Chagas Tests: Development and Standardization

1. Chagas Tests:Development and Standardization Gláucia Paranhos-Baccalà Emerging Pathogens Department bioMérieux International Biological Reference Preparations for Chagas Diagnostic Test WHO – Geneva January, 26th and 28th 2009

2. ALGORITHMIC FOR CHAGAS DISEASE DIAGNOSIS FOR CLINICAL LABORATORIES Clinical signs symptoms or epidemiological evidence of Chagas disease Acute Chronic Parasite detection Serology Reference Centers Serology two tests – WHO recommendation Clinical Laboratories - HA IFA ELISA Positive “Chagas Disease” Negative Negative Inconclusive Positive PCR Reference Centers “Non Chagas disease” “Chagas disease” Repeat Inconclusive Confirmatory test PCR or WB

3. Chagas Tests: Diagnosis Eu Market Blood Screening • DirectTransmission through transfusions, by organ transplantation or laboratory accident Adequate cost tests High Sensitivity Good Specificity Needs for Chagas test Antibody detection

4. Epidemiology In France: In 3 years 14 cases against 5 in 30 years. • Number of migrants from Latin or Central America not well known in EU Real emergence or cluster effect?

5. Chagas Tests in EU: Antibody Detection

6. T. cruzi antigens preparations for diagnostic tests based on Ab detection • Crude epimastigote extracts • Epimastigote alcaline extractions • Purified epimastigotes fractions, … Antigen preparation could present variations Batch to batch changes Lost of some epimastigotes epitopes or strain specific?

7. Vironostika® bioMérieux

8. 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: citrate, heparine, oxalate ou EDTA. Epimastigotes antigens Results: 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 (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. The Chagas disease immunoassay standardization test for antibody detection at bioMérieux • is 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 panelis: 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 bioMérieux • Antigen 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 bioMérieux Human 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. Stop solution : Sulphur acid 2N spectophometer + Substrat : Tetramethylbenzidine TMB + Ab anti IgG humaine*Peroxydase Ab anti T. cruzi Ag from T. cruzi The Chagas disease immunoassay standardization test for antibody detection at bioMérieux • Interpretation: 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.

14. Confirmatory tests for Chagas Disease Blood Donors and co-endemic areas Western Blot: TESA cruzi WB (bioMérieux) • - Trypomastigote excreted-secreted antigens

16. TESAcruzi - Parameters Serum panel bioMérieux + serum from blood banks

17. APPLICATIONS

18. SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILDREN N = 100,000 Blood samples on filter paper Central Laboratory ELISA - IIF Reference Laboratory 10% of blood samples + Reagents + Inconclusives IIF, ELISA and IHA Reagents or inconclusives – TESA cruzi Results Reagents samples Identification of childen + contactants Venous Blood

19. SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILDREN 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%)

20. USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION 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 from Ocobaya – South Yungas - La Paz IIF IgG –IMUNOCRUZI IHA – HEMACRUZI ELISA – BIOELISACRUZI IIF IgG Leishmania ssp TESAcruzi as confirmatory test

21. USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIA SEROLOGICAL TESTS IN L. DONOVANI T. CRUZI CHAGASII ELISA IIF IHA IIF 22 REAGENT 21 22 18 INCONCLUSIVE 1 115 NON REAGENT 115 115 115

22. USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIA SEROLOGICAL TESTS IN T. CRUZI L. DONOVANI CHAGASII ELISA IIF IHA TESACRUZI IIF REAGENT 22 21 22 21 18 INCONCLUSIVE 1 4 NON REAGENT 115 115 115 116 115

23. Thank you! Emerging Pathogens Department bioMérieux SA Tour CERVI IFR 128 BioSciences Lyon Gerland

24. CONGENITAL CHAGAS DISEASE ALGORITHMIC MOTHER SEROLOGY REAGENT SEARCH T.CRUZI IN NB TWO SAMPLES DURING THE FIRST MONTH NEGATIVE POSITIVE SEARCH ANTI T.CRUZI IN NB. TWO SAMPLES DURING THE FIRST MONTH SEROLOGY DURING THE FIRST 6 MONTHS TREATMENT Reagent Non Reagent PILOT PROGRAM IN TUCUMÁN, ARGENTINE PRENATAL DIAGNOSIS TREATMENT END THE FOLLOW UP REMEMBER: WITH EARLY DIAGNOSIS AND PROMPT TREATMENT OF CONGENITAL CHAGAS DISEASE THE PROBABILITY OF CURE IN NEWBORN IS 100%

25. Vironostika® Kit Composition: