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)