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.
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Transcript 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.
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,…)