Chagas disease in Spain:Chagas disease in Spain:Experience from Experience from
a reference laboratorya reference laboratory
Teresa GárateTeresa GárateServicio de ParasitologíaServicio de Parasitología
Centro Nacional de Centro Nacional de MicrobiologíaMicrobiología
Second WHO consultation: Development Second WHO consultation: Development of a WHO reference panel for the of a WHO reference panel for the control of Chagas diagnostic testscontrol of Chagas diagnostic tests
Geneva, 27 – 28 January 2009Geneva, 27 – 28 January 2009
General Role of the Parasitology Lab
Parasitology Lab and Chagas’ disease in Spain
Experience from a Experience from a Parasitology Reference Parasitology Reference
LaboratoryLaboratory
National National Microbiology Microbiology
Centre Centre
Parasitic diseasesParasitic diseasesAUTOCTHONOUS Leishmaniasis
Toxoplasmosis
Cryptosporidiasis
Giardiasis
Amebiasis
Fasciolosis
Hydatidosis
Anisakiasis
Toxocariosis
Trichinellosis
IMPORTED Malaria
Other Leishmaniasis
Chagas disease Sleeping sickness
Cysticercosis
Schistosomiasis
Filariasis/Oncho
Intestinal parasites
The Parasitology Lab works with Spanish The Parasitology Lab works with Spanish hospitals and blood donor centers on Chagas hospitals and blood donor centers on Chagas
disease diagnosisdisease diagnosis
Other Activities related with Other Activities related with T. T. cruzicruzi diagnosisdiagnosis
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
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Microscopic examination
XenodiagnosisIFAT
ELISA
PCRCommercial
assays (rapid tests)
Implementation of Implementation of T. cruziT. cruzi Diagnostic ToolsDiagnostic Tools in Parasitology Labin Parasitology Lab
Real time PCR
Culture
Evaluation of serological Evaluation of serological tests:tests:
In house In house ELISAELISA and 3 serum and 3 serum panelspanels
0.0
0.5
1.0
1.5
2.0
2.5
3.0
DO 40
5nm
Panel 1 Panel 2 Panel 3
Positive IFAT Negative IFAT
Chagasic Healthy Chagasic Healthy Chagasic Healthy(n = 13) (n = 15) (n =31) (n = 115) (n = 38) (n = 30)
Positive 11 0 30 2 38 0
Negative 2 15 1 113 0 30
Panel 2 Panel 3ELISA in
house
Panel 1
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
Evaluation of serological Evaluation of serological tests:tests:
In house In house ELISAELISA and 3 and 3 serum panelsserum panels
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
Characteristics of serum samples of Characteristics of serum samples of the 3 panels used in the evaluation the 3 panels used in the evaluation
of the In house of the In house ELISAELISA
CHAGAS HEALTHY VI SCERAL LEI SHMANIASI S
BLK Chagas test 25/25 (100%) 1/10 (10%) 5/9 (56%)
Biokit ELI SA 25/25 (100%) 1/10 (10%) 3/9 (33%)
I D-PaGI A 30/31 (97%) 1D/23 (4%) 4D/52 (8%)
BLK Chagas test Biokit ELI SA I D-Pagia
Sensitivity 100 100 97
Specificity 90 90 96
PPV 96 96 97
NPV 100 100 96
Evaluation of Evaluation of serological tests:serological tests:Commercial Commercial ELISAsELISAs
38
Non Chagasic 2
28
Spain 22
Bolivia 19
Brazil 20
Argentina 14
Ecuador 20
30
30
Characteristics of Sera Nº of sera
Panel 1 (QPanel, Brasil)
Chagasic
Chagasic patients diagnosed in Spain
Healthy individuals
Malaria
Panel 2 (Parasitology, CNM, ISCIII)
Panel 3 (Parasitology, CNM, ISCIII)
Visceral leishmaniasis
Characteristics of serum samples Characteristics of serum samples used in the evaluation of used in the evaluation of
commercial testscommercial tests
Evaluation of Commercial Evaluation of Commercial ELISAs: ELISAs: SensitivitySensitivity
(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)
SensitivityChagasicNº of positive
(total sera)
a The specificity was calculated into account results of healthy controlb The specificity was calculated into account results of healthy control and other pathologies.
Evaluation of Commercial Evaluation of Commercial ELISAs: ELISAs: SpecificitySpecificity
(CI 95%) (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)
Other pathologiesSpecificity 2b
Nº of positive (total sera)
Nº of positive(total sera)
Non ChagasicSpecificity 1a
Molecular diagnosis of Molecular diagnosis of T. T. cruzicruzi
kDNA-PCRkDNA-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
Control assay of limit Control assay of limit detectiondetection
Samples of population Samples of population at-riskat-risk
Diagnostic sensitivity and specificityDiagnostic sensitivity and specificity
PCR Positive Negative Total
Acute patients 11 0 11
Chronic patients 471 295 766
Seronegative subjects 0 1227 1227
Total 482 1522 2004
Diagnostic index in chronic phase CI (95%)
Sensitivity 61.5 58 65
Specificity 100 99.96 100
Positive predictive value 100 99.89 100
Negative predictive value 80.62 78.6 82.64
Molecular diagnosis of Molecular diagnosis of T. T. cruzicruzi
Chagas disease cases in Chagas disease cases in SpainSpain
IMPORTED
Immigration from endemic area
Long stays in endemic area
AUTOCTHONOUSAUTOCTHONOUS
Blood transfusion
Congenital transmission
Organ transplant
Laboratory accidents
Latin American population Latin American population in Spainin Spain
On December 2008: > 1.8 million415535
284043236048
196946123173119209
669506140761069
48939268
EcuadorColombia
BoliviaArgentina
PeruBrazil
ParaguayUruguay
VenezuelaChile
Rest of SA
Year of Year of detectiondetection
Year of Year of DonationDonation
N º of N º of recipientsrecipients
Nº of Nº of infectedinfected Ref.Ref.
1992 ? 1? 1 Villalba et al., 1992
Reports of Transfusional Reports of Transfusional Chagas cases in SpainChagas cases in Spain
2005 2004 1 1Fores et al., 2007; Flores-Chavez et al., 2008
2007 2005-2007 9 2 Perez et al., 2008; CNM
2006 1994-2003 10 1 Abalo et al., 2007, CNM
National Haemovigilance System
2008 2004 1 1 Ibarra et al., 2008, CNM
Madrid recipient
Coruña recipient
Málaga recipient 1
Málaga recipient 2
País Vasco
recipientOrigin Spanish Spanish Spanish Moroccan Spanish
Age 27 55 33 57 4
Clinical status before transfusion
Leukemia Hepatic transplant
Bone marrow aplasia
Choroid plexus
papilloma
Cardiac surgery
Phase of Chagas disease and symptoms
AcuteFUO
Multiorganic failure
ChronicAsympto
matic
AcuteFUO
ChronicRight bundle branch block
ChronicAsympto
matic
Treatment Yes Yes Yes Yes Yes
Follow upAfter treatment
Fatal dead
person
S (+) PCR (-)
S (-) PCR (-)
S (+) PCR (-)
S (+) PCR (-)
Transfusional Chagas Transfusional Chagas CasesCases
Characteristics Characteristics of Infected of Infected
donors:donors:A CoruñaA Coruña MadridMadrid MalagaMalaga País VascoPaís Vasco
OriginOrigin Bolivia Brazil Bolivia Bolivia
SexSex Male Female Male Male
AgeAge 62 58 53
ELISAELISA 2.3 2.2 2.3
IFATIFAT > 1/160 > 1/160 > 1/160
PCRPCR PP N/PN/P PP
TreatmentTreatment No Yes Yes
PermanencPermanence in Spaine in Spain 42 years 1 year 3 years
Malaga
Madrid
A CoruñaPaís Vasco
Newborns Positive PCR
2002 12003 32004 2 1*2005 42006 122007 78 32008 91 5Total 191 9
Congenital Chagas Congenital Chagas diseasedisease
Transmission index: ~ 4,5%* Post mortem diagnosis
Newborns examined during 2002 - 2008
In Spain ≈ 990 000 women of childbearing ageBolivia: 132 421 women
Argentina: 94 794 women
Alvar J. 1983 Laboratorio 76(456): 645-648
Laboratory Accidents Laboratory Accidents and Chagas diseaseand Chagas disease
Chagas disease cases in Chagas disease cases in SpainSpain
IMPORTED
Immigration from endemic area
Long stays in endemic area
AUTOCTHONOUSAUTOCTHONOUS
Blood transfusion
Congenital transmission
Organ transplant
Laboratory accidents
Chagas disease in Chagas disease in blood donors at- risk blood donors at- risk
C. T. Cruz Roja de Madrid
Prevalence % 1,23
2890
779500
1250
2000
2750
361 4
0
100
200
2002-2006 2003-2006 2006-2008
357
C.C. de Sangre y Tejidos de Asturias
0,13
2890
779500
1250
2000
2750
361 4
0
100
200
2002-2006 2003-2006 2006-2008
357
Asturias
Madrid
14581164906
4306832975
2516913474
1084089978476
3958
0 30000 60000 90000 120000 150000
EcuadorColombia
PeruBolivia
ArgentinaBrazil
VenezuelaParaguay
ChileUruguay
Madrid
43582721
19031886
915549
435373370
310
0 1000 2000 3000 4000 5000
EcuadorColombiaArgentina
BrazilVenezuelaParaguayUruguay
PeruChile
Bolivia
Asturias
MadridMadrid
AsturiasAsturias
Profile of immigrant populationProfile of immigrant population
2006: ≈ 17 000 2006: ≈ 17 000 immigrantimmigrant2008: ≈ 20 000 immigrant2008: ≈ 20 000 immigrant
2006: ≈ 400 000 2006: ≈ 400 000 immigrantimmigrant2008: ≈ 500 000 immigrant2008: ≈ 500 000 immigrant
Prevalence in blood Prevalence in blood donors at-risk (2002-donors at-risk (2002-
2006)2006) P N Prevalence %
Nicaragua 1 16 6.25Colombia 1 776 0.13Venezuela 1 249 0.40Ecuador 1 908 0.11Peru 2 258 0.78Bolivia 37 203 18.23Brazil 2 221 0.90End. Zone 3 207 1.45Non End. Zone 1 307 0.33ND 3 245 1.22Spain 1 277 0.36
P = SeropositiveN = Seronegative
??
6 5 27 40 75 131219 285
398476
815
1137
1 0 2 7 3 6 26 46 90138
361464
0
200
400
600
800
1000
1200
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
SERONEGATIVE SEROPOSITIVE
SeroprevalenceSeroprevalence in in population at-risk: population at-risk:
24%24%
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008Prevalence % 14 0 7 15 4 4 11 14 18 22 31 29
≈ 1144 Chagasic patients
1
3 79
150
10
4
16
571
1
5
1
2
4
7
1
16
35
33
10
7
528
40
52
8
4
40
3
11
1 59
22
1
3
1
15
Distribution of cases Distribution of cases in Spainin Spain
2289
528
0
500
1000
1500
2000
2500
Seronegative Seropositive
MadridPrevalence
19 %
598
79
0
200
400
600
800
Seronegative Seropositive
AlicantePrevalence
12 %
173
150
50
100
150
200
Seronegative Seropositive
Almeria
Prevalence8 %
0%1%2%3%4%5%
ARGE
NTI
NA
BOLI
VIA
BRAZ
ILCH
ILE
PARA
GUAY
URU
GUAY
COLO
MBI
AEC
UADO
RPE
RUVE
NEZ
UEL
AGU
ATEM
ALA
HON
DURA
SN
ICAR
AGUA
SALV
ADO
RM
EXIC
OSP
AIN
60%
80%
100%
Origin of seropositive Origin of seropositive populationpopulation
ConclusionsConclusions 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.
UNIDAD LEISHMANIA Y CHAGASUNIDAD LEISHMANIA Y CHAGASCarmen CañavateCarmen CañavateJavier NietoJavier NietoMercedes RodriguezMercedes RodriguezIsrael CruzIsrael CruzElena BodasElena BodasMarta HernándezMarta HernándezRubén GonzálezRubén GonzálezEmilia GarcíaEmilia GarcíaCarmen ChicharroCarmen ChicharroMaría FloresMaría Flores
Thank youThank you
Organ transplant and
lab incident
Months Years
Transfusional and congenital
Chagas
Acute Chronic phase
Immigration and long stays in endemic area
Chagas disease in Chagas disease in SpainSpainAUTOCTHONOUSAUTOCTHONOUS
IMPORTEDIMPORTED
??
0
1
2
3
4
5
6
DO/CO
Level of anti-Level of anti-T. cruzi T. cruzi IgGIgG in in samples of patients with samples of patients with
positive positive PCRPCR
Acute case 1(Madrid recipient)
Acute case 2(Málaga recipient)