1. PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR SEROLOGICAL REFERENCE RESULTS OF THE FEASIBILITY STUDIES WITH SERA FROM TC I REGION RESULTS OF THE FEASIBILITY STUDIES WITH SERA FROM TC I REGION DR. CARMEN GUZMAN BRACHO Instituto de Diagnóstico y Referencia Epidemiológicos

2. PROJECT ORGANIZATION WHO, JULY 2007. –Develop a reference panel. –Commitment to collect and evaluate sera from TcI region accepted. National Center of Blood Transfusion, Aug 2007. –Selection of a Blood Transfusion Center. State Center of Blood Transfusion in Hidalgo, Sep 2007. –Participate in the writing of the Protocol, responsibilities and consent form. –Make contact with donors in home towns. –Transfer donors to Pachuca City for blood collections. –Collect Plasma units. Institute of Epidemiological Diagnosis and Reference –Coordinate the Project. –Select blood donors. –Evaluate individual sera samples and mixtures. –Analyze results. –Deliver positive and negative plasma units to Fundacao Pró-Sangre Hemocentro de Sao Paulo, Jun 2008.

3. MAIN CONDITIONS Mexican donors. Final volume: 1.5 – 3.0 L Since ethical and operative problems with a single donor raise, it is suggested that a pool of positive sera is more realistic. Negative sera are necessary to dilute reactive samples. All samples must be negative to viral markers (HIV, HBV, HCV) and syphilis.

4. HIDALGO STATE LABORATORY OF PUBLIC HEALTH HIDALGO STATE CENTER OF BLOOD TRANSFUSION SELECTION OF CANDIDATES DATA BASE CONFIRMED CASES –SCREENING TEST FOR POSITIVES ELISA (BIOKIT) –CONFIRMATION TESTS OF POSITIVES ELISA recombinant (BIOKIT) ELISA crude (INTERBIOL) HAI (INTERBIOL) IFI (InDRE)

5. INITIAL RESULTS FOR SELECTED CANDIDATES 10 mL of serum from 10 candidates were collected to confirm tests results

6. FINAL RESULTS FOR SELECTED CANDIDATES According to the OD/CO index, 6 serum samples were selected to evaluate reactivity of mixtures ReactivityOD/CO LOW2.81 – 3.55 MEDIUM4.19 – 5.81 HIGH7.74 – 8.39

7. COMPOSITION OF MIXTURES Individual sera data Mixtures data

8. REACTIVITY OF THE MIXTURES TWO REPLICATES PER PLATE TWO RUNNINGS REACTIVITY IN ELISA MEXICAN CRUDE ANTIGEN WITH SERA MIXTURES FROMT. cruzi INFECTED MEXICAN INDIVIDUALS 0 1 2 3 4 5 6 7 8 12345678910 DILUTION OD/CO MEZCLA1MEZCLA2MEZCLA3MEZCLA4 Low reactivity

9. REACTIVITY OF SERA MIXTURES BY DILUTION WIDTH CI 95% = 0.68 WIDTH CI 95% = 1.37

10. WHEN ADDING ANOTHER SERUM TO THE MIXTURE….. Mixture = 2 high + 1 medium + 1 low

11. MEXICAN SERA BELONG TO T. cruzi I 1.54 of 56 isolates studied coming from 8 provinces of the Mexican Republic were TcI. 2.Given the prevalence of seropositive individuals and of disease cases in the country, T. cruzi I stocks can be considered the primary agents of Chagas’ disease in Mexico –Bosseno et al. Predominance of Trypanosoma cruzi lineage I in Mexico. J Clin Microbiol. 2002 feb;40(2):627-32. –Sánchez-Guillén et al. Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity.Mem Inst Oswaldo Cruz. 2006 Sep;101(6):585-90. PROTOCOL NUMBERREACTIVITY DATA FOR TRACEABILITY CHAIN No InDREFOLIO CETSUNIT NUMBERAGE 3HIGH5731130380160448 4MEDIUM155231780160343 SERA SENT TO BRAZIL

12. Complementary tests

13. CONCLUSIONS Mixture 1 and 2 turned out to be the best. Optimal dilution was found at 1:10. We got 200 mL of plasma from both donors included in mixture 2. Plasma units were sent to Brazil (M. Otani) coded 801603 and 801604. Finally, 10 plasma units of seronegative individuals to prepare final dilution were delivered to Brazil.

14. MANY THANKS!