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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

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

Chagas Tests: Diagnosis

Eu Market Blood Screening

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Direct Transmission through transfusions

, by organ transplantation or laboratory accident

Needs for Chagas test

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Antibody detection

Adequate cost tests High Sensitivity Good Specificity

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Epidemiology

In France: In 3 years against 14 cases 5 in 30 years.

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Number of migrants from Latin or Central America not well known in EU Real emergence or cluster effect?

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Chagas Tests in EU:

Antibody Detection * * Company Lemos Ortho Ingen / Adaltis Dade Behring Name ELISA cruzi® Bioelisa Chagas® Chagatek Elisa®

T

.cruzi ELISA Test System-1 ® EIAgen T.cruzi Ab ® ELISA Novagnost® Serological test Total parasite extract Recombinant antigens Total parasite extract Total parasite extract Total parasite extract Total parasite extract

T. cruzi antigens preparations for diagnostic tests based on Ab detection

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Crude epimastigote extracts

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Epimastigote alcaline extractions

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Purified epimastigotes fractions, …

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Antigen preparation could present variations Batch to batch changes Lost of some epimastigotes epitopes or strain specific?

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bioMérieux

Vironostika®

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Kit Elisa Cruzi: for antibody detection

Kit: Ref: ELISA CRUZI 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: adsorbance index : Positif / Doubts / Negatif .

1 < Retest < 0,8 Timing: Controls : 70 minutes of incubation.

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).

Chagas Test Standardization for antibody detection

The following steps are used for standardization:

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Sera panel set-up (negative and positive controls);

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Antigen production and titers: batch to batch;

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anti-IgG human conjugate;

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Cut-off;

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The test interpretation.

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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:

1.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.

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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

sera samples.

is: Each lot of Chagas kit is tested with about 80 Leishmaniasis

This test has been calibrated against in house standards. test.

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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.

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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.

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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.

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.

Stop solution : Sulphur acid 2N + spectophometer Substrat : Tetramethylbenzidine TMB + Ab anti IgG humaine* Peroxydase Ab anti T. cruzi Ag from T. cruzi

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Confirmatory tests for Chagas Disease

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Blood Donors and co-endemic areas Western Blot: TESA cruzi WB (bioMérieux) - Trypomastigote excreted-secreted antigens

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Trypomastigote Excreted-secreted antigens TESA = TRYPOMASTIGOTE EXCRETED SECRETED ANTIGENS

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TESAcruzi - Parameters Serum panel bioMérieux + serum from blood banks Sensitivity Specificity PPV PNV Efficiency Kappa index N positives N negatives 100% 99,5% 98,7% 100% 99% 0,99 795 1946

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APPLICATIONS

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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

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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%)

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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

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USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIA SEROLOGICAL TESTS IN REAGENT INCONCLUSIVE NON REAGENT

T. CRUZI

ELISA 22 115 IIF 21 1 115 IHA 22 115

L. DONOVANI CHAGASII

IIF 18 115

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USE THE WESTERN BLOTTING TECHNIQUE AS CONFIRMATORY TESTS FOR Trypanosoma cruzi INFECTION IN ENDEMIC AREA FOR LEISHMANIASIS IN BOLIVIA REAGENT INCONCLUSIVE NON REAGENT ELISA 22 115 IIF 21 1 115

T. CRUZI

SEROLOGICAL TESTS IN

L. DONOVANI CHAGASII

IHA 22 115 TESACRUZI 21 116 IIF 18 4 115

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Emerging Pathogens Department bioMérieux SA Tour CERVI IFR 128 BioSciences Lyon Gerland

CONGENITAL CHAGAS DISEASE ALGORITHMIC MOTHER SEROLOGY REAGENT NEGATIVE SEARCH T.CRUZI IN NB TWO SAMPLES DURING THE FIRST MONTH SEARCH ANTI T.CRUZI IN NB.

TWO SAMPLES DURING THE FIRST MONTH POSITIVE SEROLOGY DURING THE FIRST 6 MONTHS TREATMENT TREATMENT

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Reagent Non Reagent PILOT PROGRAM IN TUCUMÁN, ARGENTINE PRENATAL DIAGNOSIS END THE FOLLOW UP REMEMBER: WITH EARLY DIAGNOSIS AND PROMPT TREATMENT OF CONGENITAL CHAGAS DISEASE THE PROBABILITY OF CURE IN NEWBORN IS 100%

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Vironostika® Kit Composition: