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    Chagas Tests-Development and Standardization.ppt

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    Chagas Tests-Development and Standardization.ppt

    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

    11、ated against in house standards. test.,Antigen titers: bioMrieux used as antigen a crude extract obtained from alkaline extraction from T. cruzi II epimastigotes forms. The total protein of the parasite is estimated by a colorimetric method in each lot of the T. cruzi production. Evaluation with lot

    12、 to lot antigen production in terms of title of antigen dilution with the Chagas panel sera and a target value obtained from an already tested lot. The stability of the antigen production is also evaluated during the time and temperature. The antigen preparation should present NO variation between s

    13、erum or lot to lot. The cut-off is evaluated for each antigen production after stability and must be have a variation with less than 10%. At each antigen preparation three pilot lots are produced.,The Chagas disease immunoassay standardization test for antibody detection at bioMrieux,Human conjugate

    14、: The mouse monoclonal anti-human IgG antibodies conjugated to enzyme is commercially acquired and analyzed in each lot to lot to assure the performance of the Chagas disease kit production.,The Chagas disease immunoassay standardization test for antibody detection at bioMrieux,The cut-off: The cut

    15、off is calculated from results obtained from negative sera panel. Negative sera panel should included positive sera from possible cross-reactive infections. The exact cut-off and indeterminate values are determined by by Roc curve and Youden coefficient.,Interpretation: The test interpretation in gi

    16、ven in the instructions. The instructions is based on the ratio: optical density/cut off: reactivity index: positive results for 1 and negative results for 1.,The Chagas disease immunoassay standardization test for antibody detection at bioMrieux,Blood Donors and co-endemic areas,Confirmatory tests

    17、for Chagas Disease,Western Blot: TESA cruzi WB (bioMrieux) - Trypomastigote excreted-secreted antigens,TESAcruzi - Parameters Serum panel bioMrieux + serum from blood banks,APPLICATIONS,SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILDREN,Blood samples on filter paper,N = 100,000,C

    18、entral Laboratory ELISA - IIF,Reference Laboratory 10% of blood samples + Reagents + Inconclusives IIF, ELISA and IHA Reagents or inconclusives TESA cruzi,Results,Identification of childen + contactants,Reagents samples,Venous Blood,SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILD

    19、REN,Central Laboratory 80,000 blood samples ELISA - IIF,Reference Laboratory 8,788 10% of blood samples + Reagents + Inconclusives,Indirect Immunofluorescence -1/40 - 313 (3.6%) ELISA - 166 (1.9%) TESAcruzi 77 (0.9%),USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFE

    20、CTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIA,40.1 % of Bolivia population lives in areas with high presence of the vector and 40% of them are infected with T. cruzi (24% cardiac lesions and 16% digestive forms),CHAGAS DISEASE AND LEISHMANIASIS ARE COENDEMIC IN SOME AREAS,N= 137 serum samples f

    21、rom Ocobaya South Yungas - La Paz,IIF IgG IMUNOCRUZI IHA HEMACRUZI ELISA BIOELISACRUZI IIF IgG Leishmania ssp TESAcruzi as confirmatory test,Thank you!,Emerging Pathogens DepartmentbioMrieux SA Tour CERVI IFR 128 BioSciences Lyon Gerland,REMEMBER: WITH EARLY DIAGNOSIS AND PROMPT TREATMENT OF CONGENITAL CHAGAS DISEASE THE PROBABILITY OF CURE IN NEWBORN IS 100%,Kit Composition:,


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