Dr Azzedine ASSAL French Blood Services (EFS) Second WHO consultation on International Reference Preparation for Chagas Diagnostic Tests January 27 & 28.

Dr Azzedine ASSAL

French Blood Services (EFS)

Second WHO consultation on International Reference Preparation for

Chagas Diagnostic Tests

January 27 & 28 January, 2009

TESTS USED IN BLOOD SCREENING

Background

• The choice of a Chagas disease screening assay or strategy for TT prevention is far from straightforward

• Recommendation of the PAHO (1994): parallel use of at least 2 different serological tests in Chagas disease screening in blood donations (Lack of sensitivity and specificity) .

• Recommendations of WHO, 2002: one ELISA is recommended for blood bank screening

Control of Chagas DiseaseSecond report of the WHO expert Committee

Geneva 2002

Screening Strategies

Endemic Countries :Brazil: until 2002: 2 tests (>70% ELISA + IHA)Brazil: since 2003: 1 test ELISAArgentina 2004: 2 testsCosta Rica 2006: 2 tests (ELISA rec + Lys)

Non endemic countriesUK 1999 to 2005: 1 ELISA Lys, from 2006 ELISA rec USA 2007: 1 test (ELISA Lys) France 2007: 2 tests (ELISA rec + Lys) Spain 2008: 2 tests (ELISA rec + Lys)

Amadeo Sáez-Alquezar. Fondation Mérieux. May 2008.

Ideal screening serological test

100 % sensitivity 100 % specificity Reproducible Easy to perform Fast and automated Non subjective reading Not expensive

The ideal test does not exist

Different strategies for blood banks

Using only one test

High sensitivity test (IgG + IgM)

Use a whole parasite Lysate test (mixture of

parasite antigens)

Using 2 tests

1 Lysate ELISA + 1 rec ELISA

IFI + ELISA

French Screening Strategy

Commercial assays available : IHA or other

agglutination tests, ELISA, IFA.

French strategy: Screening based on 2 parallel

ELISAs (Crude and recombinant antigens).

IFA as an alternative test (“confirmation”) test in

case of positivity or discrepancy between the 2 ELISAs.

1) ELISAs• Recombinant ELISAs Bioelisa Chagas (Biokit, Spain). CE mark.• Crude ELISAs ELISA Cruzi (BioMérieux). No CE mark. Chagatek Elisa (Lemos, Argentina), No CE mark. T.cruzi ELISA Test System–1 (OCD). CE mark. EIAgen Trypanosoma Cruzi Ab (manufactured by Adaltis and distributed by Ingen,France). CE mark.

2) IFA Immunofluor Chagas (Biocientifica. Argentina). CE mark.

EVALUATED ASSAYS

EVALUATED FEATURES

FEASIBILITY

CLINICAL SENSITIVITY

SPECIFICITY

REPRODUCIBILITY

Reference material for test evaluation

Ideally

Sensitivity evaluation Strong positive samples Borderline samples Discordant samples Samples with reactivity against main strains of

the 2 lineages of T. cruzi

Specificity evaluation “True” negative samples Potential cross-reactive samples (leishmania, T.

Rangeli, other protozoans)

Material and methodsPanels and samples

• BBI panel : 14 positive samples + 1 negative sample

• Dilutions of Positive Control (Accurun, Ingen)

• Brazilian donor Panel (Blood Bank Sao Paulo): 36

samples of positive and negative donors, tested with

ELISA, IHA et IFA.

• Patient samples (French Guyana) 35 positive and

negative samples, tested with ID PaGia (Diamed), Biokit

ELISA and PCR

• French Blood donors for specificity study.

RESULTS

• Sensitivity / BBI Panel

• The 14 samples are detected positive by

all the kits.

3,95 5,65 9,07

Ingen

7,276,03

ELISA Cruzi BM Chagatek BMOrtho

ChagasBiokit Chagas

Average S/CO of positive samples

Sensitivity / Brazilian donor panel

• Negative samples: No discrepancies with Brazilian data.

0,06 0,1 0,62

ELISA Cruzi BM Chagatek BMOrtho

ChagasBiokit Chagas Ingen

0,150,07Average S/CO of negative samples

RESULTS (2)


• 24 non negative samples

• 20 positives in concordance with Brazilian data.

• 4 discrepant samples.

- -

ELISA IHA IFI

+ 1,53

+ 1,14

+ 1,08

+ 0,94

+

-

- -

-

+ 1/ 20

Sample N° 8

Sample N° 10

Sample N° 2

Sample N° 1

RESULTS (3)


The 4 discrepant results of Brazilian lab are discrepant with EFS tests as well

Pos au 1/30

Nég au 1/60

+ 3,46

+ 3,03

-

-

Pos au 1/60

-

+ 1,47

+ 2,07

+ 0,95

-

+ 1,66

-

+ 1,20

-

-

IIFA

EFS results

-

-

+ 2,38

+ 1,71

+ 4,17

+ 1,24

Biokit Chagatek Ortho Ingen

+

-

- -

-

+ 1/ 20

-

ZG

+ 1,53

+ 1,14

+ 1,08

+ 0,94

- -

ELISA IHA IFAElisa Cruzi

BM

Brazilian results

Sample N° 8

Sample N° 10

Sample N° 2

Sample N° 1

RESULTS (4)

Conclusion on the sensitivity of Brazilian donor panel

• Good overall sensitivity of all the kits

• Follow up of Brazilian discrepant samples

showed that the discrepant samples were

false positive samples

RESULTS (5)

A set of 35 negative and positive patient samples (Dr Christine Aznar. Laboratory of Parasitology, Cayenne Hospital, French Guyana).

Tested by 3 different assays in Guyana:

• Agglutination test (ID-PaGIA, Diamed, France).

• ELISA (Bioelisa Chagas, Biokit).

• In-house PCR.

Blind testing before result comparison with Guyana data.

Guyana patient samples

RESULTS (6)

Out of the 35 samples tested:

10 samples negative in agreement with Guyana's results

7 samples could not be interpreted (incomplete data)

18 samples expected to be positive according to Guyana’s

data.French Guyana

results

10 negative samples

18 confirmed positive samples

EFS IFA

10/10 10/10

Bioelisa Chagas (Biokit)

Elisa Cruzi (bioMérieux)

Ortho Clinical Assay

16/18 (88.9 %)

Chagatek Elisa (Lemos)

18/18 (100 %)

10/10 10/10 10/10

14/18 (77.8 %)

9/18 (50 %)

9/18 (50 %)

Guyana patient samples (2)

• Dilution series of Accurun

• Tested in 8 replicates per run, during 3 different

days (24 values)Ortho

ChagasBiokit Chagas

1,72 1,62 1,27

1/4

ELISA Cruzi BM

22,35

Chagatek BM

4,72

0,33 0,14 0,25 0,06

1/4 1/4 1/4

1,49

7,95 15,68

Ecart Type

VC en %

Last positive dilution

Average S/CO value

Reproducibility

Tested on a limited number of donations (limited number of kits).

100100 100 100

Chagatek BM

Ortho Chagas

Biokit Chagas

439 525 525 518

ELISA Cruzi BM

Number of samples tested

Specificity (%)

Specificity

Distribution of negative sample signals (S/CO)

Elisa Cruzi BioMérieux

0

100

200

300

400

500

Ratio

Nbr

e d'

écha

ntill

ons

Nbre d'échantillons entre 455 35 3 0 2 3 0 0 1 0

0 et 0,1 0,1 et 0,2 0,2 et 0,3 0,3 et 0,4 0,4 et 0,5 0,5 et 0,6 0,6 et 0,7 0,7 et 0,8 0,8 et 0,9 0,9 et 1

Ortho

0

100

200

300

400

500

Ratio

Nb

re d

'éc

ha

nti

llo

ns

Nbre de valeurs entre 409 78 6 3 1 0 1 0 0 0



Biokit

0

100

200

300

400

500

Ratio

Nb

re d

'éch

anti

llon

s




Chagatek Lemos

0

100

200

300

400

Ratio

Nb

re d

'éch

anti

llo

ns




Ingen

0

20

40

60


Ratio

Nb

re d

'éch

anti

llon

s

Nbre de valeurs entre 0 1 9 32 39 34 26 15 19 7


10 % d ’échantillons réactifs initiaux


SELECTED TESTS

• ELISA assays• Bioelisa Chagas (Biokit, Spain).

• ELISA Cruzi (BioMérieux, Brazil).

• Immunofluorescence Assay

Immunofluor Chagas (Biocientifica, Argentine).

• Implementation date: May 2nd, 2007.

Measures taken to prevent T. cruzi Transfusion transmitted infections.

• Temporary deferral, for 4 months of

travelers or residents returning from endemic

areas.

• Screening for antibodies to T. cruzi in

targeted at risk blood donors.

At risk blood donors

• Donors born in endemic areas

• Travelers and residents returning from

endemic areas

• Donors born in France from a mother born in

risk areas

• Donors who underwent blood transfusion

Donor screening algorithm

RR ELISA

Control2 RR

ELISA

yes

Confirmed positive

IFA +

yes

• Permanent deferral• Referring Physician • Look Back procedure: tracing recipients

Eligible donor Accepted donations

•Probable false positive• Permanent deferral• Referring Physician

Noinconclusive

to be Controlled 3 months later

Temporary deferral

Blood components discarded

yesIFA +

yes

No

Inconclusive

No

Negative

No

Seroprevalence in French Donors

Period: May 2, 2007 to February 29, 2008

Collected donations

Tested donations

Negative donations

Positive donations

Inconclusive results

Number of donations

(Percentage)2,143,740 97,618

(4.55 %)

96,625

(99 %)

4

(0,004 %)

1 / 24,404

989

(1 %)

N.B.= Seroprevalence in UK: 1/ 24,300 from 1999 to 2007

Positive Donors in France

• 2 first-time Bolivian donors

• 2 donors from San Salvador

One first-time donor

One repeat donor: only 2 previous donations transfused to recipients who died from underlying diseases.

French Inconclusive results

Discrepancy Number percentage

2 ELISA positive IFA negative

2 0.2 %

2 ELISA positive IFA equivocal

1 0.01 %

2 discrepant ELISA IFA positive

19 2.3 %

2 discrepant ELISA IFA negative

787 94.6 %

2 discrepant ELISA IFA equivocal

23 2.8

Total 832 donations

Control of French Inconclusive results

• 465 donors with inconclusive results

could be controlled.

• Out of these 213 (46 %) were found

negative.

Reevaluation of Ortho testTobler LH et al. Evaluation of a new enzyme-linked immunosorbent

assay for detection of Chagas antibody in US blood donors. Transfusion January 2007;47:90-96

Reevaluation of Ortho test

• Cut off calculation of Ortho test modified: better sensitivity

• Same sensitivity with BBI panel and Brazilian samples

• Good sensitivity with 53 Mexican samples: higher S/CO than those obtained with BioMérieux and Biokit kits

• Specificity evaluated on 4000 donations:

• 1 non repeated reactive sample

• 2 repeat reactive samples (specificity: 99.95 % )

Reevaluation of Ortho test

Patient panel

SamplesS/CO 2006

S/CO 2009

BioMérieux Biokit Ortho Ortho

VTLA 1.22 2.54 0.602 1.36

VTJE 1.66 1.46 0.546 1.23

Da Sis 1.36 3.19 0.346 1.03

VTVE 2.91 1.01 0.836 1.70

Only 4 samples left

Conclusions

• Current serological tests (ELISAs) have good performance

• Performance continuously improved by manufactures under stringent Quality Control procedures

• Current screening strategy results in Large

number of Indeterminate results (false

positive ?).

Conclusions (2)

• Revision of screening strategy in France

• Screening strategy should be simplified

Screening with a single ELISA sufficient

Replace IFA by true confirmatory assays

(Western Blot, immunoblot , RIPA,…)

Dr Azzedine ASSAL French Blood Services (EFS) Second WHO consultation on International Reference Preparation for Chagas Diagnostic Tests January 27 & 28.

Documents

elisa slide

samples of positive

specificity slide

blood screening slide

brazilian discrepant

tests elisa rec

biokit elisa

test elisa argentina