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

Vivax malaria paradigm

Benign tertian fever

Drug sensitive

Easily controlled

Malariatherapy

Wagner von Jauregg : 1917

Syphilis (treponema pallidum) Plasmodium vivax5-1Oml blood from malaria patient

8 to 12 malaria crisis …

5 % death

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 hypnozoïtesadaptée au climat local – reviviscence

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)

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

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

Plasmodium vivax

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)

Formes cliniques graves

• Pas de séquestration, Pas d’hyperparasitémie– Mécanismes ?

• Atteinte Pulmonaires : SDRA

• Atteinte cérébrale

• Hémorragies rétiniennes

• Rupture de rate

• Anémie sévère

P. vivax: drug sensitive parasite ?

• Standard first choice drug: chloroquine

• Chloroquine resistance since 1989

19891990

1995

1995

19911995

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)

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

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

Pvmdr1 GenBank : AY618622

one exon (4392 nucleotides) 1464 aa

68% homology P. falciparum mdr1

P. vivax chloroquine resistancePlasmodium falciparum

pfcrt

pfmdr1

Plasmodium vivax

pvcg10

Pvmdr1

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

1076F 1076L976F976Y

976Y

976F

976F

P. vivax mdr1 SNPs

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

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 Monbrison

2, Sara Brega

2, Laetitia Barbollat

2, Ivo Müller

3, John C.

Reeder3, Hans-Peter Beck

1, Stéphane Picot

2, Blaise Genton

1*

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 sulphadoxineper 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)

S tu d y site

N orth C oast

area

(M adang

P rovince)

K arim u i

area

(S im bu

P rovince)

S ou th W osera

area

(E ast S epik

P rovince)

T o tal

C h aracteristics 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)

S ex : fem ales/n (% ) 20 (58 .8) 17 (39 .5) 9 (36 .0 ) 47 (45 .2)

T em 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 oglob in (m ean (95% C I), g /d l) 10 .2 (9 .4-

11 .0)

10 .6 (10 .0 -

11 .2) 9 .2 (8 .6-9 .8 )

10 .1 (9 .7 -

10 .5 )

P arasite density (geom etric m ean

(range), per µ l)

4677 (300-

41280)

3437 (40-

36600)

4964 (160-

50640)

4182 (40 -

50640)

C lass no (% )

A dequate clin ical and parasito logical

response (A C P R ) 24 (70 .6) 40 (93 .0) 27 (100) 91 (87 .5)

T reatm en t fa ilu re (T F) 10 (29 .4) 3 (7 .0 ) 0 (0 ) 13 (12 .5)

3932

79

59 61

89

6

16 1153 59

1525 28

pvdhfr 57 pvdhfr 58 pvdhfr 61 pvdhfr 117 pvmdr1 976

pre

va

len

ce

(%

) Pure wild-type Mixed: mutant plus wild-type Pure mutant

Polymorphic SNP a sites in Treatment outcome

pvdhfr and pvmdr1 P(%)b ACPR

c TF

d ORe CI

f P

g

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 976Y l 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

pvdhfr/pvmdr1 genotypesa

Treatment

respose

No of pvdhfr

mutations

pvmdr1

polymorphism

No

(x) ACPR

b TF

c OR

d CI

e

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

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

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 - Primaquine0.50 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 – Chloroquinedose 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 - Primaquine0.25 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 – ChloroquineTotal 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

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

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

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%

Geographic Distribution of G6PD Variants

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

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…

NONO..

NONO..

TNF, IFN..

TNF

R.O.S

R.O.S

..PRBC sequestration

B.B.B. rupture

brain hemorrhages

Rosetting

oedema

ICAM1ICAM1

Neurovascular Pathology & Cerebral Malaria