1. WHO Consultation Meeting 27-28 January 2009 Quality Control of Chagas diagnostics immunoassays: Assay characteristics and manufacturers reference panels. Gustavo A. Capriotti, Biochemist, R&D Manager

2. WHO Consultation Meeting 27-28 January 2009 Antigens used in Conventional Tests for T. cruzi infection: Whole Extracts or Semipurified Fractions of parasite (epimastigote) Purified Proteins Synthetic Peptides Recombinant Antigens

3. WHO Consultation Meeting 27-28 January 2009 Conventional serological tests Indirect hemagglutination (IHA) Parasite lysate ELISA Parasite lysate Recombinant antigens

4. WHO Consultation Meeting 27-28 January 2009 Conventional serological tests Performance Method Sensitivity Specificity IHA > 97% > 98% ELISA > 98% > 99%

5. WHO Consultation Meeting 27-28 January 2009 Kits Chagatest HAI Chagatest HAI screening A-V Chagatest ELISA (lysate) Chagatest ELISA recombinante v 3.0 (FDA 510k and CE)

6. WHO Consultation Meeting 27-28 January 2009 Kits New Chagatest ELISA recombinante v.4.0 (approved in LA/CE market) Chagatest ELISA recombinante for dried blood spot samples (approved in RA) Rapid test (in development) Colorimetric PCR (in development) Quantitative PCR (to start development this year)

7. WHO Consultation Meeting 27-28 January 2009 Chagatest ELISA recombinante v.4.0 Recombinant Antigens Highly sensitive and specific mixture Proteins present in the trypomastigote stage Proteins preserved in different parasite strains/linages System with six recombinant Ags SAPA, Ag1, Ag2, Ag13, Ag30, Ag36

8. WHO Consultation Meeting 27-28 January 2009 Chagatest ELISA recombinante v.4.0 Recombinant Antigens Chronic Ag1 Ag2 Ag30 Ag13 Ag36 Ag2 Ag13 SAPA Ag13 Ag 36 SAPA Acute Congenital

9. WHO Consultation Meeting 27-28 January 2009 SENSITIVITY International performance panels PANELSORIGIN DETECTED REACTIVE SAMPLES SENSITIVITY % PMT 201BBI, USA14/14100% PP 0404Q Panel, Brazil16/16100% PP 0405Q Panel, Brazil16/16100% PP 0406Q Panel, Brazil 16/16 100% Reactive sample panels Pediatric samplesEndemic area100/100100% Pediatric samplesRosario115/11699.14% Chagatest ELISA recombinante v.4.0

10. WHO Consultation Meeting 27-28 January 2009 SPECIFICITY SAMPLESSPECIFICITY % 1192 Blood Bank samples99.66 % 477 samples from different Health Centers99.57 % 474 samples from high prevalence population98.30 % 491 samples with different clinical conditions98.37 % Chagatest ELISA recombinante v.4.0

11. WHO Consultation Meeting 27-28 January 2009 Chagatest ELISA recombinante v.4.0 Interference with other pathologies Parasites Leishmaniasis1/10 Amebiasis0/7 Toxocariasis0/6 Toxoplasmosis0/5 Hydatidosis0/5 Teniasis0/2 Infectious disease Hepatitis B0/22 Hepatitis C0/28 Syphilis0/19 HIV0/25 Other pathologies Lymphoma0/1 Myeloma0/1 Lung tuberculosis0/4 Autoimmune disorders 1/4

12. WHO Consultation Meeting 27-28 January 2009 Standardized system uses a perfectly defined antigen composition. Antigens expressed in the infected trypomastigote stage of the parasite. Highly preserved antigens in different strains of the parasite. SAPA antigen, acute and congenital infection marker. Recombinant antigens Advantages in serological diagnosis

13. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? - At production level Recombinant antigens Recombinant antigens well characterized Perfectly defined antigen mix Parasitic Lysate Characterized lysate by WB Parasite culture under strict growth conditions. Well defined WCB & MCB

14. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? The recombinant antigens are tested separately using an ELISA technique with an internal panel of 9 positive samples specific for each antigen and 4 negative samples.

15. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? ELISAs Calibration 10 weak positive samples (IP between 1.0 and 2.0) 10 medium positive samples (IP between 2.0 and 4.0) 10 strong positive samples (IP < 4.0) Note: samples diluted in negative or bovine serum may be used 20 negative samples

16. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? ELISAs Calibration A titer verifying the IP coefficient within a range of 0.9 – 1.2 must be selected. In addition, all negative samples must yield negative results.

17. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? ELISAs Titer verification - Internal panel of reactive samples evaluation: internal panel of 32 samples including weak, medium and strong. Acceptance criteria: the individual IP coefficient of each sample must be within a range of 0.9 – 1.2

18. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? ELISAs Titer verification -Specificity: 200 sera / fresh plasmas Acceptance criteria: > 99%. If < 99%, the conjugate is diluted and retested.

19. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? ELISAs Final test -Internal panel: 2 positive control sera, 3 negative control sera, 6 weak positive, 10 medium and 10 strong samples. -Commercial panels: Chagas performance panel (QPanel, Brazil); BBI Panel

20. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? Hemagglutination -Titration of red blood cells sensitization. The titers are tested with an internal panel of 15 positive sera. Some of them diluted. Panel with 10 negative sera. Acceptance criteria: a titer where diluted sera match background titer of each sample is selected. Negative sera must yield negative results.

21. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? Hemagglutination Lot preparation - Internal panel of 20 positive sera - 100 negative sera

22. WHO Consultation Meeting 27-28 January 2009 How do we ensure Standardization? Hemagglutination Final verification - Chagas performance panel (QPanel) - BBI panel

23. WHO Consultation Meeting 27-28 January 2009 Reference Panel Considerations (proposal 2007): Sample: SERUM (not plasma) Number: 10-12 (representing a range of reactivities from non- reactive to strongly reactive) Not inactivated by heat (preferably aseptically filtered, photoinactivation, UV or irradiation) Preferable without preservatives Representative from different disease stages and geographic regions Selection made based on: IHA, ELISA, Immunoblot For analytical sensitivity: diluted samples can be used For clinical sensitivity: undiluted samples

24. WHO Consultation Meeting 27-28 January 2009 International Biological Reference Preparation for Chagas (2009) A known reactivity standard is required to yield consistency lot to lot To have a primary Standard of 2 reactive sera, as suggested, seems a good alternative. This will allow to determine the analytical sensitivity for each lot, as being used for other international standards.