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)

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 Disease Second report of the WHO expert Committee Geneva 2002

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.

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

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.

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.

FEASIBILITY CLINICAL SENSITIVITY SPECIFICITY EVALUATED FEATURES 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 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.

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

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

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

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

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

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.

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.

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

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

Distribution of negative sample signals (S/CO)

Distribution of negative sample signals (S/CO)

Distribution of negative sample signals (S/CO)

Distribution of negative sample signals (S/CO)

Distribution of negative sample signals (S/CO) 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.

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

Donor screening algorithm RR 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

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

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

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 832 donations

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

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

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 % ) d

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

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)

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