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Relationship between entomological inoculation rate, Plasmodium falciparum prevalence rate, and incidence of malaria attack in rural Gabon N. Elissa 1, F. Migot-Nabias 2, A. Luty 3, A. Renaut 4, F. Toure´, M. Vaillant 5, M. Lawoko, P. Yangari, J. Mayombo, F. Lekoulou, P. Tshipamba, R. Moukagni, P. Millet 6, P. Deloron * International Center for Medical Research, BP 769 Franceville, Gabon Received 7 May 2002; received in revised form 12 August 2002; accepted 7 October 2002
Abstract To assess the relationships between variations of Plasmodium falciparum transmission and those of peripheral parasitaemia prevalence or malaria attack incidence rates in regions with limited fluctuations of transmission, we conducted a follow-up in two Gabonese populations. Entomological surveys were carried out from May 1995 to April 1996 in Dienga, and from May 1998 to April 1999 in Benguia. In Dienga, malaria transmission was seasonal, being not detected during two 3-month periods. Mean entomological inoculation rate (EIR) was 0.28 infective bite/person/night. In Benguia, malaria transmission was perennial with seasonal fluctuations, mean EIR being 0.76 infective bite/person/ night. In Dienga, 301 schoolchildren were followed from October 1995 to March 1996. Clinical malaria attack was defined as fever associated with /5000 parasites/ml of blood. P. falciparum prevalence varied from 28 to 42%, and monthly malaria attack incidence from 30 to 169. In Benguia, the entire population (122 persons) was followed from November 1998 to April 1999. Prevalence varied from 22 to 50%, and monthly malaria attack incidence from 52 to 179. In each area, entomological variations were not related to parasite prevalence, but preceded malaria attack incidence with 1- or 2-month time lag, corresponding to the pre-patency period that differs in the two populations, possibly according to differences in immunity related to parasite transmission. # 2002 Elsevier Science B.V. All rights reserved.
* Corresponding author. Present address: IRD UR010 Mother and Child Health in the Tropics, Faculte´ de Pharmacie, Laboratoire de Parasitologie, 4 Avenue de l’Observatoire, 75006 Paris, France. Tel.: /33-1-5373-9621; fax: /33-1-4216-2654 E-mail address:
[email protected] (P. Deloron). 1 Present address: DASS-Service de lutte antivectorielle, 97600 Mamoudzou, Mayotte. 2 Present address: Institut de Recherche pour le De´veloppement (IRD), UR010 Mother and Child Health in the Tropics, BP 1386 Dakar, Se´ne´gal. 3 Present address: Human Parasitology, Institute for Tropical Medicine, University of Tu¨bingen, Wilhelmstrasse 27, 72074 Tu¨bingen, Germany. 4 Present address: Bureau International de Gestion et d’Etude de Projets Intersectoriels en De´veloppement, 29750 Loctudy, France. 5 Present address: Universite´ Bordeaux 2, 33076 Bordeaux Cedex, France. 6 Present address: Universite´ Bordeaux 2, 33076 Bordeaux Cedex, France. 0001-706X/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 2 ) 0 0 2 6 6 - 8
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Keywords: Anopheles ; Malaria; Malaria attack incidence; Parasite prevalence; Plasmodium falciparum ; Transmission; Gabon
1. Introduction Gabon is an endemic area for malaria where little work related to the dynamics of neither Plasmodium species nor vector species has been done. Given the lack of data from malaria control and survey program, the impact of malaria in the population remains difficult to assess. However, in this country, in which P. falciparum resistance to chloroquine may be as high as 90% in some areas (Kremsner et al., 1994; Philipps et al., 1998), malaria and its complications in early childhood are likely to cause many casualties (Kremsner et al., 1995). In some epidemiological situations, malaria transmission intensity, as assessed by entomological parameters, has been associated with parasite prevalence, malaria-associated symptoms, and death in children (Trape and Greenwood, 1994; Beier et al., 1999; Pinto et al., 2000). In African countries where transmission is highly seasonal, with clearly distinct high and low (or no) transmission seasons, such as Senegal and Kenya, the number of subjects presenting with malariaassociated symptoms has been correlated with the rate of transmission (Beadle et al., 1995)., In equatorial African areas, previous reports indicated that malaria morbidity also varies throughout the year in parallel with the transmission intensity at a given site (Carme, 1996; Sylla et al., 2001), but no differences in morbidity are observed between two distinct locations, eventually with very different global EIR. For example, in peripheral and central Brazzaville, Popular Republic of Congo, the malaria transmission level appeared not to be related to the incidence of malaria attacks (Trape et al., 1987; Carme et al., 1992). Similar observations were recently made in Cameroon (Bonnet et al., 2002). Most of equatorial areas are characterized by rainy seasons interrupted by short dry seasons, which may reduce, but to only a limited extent, the anopheline density and the level of malaria transmission. Consequently, malaria transmission is usually perennial, eventually with some seasonal variations in in-
tensity. There is a critical need to understand the relationship between EIR and malaria disease. Therefore, two areas with distinct transmission dynamics and distinct age population were chosen to conduct the study. The objective of this study was not to compare the two populations nor the two villages, but to investigate simultaneously, in two epidemiological settings, parasite prevalence, malaria attack incidence and transmission intensity.
2. Material and methods 2.1. Study areas Gabon, located in the African equatorial forested zone, is a region where malaria is endemic. Climate is equatorial with four seasons (Figs. 1 and 2): a long dry season from July to August characterized by an insignificant precipitation of rains and a short dry season (January / February) during which the precipitation declines in comparison to the strong rains of the two rainy seasons (September /December and March /June). The mean annual daily temperature is 25.2 8C. Two villages from Southeast Gabon were selected for the surveys: Dienga and Benguia. Located in a savanna area surrounded by rainforest, Dienga is situated 100 km South West of Franceville, in the Ogooue´ Lolo Province, close to the Popular Republic of Congo border. Population is mainly from the Nzebi ethnic group, living in houses made of wood walls and corrugated iron roofs. The population accounts for 1,182 inhabitants. Thanks to the Ministry of Health from Gabon, we could organize a CIRMF malariastudy base in a disaffected dispensary where two health workers trained within the village and two CIRMF personnel were present permanently. Benguia, a small Pygmy village of 122 inhabitants, is situated 15 km West of Franceville, the capital of the Haut Ogooue´ Province. Savanna occupies mainly the landscape with many forest galleries.
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Fig. 1. Seasonal variations of rains, entomological, and parasitological parameters in Dienga, Gabon, 1995 /96. Top: variations of rains (bars) and entomological inoculation rate (EIR) (line). Bottom: variations of EIR (bars), P. falciparum parasite prevalence rate (continuous line), and P. falciparum malaria attack incidence rate (dotted line) in schoolchildren. Data on rains are not available in April.
The village is bordered on the one side by a road and on the other side by a river with its galleryforest. Houses are made of corrugated iron walls and roofs. 2.2. Entomological survey To estimate human-biting rates, mosquitoes were collected using all-night, voluntary humanbait catches indoors and outdoors at three different places chosen randomly every 2 weeks in each of the two study zones. The night was divided into two 6-h periods (18:00/00:00 and 00:00 /06:00). The catchers, with the aid of torches, collected the mosquitoes which landed on their naked legs and placed them at hourly intervals inside hemolysis
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Fig. 2. Seasonal variations of rains, entomological, and parasitological parameters in Benguia, Gabon, 1998 /99. Top: variations of rains (bars) and entomological inoculation rate (EIR) (line). Bottom: variations of EIR (bars), P. falciparum parasite prevalence rate (continuous line) and P. falciparum malaria attack incidence rate (dotted line) in the population.
tubes. These tubes were then put inside ‘hourbags’. Two night-catches (when it was possible) were carried out in every month from May 1995 to April 1996 in Dienga and from May 1998 to April 1999 in Benguia. Mosquito identification (Gillies and De Meillon, 1968; Gillies and Coetzee, 1987) and female anopheline dissection were carried out at the medical entomology laboratory the morning after collection. All the dissections were carried out with respect to hour and place. Sporozoite infection was assessed from the observation of sporozoites in freshly dissected salivary glands of female anophelines. In each area, data were analyzed by taking into account all anophelines species that were potential malaria vectors. The entomological inoculation rate (EIR) was obtained by multiplying the human biting rate (ma)
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by the sporozoite index (s). EIR was expressed as the number of infected bites per person per night (ib/p/n).
In conducting this research, protocols involving human subjects were approved by the Ethical committee of the International Center for Medical Research.
2.3. Parasitological and clinical follow up 3. Results In Dienga, all 301 children (7 /17 years old, mean age /10.4 years) of the primary school were followed longitudinally from October 1995 to March 1996 to evaluate the frequency of malaria attacks. This follow up has been previously described in detail (Deloron et al., 1999). Each school-day morning, the axillary temperature of each child was recorded at school between 08:00 and 10:00 h. Children who were absent from school were visited at home. On Saturdays and Sundays, a passive case detection was carried out at the field base. In Benguia, the whole population of the village (1 month /75 years old, mean age / 26.6 years) was followed from November 1998 to April 1999. Axillary body temperature was recorded twice a week between 15:00 and 19:00 h. In both areas, in all cases of fever (axillary temperature /37.5 8C), a complete clinical examination was performed and a thick blood smear was made and rapidly examined for malaria parasites. In addition, regardless of the clinical status, thick blood smears were collected fortnightly from each subject from both areas (11 times in Dienga and 10 times in Benguia) to assess for the presence of asymptomatic malaria infection. Slides were stained with Giemsa and examined against 200 leukocytes if positive, or against 400 leukocytes prior to being declared negative. Parasite densities were recorded as the number of parasites per microliter of blood, assuming an average leukocyte count of 8000/ml. As detailed elsewhere (Deloron et al., 1999), P. falciparum malaria attack was defined as the presence of fever and more than 5000 P. falciparum parasites/ml of blood and no apparent other cause of fever. Children presenting with a malaria attack were given an antimalarial curative treatment. Entomological, parasitological and clinical data were pooled by calendar month in order to calculate monthly EIR, P. falciparum prevalence rates and P. falciparum malaria attack incidence rates.
3.1. Malaria transmission At Dienga from May 1995 to April 1996, we carried out catches on 22 nights with 65 catchers. A total of 555 mosquitoes were captured of which 98.2% were anophelines. The estimated annual mosquito biting density was 3125 bites per person, i.e. an average of about 8.5 bites per person per night (b/p/n). Four anopheline species were identified, including 523 An. gambiae s.l. (96.0%), 11 An. moucheti (2.0%), 10 An. hancocki (1.8%), and one An. funestus (0.2%). Among these, 17 An. gambiae s.l. and one An. hancocki had sporozoites in their salivary glands. These two species were present throughout the year except in September 1995, with peak densities in June 1995 (70.0 b/p/n), and January 1996 (19.3 b/p/n). The estimated sporozoite rate in potential vectors was 2.7%. Mean inoculation entomological rate (EIR) was 0.28 infective bite/person/night (ib/p/n) corresponding to an infective bite every 3.6 days. However, malaria transmission was highly seasonal showing two peaks of 0.49 ib/p/n in July 1995 and 1.83 ib/p/ n in January 1996, separated by two 3-month periods during which transmission was not detected (Fig. 1). Each person was exposed to 82 infected bites (0.66 ib/p/n, one bite every 1.5 days) during the four months of the dry seasons and 32 infective bites per person (0.07 ib/p/n, one bite every 15 days) during the eight months of the rainy seasons. At Benguia, in 63-person nights of captures over a 12-month period (from May 1998 to April 1999), 8560 mosquitoes were caught, of which 8527 were anophelines belonging to ten different species (Elissa et al., 1999). The estimated annual mosquito biting density was 49,594 bites per person, i.e. an average of about 135.9 b/p/n. A total of 8,419 Anopheles were potential vectors belonging to the species An. funestus 5772 (68.6%); 1012 An.
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gambiae s.l. (12.0%); 1,090 An. nili (12.9%); and 545 An. moucheti (6.5%). After the dissection of 6120 Anopheles , only An. funestus (30/4047) and An. gambiae s.l. (5/760) showed sporozoites in their salivary glands. The estimated sporozoite rate in potential vectors was 0.6%. For the whole period, no significant difference in the sporozoite rates was observed between An. funestus and An. gambiae s.l. The transmission was perennial with a mean EIR of 0.76 ib/p/n, corresponding to an infective bite per person every 1.3 day, but reaching 1.50 ib/p/n in November 98 and falling to 0.20 ib/m/n in February 99 (Fig. 2). No huge difference was observed between rainy and dry seasons, as each person was submitted to 211 infected bites during the 8 months of the rainy seasons (EIR / 0.91 ib/p/n), and to 58 infective bites during the 4 months of the dry seasons (EIR /0.47 ib/p/n). 3.2. Clinical and parasitological impact in the populations In Dienga, 301 children were followed, including 171 boys and 130 girls, with a mean age (9/S.E.M.) of 10.4 (9/0.2) years. During the follow-up, fortnightly thick blood smears were performed 11 times for detection of asymptomatic infections. During those, 2395 blood smears were done. Overall, 809 (33.8%) blood smears were positive for P. falciparum . The prevalence rate remained relatively constant during the follow-up, its monthly variations being limited in the 28/42% range (Fig. 1). P. malariae was also present at a low rate, varying from 0.9 to 3.1% of the subjects. A total of 154 falciparum malaria attacks were identified in 106 children (among those 26, 6, 2 and 1 presented with 2, 3, 4 and 5 attacks, respectively). The monthly malaria attack rate increased from 33 in October at the beginning of the follow-up, to 169 in February, then decreased to 113 in March. In Benguia, the whole population of 122 subjects was followed, including 64 males and 58 females, with a mean age (9/S.E.M.) of 26.6 (9/1.8 years). During the follow-up, fortnightly thick blood smears were performed ten times for detection of asymptomatic infections. Overall, the mean rate of P. falciparum prevalence was 34.3%,
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however this rate varied from a low of 21.9% in December to a high of 50.0% in January (Fig. 2). P. malariae was present from November to January, varying from 3.1 to 6.7% of the subjects, and was not observed thereafter. Fifty-eight malarial attacks were observed during these 6 months. Monthly incidence rate of malaria attack varied from 93 in November, to a high 179 in January, and then decreased to 52 in April. 3.3. Relationships between transmission and clinical or parasitological impact in the populations In both villages, EIRs were linked neither to P. falciparum prevalence rates from the same month nor to those from one or two months later (Figs. 1 and 2). Conversely, curves of EIRs and P. falciparum malaria attack incidence rates were superposed with nevertheless a shift in time. In Dienga, EIRs preceded P. falciparum malaria attack incidence rates from 1 month, while in Benguia, a 2-month delay was required between EIR and appearance of symptoms.
4. Discussion In Dienga, malaria seems to be transmitted according to two peaks (a major peak in December to March, and a minor peak in July /August) during the year, separated by two 3-month periods during which transmission is not detected. This is likely to be related to the fact that malaria transmission is ensured almost exclusively by An. gambiae s.l., the disappearance of which is frequent during the dry season. Richard et al. reported a sudden disappearance and an explosive reappearance of An. gambiae s.l., for which breeding sites disappear or become unsuitable for larval development during the dry season (Richard et al., 1988). During the dry season, the temperature drop in such a way that sporogonic development may be slowed (Trape et al., 1992). Conversely, in Benguia, An. funestus is the major vector, but An. gambiae s.l. is also present and acts as a complementary malaria vector, and the transmission seasons are extended over a longer period than if only one of these species was present
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and biting by one or both of the two main vectors was observed each month of the year. Infected mosquitoes were caught every month of the study, and transmission was perennial although its intensity varied all over the year. In the same villages, cohorts were clinically and parasitologically followed up during several months during which the malaria transmission was highly variable. The purpose of our study was not to compare these two populations, but to assess the relationships between anopheline transmission level and malaria indices in each of two different epidemiological settings. Indeed, in Dienga, the EIR varied from 0 ib/p/n at initiation of the follow up, then increased to 1.83 ib/p/n in January, and decreased to 0.32 ib/p/n at the end of the follow up in March. Similarly, in Benguia, the EIR varied from 1.50 ib/p/n at initiation of the follow up, then decreased to a low 0.20 ib/p/n in February and increased again to 1.30 ib/p/n at the end of the follow up in April. During these periods, falciparum malaria prevalence rates variations were relatively modest and could not be related to the malaria transmission levels, in spite of the observation of an increased prevalence rate in Benguia following the peak of EIR (Fig. 2), which was not found in Dienga (Fig. 1). Such observation contrasts with data previously obtained from countries with marked high and low transmission seasons (Trape et al., 1992; Beadle et al., 1995). One explanation for this discrepancy is that anopheline mosquitoes are present throughout the year, and that entomological variations in our study areas are of too short duration to have a measurable impact on the malaria prevalence rates in the human population (Smith et al., 1993). Moreover, human populations from endemic areas may harbor malaria parasites for several months, their immune response being able to control parasite density to a low level without developing clinical symptoms, but being unable to clear parasitemia (Gravenor et al., 1995). During the same periods, and conversely to the absence of relationship between prevalence rates and EIR, the P. falciparum malaria attack incidence rates were highly variable from month to month in both villages, and changed parallel to the EIR from the preceding month in Dienga, and from two months
before in Benguia. This time lag may correspond to the duration of the exo-erythrocytic parasite cycle, and to the time necessary for the parasite density to reach a sufficient level to produce symptoms of malaria in the host. The duration of this time lag depends on the efficiency of several individual factors that may limit the likelihood that a sporozoite inoculation will originate a parasite hepatic cycle, followed by a blood infection (Beier et al., 1994; Dieye et al., 1997). Immunity against erythrocytic parasitic stages is able to extend the time necessary for the parasite density to reach the pyrogenic threshold (Rogier and Trape, 1993). Enrolment criteria and epidemiological features of malaria transmission may take into account for these differences in time lag. Nevertheless, in both contexts, the incidence of malaria attacks and the entomological inoculation rate are highly related. This is not surprising as the likelihood for an individual to be infected by a P. falciparum strain against which he has no protective immunity, or by a virulent strain more likely to be responsible of symptoms, increases with the number of parasite strains brought during a given period of time, and with the sporozoite inoculation rate. Although the immune response of our study populations may limit the variations of the parasite prevalence rate according to the variations of transmission, it seems not to be effective enough in impairing the appearance of the malaria disease.
Acknowledgements We thank Dr Je´roˆme Reltien, He´le`ne Tiga, and Hoppe Lewobo for help in collecting data in Dienga. The Dienga study was partially supported by a North /South Network INSERM grant (94NS1). CIRMF is financed by the Gabonese government, ELF Gabon, and the French Ministry of Foreign Affairs.
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