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
Positive
“Chagas Disease”
Negative
PCR
Serology two tests – WHO recommendation
Clinical Laboratories - HA IFA ELISA
Inconclusive
“Non Chagas disease”
“Chagas disease”
Repeat
Confirmatory test
PCR or WB

3. Chagas Tests: Diagnosis
Eu Market Blood Screening
Direct Transmission 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. Total parasite extract
Chagas Tests in EU: Antibody Detection
Company
Name
Serological test *
ELISA cruzi®
Total parasite extract
Bioelisa Chagas®
Recombinant antigens
Lemos
Chagatek Elisa®
Ortho
T.cruzi ELISA Test
System-1®
Ingen / Adaltis
EIAgen T.cruzi Ab®
Dade Behring
ELISA Novagnost®

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. bioMérieux
Vironostika®

8. Kit Elisa Cruzi: for antibody detection
Ref:
Number of tests: 192 tests
2 microplates of 96 tests: barrettes de 8 wells.
Human serum or plasma: citrate, heparine, oxalate ou EDTA.
Epimastigotes antigens
Results: Positif / Doubts / Negatif.
adsorbance index : < 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. This test has been calibrated against in house standards. test.
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 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 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. 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.
Ag from T. cruzi
Ab anti T. cruzi
Ab anti IgG humaine*Peroxydase
Substrat : Tetramethylbenzidine TMB +
Stop solution : Sulphur acid 2N
spectophometer

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
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/ (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 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
T. CRUZI
L. DONOVANI
CHAGASII
ELISA
IIF
IHA
REAGENT 21 22 18
INCONCLUSIVE 1
NON REAGENT
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
REAGENT 22 21 18
INCONCLUSIVE 1 4
NON REAGENT
115
116

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

25. Vironostika®
Kit Composition: