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Dr Azzedine ASSAL

Second WHO consultation on International Reference Preparation for Chagas Diagnostic Tests January 27 & 28 January, 2009 TESTS USED IN BLOOD SCREENING . Dr Azzedine ASSAL. French Blood Services (EFS) . Background .

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Dr Azzedine ASSAL

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  1. Second WHO consultation on International Reference Preparation for Chagas Diagnostic Tests January 27 & 28 January, 2009 TESTS USED IN BLOOD SCREENING 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. 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) Amadeo Sáez-Alquezar. Fondation Mérieux. May 2008.

  5. 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

  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. EVALUATED FEATURES • FEASIBILITY • CLINICAL SENSITIVITY • SPECIFICITY • 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. RESULTS (2) • Sensitivity / Brazilian donor panel • Negative samples: No discrepancies with Brazilian data.

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

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

  16. 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. 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. 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 algorithm No Control 2 RR ELISA No Negative yes inconclusive to be Controlled 3 months later No IFA + yes yes yes IFA + No Inconclusive Temporary deferral Blood components discarded Eligible donor Accepted donations RR ELISA Confirmed positive • 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 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.

  32. FrenchInconclusive results Total 832 donations

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

  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

  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: 99.95 % )

  36. Reevaluation of Ortho test Patient panel Only 4 samples left

  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 ?).

  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,…)

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