PLASMODIUM FALCIPARUM-ISOLATES
FROM CAMEROONIAN PREGNANT WOMEN DO NOT ROSETTE
MAUBERT B.*, **, ***, FIEVET N.*, **, TAMI G.*, BOUDIN C.*, ** & DELORON P.**, ****
S u m m a r y :
The placenta of pregnant women is frequently parasitized by erythrocytes infected by mature stages of Plasmodium falciparum (IE), a phenomenon associated with low birth weight of the offspring. The cytoadherence phenotype of the parasites from pregnant women suggests that placental sequestration may result from cytoadherence to the syncytiotrophoblast. However, as anatomopathological studies report that cytoadherence in the placenta is a rare event, we investigated whether placental parasites may sequester by forming rosettes with uninfected erythrocytes, another possible sequestration mechanism. Parasites from placental blood as well as parasites from the peripheral blood of pregnant and non pregnant subjects were assessed for their ability to rosette. In non pregnant subjects, the rosetting capacity of parasites was as reported in litterature while, except in one case, parasites from pregnant women did not rosette. W e conclude that the lack of rosetting is a new feature of lEs from pregnant women and that rosetting cannot be involved in the placental sequestration of lEs.
K E Y W O R D S : malaria, Plasmodium falciparum, human, placenta, pregnancy, rosette.
ABBREVIATION : IE, Plasmodium falciparum-infected erythrocyte.
Résumé : PLASMODIUM FALCIPARUM NE FORME PAS DE ROSETTES CHEZ LES FEMMES ENCEINTES CAMEROUNAISES
Chez la femme enceinte en zone d'endémie malarique, le placenta séquestre les hématies parasitées par les stades matures de Plasmodium falciparum, ce qui entraîne un faible poids de naissance. Il a été suggéré que la cytoadhérence au
syncytiotrophoblaste pouvait être impliquée dans la séquestration placentaire, mais le rôle des autres mécanismes de séquestration n'a pas été étudié. Nous avons recherché si la séquestration pouvait résulter de la formation de rosettes des hématies parasitées avec des hématies non parasitées. Nous avons mesuré la
capacité à former des rosettes d'isolats de P. falciparum provenant du sang placentaire et du sang périphérique de femmes enceintes, ainsi que du sang périphérique de sujets non enceintes. Chez les sujets non enceintes, la fréquence et l'intensité de la formation de rosettes étaient conformes aux valeurs rapportées dans d'autres études, alors que, hormis un cas, les parasites des femmes enceintes ne formaient pas de rosettes. En conclusion, la séquestration placentaire des hématies parasitées par
P. falciparum ne peut pas résulter de la formation de rosettes et le manque de capacité à former des rosettes est une nouvelle caractéristique des isolats des femmes enceintes.
MOTS CLÉS : paludisme, Plasmodium falciparum, humain, placenta, grossesse, rosette.
I
n a r e a s in w h i c h Plasmodium falciparum malariais e n d e m i c , p l a c e n t a l malaria is frequent a n d a s s o - c i a t e d with l o w birth w e i g h t o f t h e b a b y , a major c a u s e o f neonatal morbidity, thus posing a public health p r o b l e m (for r e v i e w , s e e B r a b i n , 1 9 9 1 a n d M e n e n d e z , 1 9 9 5 ) . In the p l a c e n t a , most o f infected erythrocytes ( I E ) c o n t a i n m a t u r e s t a g e s o f t h e parasite, w h i l e IEs from
t h e p e r i p h e r a l b l o o d o f t h e s a m e w o m a n c o n t a i n s o n l y y o u n g s t a g e s ( G a r n h a m , 1 9 3 8 ) . T h e i n v o l v e m e n t o f c y t o a d h e r e n c e as a m e c h a n i s m for the s e q u e s t r a t i o n o f IEs in the p l a c e n t a h a s b e e n r e p o r t e d ( F r i e d & Duffy, 1 9 9 6 ; M a u b e r t et a l , 1 9 9 7 ) , but o t h e r potential m e c h a - n i s m s h a v e not b e e n a s s e s s e d . R o s e t t e s ( b i n d i n g o f at least t w o u n i n f e c t e d e r y t h r o c y t e s to an infected ery- t h r o c y t e ) are large c e l l u l a r structures that facilitate the s e q u e s t r a t i o n o f IEs in m i c r o v a s c u l a t u r e ex vivo (Kaul et a l , 1 9 9 1 ) . W e i n v e s t i g a t e d w h e t h e r malaria parasites
from p r e g n a n t w o m e n c o u l d form rosettes, that c o u l d b e t r a p p e d in t h e p l a c e n t a l intervillous s p a c e s . F o r this p u r p o s e , w e c o m p a r e d the rosetting ability o f parasites from p r e g n a n t a n d n o n p r e g n a n t s u b j e c t s .
METHODS AND RESULTS
P
a r a s i t e s f r o m n o n p r e g n a n t s u b j e c t s w e r e c o l l e c t e d f r o m n i n e w o m e n a n d five m e n a t t e n d i n g at t h e M e s s a d i s p e n s a r y , Y a o u n d e ,* O r g a n i s a t i o n d e C o o r d i n a t i o n p o u r la Lutte c o n t r e l e s E n d é m i e s e n A f r i q u e C e n t r a l e ( O C E A C ) , P B 2 8 8 , Y a o u n d e , C a m e r o o n .
** Institut F r a n ç a i s d e R e c h e r c h e S c i e n t i f i q u e p o u r l e D é v e l o p p e m e n t e n C o o p é r a t i o n ( O R S T O M ) .
*** Institut N a t i o n a l d e la S a n t é e t de la R e c h e r c h e M é d i c a l e U 1 3 / I n s - titut d e M é d e c i n e e t d ' É p i d é m i o l o g i e A f r i c a i n e , H ô p i t a l B i c h a t - C l a u d e - B e r n a r d , 4 6 , r u e H e n r i - H u c h a r d , 7 5 0 1 8 Paris, F r a n c e .
**** P r é s e n t a d d r e s s : P h i l i p p e D e l o r o n , U n i t é d e P a r a s i t o l o g i e M é d i - c a l e , C e n t r e I n t e r n a t i o n a l d e R e c h e r c h e M é d i c a l e d e F r a n c e v i l l e , B P 7 6 9 , F r a n c e v i l l e , G a b o n .
C o r r e s p o n d e n c e : B . M a u b e r t I M E A / I N S E R M U 1 3 , H ô p i t a l B i c h a t , 46, r u e H e n r i - H u c h a r d , 7 5 0 1 8 Paris, F r a n c e . T e l . : ( 3 3 ) 0 1 4 0 2 5 63 65 - F a x : (33) 0 1 4 0 2 5 63 5 1 . e - m a i l : m a u b e r t @ b i c h a t . i n s e r m . f r .
P a r a s i t e , 1 9 9 8 , 5, 2 8 1 - 2 8 3
Note de recherche 281
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1998053281
M A U B E R T B . , F I E V E T N., T A M I G . , B O U D I N C . & D E L O R O N P .
C a m e r o o n . T h e y p r e s e n t e d with acute but uncompli- cated P. falciparum malaria, a peripheral parasitaemia higher than 0.2 %, and w e r e 22.2 ± 6.6 years old ( m ± S D ) . B l o o d samples w e r e drawn and parasites w e r e c r y o c o n s e r v e d in liquid nitrogen. B e f o r e the assay, samples w e r e t h a w e d and cultured for less than o n e cycle to pigmented stages (late trophozoites to y o u n g schizonts) according to the standard m e t h o d (Trager & J e n s e n , 1 9 7 6 ) . A m o n g those 14 isolates, parasites from 12 s h o w e d mature stages within the first life cycle, and rosetting assay w a s performed using the s t a n d a r d p r o c e d u r e ( C a r l s o n & W a h l g r e n , 1 9 9 2 ) . Briefly, 5 0 µl o f the parasite culture w a s m i x e d with 50 µl o f a 0.01 % acridine o r a n g e solution. 10 µl o f the suspension w e r e distributed under a 22 x 22 m m coverslip and at least 2 0 0 IEs w e r e e x a m i n e d under f l u o r e s c e n c e m i c r o s c o p y using x 5 0 0 magnification.
T h e n u m b e r o f rosettes (i. e. IEs that b o u n d at least two uninfected erythrocytes) was determined. Eight out o f the 12 samples studied formed rosettes, the percent o f IEs forming rosettes being: 1, 1, 1, 5.5, 6.5, 7.5, 14.5, and 1 7 . 5 . T h e s e figures are in line with other reports (Hasler et al, 1 9 9 0 ; Wahlgren et al, 1 9 9 0 ) , and the rosetting ability w a s not related to s e x .
W e a s s e s s e d rosette formation in placental b l o o d from parasitized placentas c o l l e c t e d after delivery in t w o m a t e r n i t y h o s p i t a l s ( N k o l n d o n g o a n d E t o u d i ) o f Y a o u n d e . T h e i r a g e was similar to the o n e o f n o n pre- gnant subjects ( 2 1 . 5 ± 5.6 years). An incision w a s m a d e o n the maternal face o f the placenta and a drop o f placental b l o o d w a s diluted to a 5 % hematocrit in P. falciparum culture m e d i u m c o n t a i n i n g acridine o r a n g e . Rosetting rate was assessed as a b o v e . None o f the 23 infected placental b l o o d studied presented with rosettes.
As the a b s e n c e o f rosettes in placental b l o o d may b e related to alteration o f the IEs during labor, w e stu- died parasites from the peripheral b l o o d o f pregnant w o m e n . T w e n t y five w o m e n ( a g e : 2 2 . 8 ± 4 . 6 ) pre- senting with a b l o o d parasites w e r e b l e d within o n e hour after delivery and parasites w e r e c r y o c o n s e r v e d . Rosetting assay w a s performed as m e n t i o n e d for n o n pregnant subjects. After in vitro culture, 16 samples s h o w e d mature parasites and at least 0.2 % parasite density. S e v e n w e r e the peripheral counterpart o f stu- died placental parasites and nine w e r e from other w o m e n . O n l y o n e formed rosettes and the p e r c e n t a g e o f rosette-forming IEs w a s l o w ( 2 % ) .
DISCUSSION
T
h e frequency o f rosetting was higher in para- sites from non-pregnant ( 8 / 1 2 ) than in peri- pheral b l o o d from pregnant subjects ( 1 / 1 6 )(Fisher's e x a c t test p = 0 . 0 0 1 ) , and than in placental b l o o d ( 0 / 2 3 ) ( F i s h e r ' s e x a c t test p < 0 . 0 0 0 1 ) . As rosetting assay with parasites from peripheral b l o o d w a s performed in a medium free o f i m m u n e serum, the difference in rosetting ability c a n n o t result from a difference in antibodies against rosettes.
In non-pregnant host, rosetting facilitates the s e q u e s - tration o f parasites ex vivo (Kaul et al., 1 9 9 1 ) . H o w e v e r t h e i m p l i c a t i o n o f r o s e t t i n g in h u m a n p a t h o l o g y remains uncertain as studies c o n d u c t e d in G a m b i a (Carlson, 1 9 9 3 ) and in Kenia ( R o w e et al., 1 9 9 5 ) found an association b e t w e e n rosetting and the d e v e l o p m e n t o f severe or cerebral malaria, while n o s u c h relation- ship was found in a larger study carried out in Papua N e w G u i n e a (Al-Yaman et al., 1 9 9 5 ) . Although pre- gnant w o m e n are at increased risk for malaria ( D i a g n e et al., 1 9 9 7 ) , our data demonstrate that rosetting is not involved in pregnancy-related malaria morbidity w h i c h has to b e e x p l a i n e d b y other m e c h a n i s m s . Parasites from pregnant and n o n pregnant w o m e n w e r e recently b e e n s h o w n to differ in their c y t o a d h e r e n c e p h e n o - type (Fried & Duffy, 1996). Our data s h o w that the lack o f rosetting ability is a n o t h e r feature o f parasites from pregnant w o m e n and that rosetting c a n n o t contribute to parasite concentration in the placenta.
ACKNOWLEDGEMENTS
W
e are indebted to Anne D a b a n and the staff o f the maternity clinics o f N k o l n d o n g o a n d E t o u d i , a n d e s p e c i a l l y t o O d e t t e N k o u o and Sister Anne-Philippe. This w o r k w a s sup- p o r t e d b y Institut N a t i o n a l d e la S a n t é et d e la R e c h e r c h e Médicale (INSERM) and Institut Français d e R e c h e r c h e Scientifique p o u r le D é v e l o p p e m e n t e n C o o p é r a t i o n ( O R S T O M ) and Organisation d e Coordi- nation p o u r la Lutte contre les E n d é m i e s e n Afrique Centrale (OCEAC). Bertrand Maubert was a recipient o f a fellowship o f the F r e n c h Ministère de l'Éducation N a t i o n a l e , d e l ' E n s e i g n e m e n t S u p é r i e u r et d e la R e c h e r c h e .REFERENCES
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Reçu le 17 février 1998 Accepté le 25 avril 1998
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Note de recherche 2 8 3
