Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2002092187
MOLECULAR CHARACTERISATION OF AIRPORT MALARIA: FOUR CASES IN FRANCE DURING SUMMER 1 9 9 9 JAFARI S.*, DURAND R.*, LUSINA D.** & LE BRAS J . *
Summary:
Résumé : CARACTÉRISATION MOLÉCULAIRE DE QUATRE CAS DE PALUDISME D'AÉROPORT OBTENUS EN FRANCE AU COURS DE L'ÉTÉ 1999
Four airport malaria cases have been observed in the vicinity of the Roissy-Char!es-de-Gaulle International Airport, Paris, France. These cases were geographically very close to each other and clustered in a short period of time during the summer of 1 9 9 9 . The phenotype and genotype of the Plasmodium falciparum isolates obtained from these patients were determined in order to know whether a single mosquito could have infected more than one subject. The genomic characterisation of isolates was performed using the polymorphic markers merozoite surface protein 1 (Mspl) and merozoite surface protein2 (Msp2) genes, the K and ɷrepeats domains of cg2 and the dihydrofolate reductase (DHFR) genotypes. Results showed identical genotypes for isolates 1, 2 and 4 whereas the genotype of isolate 3 differed at one locus. The molecular analysis was consistent with the hypothesis that all patients could have been bitten by the same mosquito and that patient 3, may have received a different clone and an additional species. In vitro susceptibility data did not confirm or rule out this hypothesis because isolates had the same profile of susceptibility to the tested drugs.
Quatre cas de paludisme d'aéroport ont été observés au voisinage de l'aéroport Roissy-Charles de Gaulle de Paris. Ces cas, regroupés sur une courte période de l'été 1999, étaient géographiquement très proches. Le phénotype et le génotype des isolats de Plasmodium falciparum de ces patients ont été déterminés dans le but de savoir si un seul moustique a pu infecter plus d'un sujet. Le génotypage des isolats a été effectué au moyen de l'étude des marqueurs polymorphes merozoite surface protein1(Msp1) et merozoite surface protein 2 (Msp2), des répétitions K et ɷdu gène cg2, et du génotype de la dihydrofolate réductase (DHFR). Les résultats ont montré un profil moléculaire identique pour les isolats 1, 2 et 4 et un génotpye différent pour l'isolat 3 à un locus. L'analyse moléculaire est en accord avec l'hypothèse d'une contamination par le même moustique des cas 1, 2, 3 et 4. Cependant, le cas 3 pourrait avoir reçu un clone différent et une espèce associée. L'étude de la sensibilité in vitro n'a pu infirmer ni confirmer cette hypothèse dans la mesure où les isolats ont présenté le même profil de sensibilité aux antipaludiques testés.
KEY W O R D S : airport malaria, Plasmodium falciparum, Plasmodium malariae, genotyping, in vitro susceptibility.
MOTS CLÉS : paludisme d'aéroport, Plasmodium falciparum, Plasmodium malariae, génotypage, sensibilité in vitro.
INTRODUCTION
A
spontaneously.
irport malaria is a c q u i r e d through the bite an
infective
Anopheles
mosquito
of
transported
b y a e r o p l a n e s from e n d e m i c areas ( G i a c o m i n i ,
1998;
Guillet
et al.,
T h e m a n died a n d t h e w o m a n
falciparum.
1 9 9 8 ) . T h e first t w o c a s e s
were
retrospectively reported in a F r e n c h illegitimate c o u p l e in 1 9 6 9 ( D o b y & G u i g u e n ,
1 9 8 1 ) . T h e man a n d the
w o m a n lived in the s a m e street n e a r the B o u r g e t air port. T h e y h a d n o c o m p a t i b l e travel history; they did not receive a recent b l o o d transfusion and did not take intravenous drugs. B o t h w e r e infected b y
Plasmodium
malaria h a v e b e e n reported in W e s t e r n E u r o p e , 2 8 o f which
** Laboratoire de Biologie Médicale, Centre Hospitalier Robert Bal langer, 93602 Aulnay-sous-Bois, France. Correspondence: S. Jafari, Laboratoire de Parasitologic Hôpital Bichat-Claude Bernard. 46. Rue Henri Huchard. 75877 Paris cedex 18, France. Tel.: +33 (0)1 40 25 78 97 - Fax: +33 (0)1 40 25 67 63. E-mail:
[email protected]
occurred
in
France
(Lusina
et a l ,
2000).
A
r e v i e w o f the history o f t h e c a s e s s h o w s that airport malaria mostly o c c u r r e d simultaneously in m o r e than o n e individual in t h e s a m e area ( I s a a c s o n , 1 9 8 9 ) . In m a n y c a s e s , the m e m b e r s o f t h e s a m e family living in close
proximity
to
an
international
airport
were
involved as w e r e p e r s o n n e l w o r k i n g o n airstrips or in air terminals ( B o u v i e r et al., G i a c o m i n i et al, hypothesis person
* Centre National de Référence pour la Chimiosensibilité du Palu disme, Assistance Publique-Hôpitaux de Paris. Laboratoire de Para sitologic Hôpital Bichat-Claude Bernard, 46, Rue Henri Huchard. 75877 Paris cedex 18, France.
recovered
S i n c e then, 7 3 other c a s e s o f airport
of one
has
molecular
mosquito
already
review (Isaacson,
1 9 9 0 ; Majori et al,
1997 ; Van D e n E n d e et al, been
biting
more
suggested
in
1990;
1998).The a
than
one
previous
1 9 8 9 ) . A p h e n o t y p i c a n d a limited
study o f P. falciparum
isolates w a s
also
consistent with this h y p o t h e s i s for t w o c a s e s o b s e r v e d in 1994 (Eldin d e P é c o u l a s et al,
1 9 9 6 ) . E v i d e n c e that
a s a m e m o s q u i t o m a y lead to several airport malaria c a s e s c o u l d have practical implications in terms o f alert procedures and management
o f t h e relatives or c o l
l e a g u e s o f a patient.
187
Four recent airport malaria c a s e s have b e e n o b s e r v e d in the vicinity o f the Roissy-Charles de Gaulle Inter national Airport, Paris, France (Lusina et al, 2 0 0 0 ) . T h e s e c a s e s w e r e geographically very c l o s e to e a c h o t h e r and clustered in a short period o f time during the s u m m e r o f 1 9 9 9 . T h e p h e n o t y p e and g e n o t y p e o f the P. falciparum isolates o b t a i n e d from these patients w e r e d e t e r m i n e d in order to k n o w w h e t h e r a single m o s q u i t o could have infected m o r e than o n e subject.
MATERIALS AND METHODS
V
e n o u s b l o o d samples w e r e collected in EDTA Vacutainer® tubes ( B e c t o n Dickinson, USA) and maintained at 4° C. T h e m i c r o s c o p i c observa tion of thin b l o o d smears s h o w e d that all patients were infected with P. falciparum ; o n e case (patient 3 ) pre sented another malaria e p i s o d e due to P. malariae 3 3 days after t h e initial P. falciparum infection. Patient 1, a 30-year old man, was admitted to hospital on 27 July 1999. He had d e v e l o p e d fever with cough and diarrhoea from 20 July. B l o o d smears s h o w e d 3 3 . 5 0 0 parasitized red b l o o d cells/µl (parasitemia of 0.8 % ) . Patient 1 recovered rapidly after quinine IV t r e a t m e n t . P a t i e n t 2, a 2 9 - y e a r old w o m a n , w a s admitted on 5 August, having suffered from a fever for six clays. B l o o d smears s h o w e d 2 0 9 . 0 0 0 parasitized red blood cells/pl (parasitemia of 5.5 % ) . Patient 2 received quinine IV and recovered. Patient 3, a 43-year old man, was hospitalised on 11 August, having suffered from a fever for a w e e k . B l o o d smears s h o w e d 8 6 . 0 0 0 para sitized red blood cells/µl (parasitemia of 2 % ) . T h e out c o m e was favourable under quinine IV treatment for all the four patients. Patient 3 developed a n e w malaria e p i s o d e which was clue to P. malariae as m e n t i o n e d before. Patient 4, a 25-year old w o m a n , consulted a physician on 30 July for digestive problems with fever. A viral infection was misdiagnosed and patient 4 was hospitalised o n 28 August for fever and vomiting. A pancytopenia w a s observed, b l o o d smears s h o w e d a parasitemia of 5 %. Patient 4 received quinine IV and recovered. T h e clinical presentation and epidemiolo gical aspects of these cases have b e e n reported in details e l s e w h e r e (Lusina et al, 2 0 0 0 ) . G e n o m i c DNA extraction was performed as previously described (Durand et al., 1 9 9 7 ) . T h e amplifications o f block 2 o f merozoite surface protein 1 (Msp1) and merozoite surface protein 2 (Msp2) g e n e s w e r e per formed as previously described ( Randford-Cartwright el al., 1993). Size polymorphisms o f m s p 1 and msp2 g e n e s PCR products w e r e resolved on a 2 % agarose gel stained with ethidium bromide. PCR products were purified using QIAQuick PCR Purification Kit® (Qiagen).
188
Msp1 and msp2 PCR products w e r e then s e q u e n c e d using an ABI PRISM® 3 1 0 Genetic Analyser and an ABI PRISM® Big Dye Terminator Cycle s e q u e n c i n g Kit (Perkin-Elmer Cetus). T h e g e n o m i c characterisation o f isolates was c o m p l e t e d by the study of the cg2 and the dihydrofolate reductase ( D H F R ) g e n o t y p e s . T h e K and ɷrepeats domains of cg2 w e r e amplified as pre viously described (Durand et al, 1 9 9 9 ) . Size poly morphisms o f PCR products w e r e resolved o n a 2.5 % agarose gel stained with ethidium bromide. T h e deter mination o f the nature of DHFR c o d o n 108 w a s per formed b y PCR followed by restriction fragment length polymorphism (PCR-RFLP) (Eldin de Pécoulas et al, 1 9 9 5 ) . T h e in vitro susceptibility to chloroquine, qui nine and halofantrine o f isolates w a s determined on patient parasitized red b l o o d cells within three days o f sampling using the semi-microtest method (Le Bras & Deloron, 1 9 8 3 ) . T h e 50 % inhibitory concentration ( I C ) o f tritiated hypoxanthine uptake w a s calculated by non-linear regression analysis. T h e in vitro sus ceptibility to chloroquine w a s defined as I C < 8 0 nM. Threshold values for susceptibility to quinine and halo fantrine w e r e defined respectively as IC < 8 0 0 nM and I C < 6 nM. R
50
5 0
50
5 0
RESULTS he data obtained for isolates 1, 2 and 3 reflected in vitro resistance to c h l o r o q u i n e and susceptibility to quinine and halofantrine (Table I). T h e semi-microtest was a failure for the fourth isolate d u e to the delayed shipment o f the sample. Msp1 and msp2 PCR products s h o w e d an identical pro file on the agarose gel in all the cases. All isolates w e r e polyclonal as t w o bands w e r e o b s e r v e d ( o n e of the bands appeared with a higher intensity) on the m s p 1 and msp2 profiles. Results o f DNA s e q u e n c i n g sum marized in the T a b l e I s h o w e d identity in block 2 repeat region o f m s p 1 g e n e which b e l o n g e d to the K1 allelic family for all the four isolates while the poly morphic central domain o f msp2 g e n e s h o w e d iden tity in isolates 1, 2 and 4 w h i c h b e l o n g e d to the 3D7/CAMP allelic family with seven repeats o f the h e x a n u c l e o t i d e G G T G C T in b l o c k 2 (Smythe et al., 1993). T h e polymorphic central domain o f msp2 in iso late 3 was a subtype o f the 3D7/CAMP allelic family with eight repeats of the hexanucleotide G G T G C T in block 2. T h e cg2 K and ɷpatterns of all the four iso lates s h o w e d identical multiple b a n d s on the agarose gel which confirmed the polyclonality observed with the mspl and msp2 profiles. T h e bands corresponding to K l 4 and ɷ16 cg2 repeats domains had a higher intensity. Analysis o f the s e q u e n c e s indicated that all the isolates had the "resistant" ɷ16 and K l 4 cg2 repeats T
Characteristics
Case 1
Case 2
Case 3
Case 4
Susceptibility (1) Chloroquine Quinine Halofantrine
271 n m 557 n m 0.88 nm
113 n m 339 n m 1.22 nm
311 nm 473 n m 0.92 nm
ND ND ND
DHFR (2) 108
Ser
Ser
Ser
Ser
cg2 K repeats (3) ɷrepeats (4)
K14 + KX ɷ16 + ɷx
msp1 (5)
msp1**
msp2 (6)
msp2** 7 repeats GGTGCT
K14 + K X ɷ16 + ɷx
Kl4 + KX ɷ16 + ɷx
Kl4 + KX ɷ16 + ɷx
msp1*
msp1*
msp1*
msp2" 7 repeats GGTGCT
msp2** 8 repeats GGTGCT
msp2** 7 repeats GGTGCT
(1) 50 % inhibitory concentrations (IC ) in nM. Threshold values for resistance to Chloroquine > 100 nM; Quinine > 600 nM; halofantrine > 6 nM. (ND = not d e t e r m i n e d ) . ( 2 ) Wild type a m i n o acid residue associated with antifol-susceptibility is Ser108. Mutant c o d o n s associated with antifol-resistance is A s n l 0 8 . (3) a n d (4) K14 a n d ɷ16; profiles of migration o n the agarose gel w e r e identical in all the cases and s h o w e d polyclonality. (5) msp1: the profile of migration o n the agarose gel w a s identical in all the cases. S e q u e n c e analysis s h o w e d the s a m e polymorphism for all the cases. (6) msp2: the profile of migration on the agarose gel w a s identical in all the cases. Sequence ana lysis s h o w e d identity for cases 1, 2 a n d 4 a n d a distinct polymorphism in case 3 * K1 allelic family. ** 3D7/CAMP allelic family. 50
Table I. - Drug susceptibility, point mutations in the DHFR g e n e a n d repeat d o m a i n s of the cg2, mspl malaria cases.
t y p e ( D u r a n d et al, 1999). A n a l y s i s of c o d o n 108 of t h e D H F R g e n e s h o w e d a w i l d t y p e S 1 0 8 c o d o n in all four i s o l a t e s .
DISCUSSION
R
e s u l t s s h o w e d i d e n t i c a l g e n o t y p e s for i s o l a t e s 1, 2 a n d 4 w h e r e a s t h e g e n o t y p e of i s o l a t e 3 dif f e r e d at o n e l o c u s . T h e m o l e c u l a r a n a l y s i s w a s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t all p a t i e n t s c o u l d h a v e b e e n bitten by the s a m e m o s q u i t o a n d that p a t i e n t 3 , m a y h a v e r e c e i v e d a different c l o n e a n d a n a d d i t i o n a l s p e c i e s . In vitro s u s c e p t i b i l i t y d a t a d i d n o t c o n f i r m o r r u l e o u t this h y p o t h e s i s b e c a u s e i s o l a t e s 1, 2 a n d 3 h a d t h e s a m e p r o f i l e of s u s c e p t i b i l i t y t o t h e tested drugs. T h e difference b e t w e e n t h e IC values of c a s e s 1 a n d 2 m a y reflect t h e p r e d o m i n a n c e of o n e c l o n e of t h e s e i s o l a t e s u n d e r c u l t u r e c o n d i t i o n s . T h e d i f f e r e n c e in t h e g e n o t y p e of t h e s e c a s e s w a s d e t e c t e d b y a n a l y s i n g t h e highly p o l y m o r p h i c s e q u e n c e in msp2 g e n e , w h i l e a n a l y s i s of msp 1, cg2 a n d DHFR w a s n o t c o n t r i b u t o r y . W e c h o s e t o amplify b l o c k 2 of t h e m s p 1 a n d t h e msp2 g e n e s b e c a u s e of t h e i r h i g h d e g r e e of p o l y m o r p h i s m . As p r e v i o u s l y d e s c r i b e d , b l o c k 2 of t h e msp2 gene h a s g r e a t e r variability in l e n g t h , a m i n o a c i d c o n t e n t a n d n u m b e r of r e p e a t s t h a n t h a t of m s p 1 ( S m y t h e et ai, 1 9 9 1 ; S n e w i n et al, 1994). T h e a g a r o s e g e l that w e u s e d h a d n o t a sufficient r e s o l u t i o n t o d e t e c t a l e n g t h d i f f e r e n c e of six n u c l e o t i d e s . A p o l y 50
a n d msp2 g e n e s from four airport
a c r y l a m i d e g e l c o u l d b e u s e d , b u t a s different msp2 alleles m a y exceptionally h a v e t h e s a m e molecular weight, D N A s e q u e n c i n g a p p e a r e d to b e t h e best c h o i c e . T h e a g a r o s e g e l , s h o w e d t h a t t h e four isolates h a d extra b a n d s for msp2 gene, indicative of p o l y c l o n a l i n f e c t i o n s . H o w e v e r , t h e p o s s i b i l i t y of t h e a b s e n c e of d e t e c t i o n of a m i n o r i t y c l o n e in D N A s e q u e n c i n g h a s t o b e t a k e n u n d e r c o n s i d e r a t i o n . Ideally, c l o n i n g of msp2 f r a g m e n t s f r o m e a c h i s o l a t e w o u l d b e r e q u i r e d p r i o r t o t h e s e q u e n c i n g t o d e t e c t all t h e c l o n e s e x i s t i n g in isolates. If t h e a l l e l e s f o u n d in all t h e four c a s e s w e r e identical, a u n i q u e vector w o u l d b e the most likely h y p o t h e s i s . O n t h e o t h e r h a n d , a s i n g l e o o c y s t m a y g i v e rise t o different s p o r o z o i t e s d e r i v e d from r e c o m b i n a t i o n events, driving to four meiotic p r o d u c t s of a m a t i n g in t h e m o s q u i t o g u t . N e w a l l e l e s c a n a l s o a r i s e f r o m n u c l e o t i d e r e p e a t s l i p p a g e e v e n after a selff e r t i l i z a t i o n e v e n t . D i f f e r e n t P. falciparum clones o b s e r v e d in different m a l a r i a c a s e s c o u l d t h e r e f o r e ori g i n a t e from a u n i q u e vector. T h e cg2 ɷ16 a n d K l 4 p r o files, w h i c h a r e s t r o n g l y a s s o c i a t e d w i t h r e s i s t a n c e t o c h l o r o q u i n e , w e r e o b s e r v e d for all isolates. T h e s e d a t a w e r e c o n s i s t e n t w i t h t h e in v i t r o a s s a y s h o w i n g resis t a n c e t o c h l o r o q u i n e for t h e t h r e e s u c c e s s f u l a s s a y s . T h e p o l y m o r p h i s m of t h e cg2 g e n e w a s p r e v i o u s l y d e s c r i b e d e s s e n t i a l l y in i s o l a t e s s u s c e p t i b l e t o c h l o r o q u i n e . I n t h e p r e s e n t s t u d y , its i n t e r e s t w a s l i m i t e d . A m o n g all t h e a i r p o r t m a l a r i a c a s e s r e p o r t e d t o d a t e , a s s o c i a t i o n s of t w o p l a s m o d i u m s p e c i e s h a v e n o w b e e n d e s c r i b e d t w i c e . T h e first o n e i n v o l v e d P.falci-
189
parum and P. ovale (Giacomini, 1 9 9 8 ) . T h e associa tion b e t w e e n P. falciparum and P. malariae has not b e e n previously observed in an airport malaria case. T h e late onset o f the P. malariae e p i s o d e in patient 3 could b e d u e to a longer incubation period for that species. T h e P. malariae species diagnosis w a s confir m e d b y a species-specific PCR method. T o our k n o w l e d g e , only o n e work had previously per formed g e n o m i c analysis o f airport malaria isolates (Eldin d e Pécoulas et al, 1 9 9 6 ) . This study suggested identity o f strains originating from t w o patients b a s e d o n their profiles o n the agarose gel, but DNA s e q u e n cing w a s not d o n e . Entomological studies have s h o w n that Anopheles d o not fly very far, usually five km from their birth place (Belding, 1 9 6 5 ) . Patient 1 lived at a distance o f 2.8 km from the C D G airport and w o r k e d at the airport. Patients 2, 3 and 4 lived at a distance o f four k m from the C D G airport in close proximity from e a c h other ( 2 0 0 m ) . Patients 2, 3 a n d 4 w e r e not airport e m p l o y e e s a n d did not g o to the airport in the previous w e e k s . Imported a n o p h e l e s m a y also b e secondarily transported b y motor vehicles or luggages at s o m e distance from the airport, as it was already des cribed (Whithfield, 1 9 8 4 ) . Climatic conditions allowed the survival o f imported a n o p h e l e s but w e r e probably insufficient for a local cycle. Besides, the only poten tial vectors found in the Paris region are Anopheles messae a n d Anopheles claviger. Neither has ever b e e n associated with P. falciparum (Giacomini et al., 1 9 9 7 ) . Moreover, it may b e stressed out that native malaria had totally disappeared in the Paris region b e f o r e the beginning o f the 2 0 century ( B r u c e Chwatt, 1 9 8 0 ) . Since the first descriptions in 1 9 6 9 , airport malaria c a s e s are increasingly reported in Europe (Guillet et al, 1 9 9 8 ; Lusina et al, 2 0 0 0 ) . This could reflect the growth o f traffic with the airports located within an area w h e r e P. falciparum is actively transmitted or the limits o f preventing measures. W h e n climatic condi tions are favourable, w h i c h is the c a s e in particularly hot s u m m e r s in Europe, the imported m o s q u i t o e s may remain infectious up to several w e e k s . Hot tem peratures not only allow the survival o f imported m o s q u i t o e s but also stimulate their activity. In addi tion, it has b e e n s h o w n that a mosquito infected with P. falciparum c o u l d discharge sporozoites e v e n after more than 1 0 blood meals (Belding, 1965). A recent study in 1997, revealed airport malaria c a s e s d u e to the p r e s e n c e o f i n f e c t i v e A n o p h e l e s in a i r p l a n e s ( C i m e r m a n et al, 1 9 9 7 ) . E v i d e n c e that a s i n g l e imported infectious a n o p h e l e s may lead to several air port malaria cases, s o m e o f w h i c h could b e fatal, is an argument to strengthen insecticidal p r o c e d u r e s o n flights at risk and to reinforce controls o f flights. Every airport malaria case should b e rapidly reported to health authorities in order to alert local physicians w h o practise in the airport area. t h
190
ACKNOWLEDGEMENTS
W
e are grateful to Dr M. Liance for providing o n e o f the isolates. W e thank Dr N. J . Pieniazek for his help. This work w a s sup ported b y the French Ministry o f Health (Direction d e la Veille Sanitaire), Z e n e c a Pharma, and the A g e n c e Universitaire d e la F r a n c o p h o n i e . S.J. is supported by a WHO/TDR Research Training grant.
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Reçu le 27 août 2001 Accepté le 7 janvier 2002
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