Proposed candidate materials and results of the feasibility

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Proposed candidate materials and results of the feasibility studies for a WHO serological reference panel T. cruzi type II Márcia Otani Fundação Pró-Sangue Hemocentro de São Paulo

Raw material Epidemiological data ID Age Gender State Collected date Plasma volume 1 103362946 31 Female Minas Gerais Januaria, for 28 years 2007 -02 -15 200 2 103350016 58 Male Chile Canela, 2007 -03 -16 220 3 103402366 51 Female Pernambuco Buique, for 40 years 2007 -05 -01 180 4 801603 43 Male Hidalgo, México 2008 -02 -08 202 5 801604 48 Male Hidalgo, México 2008 -02 -08 202

4 y 5 3 1 2

Epidemiological Questionnaire 1 2 3 1 -Have you ever heard about Chagas Disease? Y Y Y 2 -Do you know how to recognize a kissing bug? Y Y Y 3 -Have you ever lived in a house constructed of wood and mud? N N Y 4 -Have you had contact with a kissing bug? N Y Y 5 -Have any of your relatives ever had Chagas disease? Y N Y 6 -Did you live in the same house with a relative who is positive for Chagas Disease? N N Y 7 -Do you know any case of Chagas Disease in the region where you have born or where you have lived? N Y Y 8 -Have you ever received blood transfusion? N N N 9 -Have you ever been to any Chagas endemic region? N N N 10 -Have you ever had any clinical symptoms for Chagas Disease ? N N Y 11 -Are you under medical treatment ? N N Y 12 -Have you ever done an electrocardiogram (EKG)? Y Y Y

Raw material - Reactivity Chagatek OD/CO Bioschile OD/CO Imuno cruzi ID Elisa cruzi OD/CO Titre Used volume 103362946 9. 36 6. 19 4. 02 1/160 80 ml 200 ml Epd: 1/128 Epd: 1/32 Epd: 1/128 103350016 8. 31 6. 37 4. 24 1/320 120 ml 220 ml Epd: 1/128 Epd: 1/64 Epd: 1/128 103402366 7. 97 5. 21 3. 35 1/160 60 ml 180 ml Epd: 1/64 Epd: 1/32 Epd: 1/64 801603 4. 71 5. 03 3. 15 1/160 2 ml 202 ml Epd: 1/16 Epd: 1/32 801604 5. 37 5. 10 3. 25 1/320 2 ml 202 ml Epd: 1/64 Epd: 1/128 Epd = end point dilution (OD/CO) > 1 Prototype = 260 ml of positive + 1240 ml of negative plasma from 7 donors units. (4. 8 times diluted. )

Raw material - serial dilution OD/CO ratio = Prototype = M 2

Clinical Sensitivity and Specificity

M 2 Pure 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Elisa cruzi 4, 962 4, 792 4, 677 3, 758 2, 612 1, 896 1, 362 0, 923 0, 581 0, 435 OD/CO ratio Chagatek 5, 406 4, 774 3, 619 2, 703 1, 910 1, 297 0, 865 0, 542 0, 477 0, 413 Bioschile 3, 143 3, 042 2, 803 2, 287 2, 020 1, 616 1, 309 1, 046 0, 768 0, 579 IFI 1/80 1/40 1/20 Negative Negative

Prototype OD/CO ratio

Evaluating the Prototype • EQAS to evaluate performance in loco. • Send 1. 8 ml of the prototype serum to 25 reference centers in 16 countries in LA. • Included 4 other non diluted not pooled samples to the same panel. • Sep 2007 (OPS 0207), Apr 2008 (OPS 0108) and Sep 2008 (OPS 0208).

Cut o Data from Latin America Reference Centers – PAHO Regional EQAS

Sep 2007 Apr 2008 Sep 2008 Data from Latin America Reference Centers – PAHO Regional EQAS

Sep 07 Apr 2008 Sep 08 Data from Latin America Reference Centers – PAHO Regional EQAS

Data from Latin America Reference Centers – PAHO Regional EQAS

Data from Latin America Reference Centers PAHO Regional EQAS

Volume Prototype volume available = 1. 4 liters 2 times dilution = 2. 8 liters It is possible to obtain a total of 6 liters Units used m. L available m. L 1 80 120 2 120 100 3 60 120 4 2 200 5 2 200 Mexico Units M 2 volume available = 400 m. L 4 times dilution = 1. 6 liters

Conclusion • The mixture of 3 positive samples (from south of Latin America) diluted 4. 8 times in negative samples have produced a prototype that presented stability at – 20 ºC. • We need to define the reactivity of the cut off value. Here we have used the dilution that is IIF = 1/20. • Reactivity of the prototype diluted 2 times were 2 to 4 times de cutoff value in most of the tests evaluated and the IIF = 1/20. • Perhaps we should submit this prototype to RIPA, Western Blot and Abbott Immunoassay.

1 22 3 4 5 6 Data from Latin America Reference Centers PAHO Regional EQAS 7 8 9 10 11 12 13

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