PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR

SEROLOGICAL REFERENCE

PROPOSED CANDIDATE MATERIAL FOR A WHO PANEL FOR

SEROLOGICAL REFERENCE

RESULTS OF THE FEASIBILITY STUDIES RESULTS OF THE FEASIBILITY STUDIES WITH SERA FROM WITH SERA FROM

TC I REGIONTC I REGION

RESULTS OF THE FEASIBILITY STUDIES RESULTS OF THE FEASIBILITY STUDIES WITH SERA FROM WITH SERA FROM

TC I REGIONTC I REGION

DR. CARMEN GUZMAN BRACHOInstituto de Diagnóstico y Referencia Epidemiológicos

PROJECT ORGANIZATIONPROJECT ORGANIZATION• WHO, JULY 2007.

– Develop a reference panel. – Commitment to collect and evaluate sera from TcI region accepted.

• National Center of Blood Transfusion, Aug 2007.– Selection of a Blood Transfusion Center.

• State Center of Blood Transfusion in Hidalgo, Sep 2007.– Participate in the writing of the Protocol, responsibilities and consent

form. – Make contact with donors in home towns.– Transfer donors to Pachuca City for blood collections. – Collect Plasma units.

• Institute of Epidemiological Diagnosis and Reference– Coordinate the Project.– Select blood donors.– Evaluate individual sera samples and mixtures.– Analyze results.– Deliver positive and negative plasma units to Fundacao Pró-Sangre

Hemocentro de Sao Paulo, Jun 2008.

MAIN CONDITIONSMAIN CONDITIONS

• Mexican donors.• Final volume: 1.5 – 3.0 L • Since ethical and operative problems with a

single donor raise, it is suggested that a pool of positive sera is more realistic.

• Negative sera are necessary to dilute reactive samples.

• All samples must be negative to viral markers (HIV, HBV, HCV) and syphilis.

HIDALGOSTATE

LABORATORY OF PUBLIC

HEALTH

HIDALGOSTATE CENTER

OF BLOOD TRANSFUSION

SELECTION OF CANDIDATES

SELECTION OF CANDIDATES

DATA BASE • CONFIRMED CASES

– SCREENING TEST FOR POSITIVES • ELISA (BIOKIT)

– CONFIRMATION TESTS OF POSITIVES • ELISA recombinant (BIOKIT)• ELISA crude (INTERBIOL)• HAI (INTERBIOL)• IFI (InDRE)

INITIAL RESULTS FOR SELECTED CANDIDATES

INITIAL RESULTS FOR SELECTED CANDIDATES

10 mL of serum from 10 candidates were collected to confirm tests results

NUMBER

Recombinant antigen

Recombinant antigen

Crude antigen HAI IFI

DATE OF BLOOD SAMPLING FOR

EVALUATION

1 POSITIVE POSITIVE POSITIVE NR POSITIVE 24-Sep-072 POSITIVE POSITIVE POSITIVE 128 24-Sep-073 POSITIVE POSITIVE POSITIVE 64 04-Oct-074 POSITIVE POSITIVE POSITIVE 64 04-Oct-075 POSITIVE POSITIVE POSITIVE 16 02-Oct-076 POSITIVE POSITIVE POSITIVE 16 08-Oct-077 POSITIVE POSITIVE POSITIVE 64 19-Oct-078 POSITIVE POSITIVE POSITIVE 8 09-Oct-079 POSITIVE POSITIVE POSITIVE 128 22-Oct-07

10 POSITIVE POSITIVE POSITIVE 128 22-Oct-07

BIOKIT BIOKIT INTERBIOL INTERBIOL InDRE

SCREENING TEST

CONFIRMATORY TESTS

Mexican strains

Titration methodsEnzime Inmuno Assay

FINAL RESULTS FOR SELECTED CANDIDATES

FINAL RESULTS FOR SELECTED CANDIDATES

According to the OD/CO index, 6 serum samples were selected to evaluate reactivity of mixtures

Reactivity OD/CO

LOW 2.81 – 3.55

MEDIUM 4.19 – 5.81

HIGH 7.74 – 8.39

NUMBER HAI-InDRE ELISA-InDRE OD/CO CLASSIFICATION

1 POSITIVE 1.1 3.55 LOW REACTIVE2 POSITIVE 0.7 2.26 LOW REACTIVE3 POSITIVE 2.4 7.74 HIGH REACTIVE4 POSITIVE 1.4 4.52 MEDIUM REACTIVE5 POSITIVE 1.3 4.19 MEDIUM REACTIVE6 NEGATIVE 0.27 0.87 DISCARD7 POSITIVE 1.3 4.19 MEDIUM REACTIVE8 POSITIVE 2.6 8.39 HIGH REACTIVE9 POSITIVE 1.8 5.81 MEDIUM REACTIVE

10 POSITIVE 0.87 2.81 LOW REACTIVE

UGC 0.11 0.5 NEGATIVE

CUT OFF 1:8 CUT OFF = 0.31

COMPOSITION OF MIXTURES

COMPOSITION OF MIXTURES

NUMBER HAI-InDRE ELISA-InDRE OD/CO CLASSIFICATION

2 POSITIVE 0.7 2.26 LOW REACTIVE10 POSITIVE 0.87 2.81 LOW REACTIVE7 POSITIVE 1.3 4.19 MEDIUM REACTIVE4 POSITIVE 1.4 4.52 MEDIUM REACTIVE3 POSITIVE 2.4 7.74 HIGH REACTIVE8 POSITIVE 2.6 8.39 HIGH REACTIVE

SERA MIXTURE

SERUM INCLUDED

mL

M1 2, 3 LOW - HIGH (2.26 - 7.74) 1.0 (0.5 +0.5)M2 4, 3 MEDIUM - HIGH (4.52 - 7.74) 1.0 (0.5 +0.5)M3 10, 8 LOW - HIGH (2.81 - 8.39) 1.0 (0.5 +0.5)M4 7, 8 MEDIUM - HIGH (4.19 - 8.39) 1.0 (0.5 +0.5)

COMPOSITION (OD/CO - OD/CO)

Individual sera data

Mixtures data

REACTIVITY OF THE MIXTURES

REACTIVITY OF THE MIXTURES

• TWO REPLICATES PER PLATE• TWO RUNNINGS

REACTIVITY IN ELISA MEXICAN CRUDE ANTIGEN WITH SERA MIXTURES FROM T. cruzi INFECTED MEXICAN INDIVIDUALS

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10DILUTION

OD

/CO

MEZCLA1 MEZCLA2 MEZCLA3 MEZCLA4

Low reactivity

M1 M2 M3 M4 M1 M2 M3 M41 volume / volume 1000 original mixture 2.082 2.149 2.29 2.355 6.72 6.93 7.39 7.602 100 mL OM + 100 mL neg 200 2 1.744 1.888 1.777 1.963 5.63 6.09 5.73 6.333 100 mL OM + 200 mL neg 300 3 1.697 1.747 1.609 1.777 5.47 5.64 5.19 5.734 100 mL OM + 300 mL neg 400 4 1.589 1.635 1.475 1.522 5.13 5.27 4.76 4.915 50 mL OM + 200 mL neg 250 5 1.438 1.602 1.167 1.37 4.64 5.17 3.76 4.426 50 mL OM + 250 mL neg 300 6 1.222 1.359 1.079 1.225 3.94 4.38 3.48 3.957 50 mL OM + 350 mL neg 400 7 1.198 1.372 1.026 1.03 3.86 4.43 3.31 3.328 40 mL OM + 360 mL neg 400 10 1.006 1.141 0.853 0.979 3.25 3.68 2.75 3.169 20 mL OM + 980 mL neg 1000 50 0.406 0.504 0.316 0.343 1.31 1.63 1.02 1.11

10 10 mL OM + 990 mL neg 1000 100 0.258 0.345 0.226 0.259 0.83 1.11 0.73 0.84

OD ELISA InDRE OD / COCOMPOSITION mL DILUTION

REACTIVITY OF SERA MIXTURES BY DILUTION

REACTIVITY OF SERA MIXTURES BY DILUTION

WIDTH CI 95%= 0.68

WIDTH CI 95% = 1.37

DIL

100.0050.0010.007.006.005.004.003.002.001.00

Abs

/Co

8

7

6

5

4

3

2

1

0

MEZCLA1 (M1)

Fit for M1

95% LCL

95% UCL

DILUCION 1/x

100.0050.0010.007.006.005.004.003.002.001.00

Abs

/Co

10

9

8

7

6

5

4

3

2

1

0

MEZCLA (M3)

Fit for M3

95% LCL

95% UCL

DILUCION 1/x

100.0050.0010.007.006.005.004.003.002.001.00

Abs

/Co

10

9

8

7

6

5

4

3

2

1

0

MEZCLA (M4)

Fit for M4

95% LCL

95% UCL

DILUCION 1/x

100.0050.0010.007.006.005.004.003.002.001.00

Abs

/Co

8

7

6

5

4

3

2

1

0

MEZCLA2 (M2)

Fit for M2

95% LCL

95% UCL

WHEN ADDING ANOTHER SERUM TO THE MIXTURE…..

WHEN ADDING ANOTHER SERUM TO THE MIXTURE…..

DILUCION 1/x

100.0050.0010.007.006.005.004.003.002.001.00

Abs

/Co

6

5

4

3

2

1

0

MEZCLA 5 (M5)

Fit for M5

95% UCL for M5

95% LCL for M5

DILUCION 1/x

100.0050.0010.007.006.005.004.003.002.001.00

Abs

/Co

6

5

4

3

2

1

0

MEZCLA 6 (M6)

Fit for M6

95% LCL for M6

95% UCL for M6

Mixture = 2 high + 1 medium + 1 low

M1 M2 M5 M6 M1 M2 M5 M6original mixture 1.64 1.78 1.72 1.80 5.28 5.74 5.54 5.81

2 1.30 1.19 1.50 1.42 4.19 3.84 4.83 4.573 1.27 1.34 1.28 1.28 4.09 4.32 4.14 4.114 1.21 1.28 1.20 1.10 3.91 4.12 3.86 3.545 1.07 1.15 1.12 0.99 3.45 3.70 3.61 3.196 0.93 1.02 1.04 0.93 2.99 3.30 3.34 2.987 0.77 0.88 0.88 0.85 2.49 2.85 2.83 2.74

10 0.71 0.76 0.76 0.66 2.30 2.45 2.45 2.1250 0.30 0.28 0.33 0.26 0.97 0.90 1.05 0.85

100 0.22 0.24 0.26 0.21 0.69 0.76 0.84 0.69

DILUTIONOD ELISA InDRE OD / CO

MEXICAN SERA BELONG TO T. cruzi I

MEXICAN SERA BELONG TO T. cruzi I

1. 54 of 56 isolates studied coming from 8 provinces of the Mexican Republic were TcI.

2. Given the prevalence of seropositive individuals and of disease cases in the country, T. cruzi I stocks can be considered the primary agents of Chagas’ disease in Mexico

– Bosseno et al. Predominance of Trypanosoma cruzi lineage I in Mexico. J Clin Microbiol. 2002 feb;40(2):627-32.

– Sánchez-Guillén et al. Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity.Mem Inst Oswaldo Cruz. 2006 Sep;101(6):585-90.

PROTOCOLNUMBER REACTIVITY

DATA FOR TRACEABILITY CHAIN

No InDRE FOLIO CETS UNIT NUMBER AGE

3 HIGH 573 11303 801604 48

4 MEDIUM 155 2317 801603 43

SERA SENT TO BRAZIL

PAGE-SDS 10%. MW (kDa) , 1. POS, 2. NEG, 3. LEISH, 4. S3, 5. S4, 6. M2, 7. 1/7, 8. 1/10,

250150

10075

50

37

25

kDa 1 2 3 4 5 6 7 8

PAGE-SDS 10%. MW (kDa) , 1. POS, 2. NEG, 3. LEISH, 4. S3, 5. S4, 6. M2, 7. 1/7, 8. 1/10,

250150

10075

50

37

25

kDa 1 2 3 4 5 6 7 8

250150

10075

50

37

25

kDa 1 2 3 4 5 6 7 8

DISTANCE (PIXELS)

INV

ER

TE

D G

RA

Y V

AL

UE

S3

S4

M2

1/7

1/10

DISTANCE (PIXELS)

INV

ER

TE

D G

RA

Y V

AL

UE

S3

S4

M2

1/7

1/10

SERUM IFA

S3 1:256

S4 1:256

M2 1:256

DIL 1/7 1:64

DIL 1/10 1: 32

DIL 1/50 NEG

SERUM IFA

S3 1:256

S4 1:256

M2 1:256

DIL 1/7 1:64

DIL 1/10 1: 32

DIL 1/50 NEG

Complementary testsComplementary tests

CONCLUSIONSCONCLUSIONS

• Mixture 1 and 2 turned out to be the best.• Optimal dilution was found at 1:10.• We got 200 mL of plasma from both

donors included in mixture 2.• Plasma units were sent to Brazil (M. Otani)

coded 801603 and 801604.• Finally, 10 plasma units of seronegative

individuals to prepare final dilution were delivered to Brazil.

MANY THANKS!