Polymorphisme parasitaire et accès graves en zone périurbaine - Conférence de la 5e édition du Cours international « Atelier Paludisme » - Ronan JAMBOU - Institut Pasteur de Dakar - rjambou@pasteur.sn

1. High polymorphism of parasites isolates is
associated with Cerebral Malaria in Dakar.
Bob NS, Diop BM, Marrama L, MT Ekala, Tall A, Ka B, Hovette
Ph, 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

2. 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

3. mechanic hypothesis

4. 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
4
Inflammation
Local /
general

5. 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

6. 15°
14°
13°
16°N
Dakar
17°W 16° 15° 14° 13° 12°
SENEGAL
Area of Study
Seasonal transmission
3 M inhabitants
An arabiensis

7. 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

8. 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

9. 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
Nombrede
J F M A M J J A S O N D
Mois

10. 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

11. 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

12. TAGCATGTTTGGTATAGG
GGTTAAATCAGGACAACAT
TAGGGAACATCATAAGGA
TAGCATGTTTGGTATAGG161Chr6TAA109
GAGTAATA TGAACATGT
AATGGCAACACCATTCAAC
ATGGGTTAAATGAGGTACA
GAGTAATA TGAACATGT112Chr6TAA87
ATCATGCATTTCAGTCTGAGG
TCTGCTTGTTCCTTCTTT
CTTTCATCGATACTACGA
ATCATGCATTTCAGTCTGAGG168Chr12PFPK2
TTATGTTGGTACCGTGTA
TGTTAATCGTAGGGATAA
GATCTCAACGGAAATTAT
TTATGTTGGTACCGTGTA98Chr12PFGG377
TATTAATAATACTCAAAGC
GAATAAACAAAGTATTGCT
GTACATATGAATCACCAA
TATTAATAATACTCAAAGC70Chr11ARA2
ATGATGTGCAGATGACGA
GTGCATTCAATAATTCTA
TTCTAAATAGATCCAAAGATG
ATGTGCAGATGACGA87Chr102490
CTTTAGTAGTAGTAATAATAC
TAGAAACAGGAATGATACG
ACAAAAGGGTGGTGATTCT
CTTTAGTAGTAGTAATAATAC183Chr5TAA42
GTAATATTTAAAAAGAGAAG
TTTGTAATATTTAAAAAGAGAAG
ATGTGTAAGGAGATAGTATA
GTAATATTTAAAAAGAGAAG112Chr77A11
TGACTCTTTGATTATATACC
CAAAAGAAGTAATATATGTGCCC
CAAAAGAAGTAATATATGTGC
TGACTCTTTGATTATATACC133Chr9BM27
Primers 2Primers 1SizeChromosomeName
Microsatellites and primers used in this study

13. DHFR
1850 bp
Sequencing of Pfdhfr and exon 2 Pfcrt
dhfr ts
PfCRT
Exon 2
PCR amplification
Sequencing
250 bp

14. Hôpital Principal de DakarHôpital Principal de Dakar
59 patients : 20 CM / 3959 patients : 20 CM / 39 mildmild malariamalaria
2727 womenwomen / 32/ 32 menmen
age : 2 to 67age : 2 to 67 yearsyears
CHNCHN FannFann
15 patients : 12 CM / 315 patients : 12 CM / 3 mildmild
5 W et 10 M5 W et 10 M
age 16 to 42age 16 to 42 yearsyears
Centre de SantCentre de Santéé GuediawayeGuediawaye
104 patients : 2 CM / 102104 patients : 2 CM / 102 mildmild malariamalaria
66 W et 38 M66 W et 38 M
age 3 to 65age 3 to 65 yearsyears
PATIENTS

15. 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

16. MUTATIONS in DHFR and PfCRT
+++triple
+++++two
+-+one
NRImutant
SCNwild
1085951codon
MM n= 28
CM n= 16
MM n= 102
CM 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

17. Multiple Infections
0
10
20
30
40
50
60
70
80
90
100
Cerebral
Malaria
Mild Mal
(hosp)
Mild Mal.
(dispens)
0
0,5
1
1,5
2
2,5
Percent of multi-infection
n°population/ patients

18. Multiple Infections (by microsatellite)
0
5
10
15
20
25
30
35
40
45
50
TA
A42
PFPK
2
m
2490
AR
A2
TA
A87
B
M
27
TA
A109
PFG
377
7A11
Cerebral Malaria
Mild Mal (hosp)
Mild Mal. (dispens)
TAA109, PFG377, 7A11, ARA2 = highly
polymorphic in hospitalized patients

19. Number of alleles to describe
50% of the patients
Average = 2 for CM / 1 for MM
Total number of alleles/µSat
Average = 8 for CM and MM
= same
0
2
4
6
8
10
12
14
16
18
TAA42
PFPK
2
m
2490
A
R
A2
TAA87
B
M
27
TAA109
PFG
377
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
TAA42
PFPK
2
m
2490
A
R
A2
TAA87
B
M
27
TAA109
PFG
377
7A11
Number of alleles ot describe
75% of the patients
Average = 3 for CM / 2 for MM
All µSat = more polymorphic
in hospitalized patients

20. 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
Frequency
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
?

21. No impact ofNo 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 andNo correlation between parasiteamia or age and
polymorphismpolymorphism
Fever more than 40Fever more than 40°° associated with multiple infectionsassociated with multiple infections
Isolates with medium parasiteamiaIsolates with medium parasiteamia [100 et 1000[100 et 1000 tropho./tropho./µµll]]
associated with more polymorphic isolates and with CMassociated 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

22. 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 ??

23. 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

24. 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