Plasmodium falciparum-isolates from Cameroonian pregnant

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1998053281
PLASMODIUM
FALCIPARUM-ISOLATES
FROM CAMEROONIAN PREGNANT WOMEN DO NOT ROSETTE
,
,
,
,
,
MAUBERT B.* ** ***, FIEVET N.* **, TAMI G.*, BOUDIN C.* ** & DELORON P.** ****
Summary :
The placenta of pregnant women is frequently parasitized by
erythrocytes infected by mature stages of Plasmodium
falciparum
Résumé
: PLASMODIUM FALCIPARUM NE FORME PAS DE ROSETTES CHEZ
LES FEMMES ENCEINTES CAMEROUNAISES
Chez la femme enceinte en zone d'endémie
malarique, le
(IE), a phenomenon associated with low birth weight of the
placenta séquestre les hématies parasitées
offspring. The cytoadherence phenotype of the parasites from
de Plasmodium falciparum, ce qui entraîne un faible poids de
pregnant women suggests that placental sequestration may result
naissance.
from cytoadherence to the syncytiotrophoblast. However, as
syncytiotrophoblaste
anatomopathological studies report that cytoadherence in the
placentaire,
par les stades matures
Il a été suggéré que la cytoadhérence au
pouvait
être impliquée
dans la séquestration
mais le rôle des autres mécanismes de séquestration
placenta is a rare event, we investigated whether placental
n'a pas été étudié. Nous
parasites may sequester by forming rosettes with uninfected
pouvait
avons recherché si la séquestration
erythrocytes, another possible sequestration mechanism. Parasites
avec des hématies non parasitées.
résulter de la formation
de rosettes des hématies
Nous
from placental blood as well as parasites from the peripheral
capacité à former des rosettes d'isolats
blood of pregnant and non pregnant subjects were assessed for
du sang placentaire
their ability to rosette. In non pregnant subjects, the rosetting
ainsi
capacity of parasites was as reported in litterature while, except
sujets non enceintes,
de P. falciparum
et du sang périphérique
que du sang périphérique
parasitées
avons mesuré la
de femmes
de sujets non enceintes.
la fréquence
provenant
enceintes,
Chez les
et l'intensité de la formation de
in one case, parasites from pregnant women did not rosette. W e
rosettes étaient conformes aux valeurs rapportées
conclude that the lack of rosetting is a new feature of lEs from
études, alors que, hormis un cas, les parasites des femmes
pregnant women and that rosetting cannot be involved in the
enceintes ne formaient pas de rosettes. En conclusion, la
placental sequestration of lEs.
séquestration
placentaire
dans
d'autres
des hématies parasitées par
P. falciparum ne peut pas résulter de la formation de rosettes et le
K E Y W O R D S : malaria, Plasmodium falciparum, human, placenta, pregnancy,
rosette.
ABBREVIATION : IE, Plasmodium falciparum-infected erythrocyte.
I
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.
t h e peripheral 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
n areas in w h i c h Plasmodium
falciparum
malaria
young
stages
(Garnham,
1938). The
involvement o f
is 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 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 sequestration o f
ciated with l o w birth weight o f t h e b a b y , a major
IEs in the p l a c e n t a has b e e n r e p o r t e d (Fried & Duffy,
c a u s e o f neonatal morbidity, thus posing a public health
1996;
Maubert et a l , 1 9 9 7 ) , but o t h e r potential m e c h a -
p r o b l e m (for review, s e e B r a b i n , 1 9 9 1 a n d M e n e n d e z ,
nisms h a v e not b e e n a s s e s s e d . Rosettes ( b i n d i n g o f at
1 9 9 5 ) . In the placenta, most o f infected erythrocytes ( I E )
c o n t a i n mature stages o f t h e parasite, w h i l e IEs from
least t w o
uninfected
erythrocytes to an
infected ery-
t h r o c y t e ) are large cellular structures that facilitate the
s e q u e s t r a t i o n o f IEs in microvasculature ex vivo
(Kaul
et a l , 1 9 9 1 ) . W e investigated w h e t h e r malaria parasites
from pregnant w o m e n c o u l d form rosettes, that c o u l d
* Organisation d e Coordination
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 Afrique 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 ,
** Institut F r a n ç a i s d e R e c h e r c h e Scientifique p o u r l e D é v e l o p p e m e n t
en Coopération
b e t r a p p e d in t h e placental intervillous s p a c e s . F o r this
Cameroon.
p u r p o s e , w e c o m p a r e d the rosetting ability o f parasites
from pregnant a n d n o n pregnant
(ORSTOM).
subjects.
*** Institut National 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
et d'Épidémiologie
Claude-Bernard, 4 6 , rue Henri-Huchard,
Africaine,
Hôpital
Bichat-
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 cale, Centre
International
BP 7 6 9 , Franceville, G a b o n .
Correspondence:
METHODS AND RESULTS
d e R e c h e r c h e Médicale d e Franceville,
B . Maubert
I M E A / I N S E R M U 1 3 , Hôpital
Bichat,
46, r u e Henri-Huchard, 7 5 0 1 8 Paris, F r a n c e . Tel.: ( 3 3 ) 01 4 0 2 5 63 65 Fax: (33) 0 1 4 0 2 5 63 5 1 . e-mail: m a u b e r t @ b i c h a t . i n s e r m . f r .
P
Parasite, 1 9 9 8 , 5, 2 8 1 - 2 8 3
Note de recherche
arasites
from
collected
from
attending
at
non
the
pregnant
nine
women
subjects
and
M e s s a dispensary,
five
were
men
Yaounde,
281
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.
Cameroon. T h e y presented with acute but uncomplicated P. falciparum
malaria, a peripheral parasitaemia
higher than 0.2 %, and were 22.2 ± 6.6 years old
( m ± S D ) . B l o o d samples were drawn and parasites
w e r e c r y o c o n s e r v e d in liquid nitrogen. Before the
assay, samples w e r e thawed and cultured for less than
o n e cycle to pigmented stages (late trophozoites to
young schizonts) according to the standard method
(Trager & J e n s e n , 1976). Among those 14 isolates,
parasites from 12 showed mature stages within the first
life cycle, and rosetting assay was performed using the
standard 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 was mixed with
50 µl o f a 0.01 % acridine orange solution. 10 µl o f
the suspension were distributed under a 22 x 22 m m
coverslip and at least 2 0 0 IEs were e x a m i n e d under
fluorescence microscopy 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 17.5. T h e s e figures are in line with other reports
(Hasler et al, 1 9 9 0 ; Wahlgren et al, 1990), and the
rosetting ability was not related to s e x .
W e assessed rosette formation in placental b l o o d from
parasitized placentas collected after delivery in two
maternity hospitals ( N k o l n d o n g o and Etoudi) o f
Y a o u n d e . Their age was similar to the o n e o f non pregnant subjects ( 2 1 . 5 ± 5.6 years). An incision was
m a d e on the maternal face o f the placenta and a drop
o f placental b l o o d was diluted to a 5 % hematocrit in
P. falciparum
culture medium containing acridine
orange. Rosetting rate was assessed as above. 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 studied parasites from the peripheral b l o o d o f pregnant
w o m e n . Twenty five w o m e n (age: 2 2 . 8 ± 4 . 6 ) presenting with a b l o o d parasites were bled within o n e
hour after delivery and parasites w e r e cryoconserved.
Rosetting assay was performed as mentioned 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. Seven w e r e the peripheral counterpart o f studied placental parasites and nine w e r e from other
w o m e n . Only o n e formed rosettes and the percentage
o f rosette-forming IEs was low (2 % ) .
(Fisher's exact 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
was performed in a medium free o f immune serum,
the difference in rosetting ability cannot 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., 1991). H o w e v e r
the i m p l i c a t i o n o f rosetting 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 development
o f severe or cerebral malaria, while n o such relationship was found in a larger study carried out in Papua
New Guinea (Al-Yaman et al., 1 9 9 5 ) . Although pregnant w o m e n are at increased risk for malaria (Diagne
et al., 1 9 9 7 ) , our data demonstrate that rosetting is not
involved in pregnancy-related malaria morbidity which
has to b e explained by other mechanisms. Parasites
from pregnant and non pregnant w o m e n were 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 another feature o f parasites from
pregnant w o m e n and that rosetting cannot contribute
to parasite concentration in the placenta.
ACKNOWLEDGEMENTS
W
e are indebted to Anne Daban and the staff
o f the maternity clinics o f N k o l n d o n g o
and Etoudi, and especially to Odette
N k o u o and Sister Anne-Philippe. This w o r k was supp o r t e d by Institut National 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 de
R e c h e r c h e Scientifique pour le D é v e l o p p e m e n t e n
Coopération ( O R S T O M ) and Organisation de Coordination pour la Lutte contre les Endémies en Afrique
Centrale (OCEAC). Bertrand Maubert was a recipient
o f a fellowship o f the French 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
Recherche.
REFERENCES
AL-YAMAN
F.,
GENTON
B.,
MOKELA
D.,
RAIKO
A.,
KATI
S.,
S., R E E D E R J . & A L P E R S M. Human cerebral malaria:
lack of significant association between erythrocyte rosetting and disease severity. Transactions of the Royal Society
of Tropical Medicine and Hygiene, 1995, 89, 55-58.
ROGERSON
DISCUSSION
T
282
B . J . The risk and severity of malaria in pregnant
women. Applied Field Research in Malaria Reports N° 1
TDR/FIELDMAL/1. Special Programme for Research and
Training in Tropical Diseases, 1991, p. 33.
BRABIN
he frequency o f rosetting was higher in parasites from non-pregnant ( 8 / 1 2 ) than in peripheral b l o o d from pregnant subjects ( 1 / 1 6 )
Note de recherche
Parasite, 1 9 9 8 , 5, 2 8 1 - 2 8 3
P.
FALCIPARUM-ROSETTES
DURING PREGNANCY
J. Erythrocyte rosetting in Plasmodium
falciparum
malaria, with special reference to the pathogenesis of
cerebral malaria. Doctoral Thesis, Uppsala University,
Sweden, 1993.
CARLSON
J . & W A H L G R E N M. Plasmodium falciparum
erythro­
cyte rosetting is mediated by promiscuous lectin-like inter­
actions. Journal of Experimental
Medicine, 1992, 176,
1311-1317.
CARLSON
N., R O G I E R C , C I S S E B. & T R A P E J . F . Incidence of cli­
nical malaria in pregnant women exposed to intense per­
ennial transmission. Transactions of the Royal Society of
Tropical Medicine and Hygiene, 1997, 91, 166-170.
DIAGNE
M. & DUFFY P.E. Adherence of Plasmodium
falciparum
to chondroitin sulfate A in the human placenta. Science,
1996, 272, 1502-1504.
FRIED
P.C.C. The placenta in malaria with special refe­
rence to reticulo-endothelial immunity. Transactions of the
Royal Society of Tropical Medicine and Hygiene, 1938, 32,
13-48.
GARNHAM
HASLER
DIJK
T.,
HANDUNNETTI
M.R.,
GREENWOOD
S.H.,
AGUIAR
B.M.,
J.C.,
VAN
L A L L I N G E R G.,
SCHRAVEN-
CEGIELSKI
P.
R.J. In vitro rosetting, cytoadherence and
microagglutination of Plasmodium
falciparum-infected
erythrocytes from Gambian and Tanzanian patients. Blood,
1990, 76, 1845-1852.
&
KAUL
HOWARD
D.K.,
ROTH
Jr.
E.F.,
NAGEL
R.L.,
HOWARD
RJ.
&
HAN­
S.M. Rosetting of Plasmodium
falciparum-infected
red cells with uninfected red cells enhances vasoocclusion
in an ex vivo microvascular system. Blood, 1991, 78, 812819.
DUNNETTI
B., G U I L B E R T L.J. & D E L O R O N P. Plasmodium
falciparum-infected
erythrocyte sequestration in the human
placenta: cytoadherence mechanisms involving intercellular
adhesion molecule-1 and chondroitin-4-sulfate as syncytiotrophoblast receptors. Infection and Immunity, 1997, 65,
1251-1257.
MAUBERT
C. Malaria during pregnancy: a priority area of
malaria research and control. Parasitology Today, 1995, 11,
178-193.
MENENDEZ
A., OBEIRO J . , N E W B O L D C.I. & M A R S H K. Plasmodium
falciparum
rosetting is associated with malaria severity in
Kenya. Infection and Immunity, 1995, 63, 2323-2326.
ROWE
TRAGER W. & JENSEN J.B. Human malaria parasites in conti­
nuous culture. Science, 1976, 193, 673-675.
W A H L G R E N M.,
CARLSON J.,
RUANGJIRACHUPORN W.,
CONWAY
D.,
H., M A R T I N E Z A., P A T A R R O Y O M.E. & R I L E Y E. Geo­
graphical distribution of Plamodium falciparum
erythro­
cyte rosetting and frequency of rosetting antibodies in
human sera. American Journal of Tropical Medicine and
Hygiene, 1990, 43, 333-338.
HELMBY
Reçu le 17 février 1998
Accepté le 25 avril 1998
Parasite, 1 9 9 8 , 5, 2 8 1 - 2 8 3
Note de recherche
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