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Prevalence and levels of antibodies to the circumsporozoite protein of Plasmodium falciparum in an endemic area and their relationship to resistance against malaria infection
827
Prevalence and levels of antibodies to the circumsporozoite protein of Plasmodium falciparum in an endemic area and their relationship to resistance against malaria infection Fulvio Esposito’,*, Stefania Lombard1*2p3,David Modiano3, Fidel Zavala4, Jan Reeme’, Lansina Lamizana , Mario Coluzzi3 and Ruth S. Nussenzweig4 lDipartimento di Biologia Cellulare, Universita di Camerino, 62032 Camerino (MC), Italy; *Direzione Generale Cooperazione Sviluppo, Minister0 Affari Ester& Roma, Italy; 31stituto di Parassitologia, Universita La Sapienza, Roma, Italy; 4Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10010, USA; ‘WHO Onchocercosis Control Program, Ouagadougou, Burkina Faso; eMinistere de la Sante, Centre de Lutte contre le Paludisme, Ouagadougou; Burkina -Faso -
Abstract A study on malaria transmission, prevalence of infection and anti-sporozoite antibodies was carried out in Burkina Faso (West Africa). The prevalence and the levels of antibodies to (NANIQ were found to be related to the entomological sporozoite inoculation rates measured at the same-time in a defined area. The maior inducer of anti-(NAN& antibodv nroduction under field conditions is spcrozoite inoculation by infected mosquitoes. Levels of antibodies to (NANP)3 vary considerably with age and transmission season. High levels of anti-(NANP)s antibodies raised under field conditions might offer protection against small inocula of sporozoites.
Introduction Stage- and species-specific sporozoite-neutralizing antibodies have been demonstrated in the sera of sporozoite-immunized animals and humans (NUSSENZWEIG& NUSSENZWEIG, 1985). Almost all antisporozoite antibodies in the sera of immunized animals appeared to be directed against a single antigen, the circumsporozoite (CS) protein (ZAVALA et al., 1983). Antibodies to sporozoites occur frequently in individuals living in areas of hyperendemic malaria (NARDIN et al., 1979; TAPCHAISRI et al., 1983; HOFFMAN et al., 1986; DEL GIUDICE et al., 1987). However, little is known about the factors which determine the prevalence and levels of antibodies to sporozoites and their possible protective role in malaria endemic areas. Are the prevalence and levels of antibodies to sporozoite antigens related to the rates of sporozoite inoculation by mosquitoes? Is the induction of these antibodies affected by the presence of circulating blood-stage antigens? Do antibody levels change with the seasonal variation of intensity of malaria transmission? Can antibodies to sporozoite antigens, raised by natural inoculation, exert a protective role against re-infection in a malaria endemic area? In an attempt to answer these questions, field studies were conducted in Burkina Faso in 1985.
Materials and Methods Study area and population In Burkina Faso, West Africa, a bilateral cooperaCorrespondence to Fulvio Esposito, Dipartimento di Biologia Cellulare. via Camerini 2, 62032 Camerino (MC), Italy.
tive programme for malaria control was started in 1983-under the auspices of the local Government and of the Italian Ministrv for Foreign Affairs. As Dart of the programme a longitudinal s&ey was undeitaken, involving 3212 serum samples collected from healthy volunteers of different ages (from newborns to adults more than 70 years old). The age-matched serum donors lived in a district of Ouagadougou, the capital of Burkina Faso, and in the nearby rural villages of Kuiti and Zagtouli. The study included all the inhabitants of the localities concerned who volunteered to donate blood. Only in the village of Kuiti was the number of volunteers sufficient to allow an age-specific analysis of the results. A preliminary epidemiological survey was carried out in the same area in 1984 to establish the level of malaria transmission by ROSSI et al. (1986), SABATINELLI et al. (1986) and ESPOSITO et al. (19863. These studies can be consulted for further details of the study area and population. Serum collection and determination of Plasmodium falciparum parasitaemia Sera were obtained from firmer-orick blood samples, taken approximately every-two months, starting at the beginning of the rainy season in June 1985, and stored at -20°C until processing. Blood collections were made during the third week of June, the third week of August and the third week of November 1985. At the same time. thick and thin blood smears were prepared to assess parasitaemia. Blood smears were assessed by the examination of 200 microscope fields of Giemsa-stained thick films by 2 observers. Immunoradiometric assay Anti-sporozoite antibodies were measured as antibodies to the svnthetic DeDtide (NANI%, corresDonding to 3 repeats of the repetitive pot&n of the CS protein of P. falciparum. The protocol of ZAVALA et al. (1985, 1986) was basicallv followed. First, 4 replicates ‘of 20 sera containing- different amounts of antibodies to (NANIQ were examined by immunoradiometric assay. Since the highest coefficient of variation in this series was 13%, we assayed all other serum samples singly, at a 1: 10 dilution. Throughout the studv, each serum was simultaneouslv tested in peptide-coated wells and control wells prepared by omitting the peptide. The radioactivity in the control wells, which averaged 300 counts per minute ‘ct’minj
828 and ranged from 100 to 800 ct/rnin, was subtracted from the radioactivity in the corresponding peptidecoated wells. A serum was considered positive when the ct/min value in the peptide-coated well was at least double that obtained in the corresponding control wells. By adopting this criterion, not a single positive was found in 120serafrom healthy Italian donors who had never travelled to endemic areas. To evaluate the possibility of using the ct/min values as an estimate of the antibody level, a group of 82 serawas titrated by two-fold serial dilutions. When ct/min values and titres were compared by a nonparametric method (Spearman rank correlation), the r, was 0.976 (P<
400 sera were assayed for anti-blood stage antibodies according to the standard immunofluorescence (IFA) techniques for infected red blood cells (VOLLER & O'NEILL, 1971). Chemoprophylaxis and medical care
When chemoprophylaxis was administered, the regimen adopted by the primary health care system of Burkina Faso was followed, i.e., 10 mg of chloroquine base(Bayer) per kg of body weight on a weekly basis. Chemoprophylaxis, chemotherapy (25 mg of chloroquine base per kg of body weight) and overall medical care were under the control of the local primary health care system. When this study was carried out, chloroquine resistance had not been reported in the relevant area.
collection of mosquitoesfor determining the EIR took place during the first and third week of each month. Wide variations were observed, according to the season and the locality (Fig. 1, upper panel). Exceedingly high values of EIR were recorded in Kuiti village during most of the rainy season(from July to October). The seasonal increase of the EIR was striking in all localities. Increases of as much as lOOO-fold occurred between the beginning and the end of the rainy season.The low EIR in Kuiti in June was a reflection of a low sporozoite rate (1.2%) and low mosquito density (3.0 mosquitoes per room). Relationship between EIR and antibodies to (NANP),
The relationship between the EIR and the prevalence and levels of antibodies to (NANP)s was investigated in 3 age-matched cohorts of children from 6 months to 9 years old. These children lived in the 3 areas in which the EIR was measured. The sample sizes for the urban district and Kuiti and Zagtouli villages were respectively 108, 143, and 208 children. These figures representedat least 30% of the total population of the relevant age group in each locality. The children were surveyed 3 times, at the end of June, end of August and end of November. Prevalences and levels of antibodies to (NANP)s showed a marked difference among the 3 localities and an obvious seasonal variation (Fig. 1, lower panel). An exception was in the urban district, where the medians were consistently low due to the high proportion of negative subjects. Prevalences and levels increased between the first and the second surveys; between the second and the third surveys, 10
Entomological surveys
Entomological surveys were carried out in the same study area. Indoor resting mosquitoes were caught by pyrethrum spray. In an urban district and in the villages of Kuiti and Zagtouli, 10 compounds were chosen as collection stations and maintained throughout the survey. The collected mosquito specimens were used for the determination of the entomological inoculation rate (EIR). The EIR in each area was calculated by dividing the geometric mean of the number per room of indoor resting, human blood-fed mosquitoes by the number of humans per room, and multiplying the resulting fraction by the percentageof mosquitoes with P. falciparum CS protein in dissected salivary glands, according to the procedure described by LOMBARDIet al. (1987). Results Entomological inoculation rates (EIR) in the study area
The numbers of notentiallv infective mosauito bites per man per night, i.e. EIR, were measured from June to November 1985in the study area, in order to compare the seasonality and the intensity of transmission in an urban district and 2 rural villages. The
June
July
Aug.
Sept.
Oct.
NOV.
Fig. I. Seasonal variations of entomological inoculation rate and of prevalences and levels of antibodies to (NANP)S in 3 areas with different intensities of malaria transmission. A, Kuiti; 0, Zagtouli, 0, urban district. In the lower panel, open symbols refer to levels and closed symbols refer to prevalence of antibodies.
829 levels, but not prevalences, paralleled the decreasein the intensity of transmission, as can be seen by comparing the upper and lower panels of Fig. 1. The seasonalvariation of the antibodies to sporozoites and their differences in the studied localities suggestedsome relationship with the EIR. As shown in Fig. 2, the levels (upper panel) and the prevalences (lower panel) of the antibodies to (NANP)3, determined in the surveys of August and November, were related to the number of infective bites presumably received by each individual in the 2 month period preceding the surveys. The samekind of analysis was not possible for the June survey, since in the months preceding the survey the number of mosquitoes caught did not allow a reliable calculation of the EIR. The level of antibodies to (NANQ3 seemed to reflect particularly changesin high values of EIR (Fig. 2, upper panel). On the other hand, prevalence of antibodies seemedto be a more sensitive indicator for changes in low EIR (Fig. 2, lower panel). Antibodies to (NANI’), itant blood infection
intense transmission, the secondsurvey (August) near the neak. and the third (November) at the time of decl&ing’transmission. &mediately &er the baseline survey in June, one group, consisting of 40 children, was treated with a therapeutic dosageof chloroquine, clearing their blood of parasites. From then on, these children received weekly chemoprophylaxis. As control, another group of 37 children was closely followed and treatment was given only to those who presented malaria symptoms. The prevalence of parasitaemia in June (baseline) was well matched in the 2 groups (Fig. 3, upper panel). In August, at the peak of the transmission season, the prevalence reached 86.5% in the control
and their relationship to concom-
The prevalence and levels of antibodies to (NANQ3 were studied in 2 groups of children, from 6 to 30 months of age, both living in Kuiti village, but on the opposite sides of a rice field. The first baseline survey (June) was carried out before the beginning of
E m$ b L
a
50 -i 0.01
,
, I , I I I , I I I I 0.03 0.06 0.16 0.40 1.00 2.51 Entomological inoculation rate (geometric mean)
Fig. 2. Relationship between entomological inoculation rate (measured over the two month period preceding blood collections) and antibodies to (NANP)+ The vertical bars represent the standard error of the median in the upper panel and the 95% confidence intervals in the lower panel. 0, Kuiti; 0, Zagtouli; 0, urban district. Closed symbols refer to the August survey, open symbols to the November survey.
June
August
November
Fig. 3. Relationship between P. falctparum parasitaemia and armbodies to (NANI’),. Solid bars refer to children undergoing chemoprophylaxq shaded bars refer to children without chemoprophylaxis. Baseline data are on the left of the broken line.
I
4
8
12 Age group
I
I
16 20 (years)
I
I
24
I
I
28
Fig. 4. Levels of antibodies to (NANP)3 in relation to age and season, in Kuiti village. Corresponding entomological inoculation rates are shown in Fig. 1. Vertical bars represent the standard error of the median. 0, June, 0, August; 0, November.
group and fell to 12.5% in the group under chemoprophylaxis. The small number of positives in this latter group in August was due to children failing to present themselves for the 2 chemoprophylactic treatments before blood collection. In November, the prevalence of parasitaemia remained high in the control group (89.2%) and was zero in the group under chemoprophylaxis. The corresponding trend of prevalence of antibodies to (NANP)s is shown in the middle panel of Fig. 3. The antibody prevalence was almost identical in both groups in all 3 surveys, and therefore was unrelated to the presence of blood infection. As illustrated in the lower panel of Fig. 3, the levels of antibodies to (NANIQ corresponding to the first (June) and to the second survey (August) were quite similar in the two groups. In the third survey, a difference of borderline significance (P=O.O54, Wilcoxon rank sum test) was observed. The antibodies to blood stages in the same sera, measured by IFA using infected red blood cells, showed a completely different trend. In the group under chemoprophylaxis, the levels of antibodies remained unchanged between the first and the second survey, and then showed a significant decrease (P=O*OO4)at the third survey. On the contrary, in the control group a significant increase (P=O.OOl) between the first and the second survey was followed by stabilization (data not shown). Age-related variations
of antibody levels to (NANP),
Individuals belonging to different age groups, living in Kuiti village, were monitored for anti(NANP)s antibody levels in June, August, and November (Fig. 4). The age groups were: 6-24 months (n=37); 34 years (n=47); 5-9 years (n=59); 10-19 years (n=50); 20-35 years (n=22). The entire groups, constituted by the same individuals, were examined at each survey. The median values of antibodies in each age group varied according to the transmission season. In addition, at any given time, the level of antibodies was age-related, being maximal in adults. Role of antibodies to (NANPJ3
in protection
The relationship between prevalence of P. falciparurn parasitaemia and levels of antibodies to (NANP)s
In
Oo
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4
‘
81
8
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16
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20
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00
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Age group
(years) Fig. 5. Parasitaemia prevalences in relation to anti-(NANP), antibody levels and age in Kuiti village. The age groups and their relative sizes are the same as in Fig. 4. Open squares refer to individuals possessing a level of antibodies to (NANP), below the median value for that age group, and solid circles refer to individuals with an antibody level above the median value. Vertical bars represent 95% confidence intervals. A, beginning of the transmission season (June); B, peak of the transmission (August); C, end of the transmission season (November).
was evaluated through 3 cross-sectional surveys in a cohort of individuals. Inhabitants of Kuiti village belonging to different age groups were sub-grouped according to whether they possessedlevels of antibodies to (NANP)s above or below the median value of their respective age group. The prevalences of parasitaemia of these different groups were then compared. As shown in Fig. SA, under conditions of low transmission (June), a striking difference in the prevalence of parasitaemia was observed in the 2 groups of adults. The individuals with antibody levels below the median value had a prevalence of parasitaemia of 45.4% (5 of ll), while no infection was detected in the group of 11 adults with antibody levels above the median value. No significant difference was observed in the other age groups.
831 Under conditions of maximal transmission (August), the differences among the higher and lower antibody groups were not significant even in adults (Fig. 5B). However, when transmission started to decline (November), a difference again became apparent in adults (Fig. K), i.e., 54.5% (6 of 11) were parasitized in the lower antibody group, compared to 9.1% (1 of 11) in the higher antibody group. No correlation was found between anti-blood stage IFA titres and prevalence of parasitaemia in the same individuals. The parasitaemia prevalences in adults in June were 18.1% in the higher antibody group and 27.3% in the lower antibodv moue; in November, the values were 36.4% and 2?.j”/, iespectively.
Acknowledgements
Discussion On the basis of the results presented here, a series of conclusions can be drawn. Levels and prevalences of antibodies to (NANP)3 were found to be related to the EIRs measured at the same time in the same localities. It appears, therefore, that levels and prevalence of antibodies to (NANP)3 are useful indicators of the level of malaria transmission. The same conclusions were drawn by DRUILHE et al. (1986) who, however, compared the prevalence and levels of antibodies to sporozoites with entomological data collected in the same areas many years before. The induction of antibodies to (NANP)3 is not affected by the presence of blood stage parasites. This leads to the conclusion that antibodies to (NANP)3 are mainly the result of an immune response directed against the sporozoites. The possibility cannot be excluded, however, that weakly immunogenic, crossreactive, blood-stage epitopes may contribute to a minor degree to maintaining the level of anti(NANP)3 antibodies (COPPEL et al., 1985). Our data also indicate that the presence of blood stage parasites does not cause a detectable suppression of the anti-sporozoite antibody response. The levels of antibodies to (NANP)3 show a substantial seasonal variation, even in adults living in areas of extremely high malaria transmission. Defining this variation, in different areas, will be important when planning future anti-sporozoite vaccine strategies. A cross-sectional survey like that reported here has an intrinsic limitation: when the parasite prevalence and antibody levels on a given day are established, it cannot be concluded that such antibody levels are similar to those existing when the sporozoite inoculation(s) determining such parasitaemia took place. Taking into account this limitation, our findings indicate that adults with higher levels of anti(NANP)3 antibodies are less frequently parasitized than those with lower antibody levels. This difference was obvious during the initial and late transmission periods. During the peak period of transmission, no significant diffeiencewas f&nd, in agreement with a recentlv oublished reoort from East Africa (HOFFMAX et al., 1987). it thus appears reasonable to postulate that, when the sporo%ite inoculation rates are relativelv low. individuals with high levels of antibodies td sporizoites might enjoy proiection from malaria re-infection, since only a fraction of the potentially infective bites would give rise to an actual infection.
References Coppel, R. L., Favaloro, J. M., Crewther, I’. E., Burkot, T. R., Bianco, A. E., Stahl, H. D., Kemp, D. J., Anders, R. F. & Brown, G. V. (1985). A blood stage antigen of Plasmodium falciparum shares determinants with the sporozoite coat protein. Proceedings of the LVational Academy of Sciences, USA, 82, 5121-5125. Del Giudice, G., Engers, H. D., Tougne, C., Biro, S. S., Weiss, N., Verdini, A. S., Pessi, A., DegrCmont, A. A., Freyvogel, T. A., Lambert, P.-H. & Tanner, M. (1987). Antibodies to the repetitive epitope of Plasmodium falciparum circumsporozoite protein in a rural Tanzanian community: a longitudinal study of 132 children. American Journal of Tropical Medicine and Hygiene, 36, 203-212. Druilhe, I’., Pradier, O., Marc, J.-P., Miltgen, F., Mazier, D. & Parent, G. (1986). Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection. Infection and Immuniry, 53, 393-397. Esposito, F., Lombardi, S., Tour&, Y. T., Zavala, F. & Coluzzi, M. (1986). Field observations on the use of anti-sporozoite monoclonal antibodies for determination of infection rates in malaria vectors. Parassitologia, 28, 69-77. Hoffman, S. L., Wistar, R., Jr, Ballou, W. R., Hollingdale, M. R., Wirtz, R. A., Schneider, I., Marwoto, H. A. & Hockmeyer, W. T. (1986). Immunity to malaria and naturally acquired antibodies to the circumsporozoite protein of Plasmodium falciparum. Nat England Journal of Medicine, 315, 602-606. Hoffman, S. L., Oster, C. N., Plowe, C. V., Woollett, G. R., Beier, J. C., Chulay, J. D., Wirtz, R. A., Hollingdale, M. R. & Mugambi, M. (1987). Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science, 237, 639-642. Lombardi, S., Esposito, F., Zavala, F., Lamizana, L., Rossi, I’., Sabatinelli, G., Nussenzweig, R. S. & Coluzzi, M. (1987). Detection and anatomical localization of Plasmodium falciparum circumsporozoite protein and sporozoites in the Afrotropical malaria vector Anopheles gambiae s.1. Amen’can Journal qf Tropical Medicine and Hveiene. 37. 491-494. Na&i;E~.’ fi.j Nussenzweig,, R. S., McGregor! I. A. & Bryan, J. H. (1979). Annbodies to sporozoltes: their frequent occurrence in individuals living in an area of hyperendemic malaria. Science, 206, 597-599. Nussenzweig, V. & Nussenzweig, R. S. (1985). Circumsporozoite proteins of malaria parasites. Cell, 42,401-403. Rossi. I’. . Belli. A.. Mancini. L. & Sabatinelli. G. 11986). Enqugte entbmoiogique longitudinale sur la transmission du paludisme g Ouagadougou (Burkina Faso). Parassirologia, 28, 1-15. Sabatinelli, G., Bosman, A., Lamizana, L. & Rossi, I’. (1986). Enqu@te protozoologique sur la prbvalence du oaludisme g Ouaaadougou (Burkina Faso! en ueriode de ;ransmission ma&x& F&zssitologia, is, 17-31. Tapchaisri, P., Chomcharn, Y., Poonthong, C., Asavanich,
It is a pleasure to thank Professor Sir Ian McGregor for critically reading the manuscript. We also gratefully acknowledge the excellent technical assistanceof Silvana Bagalino in Rome and Simon-Pierre Zoneo and Benoit 1. Zoungrana in Ouagadougou. The skilled a&ninistrative work by-Sharon Hecht in New York was invaluable. This study received financial support from the Dipartimento per la Cooperazione allo Sviluppo-Minister0 Affari Esteri; The Rockefeller Foundation; Fondazione Istituto Pasteur-Cenci Bolognetti; the International Atomic Energy Agency; and WHOITDR Director’s Initiative Fund. Some of these results were preliminarily presented by F. E. at the 14th Conference of the Italian Society of Parasitologists, in Piss, 21-24 May, 1986.
832 A., Limsuwan, S., Maleevan, O., Tharavanij, S. & Harinasuta, T. (1983). Anti-sporozoite antibodies induced by natural infection. AmericanJournal of Tropical Medicine and Hygiene, 32, 1203-1208. Voller, A. & Q’Neill, P. (1971). Immunofluorescence method suitable for large scale application. Bulletin of the World Health Organization, 45, 524-529. Zavala, F., Cochrane, A. H., Nardin, E. H., Nussenzweig, R. S. & Nussenzweig, V. (1983). Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. Journal of Experimental Medicine, 157, 1947-1957.
Received 16 March 1988; revised 18 May 1988; accepted for publication 18 May 1988
TRANSACTIONS OFTHEROYALSOCIETY OFTROPICAL MEDICI~YE AND
HYGIENE (1988) 82, 832
1I Short Report I1
falciparum in France: Anopheles maculipennis and A claviger are found in the Paris area and are potential vectors (GENTILINI et al., 1978) that may become
Plasmodium falciparum malaria after three years in a non-endemic area M. P. Revel’, A. Da@, A. Saint Raimond2, G. Lenoi?, M. Dank’ and M. Gentilini’ ‘Department of Parasitology and Tropical Medicine, Groupe Hospitaher PititLSalp&wi&re, Paris, France; ‘Service de Pediatrie GcWrale, Hopital des Enfants Malades, Paris, France
A 7 years old male child was admitted to hospital in March 1987at the Hopital des Enfants Malades, with a 6 d history of headache, ocular pain, fever and asthenia. On physical examination he was found to have splenomegaly and a temperature of 38.7X, but no lack of alertness. Thick and thin blood smears revealed trophozoites of Plasmodium ovale and, surprisingly, trophozoites and gametocytesof P. falciparum in the child, who had been living in Montrouge, a Paris suburb, since January 1984. He was treated successfully with quinine (quinimax), 25 mg/kg/d. The singularity of the caselay in the late manifestation of P. falciparum malaria, more than 3 years after the patient had left an endemic area, the Comoro Islands. The onset of diseasedue to P. falciparum does not normally occur later than 2 months after returning from an endemic area (GENTILINI & DUFLO, 1986). This period of 2 months has been challenged in several publications describing the onset of P. falciparum disease up to 11 months after exposure (BRUCE-CHWATTet al., 1974; CARMEet al., 1978). Periods over 3 years before onset have not been reported, as far as we know. Several factors must be considered in connection with the late manifestation in this case. An error may have been made in the identification of the parasite, but the presence of P. falciparum gametocytes excluded such a hypothesis. The boy’s school attendance record precluded the possibility of his having revisited an endemic area since his arrival in France. After investigations, blood contamination by intravenous drug abuse or transfusion were excluded. Other possible hypotheses may be advanced. (i) The existence of quiescent hepatic or extrahepatic forms of P. falciparum, capable of causing a late episode of malaria; this possibility is at present being reviewed in the light of other observations on late manifestations (CARMEet al., 1978)although classically it has not been accepted for P. falciparum infections.
Zavala, F., Tam, J. P., Hollingdale, M. R., Cochrane, A. H., Quakyi? I., Nussenzweig, R. S. & Nussenzweig, V. (1985). Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science, 228, 1436-1440. Zavala, F., Tam, T. P. & Masuda, A. (1986). Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites. Journal of Immunological Methods, 93, 55-61.
(ii) There is the further possibility of native P.
infected by biting travellers from the tropics. However, it has not been proved that mosquitoes of these speciescan transmit tropical malarial species(SHUTE, 1943).Moreover, the sporogony cycle in the mosquito requires a temperature above 18°C for at least 12 d (GENTILINI & DUFLO, 1986). These conditions were not met during the winter months preceding the boy’s illness. (iii) Another possible explanation could be infection in France from an imported Anopheles. This has becomewell known in the neighbourhood of international airports and has resulted in several cases of serious diseasein 1977-1978 (GENTILINI et al:, 1978) when the true causewas unsuspected. Proximity to an airport did not apply in our case, but Anopheles may easily be carried by the wind for a distance of 100 km and the life span of females ranges between 8 and 34 d (GENTILINI & DUFLO, 1986). (iv) A final possibility might be that infected Anopheles had been transported in the luggage of a member of the family who had arrived in France in the months preceding the boy’s illness; clothes may constitute a possible lodging for mosquitoes (GENTILINI & DUFLO, 1986). The casereported here, which is similar to others except in the length of time before the appearanceof P. falciparum infection, raises the question of the existence of quiescent forms of the parasite, although this has still to be proved. As in other known casesof malaria in France, the arrival of a member of the family from an endemic area seems to support the hypothesis of an imported Anopheles. References Bruce-Chwatt, L. J., Southgate, B. A. & Draper, C. C. (1974). Malaria in the United Kingdom. British British Medical Medical jou&l, Jourd, ii, 707-711. Carme. B.. Dams. M. & Gentilini. M. (1978). LonnCvitC de Pldsmo~ium falciparum : g propos de’ 3 cas- d’acces palustre survenus plus de 2 mois apres le depart de la zone d’endemie. Mkdicine et Maladies Infectieuses, 8, 352-354. Gentilini, M. & Duflo, B. (1986). Paludisme. In: Medecine Tropicale, 4th edition. Paris: Flammarion. Gentilini, M., Danis, M., Dallot, J. Y ., Richard-Lenoble, D. & Felix, H. (1978). Rtapparition du paludisme autochtone. Annales de MCdecine Interne, 129, 405-410. Shute, P. G. (1943). Failure to infect English specimens of Anopheles maculipennis var. atropanw with certain strains of Plasmodium of tropical origin. Journal of Tropical Medicine and Hygiene, 43, 1-4.
Received 18 April 1988; acceptedfor publication 12341~ 1988
Prevalence and levels of antibodies to the circumsporozoite protein of Plasmodium falciparum in an endemic area and their relationship to resistance against malaria infection Fulvio Esposito’,*, Stefania Lombard1*2p3,David Modiano3, Fidel Zavala4, Jan Reeme’, Lansina Lamizana , Mario Coluzzi3 and Ruth S. Nussenzweig4 lDipartimento di Biologia Cellulare, Universita di Camerino, 62032 Camerino (MC), Italy; *Direzione Generale Cooperazione Sviluppo, Minister0 Affari Ester& Roma, Italy; 31stituto di Parassitologia, Universita La Sapienza, Roma, Italy; 4Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10010, USA; ‘WHO Onchocercosis Control Program, Ouagadougou, Burkina Faso; eMinistere de la Sante, Centre de Lutte contre le Paludisme, Ouagadougou; Burkina -Faso -
Abstract A study on malaria transmission, prevalence of infection and anti-sporozoite antibodies was carried out in Burkina Faso (West Africa). The prevalence and the levels of antibodies to (NANIQ were found to be related to the entomological sporozoite inoculation rates measured at the same-time in a defined area. The maior inducer of anti-(NAN& antibodv nroduction under field conditions is spcrozoite inoculation by infected mosquitoes. Levels of antibodies to (NANP)3 vary considerably with age and transmission season. High levels of anti-(NANP)s antibodies raised under field conditions might offer protection against small inocula of sporozoites.
Introduction Stage- and species-specific sporozoite-neutralizing antibodies have been demonstrated in the sera of sporozoite-immunized animals and humans (NUSSENZWEIG& NUSSENZWEIG, 1985). Almost all antisporozoite antibodies in the sera of immunized animals appeared to be directed against a single antigen, the circumsporozoite (CS) protein (ZAVALA et al., 1983). Antibodies to sporozoites occur frequently in individuals living in areas of hyperendemic malaria (NARDIN et al., 1979; TAPCHAISRI et al., 1983; HOFFMAN et al., 1986; DEL GIUDICE et al., 1987). However, little is known about the factors which determine the prevalence and levels of antibodies to sporozoites and their possible protective role in malaria endemic areas. Are the prevalence and levels of antibodies to sporozoite antigens related to the rates of sporozoite inoculation by mosquitoes? Is the induction of these antibodies affected by the presence of circulating blood-stage antigens? Do antibody levels change with the seasonal variation of intensity of malaria transmission? Can antibodies to sporozoite antigens, raised by natural inoculation, exert a protective role against re-infection in a malaria endemic area? In an attempt to answer these questions, field studies were conducted in Burkina Faso in 1985.
Materials and Methods Study area and population In Burkina Faso, West Africa, a bilateral cooperaCorrespondence to Fulvio Esposito, Dipartimento di Biologia Cellulare. via Camerini 2, 62032 Camerino (MC), Italy.
tive programme for malaria control was started in 1983-under the auspices of the local Government and of the Italian Ministrv for Foreign Affairs. As Dart of the programme a longitudinal s&ey was undeitaken, involving 3212 serum samples collected from healthy volunteers of different ages (from newborns to adults more than 70 years old). The age-matched serum donors lived in a district of Ouagadougou, the capital of Burkina Faso, and in the nearby rural villages of Kuiti and Zagtouli. The study included all the inhabitants of the localities concerned who volunteered to donate blood. Only in the village of Kuiti was the number of volunteers sufficient to allow an age-specific analysis of the results. A preliminary epidemiological survey was carried out in the same area in 1984 to establish the level of malaria transmission by ROSSI et al. (1986), SABATINELLI et al. (1986) and ESPOSITO et al. (19863. These studies can be consulted for further details of the study area and population. Serum collection and determination of Plasmodium falciparum parasitaemia Sera were obtained from firmer-orick blood samples, taken approximately every-two months, starting at the beginning of the rainy season in June 1985, and stored at -20°C until processing. Blood collections were made during the third week of June, the third week of August and the third week of November 1985. At the same time. thick and thin blood smears were prepared to assess parasitaemia. Blood smears were assessed by the examination of 200 microscope fields of Giemsa-stained thick films by 2 observers. Immunoradiometric assay Anti-sporozoite antibodies were measured as antibodies to the svnthetic DeDtide (NANI%, corresDonding to 3 repeats of the repetitive pot&n of the CS protein of P. falciparum. The protocol of ZAVALA et al. (1985, 1986) was basicallv followed. First, 4 replicates ‘of 20 sera containing- different amounts of antibodies to (NANIQ were examined by immunoradiometric assay. Since the highest coefficient of variation in this series was 13%, we assayed all other serum samples singly, at a 1: 10 dilution. Throughout the studv, each serum was simultaneouslv tested in peptide-coated wells and control wells prepared by omitting the peptide. The radioactivity in the control wells, which averaged 300 counts per minute ‘ct’minj
828 and ranged from 100 to 800 ct/rnin, was subtracted from the radioactivity in the corresponding peptidecoated wells. A serum was considered positive when the ct/min value in the peptide-coated well was at least double that obtained in the corresponding control wells. By adopting this criterion, not a single positive was found in 120serafrom healthy Italian donors who had never travelled to endemic areas. To evaluate the possibility of using the ct/min values as an estimate of the antibody level, a group of 82 serawas titrated by two-fold serial dilutions. When ct/min values and titres were compared by a nonparametric method (Spearman rank correlation), the r, was 0.976 (P<
400 sera were assayed for anti-blood stage antibodies according to the standard immunofluorescence (IFA) techniques for infected red blood cells (VOLLER & O'NEILL, 1971). Chemoprophylaxis and medical care
When chemoprophylaxis was administered, the regimen adopted by the primary health care system of Burkina Faso was followed, i.e., 10 mg of chloroquine base(Bayer) per kg of body weight on a weekly basis. Chemoprophylaxis, chemotherapy (25 mg of chloroquine base per kg of body weight) and overall medical care were under the control of the local primary health care system. When this study was carried out, chloroquine resistance had not been reported in the relevant area.
collection of mosquitoesfor determining the EIR took place during the first and third week of each month. Wide variations were observed, according to the season and the locality (Fig. 1, upper panel). Exceedingly high values of EIR were recorded in Kuiti village during most of the rainy season(from July to October). The seasonal increase of the EIR was striking in all localities. Increases of as much as lOOO-fold occurred between the beginning and the end of the rainy season.The low EIR in Kuiti in June was a reflection of a low sporozoite rate (1.2%) and low mosquito density (3.0 mosquitoes per room). Relationship between EIR and antibodies to (NANP),
The relationship between the EIR and the prevalence and levels of antibodies to (NANP)s was investigated in 3 age-matched cohorts of children from 6 months to 9 years old. These children lived in the 3 areas in which the EIR was measured. The sample sizes for the urban district and Kuiti and Zagtouli villages were respectively 108, 143, and 208 children. These figures representedat least 30% of the total population of the relevant age group in each locality. The children were surveyed 3 times, at the end of June, end of August and end of November. Prevalences and levels of antibodies to (NANP)s showed a marked difference among the 3 localities and an obvious seasonal variation (Fig. 1, lower panel). An exception was in the urban district, where the medians were consistently low due to the high proportion of negative subjects. Prevalences and levels increased between the first and the second surveys; between the second and the third surveys, 10
Entomological surveys
Entomological surveys were carried out in the same study area. Indoor resting mosquitoes were caught by pyrethrum spray. In an urban district and in the villages of Kuiti and Zagtouli, 10 compounds were chosen as collection stations and maintained throughout the survey. The collected mosquito specimens were used for the determination of the entomological inoculation rate (EIR). The EIR in each area was calculated by dividing the geometric mean of the number per room of indoor resting, human blood-fed mosquitoes by the number of humans per room, and multiplying the resulting fraction by the percentageof mosquitoes with P. falciparum CS protein in dissected salivary glands, according to the procedure described by LOMBARDIet al. (1987). Results Entomological inoculation rates (EIR) in the study area
The numbers of notentiallv infective mosauito bites per man per night, i.e. EIR, were measured from June to November 1985in the study area, in order to compare the seasonality and the intensity of transmission in an urban district and 2 rural villages. The
June
July
Aug.
Sept.
Oct.
NOV.
Fig. I. Seasonal variations of entomological inoculation rate and of prevalences and levels of antibodies to (NANP)S in 3 areas with different intensities of malaria transmission. A, Kuiti; 0, Zagtouli, 0, urban district. In the lower panel, open symbols refer to levels and closed symbols refer to prevalence of antibodies.
829 levels, but not prevalences, paralleled the decreasein the intensity of transmission, as can be seen by comparing the upper and lower panels of Fig. 1. The seasonalvariation of the antibodies to sporozoites and their differences in the studied localities suggestedsome relationship with the EIR. As shown in Fig. 2, the levels (upper panel) and the prevalences (lower panel) of the antibodies to (NANP)3, determined in the surveys of August and November, were related to the number of infective bites presumably received by each individual in the 2 month period preceding the surveys. The samekind of analysis was not possible for the June survey, since in the months preceding the survey the number of mosquitoes caught did not allow a reliable calculation of the EIR. The level of antibodies to (NANQ3 seemed to reflect particularly changesin high values of EIR (Fig. 2, upper panel). On the other hand, prevalence of antibodies seemedto be a more sensitive indicator for changes in low EIR (Fig. 2, lower panel). Antibodies to (NANI’), itant blood infection
intense transmission, the secondsurvey (August) near the neak. and the third (November) at the time of decl&ing’transmission. &mediately &er the baseline survey in June, one group, consisting of 40 children, was treated with a therapeutic dosageof chloroquine, clearing their blood of parasites. From then on, these children received weekly chemoprophylaxis. As control, another group of 37 children was closely followed and treatment was given only to those who presented malaria symptoms. The prevalence of parasitaemia in June (baseline) was well matched in the 2 groups (Fig. 3, upper panel). In August, at the peak of the transmission season, the prevalence reached 86.5% in the control
and their relationship to concom-
The prevalence and levels of antibodies to (NANQ3 were studied in 2 groups of children, from 6 to 30 months of age, both living in Kuiti village, but on the opposite sides of a rice field. The first baseline survey (June) was carried out before the beginning of
E m$ b L
a
50 -i 0.01
,
, I , I I I , I I I I 0.03 0.06 0.16 0.40 1.00 2.51 Entomological inoculation rate (geometric mean)
Fig. 2. Relationship between entomological inoculation rate (measured over the two month period preceding blood collections) and antibodies to (NANP)+ The vertical bars represent the standard error of the median in the upper panel and the 95% confidence intervals in the lower panel. 0, Kuiti; 0, Zagtouli; 0, urban district. Closed symbols refer to the August survey, open symbols to the November survey.
June
August
November
Fig. 3. Relationship between P. falctparum parasitaemia and armbodies to (NANI’),. Solid bars refer to children undergoing chemoprophylaxq shaded bars refer to children without chemoprophylaxis. Baseline data are on the left of the broken line.
I
4
8
12 Age group
I
I
16 20 (years)
I
I
24
I
I
28
Fig. 4. Levels of antibodies to (NANP)3 in relation to age and season, in Kuiti village. Corresponding entomological inoculation rates are shown in Fig. 1. Vertical bars represent the standard error of the median. 0, June, 0, August; 0, November.
group and fell to 12.5% in the group under chemoprophylaxis. The small number of positives in this latter group in August was due to children failing to present themselves for the 2 chemoprophylactic treatments before blood collection. In November, the prevalence of parasitaemia remained high in the control group (89.2%) and was zero in the group under chemoprophylaxis. The corresponding trend of prevalence of antibodies to (NANP)s is shown in the middle panel of Fig. 3. The antibody prevalence was almost identical in both groups in all 3 surveys, and therefore was unrelated to the presence of blood infection. As illustrated in the lower panel of Fig. 3, the levels of antibodies to (NANIQ corresponding to the first (June) and to the second survey (August) were quite similar in the two groups. In the third survey, a difference of borderline significance (P=O.O54, Wilcoxon rank sum test) was observed. The antibodies to blood stages in the same sera, measured by IFA using infected red blood cells, showed a completely different trend. In the group under chemoprophylaxis, the levels of antibodies remained unchanged between the first and the second survey, and then showed a significant decrease (P=O*OO4)at the third survey. On the contrary, in the control group a significant increase (P=O.OOl) between the first and the second survey was followed by stabilization (data not shown). Age-related variations
of antibody levels to (NANP),
Individuals belonging to different age groups, living in Kuiti village, were monitored for anti(NANP)s antibody levels in June, August, and November (Fig. 4). The age groups were: 6-24 months (n=37); 34 years (n=47); 5-9 years (n=59); 10-19 years (n=50); 20-35 years (n=22). The entire groups, constituted by the same individuals, were examined at each survey. The median values of antibodies in each age group varied according to the transmission season. In addition, at any given time, the level of antibodies was age-related, being maximal in adults. Role of antibodies to (NANPJ3
in protection
The relationship between prevalence of P. falciparurn parasitaemia and levels of antibodies to (NANP)s
In
Oo
I
4
‘
81
8
II
I
12
16
I
I
20
I
I
24
00
:8
Age group
(years) Fig. 5. Parasitaemia prevalences in relation to anti-(NANP), antibody levels and age in Kuiti village. The age groups and their relative sizes are the same as in Fig. 4. Open squares refer to individuals possessing a level of antibodies to (NANP), below the median value for that age group, and solid circles refer to individuals with an antibody level above the median value. Vertical bars represent 95% confidence intervals. A, beginning of the transmission season (June); B, peak of the transmission (August); C, end of the transmission season (November).
was evaluated through 3 cross-sectional surveys in a cohort of individuals. Inhabitants of Kuiti village belonging to different age groups were sub-grouped according to whether they possessedlevels of antibodies to (NANP)s above or below the median value of their respective age group. The prevalences of parasitaemia of these different groups were then compared. As shown in Fig. SA, under conditions of low transmission (June), a striking difference in the prevalence of parasitaemia was observed in the 2 groups of adults. The individuals with antibody levels below the median value had a prevalence of parasitaemia of 45.4% (5 of ll), while no infection was detected in the group of 11 adults with antibody levels above the median value. No significant difference was observed in the other age groups.
831 Under conditions of maximal transmission (August), the differences among the higher and lower antibody groups were not significant even in adults (Fig. 5B). However, when transmission started to decline (November), a difference again became apparent in adults (Fig. K), i.e., 54.5% (6 of 11) were parasitized in the lower antibody group, compared to 9.1% (1 of 11) in the higher antibody group. No correlation was found between anti-blood stage IFA titres and prevalence of parasitaemia in the same individuals. The parasitaemia prevalences in adults in June were 18.1% in the higher antibody group and 27.3% in the lower antibodv moue; in November, the values were 36.4% and 2?.j”/, iespectively.
Acknowledgements
Discussion On the basis of the results presented here, a series of conclusions can be drawn. Levels and prevalences of antibodies to (NANP)3 were found to be related to the EIRs measured at the same time in the same localities. It appears, therefore, that levels and prevalence of antibodies to (NANP)3 are useful indicators of the level of malaria transmission. The same conclusions were drawn by DRUILHE et al. (1986) who, however, compared the prevalence and levels of antibodies to sporozoites with entomological data collected in the same areas many years before. The induction of antibodies to (NANP)3 is not affected by the presence of blood stage parasites. This leads to the conclusion that antibodies to (NANP)3 are mainly the result of an immune response directed against the sporozoites. The possibility cannot be excluded, however, that weakly immunogenic, crossreactive, blood-stage epitopes may contribute to a minor degree to maintaining the level of anti(NANP)3 antibodies (COPPEL et al., 1985). Our data also indicate that the presence of blood stage parasites does not cause a detectable suppression of the anti-sporozoite antibody response. The levels of antibodies to (NANP)3 show a substantial seasonal variation, even in adults living in areas of extremely high malaria transmission. Defining this variation, in different areas, will be important when planning future anti-sporozoite vaccine strategies. A cross-sectional survey like that reported here has an intrinsic limitation: when the parasite prevalence and antibody levels on a given day are established, it cannot be concluded that such antibody levels are similar to those existing when the sporozoite inoculation(s) determining such parasitaemia took place. Taking into account this limitation, our findings indicate that adults with higher levels of anti(NANP)3 antibodies are less frequently parasitized than those with lower antibody levels. This difference was obvious during the initial and late transmission periods. During the peak period of transmission, no significant diffeiencewas f&nd, in agreement with a recentlv oublished reoort from East Africa (HOFFMAX et al., 1987). it thus appears reasonable to postulate that, when the sporo%ite inoculation rates are relativelv low. individuals with high levels of antibodies td sporizoites might enjoy proiection from malaria re-infection, since only a fraction of the potentially infective bites would give rise to an actual infection.
References Coppel, R. L., Favaloro, J. M., Crewther, I’. E., Burkot, T. R., Bianco, A. E., Stahl, H. D., Kemp, D. J., Anders, R. F. & Brown, G. V. (1985). A blood stage antigen of Plasmodium falciparum shares determinants with the sporozoite coat protein. Proceedings of the LVational Academy of Sciences, USA, 82, 5121-5125. Del Giudice, G., Engers, H. D., Tougne, C., Biro, S. S., Weiss, N., Verdini, A. S., Pessi, A., DegrCmont, A. A., Freyvogel, T. A., Lambert, P.-H. & Tanner, M. (1987). Antibodies to the repetitive epitope of Plasmodium falciparum circumsporozoite protein in a rural Tanzanian community: a longitudinal study of 132 children. American Journal of Tropical Medicine and Hygiene, 36, 203-212. Druilhe, I’., Pradier, O., Marc, J.-P., Miltgen, F., Mazier, D. & Parent, G. (1986). Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection. Infection and Immuniry, 53, 393-397. Esposito, F., Lombardi, S., Tour&, Y. T., Zavala, F. & Coluzzi, M. (1986). Field observations on the use of anti-sporozoite monoclonal antibodies for determination of infection rates in malaria vectors. Parassitologia, 28, 69-77. Hoffman, S. L., Wistar, R., Jr, Ballou, W. R., Hollingdale, M. R., Wirtz, R. A., Schneider, I., Marwoto, H. A. & Hockmeyer, W. T. (1986). Immunity to malaria and naturally acquired antibodies to the circumsporozoite protein of Plasmodium falciparum. Nat England Journal of Medicine, 315, 602-606. Hoffman, S. L., Oster, C. N., Plowe, C. V., Woollett, G. R., Beier, J. C., Chulay, J. D., Wirtz, R. A., Hollingdale, M. R. & Mugambi, M. (1987). Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science, 237, 639-642. Lombardi, S., Esposito, F., Zavala, F., Lamizana, L., Rossi, I’., Sabatinelli, G., Nussenzweig, R. S. & Coluzzi, M. (1987). Detection and anatomical localization of Plasmodium falciparum circumsporozoite protein and sporozoites in the Afrotropical malaria vector Anopheles gambiae s.1. Amen’can Journal qf Tropical Medicine and Hveiene. 37. 491-494. Na&i;E~.’ fi.j Nussenzweig,, R. S., McGregor! I. A. & Bryan, J. H. (1979). Annbodies to sporozoltes: their frequent occurrence in individuals living in an area of hyperendemic malaria. Science, 206, 597-599. Nussenzweig, V. & Nussenzweig, R. S. (1985). Circumsporozoite proteins of malaria parasites. Cell, 42,401-403. Rossi. I’. . Belli. A.. Mancini. L. & Sabatinelli. G. 11986). Enqugte entbmoiogique longitudinale sur la transmission du paludisme g Ouagadougou (Burkina Faso). Parassirologia, 28, 1-15. Sabatinelli, G., Bosman, A., Lamizana, L. & Rossi, I’. (1986). Enqu@te protozoologique sur la prbvalence du oaludisme g Ouaaadougou (Burkina Faso! en ueriode de ;ransmission ma&x& F&zssitologia, is, 17-31. Tapchaisri, P., Chomcharn, Y., Poonthong, C., Asavanich,
It is a pleasure to thank Professor Sir Ian McGregor for critically reading the manuscript. We also gratefully acknowledge the excellent technical assistanceof Silvana Bagalino in Rome and Simon-Pierre Zoneo and Benoit 1. Zoungrana in Ouagadougou. The skilled a&ninistrative work by-Sharon Hecht in New York was invaluable. This study received financial support from the Dipartimento per la Cooperazione allo Sviluppo-Minister0 Affari Esteri; The Rockefeller Foundation; Fondazione Istituto Pasteur-Cenci Bolognetti; the International Atomic Energy Agency; and WHOITDR Director’s Initiative Fund. Some of these results were preliminarily presented by F. E. at the 14th Conference of the Italian Society of Parasitologists, in Piss, 21-24 May, 1986.
832 A., Limsuwan, S., Maleevan, O., Tharavanij, S. & Harinasuta, T. (1983). Anti-sporozoite antibodies induced by natural infection. AmericanJournal of Tropical Medicine and Hygiene, 32, 1203-1208. Voller, A. & Q’Neill, P. (1971). Immunofluorescence method suitable for large scale application. Bulletin of the World Health Organization, 45, 524-529. Zavala, F., Cochrane, A. H., Nardin, E. H., Nussenzweig, R. S. & Nussenzweig, V. (1983). Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. Journal of Experimental Medicine, 157, 1947-1957.
Received 16 March 1988; revised 18 May 1988; accepted for publication 18 May 1988
TRANSACTIONS OFTHEROYALSOCIETY OFTROPICAL MEDICI~YE AND
HYGIENE (1988) 82, 832
1I Short Report I1
falciparum in France: Anopheles maculipennis and A claviger are found in the Paris area and are potential vectors (GENTILINI et al., 1978) that may become
Plasmodium falciparum malaria after three years in a non-endemic area M. P. Revel’, A. Da@, A. Saint Raimond2, G. Lenoi?, M. Dank’ and M. Gentilini’ ‘Department of Parasitology and Tropical Medicine, Groupe Hospitaher PititLSalp&wi&re, Paris, France; ‘Service de Pediatrie GcWrale, Hopital des Enfants Malades, Paris, France
A 7 years old male child was admitted to hospital in March 1987at the Hopital des Enfants Malades, with a 6 d history of headache, ocular pain, fever and asthenia. On physical examination he was found to have splenomegaly and a temperature of 38.7X, but no lack of alertness. Thick and thin blood smears revealed trophozoites of Plasmodium ovale and, surprisingly, trophozoites and gametocytesof P. falciparum in the child, who had been living in Montrouge, a Paris suburb, since January 1984. He was treated successfully with quinine (quinimax), 25 mg/kg/d. The singularity of the caselay in the late manifestation of P. falciparum malaria, more than 3 years after the patient had left an endemic area, the Comoro Islands. The onset of diseasedue to P. falciparum does not normally occur later than 2 months after returning from an endemic area (GENTILINI & DUFLO, 1986). This period of 2 months has been challenged in several publications describing the onset of P. falciparum disease up to 11 months after exposure (BRUCE-CHWATTet al., 1974; CARMEet al., 1978). Periods over 3 years before onset have not been reported, as far as we know. Several factors must be considered in connection with the late manifestation in this case. An error may have been made in the identification of the parasite, but the presence of P. falciparum gametocytes excluded such a hypothesis. The boy’s school attendance record precluded the possibility of his having revisited an endemic area since his arrival in France. After investigations, blood contamination by intravenous drug abuse or transfusion were excluded. Other possible hypotheses may be advanced. (i) The existence of quiescent hepatic or extrahepatic forms of P. falciparum, capable of causing a late episode of malaria; this possibility is at present being reviewed in the light of other observations on late manifestations (CARMEet al., 1978)although classically it has not been accepted for P. falciparum infections.
Zavala, F., Tam, J. P., Hollingdale, M. R., Cochrane, A. H., Quakyi? I., Nussenzweig, R. S. & Nussenzweig, V. (1985). Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science, 228, 1436-1440. Zavala, F., Tam, T. P. & Masuda, A. (1986). Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites. Journal of Immunological Methods, 93, 55-61.
(ii) There is the further possibility of native P.
infected by biting travellers from the tropics. However, it has not been proved that mosquitoes of these speciescan transmit tropical malarial species(SHUTE, 1943).Moreover, the sporogony cycle in the mosquito requires a temperature above 18°C for at least 12 d (GENTILINI & DUFLO, 1986). These conditions were not met during the winter months preceding the boy’s illness. (iii) Another possible explanation could be infection in France from an imported Anopheles. This has becomewell known in the neighbourhood of international airports and has resulted in several cases of serious diseasein 1977-1978 (GENTILINI et al:, 1978) when the true causewas unsuspected. Proximity to an airport did not apply in our case, but Anopheles may easily be carried by the wind for a distance of 100 km and the life span of females ranges between 8 and 34 d (GENTILINI & DUFLO, 1986). (iv) A final possibility might be that infected Anopheles had been transported in the luggage of a member of the family who had arrived in France in the months preceding the boy’s illness; clothes may constitute a possible lodging for mosquitoes (GENTILINI & DUFLO, 1986). The casereported here, which is similar to others except in the length of time before the appearanceof P. falciparum infection, raises the question of the existence of quiescent forms of the parasite, although this has still to be proved. As in other known casesof malaria in France, the arrival of a member of the family from an endemic area seems to support the hypothesis of an imported Anopheles. References Bruce-Chwatt, L. J., Southgate, B. A. & Draper, C. C. (1974). Malaria in the United Kingdom. British British Medical Medical jou&l, Jourd, ii, 707-711. Carme. B.. Dams. M. & Gentilini. M. (1978). LonnCvitC de Pldsmo~ium falciparum : g propos de’ 3 cas- d’acces palustre survenus plus de 2 mois apres le depart de la zone d’endemie. Mkdicine et Maladies Infectieuses, 8, 352-354. Gentilini, M. & Duflo, B. (1986). Paludisme. In: Medecine Tropicale, 4th edition. Paris: Flammarion. Gentilini, M., Danis, M., Dallot, J. Y ., Richard-Lenoble, D. & Felix, H. (1978). Rtapparition du paludisme autochtone. Annales de MCdecine Interne, 129, 405-410. Shute, P. G. (1943). Failure to infect English specimens of Anopheles maculipennis var. atropanw with certain strains of Plasmodium of tropical origin. Journal of Tropical Medicine and Hygiene, 43, 1-4.
Received 18 April 1988; acceptedfor publication 12341~ 1988