1. TESTS USED IN BLOOD SCREENING 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 d
Dr Azzedine ASSAL
French Blood Services (EFS)

2. 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

3. Control of Chagas Disease Second report of the WHO expert Committee
Geneva 2002

4. Amadeo Sáez-Alquezar. Fondation Mérieux. May 2008.
Screening Strategies
Endemic Countries :
Brazil: until 2002: 2 tests (>70% ELISA + IHA)
Brazil: since 2003: 1 test ELISA
Argentina 2004: 2 tests
Costa Rica 2006: 2 tests (ELISA rec + Lys)
Non endemic countries
UK 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)
The shift of Brazilian Blood Banks to One test is based on the fact that the PAHO recommendation for using to tests dates back to a period of time were tests lacked sensitivity. Nowadays tests has better performance and undergo a series of quality controls and regulation requirements that make them more reliable.
Amadeo Sáez-Alquezar. Fondation Mérieux. May 2008.

5. Ideal screening serological test The ideal test does not exist
100 % sensitivity
100 % specificity
Reproducible
Easy to perform
Fast and automated
Non subjective reading
Not expensive
The ideal test does not exist

6. 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

7. 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.

8. EVALUATED ASSAYS 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.

9. FEASIBILITY CLINICAL SENSITIVITY SPECIFICITY EVALUATED FEATURES
REPRODUCIBILITY

10. 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)

11. Material and methods Panels 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.

12. RESULTS Sensitivity / BBI Panel
The 14 samples are detected positive by all the kits.

13. Sensitivity / Brazilian donor panel
RESULTS (2)
Sensitivity / Brazilian donor panel
Negative samples: No discrepancies with Brazilian data.

14. Sensitivity / Brazilian donor panel
RESULTS (3)
Sensitivity / Brazilian donor panel
24 non negative samples
20 positives in concordance with Brazilian data.
4 discrepant samples.

15. Sensitivity / Brazilian donor panel
RESULTS (4)
Sensitivity / Brazilian donor panel
The 4 discrepant results of Brazilian lab are discrepant with EFS tests as well

16. Conclusion on the sensitivity of Brazilian donor panel
RESULTS (5)
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

17. RESULTS (6) Guyana patient samples
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.

18. Guyana patient samples (2)
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.

19. Reproducibility Dilution series of Accurun
Tested in 8 replicates per run, during 3 different days (24 values)

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

21. Distribution of negative sample signals (S/CO)

22. Distribution of negative sample signals (S/CO)

23. Distribution of negative sample signals (S/CO)

24. Distribution of negative sample signals (S/CO)

25. Distribution of negative sample signals (S/CO)
10 % d ’échantillons réactifs initiaux

26. SELECTED TESTS ELISA assays Bioelisa Chagas (Biokit, Spain).
ELISA Cruzi (BioMérieux, Brazil).
Immunofluorescence Assay
Immunofluor Chagas (Biocientifica, Argentine).
Implementation date: May 2nd, 2007.

27. 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.

28. Donors born in endemic areas Travelers and residents returning from
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

29. Donor screening algorithmRR
ELISA
Negative No
Control
2 RR
ELISA No
yes No
inconclusive
to be Controlled 3 months later
IFA +
yes
Eligible donor Accepted donations
IFA +
yes
Confirmed positive
Temporary deferral
Blood components discarded
yes
Inconclusive No
Probable false positive
Permanent deferral
Referring Physician
Permanent deferral
Referring Physician
Look Back procedure:
tracing recipients

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

31. 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. d

32. French Inconclusive results
Discrepancy
Number
percentage
2 ELISA positive
IFA negative 2
0.2 %
IFA equivocal 1
0.01 %
2 discrepant ELISA
IFA positive 19
2.3 %
787
94.6 % 23
2.8 d
Total donations

33. Control of French Inconclusive results
465 donors with inconclusive results could be controlled.
Out of these 213 (46 %) were found negative. d

34. Reevaluation of Ortho test
Tobler 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 d

35. 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: % ) d

36. Reevaluation of Ortho test
Patient panel
Only 4 samples left
Samples
S/CO 2006
S/CO 2009
BioMérieux
Biokit
Ortho
VTLA
1.22
2.54
0.602
1.36
VTJE
1.66
1.46
0.546
1.23
Da Sis
3.19
0.346
1.03
VTVE
2.91
1.01
0.836
1.70 d

37. 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 ?).
IFA is used in France as an alternative tests. It is cumbersome, with a subjective reading and interpretation and with analytical performance close to ELISAs, it should be replaced by a real confirmatory test such as WB or IB)

38. 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,…)
IFA is used in France as an alternative tests. It is cumbersome, with a subjective reading and interpretation and with analytical performance close to ELISAs, it should be replaced by a real confirmatory test such as WB or IB)
Sreening with only one ELISA in a non endemic country with few immigrants from endemic countries is sufficient provided the test has good sensitivity and specificity