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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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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

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


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Chagas Tests: Diagnosis

Eu MarketBlood Screening

  • DirectTransmission through transfusions, by organ transplantation or laboratory accident

Adequate cost tests

High Sensitivity

Good Specificity

Needs for Chagas test

Antibody detection


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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?


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Chagas Tests in EU: Antibody Detection


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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?


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Vironostika®

bioMérieux


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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).


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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.


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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:

    • 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.


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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.

  • 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.


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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.


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Confirmatory tests for Chagas Disease

Blood Donors and co-endemic areas

Western Blot: TESA cruzi WB (bioMérieux)

  • - Trypomastigote excreted-secreted antigens


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TESAcruzi - Parameters

Serum panel bioMérieux + serum from blood banks


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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

L. DONOVANI

T. CRUZI

CHAGASII

ELISA

IIF

IHA

IIF

22

REAGENT

21

22

18

INCONCLUSIVE

1

115

NON REAGENT

115

115

115


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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

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


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Thank you!

Emerging Pathogens Department

bioMérieux SA

Tour CERVI IFR 128 BioSciences Lyon Gerland


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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%


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Vironostika®

Kit Composition:


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