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 IDAgeGenderStateCollected date Plasma volume Female Minas Gerais Januaria, for 28 years MaleChile Canela, FemalePernambuco Buique, for 40 years MaleHidalgo, M é xico MaleHidalgo, M é xico

y 5 3

123 1-Have you ever heard about Chagas Disease? YYY 2-Do you know how to recognize a kissing bug? YYY 3-Have you ever lived in a house constructed of wood and mud? NNY 4-Have you had contact with a kissing bug? NYY 5-Have any of your relatives ever had Chagas disease? YNY 6-Did you live in the same house with a relative who is positive for Chagas Disease? NNY 7-Do you know any case of Chagas Disease in the region where you have born or where you have lived? NYY 8-Have you ever received blood transfusion? NNN 9-Have you ever been to any Chagas endemic region? NNN 10-Have you ever had any clinical symptoms for Chagas Disease ? NNY 11-Are you under medical treatment ? NNY 12-Have you ever done an electrocardiogram (EKG)? YYY Epidemiological Questionnaire

Raw material - Reactivity ID Elisa cruzi OD/CO Chagatek OD/CO Bioschile OD/CO Imuno cruzi Titre Used volume ml 9.36 Epd: 1/ Epd: 1/ Epd: 1/128 1/16080 ml ml 8.31 Epd: 1/ Epd: 1/ Epd: 1/128 1/ ml ml 7.97 Epd: 1/ Epd: 1/ Epd: 1/64 1/16060 ml ml 4.71 Epd: 1/ Epd: 1/ Epd: 1/32 1/1602 ml ml 5.37 Epd: 1/ Epd: 1/ Epd: 1/128 1/3202 ml Prototype = 260ml of positive ml of negative plasma from 7 donors units. (4.8 times diluted.) Epd = end point dilution (OD/CO) > 1

Raw material - serial dilution OD/CO ratio = Prototype = M2

Clinical Sensitivity and Specificity

Elisa cruziChagatekBioschileIFI Pure4,9625,4063,143 1/80 1/24,7924,7743,042 1/40 1/44,6773,6192,803 1/20 1/83,7582,7032,287Negative 1/162,6121,9102,020Negative 1/321,896 1,297 1,616Negative 1/64 1,362 0,8651,309Negative 1/1280,9230,542 1,046 Negative 1/2560,5810,4770,768Negative 1/5120,4350,4130,579Negative M2 OD/CO ratio

Prototype

Evaluating the Prototype EQAS to evaluate performance in loco. Send 1.8ml 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 (OPS0207), Apr 2008 (OPS0108) and Sep 2008 (OPS0208).

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

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 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

Prototype volume available = 1.4 liters 2 times dilution = 2.8 liters It is possible to obtain a total of 6 liters Unitsused mL available mL Volume Mexico Units M2 volume available = 400 mL 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.

Data from Latin America Reference Centers PAHO Regional EQAS