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Second WHO consultation: Development of a WHO reference panel for the control of Chagas diagnostic tests Geneva, 27 – 28 January 2009
Chagas disease in Spain: Experience from a reference laboratory Teresa Gárate Servicio de Parasitología Centro Nacional de Microbiología
Experience from a Parasitology Reference Laboratory General Role of the Parasitology Lab Parasitology Lab and Chagas’ disease in Spain
Parasitic diseases AUTOCTHONOUS
IMPORTED
Leishmaniasis
Malaria
Toxoplasmosis
Other Leishmaniasis
Cryptosporidiasis
Chagas disease
Giardiasis
Sleeping sickness
Amebiasis
Cysticercosis
Fasciolosis
Schistosomiasis
Hydatidosis
Filariasis/Oncho
Anisakiasis
Intestinal parasites
Toxocariosis Trichinellosis
National Microbiology Centre
The Parasitology Lab works with Spanish hospitals and blood donor centers on Chagas disease diagnosis
Other Activities related with T. cruzi diagnosis Chagas Test evaluation - In house tests
- Commercial tests Control sample preparation - Serum of characterized patients - DNA from parasite strains - Blood spiked with parasites Training microbiologists in - Microhaemotocrit
- PCR - Serological result interpretation
Implementation of T. cruzi Diagnostic Tools in Parasitology Lab
Commercial assays (rapid tests)
PCR ELISA Microscopic examination
Culture
Xenodiagnosis IFAT 1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
Real time PCR
Evaluation of serological tests: In house ELISA and 3 serum panels Positive IFAT Negative IFAT
3.0
2.5
DO
405 nm
2.0 1.5
1.0 0.5 0.0
Panel 1
ELISA in house
Panel 2
Panel 3
Panel 1
Panel 2
Panel 3
Chagasic Healthy (n = 13) (n = 15)
Chagasic Healthy (n =31) (n = 115)
Chagasic Healthy (n = 38) (n = 30)
Positive
11
0
30
2
38
0
Negative
2
15
1
113
0
30
Evaluation of serological tests: In house ELISA and 3 serum panels
Diagnostic index
Panel 1
Panel 2
Panel 3
%
CI 95%
%
CI 95%
%
CI 95%
Sensitivity
85
65 to 100
97
91 to 100
100
100 to 100
Specificity
100
100 to 100
98
96 to 100
100
100 to 100
PPV
100
100 to 100
94
85 to 100
100
100 to 100
NPV
88
73 to 100
99
97 to 100
100
100 to 100
Characteristics of serum samples of the 3 panels used in the evaluation of the In house ELISA Panel 1: Sera from Bolivian individuals with positive or negative xenodiagnosis Panel 2: Sera from population with positive or negative T. cruzi IFAT. Healthy individuals from endemic and non-endemic areas Panel 3: Sera of anti-T. cruzi Specific Panel and healthy individuals from endemic and non-endemic areas
Anti-T. cruzi Specific Sera Panel (QPanel, Sao Paulo-Brazil, http://www.panel.com.br) was kindly provided by Dra. Elena Franco
Evaluation of serological tests: Commercial ELISAs CHAGAS
HEALTHY
VISCERAL LEISHMANIASIS
BLK Chagas test
25/25 (100%)
1/10 (10%)
5/9 (56%)
Biokit ELISA
25/25 (100%)
1/10 (10%)
3/9 (33%)
30/31 (97%)
1D/23 (4%)
4D/52 (8%)
ID-PaGIA
BLK Chagas test
Biokit ELISA
ID-Pagia
Sensitivity
100
100
97
Specificity
90
90
96
PPV
96
96
97
NPV
100
100
96
Characteristics of serum samples used in the evaluation of commercial tests Characteristics of Sera Chagasic Panel 1 (QPanel, Brasil) Non Chagasic
Panel 2 (Parasitology, CNM, ISCIII)
Nº of sera 38 2
Chagasic patients diagnosed in Spain
28
Spain
22
Bolivia
19
Healthy Brazil individuals
20
Argentina
14
Ecuador
20
Panel 3 Visceral leishmaniasis (Parasitology, CNM, ISCIII) Malaria
30 30
Evaluation of Commercial ELISAs: Sensitivity Chagasic Nº of positive (total sera)
Sensitivity (CI 95%)
IFAT-CNM
65 (66)
98.5 (95.5 - 100)
ELISA-CNM
66 (66)
100 (100 - 100)
Certest
66 (66)
100 (100 - 100)
Ortho
66 (66)
100 (100 - 100)
BLK
41 (42)
97.6 (93.0 - 100)
Biokit
66 (66)
100 (100 - 100)
ID-PaGIA2
61 (66)
92.4 (86 - 98.8)
ID-PaGIA3
65 (66)
98.5 (95.5 - 100)
ICT Operon
61 (66)
92.4 (86.0 - 98.8)
Evaluation of Commercial ELISAs: Specificity Non Chagasic Nº of positive (total sera)
Specificity 2b
(CI 95%)
Other pathologies Nº of positive (total sera)
Specificity 1a
(CI 95%)
IFAT-CNM
0 (97)
100 (100 - 100)
23 (60)
85.4 (79.8 - 90.9)
ELISA-CNM
0 (97)
100 (100 - 100)
18 (60)
88.6 (83.6 - 93.5)
Certest
0 (97)
100 (100 - 100)
25 (60)
84.2 (78.4 - 89.8)
Ortho
0 (97)
100 (100 - 100)
18 (60)
88.6 (83.6 - 93.5)
BLK
0 (74)
100 (100 - 100)
13 (60)
90.3 (85.3 - 95.3)
Biokit
1 (97)
99 (97 - 100)
7 (60)
94.9 (91.5 - 98.3)
ID-PaGIA2
1 (97)
99 (97 - 100)
1 (60)
98.7 (97.0 - 100)
ID-PaGIA3
2 (97)
98 (95 - 100)
2 (60)
97.5 (95.0 - 99.9)
ICT Operon
2 (97)
98 (95 - 100)
10 (60)
92.4 (88.2 - 96.5)
a
The specificity was calculated into account results of healthy control b The specificity was calculated into account results of healthy control and other pathologies.
Molecular diagnosis of T. cruzi
Control assay of limit detection
Samples of population at-risk
kDNA-PCR modified (121-122 / HUF-REV) Britto et al 1993; Cruz et al 2002; Walsh et al 1991; Dorn et al 1997; Gomes et al 1998; Wincker et al 1994; Rubio et al 2002
Molecular diagnosis of T. cruzi Diagnostic sensitivity and specificity PCR Positive Negative
Total
Acute patients
11
0
11
Chronic patients
471
295
766
0
1227
1227
482
1522
2004
Seronegative subjects Total
Diagnostic index in chronic phase
CI (95%)
Sensitivity
61.5
58
65
Specificity
100
99.96
100
Positive predictive value
100
99.89
100
80.62
78.6
82.64
Negative predictive value
Chagas disease cases in Spain AUTOCTHONOUS Blood transfusion Congenital transmission Organ transplant Laboratory accidents
IMPORTED Immigration from endemic area Long stays in endemic area
Latin American population in Spain On December 2008: > 1.8 million Ecuador Colombia Bolivia Argentina Peru Brazil Paraguay Uruguay Venezuela Chile Rest of SA
415535 284043 236048 196946
123173 119209 66950 61407 61069 48939 268
Reports of Transfusional Chagas cases in Spain Year of detection
Year of Donation
N º of recipients
Nº of infected
1992
?
1?
1
Villalba et al., 1992
Ref.
2005
2004
1
1
Fores et al., 2007; Flores-Chavez et al., 2008
2006
1994-2003
10
1
Abalo et al., 2007, CNM
2007
2005-2007
9
2
Perez et al., 2008; CNM
2008
2004
1
1
Ibarra et al., 2008, CNM
National Haemovigilance System
Transfusional Chagas Cases
Origin Age
Madrid recipient
Coruña recipient
Málaga recipient 1
Málaga recipient 2
País Vasco recipient
Spanish
Spanish
Spanish
Moroccan
Spanish
27
55
33
57
4
Bone marrow aplasia
Choroid plexus papilloma
Cardiac surgery
Clinical status before transfusion
Hepatic Leukemia transplant
Phase of Chagas disease and symptoms
Acute FUO Multiorga nic failure
Chronic Asympto matic
Acute FUO
Chronic Right bundle branch block
Chronic Asympto matic
Treatment
Yes
Yes
Yes
Yes
Yes
Follow up After treatment
Fatal dead person
S (+) PCR (-)
S (-) PCR (-)
S (+) PCR (-)
S (+) PCR (-)
País Vasco
A Coruña
Madrid
Characteristics of Infected donors:
Malaga
A Coruña
Madrid
Malaga
País Vasco
Bolivia
Brazil
Bolivia
Bolivia
Sex
Male
Female
Male
Male
Age
62
58
53
ELISA
2.3
2.2
2.3
IFAT
> 1/160
> 1/160
> 1/160
PCR
P
N/P
P
Treatment
No
Yes
Yes
Permanenc e in Spain
42 years
1 year
3 years
Origin
Congenital Chagas disease
Newborns examined during 2002 2008 Newborns
2002 2003 2004 2005 2006 2007 2008 Total
Positive PCR
1 3 2 4 12 78 91 191
1*
3 5 9
Transmission index: ~ 4,5% * Post mortem diagnosis
In Spain ≈ 990 000 women of childbearing age Bolivia: 132 421 women Argentina: 94 794 women
Laboratory Accidents and Chagas disease
Alvar J. 1983 Laboratorio 76(456): 645-648
Chagas disease cases in Spain AUTOCTHONOUS Blood transfusion Congenital transmission Organ transplant Laboratory accidents
IMPORTED Immigration from endemic area Long stays in endemic area
Chagas disease in blood donors Asturias at- risk 2890 2750 2750 2000 2000
Madrid
1250 1250
779 500 500 200 200
100 100 36 36 4
1
00 2002-2006 2002-2006
2003-2006
200
C. T. Cruz Roja C.C. de Sangre y de Madrid Tejidos de Asturias
Prevalence %
1,23
0,13
Profile of immigrant population Asturias 0
Ecuador Colombia Argentina Brazil Venezuela Paraguay Uruguay Peru Chile Bolivia
Asturias
Madrid 0
Ecuador Colombia Peru Bolivia Argentina Brazil Venezuela Paraguay Chile Uruguay
30000
1000
2000
3000
4000
5000
4358 2721 1903 1886
915 549 435 373 370 310
2006: ≈ 17 000 immigrant 2008: ≈ 20 000 immigrant
Madrid 60000
90000
120000
150000
145811 64906 43068 32975 25169 13474 10840 8997 8476 3958
2006: ≈ 400 000 immigrant 2008: ≈ 500 000 immigrant
Prevalence in blood donors at-risk (2002-2006)
?
Nicaragua Colombia Venezuela Ecuador Peru Bolivia Brazil End. Zone Non End. Zone ND Spain
P
N
Prevalence %
1 1 1 1 2 37 2 3 1 3 1
16 776 249 908 258 203 221 207 307 245 277
6.25 0.13 0.40 0.11 0.78 18.23 0.90 1.45 0.33 1.22 0.36
P = Seropositive N = Seronegative
Seroprevalence in population at-risk: 24% 1200
1137
≈ 1144 Chagasic patients
1000
815
800
600 400 200
0
6 1
5 0
27
1997
1998
1999
2
40
7
2000
75 3
6
26
46
2001
2002
2003
2004
464 361
285
219
131
SERONEGATIVE
Prevalence %
476
398 90 2005
138 2006
2007
2008
SEROPOSITIVE
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
14
0
7
15
4
4
11
14
18
22
31
29
Distribution of cases in Spain Alicante 800
Madrid 2500 2000
2289
Prevalence 19 %
400 200
1500 1000 500
598
600
Prevalence 12 %
79
0
528
Seronegative Seropositive
0 Seronegative Seropositive
Almeria 200 150
173 Prevalence 8%
100 50
15
0 Seronegative Seropositive
ARGENTINA BOLIVIA BRAZIL CHILE PARAGUAY URUGUAY COLOMBIA ECUADOR PERU VENEZUELA GUATEMALA HONDURAS NICARAGUA SALVADOR MEXICO SPAIN
Origin of seropositive population 100% 80%
60% 5%
4%
3%
2%
1%
0%
Conclusions Panel of Reference - Positive samples by different serological tests, and - Positive samples from chagasic individuals with parasitological, epidemiological and clinical evaluation. Number of positive sera/panel - 3 serum samples for kit manufacturers - 100 serum samples for evaluation of tests - 3 serum samples for quality control. Chagas in Spain - Imported and autochonous types. - Bolivian group, mainly from Santa Cruz region, shows the highest seropositivity rates and represents the highest risk for T. cruzi infection transmission.
Thank you UNIDAD LEISHMANIA Y CHAGAS Carmen Cañavate Javier Nieto Mercedes Rodriguez Israel Cruz Elena Bodas Marta Hernández Rubén González Emilia García Carmen Chicharro María Flores
Chagas disease in Spain AUTOCTHONOUS Transfusional and congenital Chagas Organ transplant and lab incident
IMPORTED Immigration and long stays in endemic area
? Months
Acute
Years
Chronic phase
Level of anti-T. cruzi IgG in samples of patients with positive PCR 6 5
4 DO/CO 3 2 1
0
Acute case 1 (Madrid recipient)
Acute case 2 (Málaga recipient)
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