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PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR SEROLOGICAL REFERENCE

PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR SEROLOGICAL REFERENCE. RESULTS OF THE FEASIBILITY STUDIES WITH SERA FROM TC I REGION. DR. CARMEN GUZMAN BRACHO Instituto de Diagnóstico y Referencia Epidemiológicos. PROJECT ORGANIZATION. WHO, JULY 2007. Develop a reference panel.

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PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR SEROLOGICAL REFERENCE

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  1. PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR SEROLOGICAL REFERENCE 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. 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) HIDALGO STATE CENTER OF BLOOD TRANSFUSION HIDALGO STATE LABORATORY OF PUBLIC HEALTH

  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

  7. COMPOSITION OF MIXTURES Individual sera data Mixtures data

  8. REACTIVITY OF THE MIXTURES REACTIVITY IN ELISA MEXICAN CRUDE ANTIGEN WITH SERA MIXTURES FROM T. cruzi INFECTED MEXICAN INDIVIDUALS 8 7 6 OD/CO 5 • TWO REPLICATES PER PLATE • TWO RUNNINGS 4 3 Low reactivity 2 1 0 1 2 3 4 5 6 7 8 9 10 DILUTION MEZCLA1 MEZCLA2 MEZCLA3 MEZCLA4

  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 • 54 of 56 isolates studied coming from 8 provinces of the Mexican Republic were TcI. • 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. 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!

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