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
• 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
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.
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.
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.
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 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.
ELISA Cruzi BM Chagatek BM
Average S/CO of
positive samples
6,03
7,27
Ortho
Chagas
Biokit Chagas
Ingen
3,95
5,65
9,07
RESULTS (2)
Sensitivity / Brazilian donor panel
• Negative samples: No discrepancies with
Brazilian data.
ELISA Cruzi BM Chagatek BM
Average S/CO of
negative samples
0,07
0,15
Ortho
Chagas
Biokit Chagas
Ingen
0,06
0,1
0,62
RESULTS (3)
Sensitivity / Brazilian donor panel
• 24 non negative samples
• 20 positives in concordance with Brazilian data.
• 4 discrepant samples.
ELISA
Sample
1
Sample
2
Sample
8
Sample
10
N°
N°
N°
N°
+
1,53
+
1,14
+
1,08
+
0,94
IHA
IFI
-
-
-
-
+
-
-
+
1/ 20
RESULTS (4)
Sensitivity / Brazilian donor panel
The 4 discrepant results of Brazilian lab are
discrepant with EFS tests as well
Brazilian results
EFS results
ELISA
IHA
IFA
Elisa Cruzi
BM
Biokit
Chagatek
Ortho
Ingen
IIFA
Sample N°
1
+
1,53
-
-
-
-
+
2,38
+
2,07
+
4,17
-
Sample N°
2
+
1,14
-
-
-
+
1,20
+
1,71
+
0,95
+
1,24
Sample N°
8
Sample N°
10
+
1,08
+
0,94
+
-
-
-
-
-
-
+
1/ 20
ZG
-
+
1,47
+
1,66
+
3,46
+
3,03
Pos au 1/30
Nég au 1/60
Pos au 1/60
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.
French Guyana
results
Bioelisa Chagas
(Biokit)
Elisa Cruzi
(bioMérieux)
Ortho Clinical
Assay
Chagatek Elisa
(Lemos)
EFS IFA
10 negative
samples
10/10
10/10
10/10
10/10
10/10
18 confirmed
positive samples
16/18
(88.9 %)
14/18
(77.8 %)
9/18
(50 %)
9/18
(50 %)
18/18
(100 %)
Reproducibility
• Dilution series of Accurun
• Tested in 8 replicates per run, during 3 different
days (24 values)
ELISA Cruzi
BM
Chagatek
BM
Ortho
Chagas
Biokit Chagas
Last positive dilution
1/4
1/4
1/4
1/4
Average S/CO value
1,49
1,72
1,62
1,27
Ecart Type
0,33
0,14
0,25
0,06
VC en %
22,35
7,95
15,68
4,72
Specificity
Tested on a limited number of donations
(limited number of kits).
ELISA Cruzi
BM
Chagatek
BM
Ortho
Chagas
Biokit Chagas
Number of samples
tested
439
525
525
518
Specificity (%)
100
100
100
100
Distribution of negative
sample signals (S/CO)
Elisa Cruzi BioMérieux
Nbre d'échantillons
500
400
300
200
100
0
Nbre d'échantillons entre
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
455
35
3
0
2
3
0
0
1
0
Ratio
Distribution of negative
sample signals (S/CO)
Nbre d'échantillons
Ortho
500
400
300
200
100
0
Nbre de valeurs entre
0 et 0,1
409
0,1 et 0,2 0,2 et 0,3
78
6
0,3 et 0,4
0,4 et 0,5
3
1
0,5 et 0,6 0,6 et 0,7
0
Ratio
1
0,7 et 0,8
0,8 et 0,9
0,9 et 1
0
0
0
Distribution of negative
sample signals (S/CO)
Biokit
Nbre d'échantillons
500
400
300
200
100
0
Nbre d'échantillons entre
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
429
45
9
7
3
2
2
0
0
1
Ratio
Distribution of negative
sample signals (S/CO)
Chagatek Lemos
Nbre d'échantillons
400
300
200
100
0
Nbre d'échantillons entre
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
317
166
9
1
0
1
1
2
1
0
Ratio
Distribution of negative
sample signals (S/CO)
Nbre d'échantillons
Ingen
60
40
20
0
Nbre de valeurs entre
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
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
0
1
9
32
39
34
26
15
19
7
Ratio
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
No
No
Negative
Control
2 RR
ELISA
inconclusive
to be Controlled
3 months later
yes
IFA +
yes
yes
IFA +
No
yes
Confirmed
positive
No
Inconclusive
Eligible donor
Accepted
donations
•Probable false positive
• Permanent deferral
• Referring Physician
Temporary
deferral
Blood
components
discarded
• Permanent deferral
• Referring Physician
• Look Back procedure:
tracing recipients
Seroprevalence in French Donors
Period: May 2, 2007 to February 29, 2008
Collected
donations
Number of
donations
(Percentage)
2,143,740
Tested
donations
97,618
(4.55 %)
Negative
donations
96,625
(99 %)
Positive Inconclusive
donations
results
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
2 ELISA positive
IFA negative
2 ELISA positive
IFA equivocal
2 discrepant ELISA
IFA positive
2 discrepant ELISA
IFA negative
2 discrepant ELISA
IFA equivocal
Total
Number
percentage
2
0.2 %
1
0.01 %
19
2.3 %
787
94.6 %
23
2.8
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 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
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
Only 4 samples left
Samples
VTLA
VTJE
Da Sis
VTVE
S/CO
2009
S/CO 2006
BioMérieux
1.22
1.66
1.36
2.91
Biokit
2.54
1.46
3.19
1.01
Ortho
0.602
0.546
0.346
0.836
Ortho
1.36
1.23
1.03
1.70
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,…)