Proposed candidate materials and Proposed candidate materials and results of the feasibility studies for a results of the feasibility studies for a WHO serological reference panelWHO serological reference panel

T.T.cruzicruzi type II type II

Márcia OtaniMárcia OtaniFundação Pró-Sangue Hemocentro de São PauloFundação Pró-Sangue Hemocentro de São Paulo

Raw materialRaw material Epidemiological dataEpidemiological data

ID Age Gender State Collected date

Plasma volume

1 103362946 31 Female Minas Gerais

Januaria, for 28 years

2007-02-15 200

2 103350016 58 Male Chile

Canela,

2007-03-16 220

3 103402366 51 Female Pernambuco

Buique, for 40 years

2007-05-01 180

4 801603 43 Male Hidalgo, México 2008-02-08 202

5 801604 48 Male Hidalgo, México 2008-02-08 202

3

1

2

4 y 5

3

T. cruzi IT. cruzi IIaT. cruzi IIbT. cruzi IIcT. cruzi IIdT. cruzi IIe

Linajes filogenéticos deTrypanosoma cruzi

Adaptado de Souto et al. 1996;Fernandes et al. 1998; Brisse et al.

2000, 2004.

T. cruzi IT. cruzi IIaT. cruzi IIbT. cruzi IIcT. cruzi IIdT. cruzi IIe

Linajes filogenéticos deTrypanosoma cruzi

Adaptado de Souto et al. 1996;Fernandes et al. 1998; Brisse et al.

2000, 2004.

1 2 3

1-Have you ever heard about Chagas Disease? Y Y Y

2-Do you know how to recognize a kissing bug? Y Y Y

3-Have you ever lived in a house constructed of wood and mud? N N Y

4-Have you had contact with a kissing bug? N Y Y

5-Have any of your relatives ever had Chagas disease? Y N Y

6-Did you live in the same house with a relative who is positive for Chagas Disease?

N N Y

7-Do you know any case of Chagas Disease in the region where you have born or where you have lived?

N Y Y

8-Have you ever received blood transfusion? N N N

9-Have you ever been to any Chagas endemic region? N N N

10-Have you ever had any clinical symptoms for Chagas Disease ? N N Y

11-Are you under medical treatment ? N N Y

12-Have you ever done an electrocardiogram (EKG)? Y Y Y

Epidemiological QuestionnaireEpidemiological Questionnaire

Raw materialRaw material - - ReactivityReactivity

ID

Elisa cruzi

OD/CO

Chagatek

OD/CO

Bioschile

OD/CO

Imuno cruzi

Titre Used

volume

103362946200 ml

9.36Epd: 1/128

6.19Epd: 1/32

4.02Epd: 1/128

1/160 80 ml

103350016220 ml

8.31Epd: 1/128

6.37Epd: 1/64

4.24Epd: 1/128

1/320 120 ml

103402366180ml

7.97Epd: 1/64

5.21Epd: 1/32

3.35Epd: 1/64

1/160 60 ml

801603202 ml

4.71Epd: 1/16

5.03Epd: 1/16

3.15Epd: 1/32

1/160 2 ml

801604202 ml

5.37Epd: 1/64

5.10Epd: 1/64

3.25Epd: 1/128

1/320 2 ml

Prototype = 260ml of positive + 1240 ml of negative plasma from 7 donors units.

(4.8 times diluted.)

Epd = end point dilution (OD/CO) > 1

Raw material - serial dilution OD/CO ratioRaw material - serial dilution OD/CO ratio

Elisa cruzi 103362946 103350016 103402366 801603 801604Dilution DO/CO DO/CO DO/CO DO/CO DO/CO

Pure 9,362 8,312 7,965 4,708 5,369 1/2 8,546 7,646 7,688 4,115 5,738 1/4 6,885 7,192 6,769 2,962 4,846 1/8 5,219 5,758 5,535 2,154 3,931

1/16 3,462 4,481 3,908 1,496 3,158 1/32 2,777 3,373 2,388 0,927 2,477 1/64 1,858 2,131 1,438 0,596 1,612 1/128 1,273 1,465 0,935 0,431 0,842 1/256 0,835 0,769 0,635 0,369 0,762 1/512 0,615 0,546 0,473 0,281 0,492

1/1024 0,465 0,335 0,358 0,262 0,415

Chagatek 103362946 103350016 103402366 801603 801604Dilution DO/CO DO/CO DO/CO DO/CO DO/CO

Pure 6,187 6,368 5,213 5,032 5,097 1/2 5,735 6,065 4,735 4,213 4,600 1/4 4,503 5,342 3,600 2,961 3,819 1/8 3,181 3,929 2,465 2,052 2,955

1/16 2,277 3,542 1,639 1,329 2,806 1/32 1,471 2,194 1,097 0,884 1,561 1/64 0,929 1,335 0,735 0,639 1,032 1/128 0,697 0,890 0,542 0,490 0,677 1/256 0,529 0,639 0,374 0,368 0,452 1/512 0,413 0,413 0,374 0,374 0,381

1/1024 0,374 0,387 0,355 0,381 0,323

Bioschile 103362946 103350016 103402366 801603 801604Dilution DO/CO DO/CO DO/CO DO/CO DO/CO

Pure 4,024 4,235 3,346 3,147 3,246 1/2 3,831 4,000 3,164 2,673 3,132 1/4 3,629 3,559 2,904 2,377 2,814 1/8 3,039 3,211 2,590 1,836 2,156

1/16 2,452 2,612 2,092 1,327 1,954 1/32 1,958 2,287 1,675 1,158 1,647 1/64 1,518 1,794 1,127 0,748 1,436 1/128 1,184 1,316 0,853 0,537 1,081 1/256 0,842 0,987 0,607 0,436 0,783 1/512 0,610 0,691 0,456 0,357 0,640

1/1024 0,463 0,515 0,346 0,325 0,456

= Prototype

= M2

Clinical Sensitivity and Specificity Clinical Sensitivity and Specificity

Chagas Test Elisa III730 T. cruzi positive blood donor samples

020406080

100120140160

O.D./C.O.

Fre

qu

ency

Elisa cruzi730 T. cruzi positive blood donor sample

0

5

10

15

20

25

30

35

40

45

0

0,6

1,2

1,8

2,4 3

3,6

4,2

4,8

5,4 6

6,6

7,2

7,8

8,4 9

9,6

O.D./C.O.

Fre

quency

Chagas Test Elisa III956 T. cruzi negative Blood donor samples

0

5

10

15

20

25

30

35

0

0,11

0,22

0,33

0,44

0,55

0,66

0,77

0,88

0,99 1,1

1,21

1,32

1,43

1,54

1,65

1,76

1,87

1,98

O.D./C.O.

Fre

quen

cy

Elisa cruzi828 T. cruzi negative blood donor sample

0

10

20

30

40

50

60

70

0

0,11

0,22

0,33

0,44

0,55

0,66

0,77

0,88

0,99 1,

1

1,21

1,32

1,43

1,54

1,65

1,76

1,87

1,98

D.O./C.O.

Fre

qu

ency

Mexico SamplesElisa cruzi

01

23

45

67

Serial Dilution

OD

/CO

801603

801604

M2

Mexico SamplesChagatek

0

1

2

3

4

5

6

Serial Dilution

OD

/CO 801603

801604

M2

Mexico SamplesTest Elisa p/ Chagas III

0

1

2

3

4

Serial Dilution

OD

/CO 801603

801604

M2

Elisa cruzi Chagatek Bioschile IFIPure 4,962 5,406 3,143 1/80 1/2 4,792 4,774 3,042 1/40 1/4 4,677 3,619 2,803 1/20 1/8 3,758 2,703 2,287 Negative

1/16 2,612 1,910 2,020 Negative 1/32 1,896 1,297 1,616 Negative 1/64 1,362 0,865 1,309 Negative 1/128 0,923 0,542 1,046 Negative 1/256 0,581 0,477 0,768 Negative 1/512 0,435 0,413 0,579 Negative

M2M2

OD/CO ratio

Brazil SamplesElisa cruzi

0123456789

10

Pure

1/2

1/4

1/8

1/1

6 1

/32

1/6

4

1/1

28

1/2

56

1/5

12

1/1

024

Serial Dilution

OD

/CO

103362946

103350016

103402366

Prototype

Brazil SamplesChagatek

0

1

2

3

4

5

6

7

Serial Dilution

OD

/CO

103362946

103350016

103402366

Prototype

Brazil SamplesTest Elisa P/ Chagas III

0

1

2

3

4

5

Pure

1/2

1/4

1/8

1/1

6 1

/32

1/6

4

1/1

28

1/2

56

1/5

12

1/1

024

Serial Dilution

OD

/CO

103362946

103350016

103402366

Prototype

Elisa cruzi Chagatek Bioschile IFIPure 5,723 3,910 3,252 1/40 1/2 5,004 2,994 2,704 1/20 1/4 3,435 1,935 2,086 Negative 1/8 2,262 1,219 1,711 Negative

1/16 1,338 0,794 1,349 Negative 1/32 0,862 0,568 0,958 Negative 1/64 0,592 0,452 0,658 Negative

PrototypePrototype

OD/CO ratio

Evaluating the Prototype Evaluating the Prototype • EQAS to evaluate performance in loco.

• Send 1.8ml of the prototype serum to 25 reference centers in 16 countries in LA.

• Included 4 other non diluted not pooled samples to the same panel.

• Sep 2007 (OPS0207), Apr 2008 (OPS0108) and Sep 2008 (OPS0208).

Data from Latin America Reference Centers – PAHO Regional EQAS

Prototype X 2 times diluted Reactivity by Kit April 2008

0

2

4

6

8

10

12

14

16

2a 3a 4a 22a 9

10b 26 18

a17

a24

a 21 21 13a 26 14

b28

b 5b 8b 7b 1b 23b

20b

16b

24a

Results

OD

/CO

Prototype Diluted 2X - - - - - DO/CO mean

Chagas Test Elisa III

Chagatest Elisa

Wiener Recomb. Chagatek

Biokit Elisa cruzi

UCR IICS Col. Chile

Bioschile

Cut offCut off

Reactivity

0

2

4

6

8

10

12

14

16

18

20

13a

17a

18b

18a

17b 21 22

b 21 19 13a

17a

18b

20b 6

14a

28a 1b 14

a28

b11

b13

a14

b

Laboratory

OD

/CO

Prototype Mean

Diluted 2X Mean

Chagatest Elisa Wiener CV = 35% Chagatest Elisa Recombinante

Wiener CV = 27%

ChagascreenCV = 9%

Chagatek CV = 31%

Data from Latin America Reference Centers – PAHO Regional EQAS

Sep 2007 Apr 2008 Sep 2008

Data from Latin America Reference Centers – PAHO Regional EQAS

Reactivity

0

1

2

3

4

5

6

7

8

9

10

23a

23a

23a 7a 7a 1b 3a 27 3a 4b 9 27 1a 2b 4a 5b 26 10

a 5a 8b 8a 10a

Laboratory

OD

/CO

Prototype Mean

Diluted 2X Mean

Elisa cruziCV = 16% Chagastest Elisa III

CV = 24%BiokitCV = 10% Test Elisa p/ Chagas

CV = 14%

Sep 2007 Apr 2008 Sep 2008

Data from Latin America Reference Centers – PAHO Regional EQAS

Reactivity

0

1

2

3

4

5

22a 22a 22b 20a 20b 20a 20b 16a 16b 16a 16b 16a 16b 16c 24a 24a 24b 24a 24b 25 25

Laboratory

OD

/CO

Prototype Mean

Diluted 2X Mean

PeroxidasaCV = 4,3 %

Colombia

Chile EIACV =11 %

Chagas Test IICSCV = 11 %

LSU-ICMRTCV = 75%

Sep 07 Apr 2008 Sep 08

Stability of the Prototype Kit / Semester

02468

101214

Test

Elis

a p/

Cha

gas

Elisa

cruz

i

Chaga

s Te

st E

lisa

III

Chaga

test

Elis

a

Chaga

test

Elis

a Rec

Bioelis

a Cha

gas

Chaga

s Te

st II

CS ELI

SA

Chaga

s EIA

Chaga

tek

ELISA

Chaga

s cr

een

Chaga

s Elis

a Per

oxida

sa

Chaga

s EIA

Colo

mbia

Test

Elis

a p/

Cha

gas

(UCR)

Chaga

s Pha

rmat

est

Kit

OD

/CO

Sep 2007 April 2008 Sep 2008

Data from Latin America Reference Centers – PAHO Regional EQAS

Data from Latin America Reference Centers – PAHO Regional EQAS

Prototype X 2 times diluted April 2008

0

2

4

6

8

10

Test

Elis

a p/

Cha

gas

Elisa

cruz

i

Chaga

s Te

st E

lisa

III

Chaga

test

Elis

a

Chaga

test

Elis

a Rec

Bioelis

a Cha

gas

Chaga

s Te

st II

CS ELI

SA

Chaga

s EIA

Chaga

tek

ELISA

Chaga

s cr

een

Chaga

s Elis

a Per

oxida

sa

Chaga

s EIA

Colo

mbia

Test

Elis

a p/

Cha

gas

(UCR)

Chaga

s Pha

rmat

est

Imun

ocru

zi IF

I

Polich

aco

HAI Cha

gas

Kit

OD

/CO

Prototype Diluted 2x

Stability of Donor Sample (not pooled not diluted)

02468

101214

Test

Elis

a p/

Cha

gas

Elisa

cruz

i

Chaga

s Te

st E

lisa

III

Chaga

test

Elis

a

Chaga

test

Elis

a Rec

Bioelis

a Cha

gas

Chaga

s Te

st II

CS ELI

SA

Chaga

s EIA

Chaga

tek

ELISA

Chaga

s cr

een

Chaga

s Elis

a Per

oxida

sa

Test

Elis

a p/

Cha

gas

(UCR)

Chaga

s Colo

mbia

Chaga

s Pha

rmat

est

Imun

ocru

zi IF

I

Polich

aco

HAI Cha

gas

Kit

OD

/CO

Sep 2007 April 2008

Stability of prototype

0

2

4

6

8

10

12

14

Test

Elis

a p/

Cha

gas

Elisa

cruz

i

Chaga

s Te

st E

lisa

III

Chaga

test

Elis

a

Chaga

test

Elis

a Rec

Bioelis

a Cha

gas

Chaga

s Te

st II

CS ELI

SA

Chaga

s EIA

Chaga

tek

ELISA

Chaga

s cr

een

Chaga

s Elis

a Per

oxida

sa

Chaga

s EIA

Colo

mbia

Test

Elis

a p/

Cha

gas

(UCR)

Chaga

s Pha

rmat

est

Imun

ocru

zi IF

I

Polich

aco

HAI Cha

gas

Kit

OD

/CO

Sep 2007 April 2008

Data from Latin America Reference Centers PAHO Regional EQAS

Prototype stability by temperature / timeElisa cruzi

4,0

4,2

4,4

4,6

4,8

5,0

5,2

5,4

5,6

5,8

6,0

nov-07 jan-08 mar-08 mai-08 jul-08 set-08 nov-08

Assay Date

Me

an

O

D/C

O

-20ºC

4 ºC

22 ºC

37 º C

Mean

Mean

Mean

Mean

Prototype volume available = 1.4 liters

2 times dilution = 2.8 liters

It is possible to obtain a total of 6 liters

Units used mL available mL

1 80 120

2 120 100

3 60 120

4 2 200

5 2 200

Volume Volume

Mexico Units

M2 volume available = 400 mL

4 times dilution = 1.6 liters

ConclusionConclusion

• The mixture of 3 positive samples (from south of Latin America) diluted 4.8 times in negative samples have produced a prototype that presented stability at –20 ºC.

• We need to define the reactivity of the cut off value. Here we have used the dilution that is IIF = 1/20.

• Reactivity of the prototype diluted 2 times were 2 to 4 times de cutoff value in most of the tests evaluated and the IIF = 1/20.

• Perhaps we should submit this prototype to RIPA, Western Blot and Abbott Immunoassay.

Reactivity

0

2

4

6

8

10

12

14

16

18

20

13a

17b

18a

24b

21

13b

18b

21

8a

28a

26

13a

23b

7a

1a

4a

27

4a

10a

1a

3b 6 26 3 7a

10a

22a

20b

16b

16c

24a

Laboraotry

OD

/CO

Prototype Mean

Diluted 2X Mean

Reactivity

0

2

4

6

8

10

12

14

16

18

20

13a

17b

18a

24b

21

13b

18b

21

8a

28a

26

13a

23b

7a

1a

4a

27

4a

10a

1a

3b 6 26 3 7a

10a

22a

20b

16b

16c

24a

Laboraotry

OD

/CO

Prototype Mean

Diluted 2X Mean

1

2

Reactivity

0

2

4

6

8

10

12

14

16

18

20

13a

17b

18a

24b

21

13b

18b

21

8a

28a

26

13a

23b

7a

1a

4a

27

4a

10a

1a

3b 6 26 3 7a

10a

22a

20b

16b

16c

24a

Laboraotry

OD

/CO

Prototype Mean

Diluted 2X Mean

4

56 7 8 9 10

11 12 13

3

1

2

Sep 2007 April 2008 Sep 2008Kit DO/CO DO/CO DO/CO

1 Chagas creen 11,947 - -2 Chagatest Elisa 9,507 8,881 8,1003 Chagatest Elisa Rec 7,470 8,882 8,2074 Chagatek ELISA 5,635 6,341 6,0635 Elisa cruzi 4,704 3,853 4,9906 Bioelisa Chagas 3,264 3,506 -7 Chagas Test Elisa III 3,023 3,088 2,8408 Test Elisa p/ Chagas 3,079 2,871 2,9019 Chagas Elisa Peroxidasa 3,048 - 3,110

10 LSU-ICMRT 4,399 0,930 -11 Chagas Test IICS ELISA 2,417 1,996 2,44012 Chagas EIA 1,920 1,588 1,43613 Chagas EIA Colombia - 1,430 1,020

Reference Serum

Data from Latin America Reference Centers PAHO

Regional EQAS