1. Holland Laboratory Tests Used In Blood Screening (II) Test Performance; Optimal Use of Reference Panels David A. Leiby, PhD Head, Transmissible Diseases WHO Consultation 27-28 January 2009 WHO Headquarters, Geneva

2. Common Serologic Assays for T. cruzi indirect immunofluoresence (IFA) indirect hemagglutination (IHA) ELISA RIPA Western/immuno blots rapid tests* * not used for blood screening

3. U.S./Canadian Transfusion Cases: 1987:California - Mexican donor 1989:New York City - Bolivian donor Manitoba - Paraguayan donor 1993:Houston - unknown donor 1999:Miami - Chilean donor 2000:Manitoba - German/Paraguayan donor 2002:Rhode Island – Bolivian donor

4. Impact of Latin American Immigration

5. LA Seroprevalence: 1996-98 % Donors Positive 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 1996 1997 1998 1/9,900 1/7,200 1/5,400 Leiby et al., Transfusion 2002;42: 549-555

6. Models for Testing/Implementation universal blood screening screening + confirmatory assays parallel testing (2-3 assays) risk-factor models birth in endemic country lived in rural area, thatched roof, vector exposure, etc. immunosuppressed patients must identify recipients at greatest risk test each donor only once/twice logistically complex needs cost/benefit analysis analysis of sensitivity

7. T. cruzi Ab Testing Sample sent from NTL for RIPA Positive, Indeterminate, NegativeIndex donation Repeat ReactiveNon-Reactive No Actions SSO collates test results and sends reports Retrieve index frozen plasma or index retention sample (IRS) Defer donor and enroll in Chagas follow-up study (CFS) Current ARC Testing Algorithm

8. Chagas Follow-Up Studies RIPA Positive Donor Donor follow-up:questionnaire 2 clot tubes 2 EDTA tubes 3 heparin tube T. cruzi Ab Testing RIPA PCR Hemoculture SSO collates test results and sends reports

9. Donor Management Practices in U.S. RRs notified/deferred supplemental testing encouraged no FDA licensed tests exists RIPA most sensitive test available donor counseling including donor follow-up studies encouraged no donor reentry refer RIPA positive donors to physicians recipient tracing for RIPA positive donors

10. T. cruzi Reactive Donors (01/29/07-11/30/08) Total CasesRIPA PosRIPA Reported 2633639 (25%)2597 17.8 million donations screened 0.015% RR rate RR from 46 states (-DE, RI) RIPA pos (25%) from 38 states (+PR, DC) 60% from FL and CA (1:3700-1:7500) Overall: 1:27,000

11. Continental U.S. Map: RIPA Positives (thru 11/30/08) Total Repeat ReactiveRIPA PositiveRIPA Negative/IndRIPA Pending/NT 29897352135 / 4574 >80% from ARC/BSL

12. 22 Month Experience – ARC Donation TypeNo. ScreenedNo. RIPA PositivePrevalence Total Donors 5,459,4962351:23,232 Allogeneic 5,046,7412251:22,430 Autologous 65,96061:10,993 Pheresis 346,79541:86,699 Total Donations 12,059,2702351:51,316 Allogeneic 10,666,4372251:47,406 Autologous 99,86361:16,644 Pheresis 1,292,97041:323,243 2,482,904 donors contributed 2,317,801 person years (only intervals between donations that were tested were counted)

13. +-% PCR (EDTA) 2210617 Hemoculture (Heparin) 91058 PCR/Hemoculture Results for RIPA Pos Donors

14. RIPA Confirmed Positive Donors (n=394)

15. Donor Demographics to 11/30/08 RIPA Pos (N=394) ~FT donors 250 (63%) ~RPT donors 144 (37%) ~Male 212 (54%) ~Female 182 (46%) ~Country of birth (N=149) Mexico 44 US 37 El Salvador 24 Bolivia 15 Honduras 6 Colombia 5 Argentina, Guatemala 4 Brazil 3 Ecuador, Nicaragua 2 Paraguay, Chile, Somalia 1 RIPA Neg/Ind (N=1490) ~FT donors 414 (28%) ~RPT donors 1076 (72%) ~Male 898 (60%) ~Female 592 (40%) ~Country of birth (N=420) US 400 India 3 China, Germany 2 Thailand, Taiwan, Cuba, Hungary, New Zealand, Barbados, Ecuador, Panama, Ukraine, Venezuela, Canada 1 Colombia 2* *21-37 years in Colombia, 1/2 with ECG irregularities and 1 with upper GI symptoms

16. Autochthonous Transmission Summary 45 potential autochthonous cases identified from screening US blood donors 37 from the ARC 7 appear to be likely cases 5 parasitemic 2 donors thoroughly investigated by CDC (MS) another 7 have other risk factors some may be false positive continued investigation needed to determine frequency and risk factor (e.g., hunting, camping, time spent outdoors, etc.) US-derived T. cruzi (USTC) study participants CDC, ARC and UBS additional questionnaire re risk; CDC and ARC IRB approved

17. RIPA Positive Donors Prior Non-reactive/Untested Donations 16 with prior S/CO values: 0.12-0.98 5 with multiple prior negative donations 8 QNS or NT donations Unlikely to be true incident infections!

18. IND/clinical trial (28/08/06 -28/01/07) PPV = 32 RIPA pos/63 RR = 51%; pos in 2 states RR rate = 63/148,969 = 0.042% Prevalence = 32/148,969 = 1:4655 Nationwide screening (29/01/07-30/11/08) PPV = 639 RIPA pos/2597 RR RIPA tested = 25%; pos in 38 states (+ PR, DC) 60% from FL and CA (1:3700-1:7500) RR rate = 2597 RRs/>17.8 x 10 6 donations = 0.015% Projected prevalence = 1:27,000 Test Performance Sensitivity of Universal Testing (Ortho ELISA PI) 860/861 = 99.88% (95% CI = 99.35% to 100.00%)

19. Proposed Chagas Reference Standard requirements must include T. cruzi I and II broad geographic reactivity pooled vs. neat? sensitivity in diluted samples targeted antibody titers stability over time specified characteristics 1 medium reactive from south 1 medium reactive from north 1 clear negative

20. Importance of Global Reference Standards test validation quality control comparisons with other tests

21. Interested Parties test manufacturers evaluate candidate antigens measure sensitivity regulatory agencies determine analytic sensitivity reproducibility/proficiency compare assays blood establishments assay evaluation quality assessment research institutions diagnostic centers facilitate comparison with other laboratories internal controls

22. Summary & Conclusions significant number of U.S. blood donors infected with T. cruzi 639 confirmed positives nationwide distribution 1:27,000 donors infected current test performance 99.88% sensitivity 25% PPV need for a global reference standard test validation quality control assay comparison reference standard characteristics broad geographic reactivity is critical

23. Acknowledgements American Red Cross Susan Stramer, SSO Greg Foster, SSO David Krysztof SSO Rebecca Townsend SSO Megan Nguyen, HL Melanie Proctor, HL Ross Herron, West Div Pamela Kahm, West Div Norma Espinoza, West Div Kay Crull, West Div Blood Systems Laboratories Sally Caglioti Frank Radar Larry Morgan