Presentation on theme: "Chagas Tests: Development and Standardization"— Presentation transcript:
1 Chagas Tests: Development and Standardization Gláucia Paranhos-BaccalàEmerging Pathogens DepartmentbioMérieuxInternational Biological Reference Preparations for Chagas Diagnostic TestWHO – Geneva January, 26th and 28th 2009
2 ALGORITHMIC FOR CHAGAS DISEASE DIAGNOSIS FOR CLINICAL LABORATORIES Clinical signs symptoms or epidemiological evidence of Chagas diseaseAcuteChronicParasite detectionSerologyReference CentersPositive“Chagas Disease”NegativePCRSerology two tests – WHO recommendationClinical Laboratories - HA IFA ELISAInconclusive“Non Chagas disease”“Chagas disease”RepeatConfirmatory testPCR or WB
3 Chagas Tests: Diagnosis Eu Market Blood ScreeningDirect Transmission through transfusions, by organ transplantation or laboratory accidentAdequate cost testsHigh SensitivityGood SpecificityNeeds for Chagas testAntibody detection
4 Epidemiology In France: In 3 years 14 cases against 5 in 30 years. Number of migrants from Latin or Central Americanot well known in EUReal emergence or cluster effect?
6 T. cruzi antigens preparations for diagnostic tests based on Ab detection Crude epimastigote extractsEpimastigote alcaline extractionsPurified epimastigotes fractions, …Antigen preparation could present variationsBatch to batch changesLost of some epimastigotes epitopes or strain specific?
8 Kit Elisa Cruzi: for antibody detection Ref:Number of tests: 192 tests2 microplates of 96 tests: barrettes de 8 wells.Human serum or plasma: citrate, heparine, oxalate ou EDTA.Epimastigotes antigensResults: Positif / Doubts / Negatif.adsorbance index : < Retest < 0,8Timing: 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 (blanc).
9 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.
10 This test has been calibrated against in house standards. test. The Chagas disease immunoassay standardization testfor antibody detection at bioMérieuxis based on the Sensitivity, Specificity 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 from 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 determined.BioMérieux 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 others 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.This test has been calibrated against in house standards. test.
11 The Chagas disease immunoassay standardization test for antibody detection at bioMérieuxAntigen titers: bioMérieux 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 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 serum 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.
12 The Chagas disease immunoassay standardization test for antibody detection at bioMérieuxHuman conjugate: 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 cut-off: The cut 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.
13 The Chagas disease immunoassay standardization test for antibody detection at bioMérieuxInterpretation: The test interpretation in given 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.Ag from T. cruziAb anti T. cruziAb anti IgG humaine*PeroxydaseSubstrat : Tetramethylbenzidine TMB+Stop solution : Sulphur acid 2Nspectophometer
14 Confirmatory tests for Chagas Disease Blood Donors and co-endemic areasWestern Blot: TESA cruzi WB (bioMérieux)- Trypomastigote excreted-secreted antigens
18 SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILDREN Blood samples on filter paperCentral LaboratoryELISA - IIFReference Laboratory10% of blood samples + Reagents + InconclusivesIIF, ELISA and IHAReagents or inconclusives – TESA cruziResultsReagents samplesIdentification of childen + contactantsVenous Blood
19 SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILDREN Central Laboratory – 80,000 blood samplesELISA - IIFReference Laboratory – 8,78810% of blood samples + Reagents + InconclusivesIndirect Immunofluorescence -1/ (3.6%)ELISA (1.9%)TESAcruzi – 77 (0.9%)
20 CHAGAS DISEASE AND LEISHMANIASIS ARE COENDEMIC IN SOME AREAS USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIA40.1 % 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 AREASN= 137 serum samples from Ocobaya – South Yungas - La PazIIF IgG –IMUNOCRUZIIHA – HEMACRUZIELISA – BIOELISACRUZIIIF IgG Leishmania sspTESAcruzi as confirmatory test
21 USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIASEROLOGICAL TESTS INT. CRUZIL. DONOVANICHAGASIIELISAIIFIHAREAGENT212218INCONCLUSIVE1NON REAGENT115
22 USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIASEROLOGICAL TESTS INT. CRUZIL. DONOVANICHAGASIIELISAIIFIHATESACRUZIREAGENT222118INCONCLUSIVE14NON REAGENT115116
23 Thank you! Emerging Pathogens Department bioMérieux SA Tour CERVI IFR 128 BioSciences Lyon Gerland
24 SEARCH ANTI T.CRUZI IN NB. PILOT PROGRAM IN TUCUMÁN, ARGENTINE CONGENITAL CHAGAS DISEASE ALGORITHMICMOTHER SEROLOGYREAGENTReagentSEROLOGY DURINGTHE FIRST 6 MONTHSSEARCH T.CRUZI IN NBTWO SAMPLES DURING THEFIRST MONTHTREATMENTEND THEFOLLOW UPNon ReagentPOSITIVESEARCH ANTI T.CRUZI IN NB.NEGATIVEPILOT PROGRAM IN TUCUMÁN, ARGENTINEPRENATAL DIAGNOSISREMEMBER: WITH EARLY DIAGNOSIS AND PROMPT TREATMENT OFCONGENITAL CHAGAS DISEASE THE PROBABILITY OF CURE INNEWBORN IS 100%