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)
Sensitivity / Brazilian donor panel
• 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)
Sensitivity / Brazilian donor panel
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
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
Distribution of negative sample signals (S/CO)
Biokit
0
100
200
300
400
500
Ratio
Nb
re d
'éch
anti
llon
s
Nbre d'échantillons entre 429 45 9 7 3 2 2 0 0 1
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
Distribution of negative sample signals (S/CO)
Chagatek Lemos
0
100
200
300
400
Ratio
Nb
re d
'éch
anti
llo
ns
Nbre d'échantillons entre 317 166 9 1 0 1 1 2 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
Distribution of negative sample signals (S/CO)
Ingen
0
20
40
60
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
Ratio
Nb
re d
'éch
anti
llon
s
Nbre de valeurs entre 0 1 9 32 39 34 26 15 19 7
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
10 % d ’échantillons réactifs initiaux
Distribution of negative sample signals (S/CO)
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,…)