Download Second WHO consultation: Development of a WHO

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)