High polymorphism of parasites isolates is associated with Cerebral Malaria in Dakar.

Bob NS, Diop BM, Marrama L, MT Ekala, Tall A, Ka B, HovettePh, Seck SY, O Mercereau-Puijalon, Jambou R

1 Institut Pasteur de Dakar, 2 Clinique des maladies infectieuses CHN Fann, 3 Institut Pasteur Paris, 4 Hôpital Principal de Dakar

Severe Malaria

Newborn

fever

dehydratation

convulsion

Metabolic disorders

= impaired consciousness

Child

Severe anemia +++

High parasiteamia +++

Cerebral attack

Adult

Respiratory distress

cerebral malaria

low parasiteamia

= delay in treatment

mechanic hypothesis

Van der heyde 2006

Activation des endothéliums = expression d’ICAM

Adhesion RBC

Adhesion leukocytes

Disruption tigh junction = leakage

hemorrhage

Apoptose EC

Adhesion platelets

Blockage capillaries

1

3

22

3

2

4

3

mechanic + immune

4

4InflammationLocal / general

Do isolates differ

MILD / CEREBRAL malaria

Which mechanism the most important ?

RATIONALE

�low transmission

�Low immunity

�Drug resistance

�Travel

�Highly virulent strains ?

� treatment retardation

Malaria in Urban area

15°

14°

13°

16°N

Dakar

17°W 16° 15° 14° 13° 12°

SENEGAL

Area of Study

Seasonal transmission

3 M inhabitants

An arabiensis

Prevalence of malaria in consultations

0

0,05

0,1

0,15

0,2

0,25

0,3

septembre octobre novembre decembre

0-1 y

1- 4 y

5-14 y

15- 49 y

> 50 y

0

0,1

0,2

0,3

0,4

0,5

0,6

septembre octobre novembre decembre

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

septembre octobre novembre decembre

part of class of age in the malaria cases

prevalence of severe malaria.

0

200

400

600

800

1000

1200

1400

M F M F M F M F M F

0-1 an 1- 4ans 5-14 ans 15- 49 ans 50ans &+

consultations by age

MALARIA AT CHN FANN

Number of cases / 2004

0 100 200 300 400 500 600

CHOLERA

HIV

malaria

Tetanos

infections

Meningitides

Tuberculosis

Others

In emergency unit, malaria = only 10% of the patients

� Number of severe malaria : 170

� Geographic origin of the patients : Dakar 92%

(urban population in Africa : 2030 = 50%)

� Seasonality of the cases

Severe Malaria at CHN Fann , 2004

MALARIA AT CHN FANN – 2004 -

Rainy season

0

5

10

15

20

25

30

35

40

No

mb

re d

e

J F M A M J J A S O N D

Mois

� Age : 15-34 ans = 64% of patients

� Sex-ratio = 1,46 (male +++)

� Delay before hospitalization > 48 hours for 68% patients

� Evolution

- Duration of hospitalization : mean 5 days [0 to 40 d]

- Total Mortality : 26,8%

MALARIA AT CHN FANN

HospitalsHospitals

(1) Clinical enrolment : cerebral disorder + fever (1) Clinical enrolment : cerebral disorder + fever

(2) Classification as (2) Classification as

-- cerebral malaria (cerebral malaria (ICT + thick smear (+) / meningitides (ICT + thick smear (+) / meningitides (--) / Glasgow) / Glasgow))

-- Mild malariaMild malaria

-- othersothers

DispensariesDispensaries

(1) mild malaria : ICT and thick smear (+) / fever / symptoms of(1) mild malaria : ICT and thick smear (+) / fever / symptoms of

malaria / no symptoms of CMmalaria / no symptoms of CM

(2) matched for age / gender / area in town, with CM(2) matched for age / gender / area in town, with CM

ENROLMENT

TAGCATGTTTGGTATAGGGGTTAAATCAGGACAACAT

TAGGGAACATCATAAGGATAGCATGTTTGGTATAGG161Chr6TAA109

GAGTAATA TGAACATGT

AATGGCAACACCATTCAAC

ATGGGTTAAATGAGGTACA

GAGTAATA TGAACATGT112Chr6TAA87

ATCATGCATTTCAGTCTGAGGTCTGCTTGTTCCTTCTTT

CTTTCATCGATACTACGAATCATGCATTTCAGTCTGAGG168Chr12PFPK2

TTATGTTGGTACCGTGTATGTTAATCGTAGGGATAA

GATCTCAACGGAAATTATTTATGTTGGTACCGTGTA98Chr12PFGG377

TATTAATAATACTCAAAGC

GAATAAACAAAGTATTGCT

GTACATATGAATCACCAA

TATTAATAATACTCAAAGC70Chr11ARA2

ATGATGTGCAGATGACGAGTGCATTCAATAATTCTA

TTCTAAATAGATCCAAAGATGATGTGCAGATGACGA87Chr102490

CTTTAGTAGTAGTAATAATACTAGAAACAGGAATGATACG

ACAAAAGGGTGGTGATTCTCTTTAGTAGTAGTAATAATAC183Chr5TAA42

GTAATATTTAAAAAGAGAAG TTTGTAATATTTAAAAAGAGAAG

ATGTGTAAGGAGATAGTATAGTAATATTTAAAAAGAGAAG 112Chr77A11

TGACTCTTTGATTATATACCCAAAAGAAGTAATATATGTGCCC

CAAAAGAAGTAATATATGTGCTGACTCTTTGATTATATACC133Chr9BM27

Primers 2Primers 1SizeChromosomeName

Microsatellites and primers used in this study

DHFR1850 bp

Sequencing of Pfdhfr and exon 2 Pfcrt

dhfr ts

PfCRT

Exon 2

PCR amplification Sequencing

250 bp

Hôpital Principal de DakarHôpital Principal de Dakar

59 patients : 20 CM / 39 59 patients : 20 CM / 39 mildmild malariamalaria

27 27 womenwomen / 32 / 32 menmen

age : 2 to 67 age : 2 to 67 yearsyears

CHN CHN FannFann

15 patients : 12 CM / 3 15 patients : 12 CM / 3 mildmild

5 W et 10 M 5 W et 10 M

age 16 to 42 age 16 to 42 yearsyears

Centre de SantCentre de Santéé GuediawayeGuediawaye

104 patients : 2 CM / 102 104 patients : 2 CM / 102 mildmild malariamalaria

66 W et 38 M66 W et 38 M

age 3 to 65 age 3 to 65 yearsyears

PATIENTS

CLINICAL DATA

57,8%31,7%35,3%patients consulting before 4 days after beguining of the symptoms

18,4 %38,5 %27,5 %patients treated before consultation

1043612762656Parasitemia (paras. / µL)

10,612,29,7Hemoglobin (mean g/L)

14 %12,5%14,7%Patients with temperature > 40°C (%)

0.81.51Sex ratio

13.7 (11)15.5 (13)14.5 (10.3)Mean age (SD)

1024234N°patients

Mild Mal. dispensary

Mild Mal Hospital

Cerebral malaria

MUTATIONS in DHFR and PfCRT

+++triple

+++++two

+-+one

NRImutant

SCNwild

1085951codon

MM n= 28CM n= 16

MM n= 102CM n= 37

DHFR CRT exon 2

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

Wild 1 mutat. 2 mutat. 3 mutat.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

CVIET CVIET/CVMNK CVMNK

CM

MM

Multiple Infections

0

10

20

30

40

50

60

70

80

90

100

CerebralMalaria

Mild Mal (hosp)

Mild Mal. (dispens)

0

0,5

1

1,5

2

2,5

Percent of multi-infection

n°population/ patients

Multiple Infections (by microsatellite)

0

5

10

15

20

25

30

35

40

45

50

TAA

42

PFPK2

m24

90

AR

A2

TAA

87

BM

27

TAA

109

PFG37

7

7A11

Cerebral Malaria

Mild Mal (hosp)

Mild Mal. (dispens)

TAA109, PFG377, 7A11, ARA2 = highlypolymorphic in hospitalized patients

Number of alleles to describe50% of the patients Average = 2 for CM / 1 for MM

Total number of alleles/µSatAverage = 8 for CM and MM

= same

0

2

4

68

10

12

14

16

18

TAA

42

PFPK2

m24

90

AR

A2

TAA

87

BM

27

TAA

109

PFG37

7

7A11

Cerebral Malaria

Mild Mal (hosp)

Mild Mal. (dispens)

0

0,5

1

1,5

2

2,5

3

3,5

TAA42 PFPK2 m2490 ARA2 TAA87 BM27 TAA109PFG377 7A11

0

1

2

3

4

5

6

TAA

42

PFPK2

m24

90

AR

A2

TAA

87

BM

27

TAA

109

PFG37

7

7A11

Number of alleles ot describe75% of the patients Average = 3 for CM / 2 for MM

All µSat = more polymorphicin hospitalized patients

TAA42

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1 2 3 4 5 6 7 8 9 10 11 12

MM

CM

2490

0

0,1

0,2

0,3

0,4

0,5

0,6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

ARA2

0

0,05

0,1

0,15

0,2

0,25

0,3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

PFPK2

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0,5

1 2 3 4 5 6 7 8 9 10 11

numéro d'allèle

TAA87

0

0,1

0,2

0,3

0,4

0,5

0,6

1 2 3 4 5 6 7 8 9 10 11

BM27

0

0,1

0,2

0,3

0,4

0,5

0,6

1 2 3 4 5 6 7 8 9 10

TAA109

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

1 2 3 4 5 6 7 8 9 10 11 12 13

Fre

qu

en

cy

PFG377

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

1 2 3 4 5 6 7 8 9 10

7A11

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

1 2 3 4 5 6 7 8 9 10 11 12

N°of Alleles

?

�� No impact of No impact of bednetsbednets on the diversity of the parasiteson the diversity of the parasites

�� No impact of treatment before consultationNo impact of treatment before consultation

�� No correlation between parasiteamia or age and No correlation between parasiteamia or age and

polymorphismpolymorphism

�� Fever more than 40Fever more than 40°° associated with multiple infectionsassociated with multiple infections

�� Isolates with medium parasiteamia Isolates with medium parasiteamia [100 et 1000 [100 et 1000 tropho./tropho./µµll]]

associated with more polymorphic isolates and with CM associated with more polymorphic isolates and with CM

Clinical features and polymorphism

No linkage between a specific allele of µSta and :

- location in the town,

- type of infection, anemia, high parasiteamia..etc

- DHFR and CRT genotypes

� Cerebral malaria associated with

- multiple infections (higher inoculation rate ?)

- lower parasiteamia ( treatment ? sequestration ?)

- higher allelic diversity

� No change in alleles repartition for the different types of

infection = no evidence of specific isolates associated

with a specific feature

CONCLUSION

So why such a polymorphism

In low transmission area

Higher diversity = more pathology ??

No malaria during winter

suburban area :

-good quality surface water =

An arabiensis

- high density of population

- low immunity

- daily transfer of workers

- Input of parasites from the rural

area at the beginning of the rainy

season = end of vacations

Transmission

Selection of strains

Treatment

Origin of the polymorphism

Many thanks for the medical staffs

Centre de Santé de Guediawaye,

Service de Maladie infectieuses CHN de Fann

Service de Maladie infectieuses Hopital principal de Dakar

Programs : RAI –FSP MAE , Paris

RESMAL-Chip, EU

Academie des Sciences, Prix Louis D

Genopole Institut Pasteur