Chagas Tests-Development and Standardization.ppt
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1、Chagas Tests: Development and Standardization,Glucia Paranhos-Baccal Emerging Pathogens Department bioMrieuxInternational Biological Reference Preparations for Chagas Diagnostic TestWHO Geneva January, 26th and 28th 2009,ALGORITHMIC FOR CHAGAS DISEASE DIAGNOSIS FOR CLINICAL LABORATORIES,Chagas Tests
2、: Diagnosis,Eu Market Blood ScreeningDirect Transmission through transfusions, by organ transplantation or laboratory accident,Adequate cost testsHigh SensitivityGood Specificity,Needs for Chagas test,Antibody detection,Epidemiology,In France:In 3 years 14 cases against 5 in 30 years.Number of migra
3、nts from Latin or Central America not well known in EUReal emergence or cluster effect?,Chagas Tests in EU: Antibody Detection,Crude epimastigote extractsEpimastigote alcaline extractionsPurified epimastigotes fractions, ,Antigen preparation could present variationsBatch to batch changesLost of some
4、 epimastigotes epitopes or strain specific?,T. cruzi antigens preparations for diagnostic tests based on Ab detection,bioMrieux,Kit Elisa Cruzi: for antibody detection,Kit: ELISA CRUZI Ref: 35117 Number of tests: 192 tests 2 microplates of 96 tests: 12 barrettes de 8 wells.Human serum or plasma: cit
5、rate, heparine, oxalate ou EDTA.Epimastigotes antigensResults: Positif / Doubts / Negatif.adsorbance index : 1 Retest 0,8 Timing: 70 minutes of incubation. Controls : 2 pos Controls and 3 neg Controls in each test.If necessary: one well reserved for the R3 diluant solution to callibrate the reader (
6、blanc).,Chagas Test Standardization for antibody detection,The following steps are used for standardization:Sera panel set-up (negative and positive controls);Antigen production and titers: batch to batch;anti-IgG human conjugate;Cut-off;The test interpretation.,is based on the Sensitivity, Specific
7、ity and Reproducibility with the following steps listed bellow:Sera panel set-up: A positive and negative sera panel with samples from a serological screening of blood donors in an hemocenter service is obtained-positive sera panel is: Each lot of Chagas kit is tested with about 200 positive sera fr
8、om chagasic patients of different endemic regions from Brazil with varying degrees of reactivity. The positive panel was evaluated in at least in two reference tests as Immunofluorescence assay, indirect hemagglutination. The titers of each positive serum included in the panel was previously determi
9、ned. BioMrieux has a positive sera panel (n=40) representing others endemic areas located at Argentina, Bolivia, Venezuela and Mexico. This precious panel is used to evaluate the final lot. negative sera panel is: Each lot of Chagas kit is tested with about 2000 negative samples and positive for oth
10、ers pathologies as: hepatitis, malaria, syphilis, lupus, HIV, HCV, HTLV-I.-potential cross-reactivity panel is: Each lot of Chagas kit is tested with about 80 Leishmaniasis sera samples.,The Chagas disease immunoassay standardization test for antibody detection at bioMrieux,This test has been calibr
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