1

Chagas Tests:Development and Standardization

Gláucia Paranhos-BaccalàEmerging Pathogens Department

bioMérieux

International Biological Reference Preparations for Chagas Diagnostic Test

WHO – Geneva January, 26th and 28th 2009

2

Clinical signs symptoms or epidemiological evidence of Chagas

disease

Acute Chronic

Parasite detection SerologyReference Centers

Positive“Chagas Disease”

Negative

PCRReference Centers

Serology two tests – WHO recommendationClinical Laboratories - HA IFA ELISA

PositiveNegative Inconclusive

“Non Chagas disease”

“Chagas disease”

Repeat

Inconclusive

Confirmatory testPCR or WB

ALGORITHMIC FOR CHAGAS DISEASE DIAGNOSIS FOR CLINICAL LABORATORIES

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 Americanot well known in EU

Real emergence or cluster effect?

5

Chagas Tests in EU: Antibody Detection

Company Name Serological test

* ELISA cruzi® Total parasite extract

* Bioelisa Chagas® Recombinant antigens

Lemos Chagatek Elisa® Total parasite extract

Ortho T.cruzi ELISA Test

System-1®

Total parasite extract

Ingen / Adaltis EIAgen T.cruzi Ab® Total parasite extract

Dade Behring ELISA Novagnost® Total parasite extract

6

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?

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

7

bioMérieux

Vironostika®

8

Kit Elisa Cruzi: for antibody detection

Kit: ELISA CRUZIRef: 35117Number of tests: 192 tests2 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

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.

-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.

The Chagas disease immunoassay standardization test for antibody detection at bioMérieux

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

11

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.

The Chagas disease immunoassay standardization test for antibody detection at bioMérieux

12

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 Chagas disease immunoassay standardization test for antibody detection at bioMérieux

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

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

The Chagas disease immunoassay standardization test for antibody detection at bioMérieux

14

Blood Donors and co-endemic areas

Confirmatory tests for Chagas Disease

Western Blot: TESA cruzi WB (bioMérieux)

- Trypomastigote excreted-secreted antigens

15

TESA= TRYPOMASTIGOTE EXCRETED-

SECRETED ANTIGENS

Trypomastigote Excreted-secreted antigens

16

TESAcruzi - Parameters

Serum panel bioMérieux + serum from blood banks

Sensitivity 100%

Specificity 99,5%

PPV 98,7%

PNV 100%

Efficiency 99%

Kappa index 0,99

N positives 795

N negatives 1946

17

APPLICATIONS

18

SEROEPIDEMIOLOGICAL SURVEY ON CHAGAS DISEASE PREVALENCE AMONG CHILDREN

Blood samples on filter paper

N = 100,000

Central Laboratory

ELISA - IIF

Reference Laboratory

10% of blood samples + Reagents + Inconclusives

IIF, ELISA and IHA

Reagents or inconclusives – TESA cruzi

Results

Identification of childen + contactants

Reagents samples

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

T. CRUZI

L. DONOVANI

CHAGASII

ELISA IIF IHA IIF

REAGENT

21 22 18

INCONCLUSIVE 1

NON REAGENT

115 115 115

22

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

Emerging Pathogens Department bioMérieux SA

Tour CERVI IFR 128 BioSciences Lyon Gerland

24

CONGENITAL CHAGAS DISEASE ALGORITHMIC

MOTHER SEROLOGYREAGENT

Reagent

SEROLOGY DURING SEROLOGY DURING THE FIRST 6 MONTHSTHE FIRST 6 MONTHS

SEARCH T.CRUZI IN NBTWO SAMPLES DURING THE

FIRST MONTH

TREATMENTEND THE

FOLLOW UP

Non Reagent

TREATMENT

POSITIVE

SEARCH ANTI T.CRUZI IN NB.TWO SAMPLES DURING THE

FIRST MONTH

NEGATIVE

PILOT PROGRAM IN TUCUMÁN, ARGENTINEPRENATAL DIAGNOSIS

REMEMBER: WITH EARLY DIAGNOSIS AND PROMPT TREATMENT OF CONGENITAL CHAGAS DISEASE THE PROBABILITY OF CURE IN

NEWBORN IS 100%

25

Vironostika®

Kit Composition: