Plasmodium vivax, un parasite pas si banal - Séances Pratiques de la 5e édition du Cours international « Atelier Paludisme » - Stéphane PICOT - Hôpital E. Herriot, Lyon, France - picot@rockefeller.univ-lyon1.fr

1. Plasmodium vivax,
un parasite pas si banal
Université Claude Bernard - Faculté de Médecine
Equipe d’accueil - IFR des Neurosciences
Radicaux libres, substrats énergétiques et physiopathologie cérébrale
Lyon - France
Université Claude Bernard - Faculté de Médecine
Equipe d’accueil - IFR des Neurosciences
Radicaux libres, substrats énergétiques et physiopathologie cérébrale
Lyon - France

2. Vivax malaria paradigm
Benign tertian fever
Drug sensitive
Easily controlled
Malariatherapy
Wagner von Jauregg : 1917
Syphilis (treponema pallidum)
Plasmodium vivax
5-1Oml blood from malaria patient
8 to 12 malaria crisis …
5 % death

3. Transmissibilité de P. vivax
• P. vivax :
– Cycle anophélien en 10 jours environ à 30°C (Pf:13j)
– Jusqu’à 15°C (Pf: 18°C)
• Stades hépatiques
– 1 « lot » de schizontes hépatiques
– 1 « lot » d’hypnozoïtes
• Périodicité de libération des hypnozoïtes
adaptée au climat local
– reviviscence

4. Définitions
• Reviviscence (relapse) : nouveau cycle érythrocytaire à
partir de la libération de mérozoïtes provenant de
schizontes hépatiques primaires
• Recrudescence (recrudescence) : réapparition d'une
parasitémie détectable à la suite d'une résistance au
traitement
• Rechute (relapse) : accès clinique secondaire, à distance
du premier épisode, pour lequel l'origine des parasites
est difficile à mettre en évidence (rupture
d'hypnozoïtes ou formes érythrocytaires résistantes au
traitement)

5. R. Carter
Trends Parasitol. 2003 May;19(5):214-9.

6. Duffy Antigen receptor for Chemokines
(DARC)
• P. vivax et P. knowlesi
• Interaction de « Duffy-binding-like domain » (DBL) avec
le récepteur DARC.
• Polymorphisme de PvDARC (Papouasie-Nouvelle-Guinée,
Colombie)
• famille des DBLs : PfEMP1 (cytoadhérence)
• DARC est absent chez la plupart des populations noires
d’Afrique.
• P. vivax est présent :
– les peuples berbéroïdes (touaregs et maures) autour du Sahara
– les éthiopiens d’origine yéménite,
– les malgaches d’origine indonésienne
– les bushmen d’Afrique de l’est (Kalahari,Tanzanie) et du sud

7. Plasmodium vivax

8. benign tertian fever ?
The Ghost of the swamp
« The ghost of a man, a sufferer from his cradle to his grave,
aged even in childhood and laying down in misery that life
which was but one disease »
John Maccinlloch, 1827
Pernicious complications
Acute respiratory distress syndrome (Tanios 2001, Lawn 2003)
Cerebral malaria (Beg 2002)
Acute disseminated encephalomyelitis (Koibuchi 2003)
Retinal haemorrhages (Choi 2004)

9. Formes cliniques graves
• Pas de séquestration, Pas d’hyperparasitémie
– Mécanismes ?
• Atteinte Pulmonaires : SDRA
• Atteinte cérébrale
• Hémorragies rétiniennes
• Rupture de rate
• Anémie sévère

10. P. vivax: drug sensitive parasite ?
• Standard first choice drug: chloroquine
• Chloroquine resistance since 1989
19891990
1995
1995
1991
1995

11. P. vivax: drug sensitive parasite ?
• Supposed to be intrinsically resistant to sulfadoxine-
pyrimethamine
➪ Role of falciparum/vivax co-infection in Thaïlande
• Hypnozoïtes clearance by primaquine
➪ Decrease in primaquine efficacy (Indonesia, PNG,
Thailande)
➪ Prophylaxis failure
– Atovaquone-proguanil: Ethiopia (Povinelli & al 2003)
– Mefloquine : French Guyana (Picot & al. 2005)

12. Easy to detect vivax drug resistance ?
• Therapeutic efficacy tests
– WHO protocols
– Relapse patterns
– Scarcity of data
• In vitro tests
– Culture of P. vivax
– Few studies and few labs.
• Molecular markers
– Available for sulfadoxine-pyrimethamine and amino-4-quinoleines
– Blood spots easy to obtain
– Real time PCR highly efficient and reproducible

13. LRM/IRM LRT FRT FST
pvdhfr
F57L/I S58R S117N/T I173LT61M
C59R S108N I164L
pfdhfr
83,5132848Total =
100Laos
1527454travellers
5644Thaïlande
12,587,5Indonesia
7129Turkey
3664Azerbaijan
Quadruple
F57L/I + S58R
T61M + S117T
Triple
S58R + S117N
+ I173L
Double
S58R +
S117N
Single
S117N
Wild
type
%
PvDHFR Folate pathways resistance

14. Pvmdr1 GenBank : AY618622
one exon (4392 nucleotides) 1464 aa
68% homology P. falciparum mdr1
P. vivax chloroquine resistance
Plasmodium falciparum
pfcrt
pfmdr1
Plasmodium vivax
pvcg10
Pvmdr1

15. Mutations ponctuelles conférant
la CQR chez P. falciparum
GAT
Asp/D
AAT
Asn/N
1042
TTA
Leu/L
1039
TAT
Tyr/Y
GAT
Asp/D
1246
TGT
Cys/C
AGT
Ser/S
1034
TAT
Tyr/Y
940
TTT
Phe/F
TAT
Tyr/Y
184
TAT
Tyr/Y
AAT
Asn/N
86
CQRCQSPositions AA
Pfmdr1
AAC
Asn/N
1079
CTT
Leu/L
TTT
Phe/F
1076
GAT
Asp/D
1291
AGT
Ser/S
1071
TTC/TTT
Phe/F
TAC
Tyr/Y
976
TAC
Tyr/Y
189
AAC
Asn/N
91
CQRCQSPositions AA
Pvmdr1
= site de mutation
TM = Transmembranaire
II° fragment amplifié
ATPTM TM
I° fragment amplifié
ATP
Mutations ponctuelles conférant
la CQR chez P. vivax

16. 1076F 1076L976F976Y
976Y
976F
976F
P. vivax mdr1 SNPs

17. Usage des codons
• A+T : 15% moins élevé que P. falciparum
• Codon préféré : 3° base
– 100 % A/T : P. falciparum
– 50 % A/T : P. vivax
• AA les plus fréquents pour les protéines :
– P vivax : Lys, Glu, Leu, Ser
– P. falciparum : Asn, Glu, Leu

18. The association of mutations in Plasmodium vivax dhfr and mdr1 and in vivo
resistance to amodiaquine or chloroquine plus sulphadoxine-pyrimethamine in
Papua New Guinea
Jutta Marfurt1
, Frédérique de Monbrison2
, Sara Brega2
, Laetitia Barbollat2
, Ivo Müller3
, John C.
Reeder3
, Hans-Peter Beck1
, Stéphane Picot2
, Blaise Genton1*
Children between 6 months and 7 years of age
axillary temperature ≥37.5°C or history of fever during the last 48 hours
and microscopically confirmed monoinfection with P. vivax (density >250 /microlitre).
Absence of danger signs for severe or complicated malaria, signs of any other
disease, malnutrition or anaemia.
Standard AQ or CQ plus SP first-line treatment
(10 mg chloroquine or amodiaquine per kg on Day 0, 1 and 2, and 25 mg sulphadoxine
per kg plus 1.25 mg pyrimethamine per kg on Day 0)
Follow-up visits were done on Day 1, 2, 3, 7, 14, and 28.
Treatment failure :
5 mg artesunate per kg on Day 1 followed by 2.5 mg artesunate per kg on Day 2 to 7,
and a single dose of 25 mg sulphadoxine per kg plus 1.25 mg pyrimethamine per kg
on Day 3)

19. Study site
N orth C oast
area
(M adang
Province)
K arim ui
area
(Sim bu
Province)
South W osera
area
(East Sepik
Province)
Total
C haracteristics n=34 n=43 n=27 n=104
W eight (m ean (95% C I), kg)
15.9 (8.0-
23.8)
13.7 (12.2-
15.2)
12.0 (10.6-
13.5)
14.1 (11.2-
16.9)
A ge (m ean (95% C I), yrs) 2.3 (1.9-2.7)
3.5 (3.0-
4.0)
3.2 (2.5-3.9)
3.0 (2.7-
3.4)
Sex: fem ales/n (% ) 20 (58.8) 17 (39.5) 9 (36.0) 47 (45.2)
Tem perature (m ean (95% C I),°C )
37.1 (36.6-
37.6)
38.6 (38.3-
38.8)
37.0 (36.4-
37.6)
37.7 (37.4-
38.0)
H aem oglobin (m ean (95% C I), g/dl)
10.2 (9.4-
11.0)
10.6 (10.0-
11.2)
9.2 (8.6-9.8)
10.1 (9.7-
10.5)
Parasite density (geom etric m ean
(range), per µl)
4677 (300-
41280)
3437 (40-
36600)
4964 (160-
50640)
4182 (40-
50640)
C lass no (% )
A dequate clinical and parasitological
response (A C PR )
24 (70.6) 40 (93.0) 27 (100) 91 (87.5)
Treatm ent failure (TF) 10 (29.4) 3 (7.0) 0 (0) 13 (12.5)

20. 39
32
79
59 61
8
9
6
16 11
53 59
15
25 28
pvdhfr 57 pvdhfr 58 pvdhfr 61 pvdhfr 117 pvmdr1 976
prevalence(%)
Pure wild-type Mixed: mutant plus wild-type Pure mutant

21. Polymorphic SNP a
sites in Treatment outcome
pvdhfr and pvmdr1 P(%)b
ACPRc
TFd
ORe
CIf
Pg
pvdhfr Mut57h
61 49 12 9.31 1.16-74.75 0.01
pvdhfr 57L 60 49 11 4.27 0.89-20.40 0.05
pvdhfr 57I 1 0 1 *
0.01
pvdhfr 58R 68 56 12 6.64 0.82-53.52 0.04
pvdhfr 61M 21 15 6 4.11 1.21-13.99 0.02
pvdhfr Mut117i
41 33 8 2.62 0.79-8.68 0.11
pvdhfr 117T 28 20 8 5.36 1.58-18.23 0.01
pvdhfr 117N 13 13 0 §
0.14
pvmdr1 976Fk
39 30 9 4.28 1.22-15.04 0.02
pvmdr1 976Yl
72 67 5 0.18 0.04-0.74 <0.01
a
SNP, single nucleotide polymorphism; pvdhfr, P. vivax dihydrofolate reductase; pvmdr1, P. vivax
multidrug resistance gene 1; b
P, prevalence; c
ACPR, adequate clinical and parasitological response;
d
TF, treatment failure; e
OR, odds ratio; f
CI, 95% confidence interval; g
calculated by standard χ2
analysis or Fisher’s exact test; h
Mut57, either 57L or 57I; i
Mut117, either 117T or 177N; k
976F
represents mutated allele; l
976Y represents wild-type allele; *
57I polymorphism was found in only
one patient who failed treatment; §
117N was not found in patients who failed treatment

22. pvdhfr/pvmdr1 genotypesa Treatment
respose
No of pvdhfr
mutations
pvmdr1
polymorphism
No
(x)
ACPRb
TFc
ORd
CIe p
(LRTf
)
Wild-type 976Yg
19 18 1 0.32
0.04-
2.62
0.22
Wild-type 976Fh
9 9 0
Single 117 976Y 3 3 0
Single 117 976F 1 1 0
Single 58 976Y 1 1 0
Single 58 976F 1 1 0
Double 57-58 976Y 18 17 1 0.34
0.04-
2.82
0.26
Double 57-58 976F 11 8 3 2.96
0.67-
13.03
0.18
Double 58-117 976Y 4 4 0
Double 58-117 976F 1 1 0
Triple 57-58-117 976Y 5 4 1 1.73
0.18-
16.79
0.65
Triple 57-58-117 976F 6 5 1 1.37
0.15-
12.72
0.79
Quadruple 57-58-
61-117
976Y 11 10 1 0.64
0.08-
5.47
0.67
Quadruple 57-58-
61-117
976F 10 5 5 10.25
2.44-
43.11
<0.01
Total (n) 100 87 13

23. Easy to treat vivax malaria ?
1. Standard treatment
2. Moderate drug resistance area (Thaïlande)
3. High risk of drug resistance area (Indonesia, PNG)
Check for G-6-PD status before primaquine, or forget it

24. Primaquine : 0.50 mg/kg/j, 14 jours
- LARIAM (250 mg)
2 cp / day, 1 day
- Primaquine (cp à 7.5 mg)
4 cp/day x 14 jours
Mefloquine : 15 mg/kg, dose unique
Contamination in high risk resistance area (Indonesia, PNG)
2 - Primaquine
0.50 mg/kg/j,
14 jours
- Chloroquine (100mg)
6 cp day 1, 6 cp day 2, 3 cp day 3
- Primaquine (cp à 7.5 mg)
4 cp/day x 14 jours
1 – Chloroquine
dose totale : 25 mg/kg/3jours.
Soit J1 : 10mg/kg ; J2 : 10 mg/kg, J3 : 5
mg/kg
Contamination in low chloroquine sensitive area (Thaïlande)
2 - Primaquine
0.25 mg/kg/d,
14 days
- Chloroquine (100mg)
6 cp day 1, 6 cp day 2, 3 cp day 3
- Primaquine (7.5 mg)
2 cp/day x 14 days
1 – Chloroquine
Total dose: 25 mg/kg/3 days.
D1 : 10mg/kg ; D2 : 10 mg/kg, D3 : 5
mg/kg
Contamination in chloroquine sensitive area:
patient 60 kg bw. G6PD test normalTreatment

25. Background on G6PD
• G6PD: pentose phosphate pathway, converts NADP+ to NADPH
• G6PD deficiency is a sex-linked genetic disorders, with full
expression in males
• Persons who are G6PD deficient are at increased risk for
experiencing hemolytic anemia when taking primaquine
• Primaquine is the only drug available that kills liver stage parasites
to prevent late malaria relapse in Plasmodium vivax, ovale
Adapted from Dennis Shanks, US army
(A - ) Variant affects approximately 10% of African Americans
Enzyme usually >10% normal
(B - ) Variant (MED) is the most common type affecting people from
Eastern Mediterranean
Enzyme usually <10% of normal
G6PD Genetic Variants

26. Mediterranean (B-) Variants
• Serious hemolysis can occur following one dose
of 15 mg primaquine base
• Patient may require blood transfusion often
hemolyzing > 50% erythrocytes
• Complications include:
– Acute Renal Failure
– High Output Cardiac Failure
– Anoxia and Death

27. The frequency of the (B-) variant differs
markedly among different populations
Caucasian 0.4%
Italians 0.5-1.0%
Hispanic 0.9%
Greek 2-9%
Sardinians 3-35%
Asian 1.8%
African American 7.6%

28. Geographic Distribution of G6PD Variants

29. Asymptomatic patient,
Long stay in high risk area
(PNG), index case
G6PD normal
Minimun dose in unknown
G6PD normal
Atovaquone/proguanil
(duration of stay + 7 days
after return)
Systematic treatment with
primaquine after return
15 mg/14 days
double dose from PNG
Primaquine 30 mg/day (0.5
mg/kg/j) :
1-2 days before stay, every
day, + 7 days after
Doxycycline, mefloquine
(duration of stay + 28 days
after return
Do NOT forget P. falciparum
chemoprophylaxis in case
of mixed transmission area
Do NOT forget P.
falciparum
chemoprophylaxis in case
of mixed transmission area
No action against
hypnozoïtes and relapses
Activity against hypnozoïtesActivity againts primary
liver schizontes and
hypnozoïtes
Activity against erythrocytic
stages
Terminal
Prophylaxis
Causal
Prophylaxis
Suppressive
Prophylaxis

30. vivax malaria paradigm
Benign tertian fever
Drug sensitive
Easily controlled
Plasmodium vivax, the ghost parasite
Mostly disappeared in Europe in the mid-20th century,
will reappeared soon…

32. NONO
..
NONO
..
TNF, IFN..
TNF
R.O.SR.O.S
..
PRBC sequestration
B.B.B. rupture
brain hemorrhages
Rosetting
oedema
ICAM1ICAM1
Neurovascular Pathology & Cerebral Malaria