1. T. cruzi Incidence Study in Blood Donors and its Implications for Selective Testing: Study Results Susan L. Stramer, PhD American Red Cross BPAC Aug 2, 2011 1

2. Acknowledgements ARC –Rebecca Townsend –Ed Notari –Roger Dodd –Jaye Brodsky Community Blood Centers of FL –Bruce Lenes –Sharon Edie –Angelica Lynn BSI –Brian Custer –Hany Kamel –Mike Busch 2

3. Major Points No observed incidence Observe low level reactivity –False positivity –Remote prior infections < 1% observed rate of transfusion transmissions Retesting policy adds no value –Complex and resource intensive

4. Background The Ortho T. cruzi EIA (Ab) is used for donor screening –ARC and BSI implemented “universal testing” Jan 29, 2007 Repeat reactive (RR) donations are further tested using a laboratory-derived test (LDT) RIPA (Quest Diagnostics) RIPA-pos donors are considered confirmed –whether at index, repeat of independent index sample or at donor follow-up Donors of RR donations are followed and tested by EIA/RIPA –RIPA pos donors also tested by parasitologic tests Hemoculture (HC) and PCR –RR donors are also asked to respond to a detailed survey regarding demographics and risk factors True pos “cases” and false pos “controls” 4

5. Retest Algorithm for T. cruzi Ab RR (Ortho EIA) Donors Independent Index or Donor follow-up (F/U) samples Sample TypeLicensed Ortho EIA Interpretation RIPA Performed (Yes/No) If RIPA Negative or Indeterminate: Independent Index or Follow-up Repeat ReactiveYes Independent Index or Follow-up NonreactiveNo If RIPA Positive: Independent Index or Follow-up Repeat ReactiveYes Independent Index or Follow-up NonreactiveYes 5 PRISM added to algorithm following licensure (April 2010)

6. Background Due to low rate of transfusion transmission identified by lookback, and donor risk being remote (22-mo experience), selective T. cruzi Ab testing implemented by all ARC and BSI regions except those included in the “Incidence Study” –8/10/09 ARC –4/1/10 BSI –1X nonreactive test result qualifies the donor for all future donations Regions having the highest prevalence in the Southwestern and South Central portion of the US (5-fold above “system”) remained on universal testing to represent “endemic travel” and “autochthonous” risk Assumption was that the highest prevalence sites would provide the greatest chance of observing donor incidence (if it were to occur) 3 ARC = S CA (Los Angeles; 006), OK/TX (049) and AR (055) Community Blood Centers of FL (S FL) Central Coast UBS (CA) 6

7. Incidence Study Protocol Continue universal testing for 5 years to extend the timeframes for detection of incident infection in repeat donors in regions with presumed increased travel to endemic areas and those with the greatest likelihood of autochthonous infection –5 million person years (pys) total –Donor observation period target a mean of 1.9 years (from first to last donation) –Incident case = seroconverter Screen RR and RIPA pos with a prior donation having a low signal (S/CO 2.0) while remaining RIPA pos; donor may be HC/PCR pos 7

8. Incidence Study Outcome Accumulate ~ 5 million pys of observation in high prevalence areas We chose 4 as the number of incident donors that would be acceptable during a 5-year study as comparable to other TTD residual risks – ~ 1 incident case per million pys The upper 95% CI for a value of 4 is ~ 10 Upper CI of 10 per 5 million pys translates to 2 per million pys Add 20% for risk in remainder of US (5-fold lower) Criterion established for study: upper limit of acceptable risk = 2.4 cases per million pys 8

9. Incidence Study Completion Study completed after 48 months –1/29/07-1/31/11 54 months in one region, Los Angeles –included in the IND prior to test kit licensure No incident/seroconverting donors were identified 9

10. Red Cross Biomedical Services 10 35 Regions collecting in 43 states and Puerto Rico 7 Divisions 5 National Testing Laboratories

11. Blood Systems Collections Areas 3 CTS laboratories 13 main regional areas collecting in UBS/BCP centers located in 18 states 11

12. Cumulative Universal Testing T. cruzi Reactive Donors by State of Residence (01/29/07 – 01/31/11) 4 5 14 233 5 6 22 116 2 17 69 1 3 2 8 488 2 PR 5 5 3 48 22 15 4 5 5 1 11 5 5 7 4 21 5 15 8 7 1 Total Cases RIPA PosRIPA Reported Total39751179 (30%)3947 ARC + CBCF25405992540 BSI1435*580*1407* 28.8 million donations screened 0.014% RR rate RR from 48 states (-DE, HI) RIPA pos (30%) from 40 states (+PR, DC) 66% from FL and CA (1:5100-1:6800) Overall: 1:24,200 1 DC 1 1 1179 48 months 12

13. Region # RIPA Pos Donors 48, 54*mos # Donations Tested IVD + IND* RIPA Pos Prevalence S CA (006)*2121,395,9641 : 6,585 OK/TX (049)15391,2631 : 26,084 AR (055)16452,2191 : 28,264 CBCF2031,033,0051 : 5,089 Central Coast UBS20 365,8551 : 18,293 Total 466 (60%)3,638,306 1 : 7,808 (4.3-fold higher) All Other ARC+BSI 31222,379,3321 : 71,729 All Total 77826,017,6281 : 33,442 T. cruzi Seroprevalence – Universal + Selective 48 months 13

14. Combined Lookback Experience Component No. Recipients Tested No. ELISA/RIPA Pos (minus other risk factors) ARC 48 mos ABC 22 mos RBC 71810 Platelets 924(1*), 2** Plasma+ Cryo 20420 Whole Blood 300 Unknown 700 Total Conf’d 1121472 (0.8% of 259 total tested) * 1 El Salvadoran recipient rec’d random donor platelets from a Brazilian donor; both donor/recipient PCR/HC (-); likely preexisting Ab in recipient **2 apheresis platelet recipients (no other risk factors) from 2 difft dtns from an Argentine donor; both donor/recipient PCR/HC (+) 14

15. Combined Lookback Experience Risk of transfusion transmission in the US as determined by tracing prior donations from RIPA-positive donors since the initiation of widespread T. cruzi Ab testing using FDA licensed screening assays and contemporary methods of component manufacture/transfusion practice is < 1% 15

16. When does transfusion transmission occur; US/Canada Case Reported# Recipients Infected Component/Medical Condition (*immunosuppressed) Donor/Birth Country/Yrs US/Canada Donor Pos Test Results (when available) Kessler et al. NYC 2010 2 – platelets (US)Argentina 40 yrs EIA S/CO 5-6; RIPA+; PCR/HC+ Young et al. Rhode Island 2007 1 – platelets (US); irrad/LR’d; neuroblastoma* Bolivia 17 yrs 1:256 IFA (13 weeks post dtn) Lane et al. Manitoba 2000 1 - 299 platelets/8 RBC units (Canada); prolymphocytic leukemia* Germany (born) Paraguay (child) 2 difft EIAs/RIPA+ Leiby et al. Miami 1999 1 – platelets (US); multiple myeloma* Chile 33 yrs EIA S/CO/RIPA+; PCR/HC+; donor & recip linked Cimo et al. Houston 1993 1 - >500 units (US); colon cancer* No donor identified Grant et al. NYC 1989 1 – platelets (US); Hodgkin’s disease* Bolivia 16 yrs 1:512 IFA Nickerson et al Manitoba 1989 1 – platelets (Canada); acute lymphocytic leukemia* Paraguay 20 yrs > 1:2000 ELISA Geiseler et al Southern CA 1987 1 – blood products (US/endemic travel); acute leukemia* Likely father (Mexico) 1:128 IFA 16

17. Course of Infection in Platelet Recipient Leiby et al, NEJM, 1999; 341(16):1237-1239 Days Post-transfusion Signal/Cutoff (s/co) 020406080100120140160 PCR & HC positive 0 1 2 3 17

18. Person-Time (Donor Observation Period) Calculations All donors with > 2 T. cruzi Ab screened donations selected during 48 months (54 months Los Angeles) Time from first => last donation for each donor calculated (observation period); expressed as pys Calculated pys separately for each analysis –Mean observation period –Total observation period –Graphs used person months to show groups of donors within specific observation times Incidence and Exact binary upper and lower 95% confidence limits (UCL, LCL) calculated 18

19. Risk/Site Number of Donors Mean Observation Period (pys) Incident Cases 95%CL (LCL;UCL) per million pys Total Observation Period (pys) Travel Risk ARC Los Angeles (006) [54 months] 244,4981.6960 (0; 8.9)414,578 UBS Central Coast (CC, 034) [48 months] 49,5911.6390 (0; 45.4)81,293 Subtotal294,0891.6860 (0; 7.4) 495,871 Autochthonous Risk ARC Southwest (OK/TX; 049) 59,0451.4200 (0; 44.0) 83,856 ARC Greater Ozarks (AR; 055) 74,9201.5500 (0; 31.8) 116,152 Subtotal ARC 133,9651.4930 (0; 18.4) 200,008 BSI Regions (11,16,20,26 & 31; TX, AR, MS, LA) Subtotal BSI 175,6111.4680 (0; 14.3) 257,773 Subtotal ARC + BSI [48 months] 309,5761.4790 (0; 8.1)457,781 Incidence Study Outcomes 19

20. Risk/Site Number of Donors Mean Observation Period (pys) Incident Cases 95%CL (LCL;UCL) per million pys Total Observation Period (pys) Overall Risk ARC, all donations (universal + selective) [48-54 months] 3,523,8941.402 0 (0; 0.75)4,941,088 BSI, all donations (universal + selective) [48 months] 698,3911.6010 (0; 3.3)1,118,274 Total4,222,2851.4350 (0; 0.61) 6,059,362 Extended Observation Periods ARC all apheresis donations [48-54 months] 492,0201.6430 (0; 4.6)808,313 BSI all apheresis donations [48 months] 275,9631.7900 (0; 7.5)494,095 Total Apheresis767,9831.6960 (0; 2.8)1,302,408 20

21. Graph_Chagas_Travel_Both 21

22. Graph_Chagas_Autoch_Both 22

23. Graph_Chagas_ALL_Both ----Selective testing---- 23

24. Graph_Chagas_ALL_APH_Both ----Selective testing---- 24

25. Possible Seroconverters Identified During Universal Testing 22 donors identified with nonreactive Ab tests who on subsequent donation at 41-532 days were found to be RR and RIPA pos (index, repeat independent sample or follow-up) –21 based on Ortho EIA testing –1 based on Abbott PRISM clinical trial Reactivity was inconsistent and none of these donors met the definition of an incident/ seroconverting donor 25

26. Course of Infection in Platelet Recipient Leiby et al, NEJM, 1999; 341(16):1237-1239 Days Post-transfusion Signal/Cutoff (s/co) 020406080100120140160 PCR & HC positive 0 1 2 3 26

27. Possible Seroconverters Identified During Universal Testing (to 8/9/09) N=18; 13/13 PCR and 14/14 HC Negative Days S/CO 27

28. Possible Seroconverters Identified by Sites Remaining on Universal Testing (from 8/10/09) N=4; 2/2 PCR and HC Negative Days S/CO 28

29. WBN Prior Donation Index Collect Date Testing Interval (days) State Ab ELISA S/CO RIPA PRISM S/CO Index PCRHC Collect Date S/CO(Index, 2 nd Index, F/U) 011LK52205 3/18/07 0.983 4/28/07 41MO 1.317*/0.038/ 1.384*/1.419 -/+/-/-0.15--/- 017GN57725 2/27/07 0.835 6/7/07 100WI 1.794*/0.455/ 0.411/0.834/0.634 +/-/+/-/-0.06-/-/- 021KE29415 5/10/07 0.475 8/21/07 103OR 1.078*/1.054*/ 1.277* -/+/-0.05-- 004L 96697 4/30/07 0.819 8/30/07 122NH 1.166*/0.811/ 0.959/1.008* +/+/-/-0.10-/- 012C 81057 7/11/07 0.802 9/5/07 56NC 1.120*/0.737/ 0.770/0.846 +/+/-/-0.06-/- 049GL34705 7/19/07 0.440 11/8/07 112OK 1.035*/0.599/ 0.705/1.050* +/-/-/+0.06-/- 053FW61740 4/14/07 0.836 12/13/07 243VA 1.096*/1.092* +/++/+1.22*NA 036FP31850 10/16/07 0.852 2/12/08 119SC 1.114*/1.116*/ 0.830/1.003* -/+/-/-0.16-/-- 020FT11144 11/19/07 0.869 4/2/08 135UT 1.021*/0.931/ 1.010* +/-/-0.05-- 011GZ71884 4/4/07 0.239 5/21/08 413MO 1.492*/1.761*/ 2.960*/0.411 -/-/+/-0.27NA- 020FW16929 1/31/08 0.689 5/29/08 119UT 1.333*/0.749/ 1.041/0.251/0.857 -/- /+/-/-0.13-/- 007FM15946 4/30/08 0.371 6/23/08 54AZ 1.291*/0.523/ 0.222/0.485 +/+/-/-0.07--/- 024GY39349 5/6/08 0.230 7/19/08 74IN 1.178*/0.839/ 1. 187/0.641/0.309 -/- /+/-/-0.48-/- 006FP69971 2/20/07 0.915 8/5/08 532CA 1.110*/1.100* +/-0.72NA 006GS26670 6/4/08 0.124 8/6/08 63CA 0.116/0.095/ 0.094/0.106 +/-/-/-1.51*/0.81-/- 022GJ11661 3/12/08 0.399 8/8/08 149PA 1.110*/0.113 +/-0.06NA W0368 12/16/08 0.961 4/29/09 134FL 1.123*/0.280/ 0.346 /0.316 +/-/Ind/-0.14-/- 038GE30113 2/10/09 0.976 5/29/09 108IN 1.083*/0.923 +/++/+0.45NA Possible Seroconverter Cases N=18 (1/29/07-8/9/09) * Denotes average of 3 S/CO values Chagas Clinical Trial sample with RR PRISM at Index Yellow highlighted S/CO = 20% elevated negative 29

30. Possible Seroconverter Cases N=4 (8/10/09-1/31/11) 3 regions and CBCF that continued with Universal Testing WBN Prior Donation Index Collect Date Testing Interval (days) State Ab ELISA S/CO RIPA PRISM S/CO Index PCRHC Collect Date S/CO (Index, 2 nd Index, F/U) 006GG00723 01/04/09 0.941 08/21/2009 229CA 1.086*/1.024*-/+3.06* NA 006GX87964 05/22/09 0.994 12/09/2009 201CA 1.083*/0.046/1.217*+/-/-0.06 -- 006LH19583 01/26/10 0.332 06/08/2010 133CA 1.203*/1.009*/0.522+/+/++/+/+2.64* -- 006KP44769 09/02/10 0.217 09/16/2010 14CA 1.663*/0.344/0.087+/-/-0.17 pend * Denotes average of 3 S/CO values Yellow highlighted S/CO = 20% elevated negative Of 18+4: 3 “yes” responses on risk questions; all followed with no seroconversion 006LH19583 born and lived in Mexico 39 yrs 006GX87964 Hispanic, born in US W036809203439 born and lived in Venezuela for 10 yrs 30

31. T. cruzi antibody reactivity Pheresis donor 006KP44769 S/CO Date 18 prior nonreactive donations RR/RIPA pos donation and retesting of regional retention sample Follow-up collected 298 days post index Assay cutoff 31

32. US-Derived T. cruzi Study (USTC) 54 eligible RR/RIPA Pos donors (48 ARC and 8 UBS); 37 enrolled 15 Defined as Concordant (CG)/22 Discordant (DG) Vector and reservoir N=28 and N=17 Vector only PR 32

33. WBN Prior Donation RIPA PRISM S/CO Index CDC Testing Leish IFA Focus, USA Collect Date S/CO IFA Epi lysate CDC In house Chagatest EIA rDNA Weiner, Argentina TESA Trypo lysate bioMx, France 011LK52205 3/18/070.983 -/+/-/-0.15Neg 017GN57725 2/27/070.835 +/-/+/-/-0.06Neg 004L 96697 4/30/070.819 +/+/-/-0.10Neg 012C 81057 7/11/070.802 +/+/-/-0.06Neg 049GL34705 7/19/070.440 +/-/-/+0.06Neg 020FW16929 1/31/080.689 -/- /+/-/-0.13Neg 007FM15946 4/30/080.371 +/+/-/-0.07Neg 024GY39349 5/6/080.230 -/- /+/-/-0.48Neg 006GS26670 6/4/080.124 +/-/-/- 1.51* /0.81 Neg Possible Seroconverter Cases N=18 (1/29/07-8/9/09) enrolled in USTC Chagas Clinical Trial sample with RR PRISM at IndexYellow highlighted S/CO = 20% elevated negative 33

34. USTC Testing Results for the Discordant Group (DG); N=22 IDIndex Ortho EIANo. RR Ortho/No. pos RIPA/IFA titerWiener EIATESAPRISM Mean Mean S/CONo. tested 1:X S/CO DG13.4 3/4 1/3≤16NEG 0.21 DG22.8 4/4 1/4≤16NEG 0.06 DG32.5 4/5 1/4≤16NEG 0.15 DG41.9 5/5 ≤16NEG 0.42 DG51.8 3/4 1/4≤16NEG 0.17 DG61.8 1/5 2/5≤16NEG 0.06 DG71.7 2/4 1/4≤16NEG 0.10 DG81.4 3/4 2/4≤16NEG 0.11 DG91.3 2/5 1/4≤16NEG 0.13 DG101.3 3/4 1/4≤16NEG 0.15 DG111.3 1/4 2/4≤16NEG 0.07 DG121.3 1/4 2/4≤16NEG 0.15 DG131.2 4/5 1/5≤16NEG 0.07 DG141.2 3/5 2/4≤16NEG 0.17 DG151.2 2/5 1/5≤16NEG 0.48 DG161.2 2/4 ≤16NEG 0.10 DG171.2 3/41/3≤16NEG N/A DG181.1 1/4 2/4≤16NEG 0.06 DG191.1 1/5 ≤16NEG 0.08 DG201.0 2/4 ≤16NEG 0.06 DG211.0 1/5 ≤16NEG 0.05 DG220.1 0/4 1/4≤16NEG 0.81 34

35. Rate of Possible Seroconverters: Universal Testing ARC + CBCF Testing Period Time Frame Total Donations No.Frequency Universal Testing 1/29/07- 8/9/09; 31 mos 17,568,120181/976,007* Cont’d Universal Testing 8/10/09- 1/31/11; 17 mos 1,414,40241/353,601* TOTAL48 months 18,982,522221/862,842 * NS; p = 0.10 Chi square and Exact measures mid-P (2-tail) 35

36. Assay Variability Biologic (test component or other agent, remote prior infection)/Analytic (cutoff) Donor follow-up of 3 donor populations derived from 246 RR/RIPA Pos ARC donors enrolled in follow-up during 48 months (651 total RIPA pos) –N=174 Consistent RR/RIPA Pos –N=57 Inconsistent RR/RIPA Pos Index Ortho EIA RR; RIPA pos index or follow up –N=22 Possible Seroconverters with 16 donors followed (15 Ortho EIA/1 Abbott PRISM triggered) Demographic analysis of 3 populations Analysis of Ortho EIA cutoff values for 3 populations Relationship of Ab S/CO values with PCR positivity –Higher S/CO values correlate with PCR positivity 36

37. Number of Donors S/CO Value Sample descriptionN Index Ortho EIA Median S/CO Index Abbott PRISM Median S/CO Consistent EIA/RIPA (+) 1744.795.55 Inconsistent EIA/RIPA (+) 571.391.25 Possible Seroconverters inconsistently EIA/RIPA (+) 21 (15)1.170.14 Distribution of T. cruzi RIPA-Positive Donation S/CO Values ARC Donors Participating in Follow-up (n=246) Mean Ortho EIA Test-of-Record Results Mean follow-up 76 days (2 - 1083) Mean follow-up 94 days (23 - 347) Mean follow-up 77 days (18 - 277) 37

38. Distribution of T. cruzi RIPA-Positive Donation S/CO Values ARC Donors Participating in Follow-up Mean Ortho EIA Test-of-Record Results Jaye’s Box and Whisker graph 4.79 1.39 1.17 N = 174* N = 57 N = 15 *p < 0.0001 Wilcoxon Rank Sums 38 % PCR + 30 4 0 HC + 8 0 0

39. VariableNo. (%) Consistent (N=156) “AA” No. (%) Inconsistent (N=50) “BB” No. (%) Possible Seroconverters (N=16) “CC” Born in endemic country 139 (89) 31 (62) ****** 2 (13) * Lived in endemic country > 1 yr 142 (91) 31 (62) ****** 2 (13) * Time in endemic country >1 yr Mean 20 yrs (1-51)Mean 21 yrs (1-39)(10 and 39) Mother/maternal grandmother born in an endemic country 141 (90) 32 (64) ****** 3 (19) * Hispanic 143 (92) 34 (68) ****** 3 (19) * House dirt floors/walls 95/142 (67)27/36 (75)1/3 (33) Lived in a rural area 102/142 (72)29/36 (81)2/3 (67) House thatched roof 40/142 (28)7/36 (19)0 (0) Age Mean 44yrs (16-82) Mean 43yrs (17-80) Mean 37yrs (17-74) Male 67 (43)30 (60)10 (63) Female 89 (57)20 (40)6 (37) Risk Factors/Demographics: RIPA-Pos Donors – 48 months Chi-square with Yates correction; *p<0.01;***p < 0.0001;3 “yes” no seroconversion 39

40. Ortho Assay Variability as defined by the Cutoff Cutoff = PC mean [0.425]; PC range = OD ≥ 0.300, ≤ 1.800 Group #Runs/#Samples Cutoff OD Mean % Runs <0.338 Mean All 2007-2010 71,1170.33850 Consistent 338 / 1740.34441 Inconsistent 111 / 570.33457 Possible Seroconverters 30 / 150.32860 Acceptable Cutoff Range: 0.1275 – 0.705 40

41. T. cruzi Ab reactivity (EIA) correlates with PCR positivity among seroreactive donors in Brazil, Honduras, US Sabino et al., accepted 2011 AABB Annual Mtg p<0.005 p<0.001p<0.0001 6.57 5.78 6.33 4.27 5.50 3.56 9 40 45191681N= 41

42. Impact of Retesting after 2 or 5 Years 2005 donor cohort (~2.7 million donors) followed from first to last donation 9-year donation data to model retest volume (~60 million donations) for a 2- or 5-year retest policy 42

43. Determination of Repeat Donation Patterns for ARC Donors in CY2005 All donors with an initial donation in CY2005 who had subsequent donations through CY2010 included Time from first => last donation calculated Plotted the number of donors within the 2005 cohort (2,678,057) who continued to donate (i.e., not yet had their last donation within the study period) 43

44. 44

45. 36,688,648 Untested donations 15,850,682 1 st Test donations 7,135,528 Retest donations 45

46. 41,773,568 Untested donations 15,850,682 1 st Test donations 2,050,608 Retest donations 46

47. Summary Ongoing 1-time testing to qualify a donor for repeat donation is “safe” as evidenced by: –Zero observed incidence in the 4-year “Incidence Study” 6 million plus pys of observation 4.22 million donors (mean obs time = 1.435 pys) –Upper 95% confidence value (0.61/million pys) yielded risk of incidence lower than that of other current infectious diseases –< 1% obs’d occurrence of transfusion transmission Risk greatest in donors with high S/CO values, consistently RIPA pos and PCR/HC pos Low frequency of donors identified during universal testing from ARC + CBCF with inconsistent reactivity who were not seroconverters –22/~19 million donations or 1/900,000 –Biologic and analytic false positives, remote prior infection? 47

48. Summary Retesting test-negative donors after 2 or 5 years adds no value relative to the low (no) risk of incidence and infrequent occurrence of transfusion transmission –Implementation of such systems would be a burden to blood centers requiring significant resources Anywhere from 500,000 to 1 million additional tests per year for the ARC; 2x for the US Competes/may prevent the implementation of other other safety initiatives –e.g., Babesia 48

49. Conclusion A selective testing strategy based on qualifying a donor by a single negative donation has high sensitivity and has significantly reduced the amount of testing required without compromising recipient safety 49