1. E011375A (03-13-01) 1 Issues Regarding Qualification/Implementation of an Antibody Test for Trypanosoma cruzi Susan L. Stramer, PhD American Red Cross Blood Products Advisory Committee Meeting April 26, 2007

2. E011375A (03-13-01) 2 Outline uClinical study design and results –8/28/06 to 1/28/07 –3 regions (West Division); SoCal, NoCal, AZ –MMWR 2/23/07; 56 (07):141-143 uImplementation: AABB Association Bulletin #06-08 uIVD results 1/29/07-4/17/07; approx 65% collected blood US –ARC, UBS, 15 other blood centers and >50 hospitals –Distribution in US and accuracy of predictions uTest performance –ELISA, RIPA, T. cruzi IFA, Leishmania IFA, “Special Protocol” uLookback results uDonor demographics –Risk factors –Possible autochthonous cases (indigenous, native)

3. E011375A (03-13-01) 3 Chagas Protocol/Assumptions uFDA request of Ortho to expand clinical studies to include areas where T. cruzi antibody prevalence previously documented –Pivotal clinical trial yield 0 conf’d pos (of 40,665 tested); 99.998% specificity (PI) –100,000 donations defined the study –Testing would continue through test licensure and implementation –SoCal, NoCal and AZ (West ARC Division) FDA requirements for donor informed consentFDA requirements for donor informed consent –Use of an information sheet with signature on BDR acknowledging receipt not allowed –Specific signature and date required on info sheet; matched to tubes that were tested Due to these requirements, other blood centers declinedDue to these requirements, other blood centers declined

4. E011375A (03-13-01) 4 ARC Prevalence Study T. cruzi Results 8/28/06-1/28/07 All sites SoCalArizonaNoCal Total Attempted Donors 189,828127,05326,60136,174 Total Tested 148,96995,66223,98129,326 Total RR Confirmed 63 (1:2365) 32 (1:4655) 50 (1:1913) 25 (1:3827) 4 (1:5995) 2 (1:11,990) 9 (1:3258) 5 (1:5865) Total (%) Refusals 40,859 (21.5%) 31,391 (24.7%) 2620 (9.8%) 6848 (18.9%) 40,859 refusals/detection rate of 1:4655 = 9 undetected donors!

5. E011375A (03-13-01) 5 The prevalence of infection with T. cruzi in the US varies by region and might now be higher than previously thought, especially in geographic areas such as Los Angeles County, where a substantial proportion of blood and organ donors have emigrated from Chagas-endemic countries. MMWR 55 (29); 2006

6. E011375A (03-13-01) 6 Implementation uAccording to AABB Assn Bull #06-08 –Collaboration with the CDC and FDA uComponent mgmt –Components from RR donors quarantined/withdrawn from the market (3 calendar days) IndexIndex Prior in-date donationsPrior in-date donations –As long as electronic records exist –Recipient tracing –Autologous unit release with approval of auto donor’s referring physician –Inventory testing (in-house or distributed) not recommended; each facility assess risk

7. E011375A (03-13-01) 7 Implementation uDonor mgmt –RRs notified/deferred –Supplemental testing encouraged, although no FDA licensed confirmatory/supplemental test exists RIPA most sensitive test, however not 100%RIPA most sensitive test, however not 100% –Leishmania testing on supplemental test unconfirmed; no mention Plasmodium or Paracoccidiodes braziliensus –Donor counseling including donor follow-up studies encouraged –No donor reentry –Refer supplemental test positive donors to knowledgeable physician Referrals from personal physician, blood center, Am Assoc Tropical Med or CDCReferrals from personal physician, blood center, Am Assoc Tropical Med or CDC –Recipient tracing from supplemental test positive donors “Licensed test for Ab detection has suitable performance characteristics for blood donor screening and as such may be useful in testing of the above individuals.”“Licensed test for Ab detection has suitable performance characteristics for blood donor screening and as such may be useful in testing of the above individuals.” uCOI and Component Labels –Component tested by a licensed test and the results are negative/nonreactive

8. E011375A (03-13-01) 8 Models for Testing/Implementation uUniversal uImmunosuppressed patients –Puts burden on hospitals to identify correct units for recipients at highest risk uGeographic models –By US census data/WHO seroprevalence by country uTest one-time-only per donor where only new donors are tested; repeat donors are questioned re risk and only “yes” responses are tested –Assumes donor understands the questions, questions may be culturally sensitive, assumes no autochthonous risk –Must be validated; each positive requires knowledge of risk and when it occurred –Logistically complex relative to sample tracking and component management –Financial benefit has not been validated –Confusing message to test kit developers

9. T. cruzi Reactive Donors by State of Residence (01/29/07 – 04/17/07) 3 5 8 56 6 12 15 10 2 4 3 6 6 4 2 3 37 4 PR Total RR Donors RIPA PosRIPA Reported ARC21341191 Blood Systems527 (2)29 (4) 1 3 2 2 4 8 6 2 7 7 1 3 2 5 3 4 1 1 1 3 3 2 3 1 1 Nos. indicate 265 RR donors by state; no RR donors or testing not occurring in 6 states; 48 confirmed pos (+2) in 17 states: 19 CA, 11 FL, 3 MD, 2 NY, UT, VA; 1 AR, CT, GA, MA, MT, NC, NJ, OR TN, WA, (AZ)

10. E011375A (03-13-01) 10 RR donations reported to the AABB; 4/24/07 7 facilities reporting; 4 sites with RR donors 272 total RR

11. E011375A (03-13-01) 11 Confirmed pos reported to the AABB website Of 228 = 21.5%

13. E011375A (03-13-01) 13 Do the states in which confirmed positive donors agree with models based on immigration?

14. N.J. - N.Y. Metro Washington, D.C. L.A. County Based on immigration and test positive rates for endemic countries

15. E011375A (03-13-01) 15

16. E011375A (03-13-01) 16 Test Performance uClinical trial (8/28/06-1/28/07) –PPV = 32 RIPA pos/63 RR=51%; pos in 2 states –RR rate = 63/148,969 = 0.042% –Prevalence = 32/148,969 = 1:4655 –Specificity = 148,906 test neg/148,906+31 false pos = 99.979% 99.997% in PI99.997% in PI uNationwide screening (1/29/07-4/17/07) –PPV = 50 RIPA pos/224 RR RIPA tested = 22%; pos in 17 states 30 of 50 (60%) RIPA pos from 2 states (S CA, FL)30 of 50 (60%) RIPA pos from 2 states (S CA, FL) –RR rate = 265 RRs (213 ARC + 52 BSL)/1,757,800 donations = 0.015% –Projected prevalence based on 22% PPV of RIPA tested = 1:30,150 –Specificity (ARC) first week = 124,934 test neg/124,934+28 false pos = 99.978% (6 pos) uOverall prevalence = 1:21,100, and 27% rate of RIPA positivity

17. Samples (ARC/BSL) N%Mean S/COS/CO Range RIPA Positive7329%3.450.93 – 7.72 RIPA Negative18171%1.420.82 – 3.89

18. E011375A (03-13-01) 18 Variability of ELISA/RIPA u3 samples in 10% negative gray zone that were RIPA pos u2 samples +/- RIPA (index serum pos/neg in ret’d plasma) uSamples ELISA reactive @ index, nonrx ret’d plasma, reactive in f/u Index ELISA Plasma ELISA Follow up ELISA RIPA index RIPA plasma RIPA f/u 0.96, 1.2, 1.1 0.85n/aposposn/a 0.93, 1.0, 0.98 n/a 0.96,0.99, 0.99 posn/apos 0.98,1.05, 1.0 0.23n/aposnegn/a 1.0, 1.1, 1.1 0.34n/aposnegn/a 1.2, 1.3, 1.4 0.72 1.43, 1.39, 1.43 pospospos 94% RIPA pos concordance = 30 ret’d plasma/32 index serum

19. E011375A (03-13-01) 19 Retesting Retrieved Frozen Plasma u51 confirmed pos donors by single to multiple RIPA => retested using ELISA or other research test; 14-31% of low level reactivity is lost; overall 67-84% reactivity retained Retrieved Plasma Results Retrieved Plasma Results Serum+-Total+-Total +34 15 (31%) 4942 7 (14%) 49 -022112 Total 34 (67%) 1751 43 (84%) 851

20. E011375A (03-13-01) 20 Confirmatory: RIPA vs IFA u54 RR samples (from which plasma units available for further testing) from the IND study were sent to Focus for IFA and titers if IFA pos (encouragement of S. Wendel) –24 (44.4%) RIPA pos; 11 IFA pos (20.4%) –16 discordant 14 IFA-/RIPA +14 IFA-/RIPA + 2 IFA+/RIPA- 2 IFA+/RIPA- –Overall agreement 38/54=70.4%38/54=70.4% Index ELISA ELISA (retr’d plasma) RIPA (x2) T. cruzi IgG IFA POS 4.35.2POS1:16 3.44.0POS1:16 5.56.4POS1:64 3.84.1POS1:32 2.72.2POS1:16 4.35.9POS1:128 3.96.3POS>1:256 0.950.47NEG1:16 3.52.4POS1:16 1.20.7NEG1:16* 5.76.1POS>1:256 *also Leish IFA reactive

21. E011375A (03-13-01) 21 Leishmania Testing uAll reactive, RIPA unconfirmed (IR in IND; RR in IVD) sent to Focus for Leishmania IgM/IgG IFA –4 species: L. donovani, L. braziliensis, L. tropica, L. mexicana uIND: 65 IRs => 36 sent for Leish testing (index, ret’d plasma unit and follow up) –31 RIPA neg; 5 RIPA pos (due to low level EIA; S/CO values of 0.93-1.22) uIVD: 104 RRs RIPA neg to date sent for Leish testing (ret’d plasma only)

22. E011375A (03-13-01) 22 Leishmania Testing u4 Leish pos donors (2 IND + 2 IVD): –17.5 yo female FT donor (CA) who was T. cruzi RIPA neg/IFA 1:16 (+) index 1:32 IgG (L. tropica)/plasma (-); plasma 1:16 IgG (L. donovani)/index (-)index 1:32 IgG (L. tropica)/plasma (-); plasma 1:16 IgG (L. donovani)/index (-) Asian American; visited maternal relatives for 2 weeks in 1996 in urban areas of Brazil (mother and grandmother lived in Brazil); no travel risk for L. tropica; follow up testing negative; mother also tested negative for T. cruzi and LeishmaniaAsian American; visited maternal relatives for 2 weeks in 1996 in urban areas of Brazil (mother and grandmother lived in Brazil); no travel risk for L. tropica; follow up testing negative; mother also tested negative for T. cruzi and Leishmania –18 yo male RPT donor (CA) plasma 1:16 IgG (L. donovani)/index (-); 3 total donationsplasma 1:16 IgG (L. donovani)/index (-); 3 total donations No travel risk; follow up testing negativeNo travel risk; follow up testing negative –71 yo female RPT donor (MA); 2 total donations; 1:16 L. braziliensis IgG; no follow up –64 yo female RPT (MI); 19 total donations; 1:20 L. braziliensis IgM and 1:40 L. donovoni IgM; no follow up uLikely all false pos for both T. cruzi by EIA and Leish by IFA uRecommendation; discontinue Leish following BPAC discussion

23. E011375A (03-13-01) 23 Other Testing Results/Procedures uIND study –Follow up of 36 donors; 16 of 32 confirmed pos –1/16 PCR positive; hemoculture pending/negative –Sample handling?? uIVD testing; pending u“Special Protocol” for PCR and hemoculture –Increase sensitivity by having regions initiate processes Pooling EDTA whole blood samples in guanidine/EDTA soln=>PCR (lyses cells and stabilizes kinetoplasts containing mutliple copies of DNA); store at 2-8CPooling EDTA whole blood samples in guanidine/EDTA soln=>PCR (lyses cells and stabilizes kinetoplasts containing mutliple copies of DNA); store at 2-8C Pooling heparinized whole blood samples, centrifuge/removal of plasma (Ab); mix buffy coat and red cells with LIT media; store at 2-8CPooling heparinized whole blood samples, centrifuge/removal of plasma (Ab); mix buffy coat and red cells with LIT media; store at 2-8C 23 samples submitted; 1 PCR pos donor to date (22 neg)23 samples submitted; 1 PCR pos donor to date (22 neg)

24. E011375A (03-13-01) 24 IND Lookback Results u32 confirmed positive donors during IND –17 repeat donors (allogeneic/directed) –140 prior donations => 170 components uOf 170 components –6 whole blood (destroyed) –3 platelets (2 destroyed, 1 transfused) –80 red cells (7 destroyed, 38 transfused, 35 pending) –81 plasma (11 destroyed, 4 transfused, 65 frac, 1 pending) uOf 1, 38 and 4 transfused components –1 platelet recipient => deceased 11 days post transfusion –38 red cell recipients => 15 living, 13 deceased, 10 unknown 11 recipients tested from 8 donors (red cells transfused 7-36 days); all ELISA/RIPA neg (7 also PCR neg)11 recipients tested from 8 donors (red cells transfused 7-36 days); all ELISA/RIPA neg (7 also PCR neg) –4 plasma recipients => deceased

25. E011375A (03-13-01) 25 IVD Lookback Results u41 confirmed positive donors since licensed test implemented –21 repeat donors (allogeneic/directed) –171 prior donations => 108 components (reported to date) uOf 108 components –17 platelets (6 destroyed, 6 transfused, 5 unknown) –49 red cells (1 destroyed, 9 transfused, 39 pending) –41 plasma (1 destroyed, 2 transfused, 27 frac, 11 pending) –1 cryo (1 pending) uOf 6, 9 and 2 transfused components –6 platelet recipients => 5 deceased, 1 living => ELISA and PCR neg (RIPA pending) –9 red cell recipients => 6 deceased, 1 unknown; 2 living => ELISA, RIPA and PCR neg (RIPA pending on one) –2 plasma recipients => 2 living => ELISA neg (RIPA and PCR pending)

26. E011375A (03-13-01) 26 Lookback Summary uRecipients from 10 RIPA pos donors tested uIND –11 red cell recipients tested from 8 donors uIVD –1 platelet recipient tested from 1 donor –2 red cell recipients tested from same donor –2 plasma recipients tested from 1 donor uTotal 16 recipients test negative to date

27. E011375A (03-13-01) 27 Lookback Significance uLookback case summary – platelets/whole blood, considered to be highest risk –1 of 4 Leiby LA and Miami (NEJM 1999) –4 of 9 Kirchhoff Mexico (Transfusion 2006) –0 of 1 Stramer (IVD, unpub) –5 of 14 = 36% uWhy not higher? –Donor must be parasitemic (intermittent) –Parasites must remain viable/infectious in component during processing/handling –Acute infections are most frequently recognized in immunosuppressed patients

28. E011375A (03-13-01) 28 Donor Demographics u61 RR IND ARC donors –32 RIPA pos 14 FT, 18 RPT14 FT, 18 RPT 19 male, 13 female19 male, 13 female 17-84 yo (mean 47 yo; median 50 yo)17-84 yo (mean 47 yo; median 50 yo) 30 allo, 1 auto, 1 platelet pheresis30 allo, 1 auto, 1 platelet pheresis Countries represented (15 donor surveys received); 11/15 endemic areas: Mexico (5), US (4), El Salvador (2), Bolivia (2), Guatemala (1), Argentina (1)Countries represented (15 donor surveys received); 11/15 endemic areas: Mexico (5), US (4), El Salvador (2), Bolivia (2), Guatemala (1), Argentina (1) u213 RR IVD ARC donors –41 RIPA pos 20 FT, 21 RPT20 FT, 21 RPT 26 male, 15 female26 male, 15 female 39 allo, 1 auto, 1 directed (FT male)39 allo, 1 auto, 1 directed (FT male) Countries represented (10 donor surveys received); 9/10 endemic areas: Mexico (4), El Salvador (3), Brazil (1), Bolivia (1), US (1)Countries represented (10 donor surveys received); 9/10 endemic areas: Mexico (4), El Salvador (3), Brazil (1), Bolivia (1), US (1)

29. E011375A (03-13-01) 29 Donor Demographics u52 RR IVD UBS donors –7 + (2) RIPA pos 6 FT, 3 RPT6 FT, 3 RPT 8 males, 1 female8 males, 1 female 7 allo, 1 platelet pheresis, 1 auto7 allo, 1 platelet pheresis, 1 auto Countries represented: Mexico (4), El Salvador (2), Guatemala (1) Venezuela (1) (1 unknown)Countries represented: Mexico (4), El Salvador (2), Guatemala (1) Venezuela (1) (1 unknown) uTotal 328 RR donors – 82 RIPA pos 40 FT, 42 RPT40 FT, 42 RPT 53 males, 29 females53 males, 29 females 76 allo, 2 platelet pheresis, 3 auto, 1 directed76 allo, 2 platelet pheresis, 3 auto, 1 directed Countries represented (N=33): Mexico (13), El Salvador (7), US (5), Bolivia (3), Guatemala (2), Venezuela (1), Argentina (1) Brazil (1)Countries represented (N=33): Mexico (13), El Salvador (7), US (5), Bolivia (3), Guatemala (2), Venezuela (1), Argentina (1) Brazil (1) 28/33 (85%) endemic areas vs 28/28 controls from nonendemic (US + 1 China)28/33 (85%) endemic areas vs 28/28 controls from nonendemic (US + 1 China)

30. E011375A (03-13-01) 30 Gender Age (yrs) Born/Lived Endemic Country Years in Endemic Country Mother Endemic Country Rural Residence Endemic Country Cardiac/GI Symptoms F50 El Salvador 35Y Thatched roof Racing heartbeat F (runner) 60 US only n/aNN Racing heartbeat M50Bolivia6YNNone M57Mexico11YNNone F (vet) 64 US born/lived in Mexico n/aNN Irreg heartbeat M76Mexico29YN Swollen ankles F (LB) 34 El Salvador 29Y Thatched roof; mud floors, bitten Visible neck veins M39Bolivia11 Y (w/cardiac symptoms) Thatched roof; mud floors, bitten None, but yes for maternal family F66Guatemala27Y Thatched roof; mud floors Extra heart sounds F57 US only n/aNNNone F (PCR+) 29Mexico15Y Thatched roof; mud floors None M59Mexico19Y Thatched roof; mud floors, bitten ECG irreg, diff. breathing M42Mexico21Y Mud floors Sticking sens esophagus, stomach

31. E011375A (03-13-01) 31 Gender Age (yrs) Born/Lived Endemic Country Years in Endemic Country Mother Endemic Country Rural Residence Endemic Country Cardiac/GI Symptoms M57Argentina12YN Racing heartbeat F (preg; f/u CDC) 33 El Salvador 18YN ECG irreg; multiple other M75Mexico20Y Dirt floor Shortness breath F (6 children tested neg) 56Mexico19Y Dirt floor Shortness breath M (PCR+) 27 El Salvador 18YN ECG irreg; multiple other F42Mexico33 Y (w/cardiac symptoms) Y Shortness of breath F57Brazil46Y Thatched roof; mud floors Shortness of breath M57 El Salvador 33Y Thatched roof; mud floors ECG irreg; multiple other F33Bolivia3 Y (w/cardiac symptoms) N Shortness of breath M50 US only n/aNn/a Trouble swallowing M28Mexico17Y Dirt floor Racing heartbeat

32. E011375A (03-13-01) 32 Possible Autochthonous Cases u61 yo female (CA) runner in Griffith Park –Griffith park zoo and other animals demonstrated to harbor T. cruzi (6 sp of triatomine bugs in CA; 18 mammal species are reservoirs) –Lived in LA entire life, high quality housing –Travel outside of the US Time share in Cancun where she spends a week 1990-1999; no campingTime share in Cancun where she spends a week 1990-1999; no camping u64 yo female (AZ) retired vet –Lived in rural Mexico where she volunteered as a vet/exposure to infectious material u57 yo female (CA) lives in rural area –Rural area recovering from fire damage –Has many pets and frequently sees raccoons, opossums, skunks on her property and adjacent property, also gardens on her property –Lived in LA entire life, high quality housing –Multiple transfusions in 1971 in CA –Asymptomatic u50 yo male (AR) –Lived in US entire life; one trip to Nassau –Asymptomatic –Only possible “risk” is time spend in Corpus Christi, TX where he slept outside for several weeks

33. E011375A (03-13-01) 33 Summary and Conclusions uPrevalence 1:21,100 –82 RIPA pos donors from 328 RR donors in 17 states during IND/IVD testing of >1.9 million donations –60% from California and Florida uLeishmania –Adds no value; only confusion! uLookback (to date) –60 transfused components from 278 mfg’d from 38 RIPA pos donors –16 recipients tested neg from 10 donors; only 1 platelet recipient uDonor Demographics –28/33 (85%) endemic areas vs 28/28 controls from nonendemic (US + 1 China) –5 possible autochthonous cases; time of infection unknown

34. E011375A (03-13-01) 34 Acknowledgements uAmerican Red Cross –Greg Foster, SSO –David Leiby, HL –Roger Dodd, HL –Ross Herron, West Div –Pamela Kahm, West Div –Norma Espinoza, West Div –Kay Crull, West Div –Muriel Nelson, IT –Mike Savin, IT uBlood Systems Laboratories –Sally Caglioti –Frank Radar –Larry Morgan uOrtho Clinical Diagnostics –Cheryl Vitow –Cindy Ballas –Brian McDonough