge for Blood Products Enhance Product Safety, Purity and Potency Avoid Product Shortages & Major Increased Costs and Critical Path opportunities exist that could improve blood product safety, efficacy and availability while minimizing disruptions to the blood system

FIVE LAYERS OF BLOOD SAFETY FDA’s approach is to optimize each safety layer: 1. Donor screening and deferral based on geographic, behavioral and medical risk factors (donor education, self deferral, donor interview) 2. Laboratory testing and deferral (HIV-1, HIV-2, HBV, HCV, HTLV-I, HTLV-II, WNV, Chagas, syphilis) 3. Deferral registries to prevent use of blood from deferred donors 4. Quarantine controls to prevent unit release pending verification of donor suitability 5. Investigation and correction of deviations

Impact on the US Public Health: Blood Safety and Availability Millions of units are transfused annually Risk of transmission of infectious disease agents through transfusion has been significantly reduced with the introduction of donor screening tests

Evolution of Testing and Deferral What happens when an emerging pathogen threatens the safety of the blood supply? We introduce deferral criteria based on epidemiologically determined risk factors (medical, geographic or behavior-related exposures) as an initial safety measure Where feasible, tests are developed, but deferral is maintained as an overlapping safeguard Test sensitivity and specificity generally improve. However, risk-based deferrals are usually retained as an overlapping safeguard, especially when data are lacking on the relative safety contribution of risk-based vs. test-based deferrals

Chagas Transmissions by Blood or Organ Donations: U.S./Canadian 1987: California – Mexican blood donor 1989: New York City – Bolivian blood donor Manitoba – Paraguayan blood donor 1993: Houston – unknown blood donor 1999: Miami – Chilean blood donor 2000: Manitoba – German/Paraguayan blood donor 2001: Three organ recipients – Central American organ donor 2002: Rhode Island – Bolivian donor 2006: Los Angeles – Heart donor traveled to Mexico Los Angeles – Heart donor born in El Salvador

Blood Screening ELISA Licensed December, 2006 ORTHO T. cruzi ELISA Test System approved for use as a blood screening assay Large blood centers implemented testing Feb. 2007. FDA recommendations for implementation under consideration. Pre-release testing of kit lots is CBER responsibility CBER Lot Release Panel developed Cross testing with manufacturers panels

T. cruzi Reactive Donors by State of Residence (01/29/07 – 01/14/09) Total Repeat Reactive RIPA Positive RIPA Negative/Ind RIPA Pending/NT 3071 749 2177 / 47 98 26 22 16 9 9 19 31 53 53 229 42 43 6 13 81 1 4 59 81 14 DC 119 65 16 27 2 38 37 17 39 8 73 482 52 26 21 96 31 24 26 46 12 35 PR 78 30 26 30 18.9 million donations screened 0.015% RR rate RR from 47 states (-DE) RIPA pos (25%) from 38 states (+PR, DC) 61% from FL and CA (1:3800-1:7600) Overall: 1:27,600 (Source: AABB) 36 57 365 * 3 RIPA pos samples represent multiple positives from auto donor and cord blood

Epidemiology of T.cruzi infection among US blood donor Confirmed positive (RIPA) donors: 25% US born 75% born in T.cruzi endemic countries 63% first time donors (increasing) 37% repeat donors (declining) Potential autochthonous cases ~42 Chagas positive cases from 47 states Look back has identified 0/88 of recipients* All donors are long term chronic asymptomatic (low parasitemia) Transfusions are rarely WB L/B have few platelet Transfusions ( > probability to transmit than RBC) *On going investigations for a few cases possible T-T but rare No true seroconversions have been identified

Current Status of T.cruzi testing in the US Continuation of universal blood screening for T.cruzi to have a better evaluation of epidemiology of the disease and performance of the tests. Need for licensed confirmatory test for donor reentry Blood established performed validation of selective testing strategy (STS) to chart future course of testing in the US Public discussion of the STS

Standardization of Serological Testing for Chagas’ Disease Validation and licensing of new tests Analytical characteristics Sensitivity, specificity and reproducibility Clinical characteristics Random donor specificity trial Known positive and High risk sensitivity trials Testing lot to lot consistency of licensed assays

T. cruzi Panel Development Purpose ∙ Lot Release Panel to test lot to lot consistency ∙ Not a Reference Panel ∙ Not an International Standard Objective ∙ Obtain T. cruzi positive specimens from various geographical locations. ∙ Emphasis on immigration patterns found in the U.S. donor pool from endemic countries.

Geographic Distribution of Panel Members * 7% 0.1% 24% 1.2% 0.4% 9.8% 10.7% 30% 3% 3.9% 17.7% 11.7% Geographic Distribution of Panel Members * * * * *Endemic countries for CBER Panel N%-WHO estimate of prevalence, Global burden of Chagas’ disease in the year 2000 DRAFT A Moncayo, F Guhl, C Stein. http://www.who.int/healthinfo/statistics/bod_chagas.pdf *

Preliminary Formulation of Panel Obtained/Purchased positive units and tested neat for titers and co-infections (e.g. HCV, HBsAg, HIV etc.) Determined acceptable units for possible panel manufacturing ∙ Highest Titers ∙ Largest Volumes ∙ Geographical Variation

Preliminary Formulation of Panel con’t Chose Acceptable Units from Following Areas ∙ Mexico, Argentina, Nicaragua, and Brazil Tested at CBER/DETTD with Serial Dilutions until Non-Reactive ∙ Ortho T. cruzi ELISA Test System Sent Serial Dilutions to Kit Manufacturers ∙ Ortho-Clinical Diagnostics ∙ Abbott Laboratories Evaluate all results to construct final panel

Candidate CBER Lot Release Panel 10 Member Panel 2 Negatives 4 geographical units at 2 dilutions ∙ Consensus High Positive (S/CO >2.0) ∙ Consensus Low Positive (S/CO ~ 1.0) 500 ml Bulk, 500 ml for Final Vialing Fill Volume 1.25 ml

Validation of Candidate Panel CBER/DETTD Tested Final Vialing ∙ Ortho T. cruzi ELISA Test System ∙ Abbott PRISM Chagas ∙ Abbott Chagas Confirmatory Assay Ortho-Clinical Diagnostics Tested Abbott Laboratories Tested

CBER Panel Testing Results Manufacturers Testing CBER Testing Lot Release Criteria CBER and Manufacturers Testing* Panel Member Screening Test A B Screening Tests Confirmatory Test 1 (Negative) 0.163 0.183 0.169 0.220 - Negative 2 (Nic. High) 1.776 5.782 1.784 5.720 + Positive 3 (Mex. High) 1.647 3.512 1.650 11.430 4 (Arg. High) 1.184 1.524 1.006 1.580 +/- 5 (Braz. High) 3.141 7.018 3.244 6.900 6 (Negative) 0.134 0.198 0.132 0.200 7 (Nic. Low) 1.131 2.641 1.100 3.560 8 (Mex. Low) 0.800 1.861 0.944 3.660 9 (Arg. Low) 0.606 0.812 0.642 1.010 10 (Braz. Low) 2.336 3.782 2.396 3.740 *Lot release criteria for the Confirmatory test will be determined after testing additional lots

Summary Selection of panel members guided by availability, antibody titer, geographic source of specimens and origins of US immigrant blood donor population. Panel dilutions guided by multiple tests on two testing platforms. 4 panel members mandatory positive (S/Co >2) 4 panel members low positive/high negative (S/Co~1) 2 negative Acceptance criteria for each panel member as negative, positive or positive/negative determined by multiple testing of the diluted specimens from final filled vials.

Acknowledgments Robert Duncan Steve Kerby Kori Francis Robin Biswas Alain Debrabant Leslyn Aaron Karen Smith Silvano Wendel- Brazil

CBER Lot Release Procedures Manufacturer submits kit lot with testing data for FDA review Manufacturer’s in-process test results Manufacturer’s release panel results Test results from 100 non-reactive specimens Manufacturer’s results from CBER panel CBER tests kit with CBER panel CBER approves/fails release of kit lot