Polymorphisme parasitaire et accès graves en zone périurbaine
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Transcript of Polymorphisme parasitaire et accès graves en zone périurbaine
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