- Open Access
Malaria infection and disease in an area with pyrethroid-resistant vectors in southern Benin
© Damien et al; licensee BioMed Central Ltd. 2010
Received: 11 July 2010
Accepted: 31 December 2010
Published: 31 December 2010
This study aimed to investigate baseline data on malaria before the evaluation of new vector control strategies in an area of pyrethroid-resistance of vectors. The burden of malaria was estimated in terms of infection (prevalence and parasite density) and of clinical episodes.
Between December 2007 and December 2008 in the health district of Ouidah - Kpomassè - Tori Bossito (southern Benin), a descriptive epidemiological survey of malaria was conducted. From 28 selected villages, seven were randomized from which a total of 440 children aged 0 to 5 years were randomly selected. Clinical and parasitological information was obtained by active case detection of malaria episodes carried out during eight periods of six consecutive days scheduled at six weekly intervals and by cross-sectional surveys of asymptomatic infection. Entomological information was also collected. The ownership, the use and the correct use of long-lasting insecticide-treated nets (LLINs) were checked over weekly-survey by unannounced visits at home in the late evening.
Mean parasite density in asymptomatic children was 586 P. falciparum asexual forms per μL of blood (95%CI 504-680). Pyrogenic parasite cut-off was estimated 2,000 P. falciparum asexual blood forms per μL. The clinical incidence of malaria was 1.5 episodes per child per year (95%CI 1.2-1.9). Parasitological and clinical variables did not vary with season. Anopheles gambiae s.l. was the principal vector closely followed by Anopheles funestus. Entomological inoculation rate was 5.3 (95%CI 1.1-25.9) infective bites per human per year. Frequency of the L1014F kdr (West) allele was around 50%. Annual prevalence rate of Plasmodium falciparum asymptomatic infection was 21.8% (95%CI 19.1-24.4) and increased according to age. Mean rates of ownership and use of LLINs were 92% and 70% respectively. The only correct use of LLINs (63%) conferred 26% individual protection against only infection (OR = 0.74 (95%IC 0.62-0.87), p = 0.005).
The health district of Ouidah-Kpomassè-Tori Bossito is a mesoendemic area with a moderate level of pyrethroid-resistance of vectors. The used LLINs rate was high and only the correct use of LLINs was found to reduce malaria infection without influencing malaria morbidity.
Despite considerable worldwide efforts made in recent years to control malaria , the disease is still a major public health problem with nearly 250 million cases and about one million deaths each year. Eighty five percent of deaths occur among children under five  from which nearly all are in sub-Saharan Africa. In 2007, malaria was declared to be the most important disease in this age group, in Benin, leading to 43% of all medical consultations and 29% of hospital admissions . The National Malaria Control Programme (NMCP) has implemented WHO/GMP's (World Health Organization/Global Malaria Programme) recommended preventive and curative strategies . These include i) Artemisinin combination therapy (ACT) which is dispensed at health centers and has recently been made available to communities for children under five years old; ii) Intermittent preventive treatment (IPT) during pregnancy; iii) Long-lasting insecticide-treated mosquito nets (LLINs) which have continued to be distributed following the nation-wide deployment among high-risk populations (i.e. children of under five and pregnant women) and iv) Indoor residual spraying (IRS) using carbamate insecticide applied in specific districts through the President's Malaria Initiative . Many studies have demonstrated that the use of insecticide treated nets reduced uncomplicated malaria episodes by at least 50% . Unfortunately, insecticide resistance in malaria vectors has dramatically increased in Africa , especially in Benin [8–10] and may seriously compromise the success of vector control management. Two studies conducted in experimental huts in South Benin, where Anopheles gambiae was resistant to pyrethroids, have reported that significant reduction in the efficacy of pyrethroids was applied either in treated nets or IRS [11, 12]. In order to manage insecticide resistance, the Centre de Recherche Entomologique de Cotonou (CREC) in collaboration with the Institut de Recherche pour le Développement (IRD) and the NMCP has evaluated successfully (WHOPES phases I and II) a new insecticide resistance management (IRM) strategy combining in the same household a LLIN and a carbamate treated plastic sheeting [13, 14]. In the context of a future community-based evaluation of this promising IRM strategy (phase III trial), the malaria burden was evaluated in a health district of southern Benin where a nation-wide distribution of LLINs to children <5 had been implemented in 2007. This study constitutes an analysis of the baseline situation of malaria in terms of infection (prevalence and parasite density) and clinical episodes. Entomological information was also collected.
Description of the study area
Density of population (People/Km2)
Open water cisterns (N)
Distance from lake (fresh water) (Km)
Distance from lagoon (brackish water) (Km)
Distance from health center (Km)
Parasitological and clinical measures
Active case detection (ACD) for malaria episodes was carried out during eight periods of six consecutive days at six weeks intervals throughout the year. Each day a nurse assisted by a local village helper trained for the study, visited the households in the sample. A physician supervised the field work. The presence or absence and state of health of each child were recorded daily on a specially prepared form (one form per household). The nurse examined and recorded data on every case of sickness detected at home. A thick blood film was taken from every sick child. Children were treated according to the clinical diagnosis made by the nurse. When malaria was suspected, the patient was treated with artemether-lumefantrine for three days according to the recommendations of WHO and NMCP [19, 20]. Cross-sectional surveys (CSS) were carried out at each monitoring clinical period (n = 8) on every asymptomatic child (confirmed by axillary temperature < 37.5°C). A thick film sample was taken on the fourth day to be sure that asymptomatic children were free of illness in preceding days. Cross-check quality controls were conducted every six weeks during the collection of field data.
Data were collected two weeks before each clinical monitoring. Adult mosquitoes were caught using Human Landing Catches (HLC) technique . In the study area, 896 human-nights of capture of human landing mosquitoes were organized every six weeks over a year period (128 nights per village; eight places per village and per night, half indoor and half outdoor). Treated nets were present in the mosquito collection sites. The mosquito species were identified using morphological characteristics according to the identification keys of Gillies & De Meillon  and Gillies & Coetzee . All mosquitoes of An. gambiae complex and Anopheles funestus group were stored in individual tubes with silicagel and preserved at -20°C for P. falciparum circumsporozoite index estimation and molecular identification.
Control of LLINs
The ownership, the use and the correct use of LLINs (Permanet® 2.0) which were distributed in October 2007 were checked over weekly-survey. The visits of the nurse were unannounced and took place in the late evening around 9.00 PM when children were expected to be asleep . The unannounced visits determined the ownership (whether the LLINs were seen during the control), the use (whether children were sleeping under it during the control) and the correct use (whether the LLINs were correctly hung and tucked and were not torn). The rates of LLINs ownership, use and correct use were calculated relative to the total number of observations.
Laboratory processing was done at the CREC, Cotonou. Parasitological infection was detected on Giemsa-stained thick smears. Asexual stages of each Plasmodium species were counted in the blood volume occupied by 200 leucocytes and parasite density was calculated by assuming 8,000 leucocytes/μL of blood. Thick smears from each village were read by the same experienced technician, under the supervision of a parasitologist. The readings of the two technicians were also compared on the same set of blood samples. Their estimations of parasite detection and parasite density did not differ significantly. Cross-check quality control was regularly done on a randomly selected sample representing 10% of all thick smears.
After scoring field-collected Anopheles mosquitoes and identifying the species of each specimen by Polymerase Chain Reaction (PCR) , the presence and relative frequency of the molecular M and S forms of An. gambiae sensu stricto (s.s) were determined according to the method of Favia . Infection of mosquitoes was determined on the head and thorax of individual vector specimens by ELISA using monoclonal antibodies against P. falciparum circumsporozoite protein (CSP) . The method of Martinez-Torrez was used for the molecular detection of the L1014F kdr allele .
Demographic, parasitological, clinical and entomological data were double entered independently in the Access 2003 database. Parasitological and clinical data were analyzed using the svy command (STATA 11.0). For each person only one blood sample per monitoring period was considered for analysis. When a pathological condition was detected, the blood sample taken during the clinical episode was retained for analysis. Parasitological data were analyzed separately in terms of prevalence of P. falciparum asexual blood forms, density of P. falciparum asexual blood forms in parasite positive blood thick films and prevalence of P. falciparum gametocytes. A generalized estimating equation (GEE) approach, which can be used with normal distributions and discrete data was used for statistical analysis of repeated measures. To take into account the interdependence of observations made on the same person, an exchangeable correlation structure was used in which the correlation between these observations made on one person at different times was assumed to be the same. The prevalence of asymptomatic malaria infections was analyzed as a binomial response by using a logistic regression model. The parasite density was log transformed for a normally distributed response and analyzed with a link function by using a linear regression model.
The association between the parasite density and the occurrence of clinical episodes was tested using a Poisson regression model, taking clinical status (pathological episode versus asymptomatic state) as the dependent variable, and parasite density as the independent variable. In this type of model, a random intercept variable is allowed to vary with subjects, and this random subject-specific intercept allows the interdependence of the observations made on the same person to be taken into account. For each pathological period, the probability that it was caused by malaria was estimated by the Attributable Fraction (AF) calculated from the odds ratios associated with the estimated parasite density in the logistic model [29, 30]. The pathological episodes were clinically defined by a high axillary temperature (≥ 37.5°C), sweats, shivers, headaches, nausea or vomiting  or by a history of fever during the 48 hours proceeding the first day of ACD or, for infants under one year of age, anorexia or any pathological condition described by the mother [32, 33]. For individuals, the number of malaria attacks over a given periods was estimated by the sum of probabilities that pathological episodes were due to malaria, depending on the parasite density. The malaria incidence rate was calculated dividing the ratio of pathological episodes attributable to malaria by the number of child-days.
The three dependent variables (i.e. prevalence rate of P. falciparum infection, mean parasite density in positive children and clinical incidence rate) were analyzed according to demographic (age groups 0-23, 24-59, 60-71 months and sex), environmental (season and villages) and sanitary (LLIN's ownership, use and good use) variables. The Chi2 test was used to compare the rate of ownership, use and correct use of LLINs. An optimum pyrogenic parasite density cut-off was calculated using the estimated AFs with a logistic model. The sensitivity and the specificity were similarly determined . The sensitivity was estimated by the ratio of malaria episodes with positive cut-off to a total of malaria episodes. The specificity was estimated by the ratio of no malaria febrile episodes with parasite density below the cut-off to the total of no malaria febrile episodes. The suitable positive Likelihood-ratio (>10), negative Likelihood-ratio (<0.1) results and Youden's J index were also determined from the model.
The human biting rate (HBR) was expressed as the number of anopheles bites per human per night. The sporozoite index was calculated as the proportion of mosquitoes found to be positive for CSP. The entomological inoculation rate (EIR) was calculated as the product of the HBR and the sporozoite index and expressed as the number of infected bites per human per year.
A total of 440 children in seven villages were parasitologically and clinically monitored during 18,262 person-days from which 402 (2.2%) were missing for the following reasons: 366 not found and 36 refusals. Ten children died during the study. The mean age of the children at inclusion was 2.1 years. The female/male ratio was 1:1. Each child in the survey was visited on an average of 42 days out of the 48. A total of 3,074 thick blood films were taken, comprising 2,838 in asymptomatic children and 236 in sick children, with an average of seven per child.
Parasitological indexes of asymptomatic children observed by CCS
Distribution of Plasmodium species according to clinical status.
Multivariate analysis of the prevalence rates of Plasmodium falciparum asymptomatic infection determined by cross-sectional surveys.
Prevalence rate of P. falciparum asymptomatic infection
N positive/N total (%)
Correct use of LLIN's
Multivariate analysis of parasite density among positive asymptomatic children observed by cross-sectional surveys.
Geometric average (95%CI)
Adjusted multiplicative factor (95%CI)
Correct use of LLIN's
Clinical malaria observed by ACD
Attributable fraction estimates of pathological episodes to falciparum malaria.
N asymptomatic children
N sick children
N clinical malaria cases
N other clinical cases (No malaria)
Pyrogenic cut-off, sensitivity and specificity estimates by using attributable fraction (sensibility = number of malaria episodes ≥ cut-off/total of malaria episodes; specificity = number of no malaria episodes < cut-off/total of no malaria episodes).
Cut-off (trophozoites of P. falciparum/μL)
N malaria episodes ≥ pyrogenic cut-off
N no malaria episodes < pyrogenic cut-off
> = 15000
Multivariate regression analysis of malaria incidence taken into account the cumulative number of monitoring days
N evocative malaria cases
N malaria cases*
Incidence per child per year (95%CI)
Adjusted Relative Risk (95%CI)
Correct use of LLIN's
Overall 13,602 mosquitoes including 115 An. gambiae sensu lato (s.l.) (65 and 50 indoor and outdoor, respectively) and 67 An. funestus (40 and 27 indoor and outdoor, respectively) were caught in the seven villages. The number of CSP positive An. gambiae s.l. and An. funestus were nine and four respectively. The aggressiveness of culicidae and malaria vectors (An. gambiae s.l. and An. funestus) were 5,541 (95%CI 2,008-15,288) and 74 (95%CI 17-318) bites per human per year. The annual EIR was 5.3 (95%CI 1.1-25.9) infected bites per human per year. The 1014F kdr allele was present in both molecular M and S forms. The frequency of this mutation was respectively 0.47 (95%CI 0.37-0.57) and 0.61 (95%CI 0.43-0.80) in the M and S forms.
Ownership and use of LLINs
The LLINs ownership rate reached 91.8% (2,769/3017; 95%IC 90.8-92.8) and remained high through the year. Use was significantly higher during the rainy season than the dry season 73% (1,062/1,451; 95%CI 70-75) and 67% (1,047/1,566; 95%CI 64-69) respectively, p = 0.0001. It significantly decreased to 31% (118/385; 95%CI 26-31) in the middle of dry season. Correct use was also the highest during the rainy season (68% (990/1,566; 95%CI 65-70)) compared to the dry season (42% (665/1,171; 95%CI 40-45)), p < 0.0001, (Figure 3C).
This prospective longitudinal study allowed the epidemiology of malaria in the health district of Ouidah-Kpomassè-Tori Bossito after the nation-wide distribution of LLINs to children in October 2007 to be characterized. Previous studies have described the epidemiology of malaria in Benin, by focusing mainly on malaria transmission  and on clinical and parasitological aspects as well in rural areas [36–38] as in the city of Cotonou . Other authors  described the process indicators, results and impact of malaria control which were useful for the implementation of the monitoring and assessment system of ''Roll Back Malaria'' in Benin. The large scale and selective distribution of LLINs in Africa in the last decade were also the subject of several studies which concerned mainly the acceptability and/or the population perception without investigating their parasitological and clinical effects [41–47]. Pyrethroid resistance in malaria vectors has been observed in many African countries . Nevertheless, no loss of effectiveness of LLINs has been reported at an operational level .
The entomological findings showed that the health district of Ouidah-Kpomassè-Tori Bossito is a mesoendemic area with a mean annual EIR of 5.3 infected bites (95%CI 1.1-25.9). This EIR was found in conjunction with the annual prevalence rate of 21.8% (95%CI 19.1-24.4) observed in young asymptomatic children [29, 49, 50]. It confirms Velema's parasitological observations in the same area twenty years ago . As regards the resistance of An. gambiae to pyrethroids, the L1014F kdr allele reached 50%, in accordance with previous studies carried out in southern Benin [8, 18]. The annual infection rate increased with age in accordance with what is usually observed in mesoendemic area . The high infection rate in the dry season could be influenced by the peak observed at the end of the rainy season (33%) just one month after the national distribution of LLINs (Figure 3A, Table 3). The parasite density of positive children did not vary with age group or season (Figure 2). These results may be attributed to the protection conferred by LLINs. In mesoendemic areas, the acquisition of immunity against malaria would develop gradually and bring about a decrease in parasitaemia with increasing age . Here, where the level of parasite exposure was reduced by treated nets, immunity may be acquired more slowly [52, 53]. The different rates of infection found in the villages can be explained by variety in their natural characteristics. The prevalence rate was the highest in Satré, Wanho, Kindjitopka and Hèkandji close to fresh water (Toho Lake), open water cisterns or swamps where breeding sites of anopheles are mostly found (Tables 1 and 3).
The calculated AF of pathological episodes to malaria helped to determine the optimum parasite pyrogenic cut-off at 2,000 P. falciparum asexual blood forms per μL. The use of AF to define the pyrogenic parasite cut-off allows the best trade-off between sensitivity and specificity level . In stable malaria areas, P. falciparum parasitaemia is dependent on the season and age, which affects the malaria-AF of pathological episodes and thus the malaria case definition according to pyrogenic parasite density cut-off [33, 55]. In the present study, the parasite density did not vary with season or age. Therefore, the AF could be considered the same whatever the season and the age group. The cut-off of 2,000 falciparum asexual blood forms per μL was close to the value of 1,000 found in mesoendemic area on children under 3 years  and to the 3,000 to 6,000 found in hyperendemic area among children aged 0 to 12 years in south of Benin respectively .
In the health district of Ouidah-Kpomassè-Tori Bossito, one pathological episode out of three was attributed to malaria. To avoid a maximum of missed cases the malaria case definition took into account signs evoking malaria or history of fever during the 48 hours preceding the first day of ACD as advised Mcguinness . Mean annual incidence rate of falciparum clinical malaria was 1.5 per child per year. In P. falciparum high-endemic area, the pyrogenic cut-off of parasitaemia in persons of a given age is similar for all Plasmodium species . Given the high parasite density, P. malariae could have been responsible for one malaria clinical case with 2,360 parasites/μL and P. ovale for two cases with a parasitaemia of (4,800 and 9,800 parasites/μL) respectively.
Use of LLINs
In 2001 before the national distribution of LLINs, in south of Benin, 4.3% of household owned a treated net (ITN) and 2.4% of children under five years old slept under ITNs . In 2006, ITNs possession was estimated to 25.6% and its utilization by the children less than 5 years was 21% in Ouidah . After the national distribution of LLINs, ownership rose to over of 90% and was continuous over of the year (Figure 3C). Throughout the 12 months of the study, two children out of three were found sleeping under LLINs during unannounced and nocturnal inspections. Some studies have already concluded that free distribution of nets via a national campaign is effective in rapidly increasing their possession and use [42, 57, 58]. This high percentage of use may have been the result of adapted sensitization to the beliefs and behaviours of the communities and to the presence of medical staff assisted by a local village helper. Indeed, the success of sensitization depended strongly on the partnership between the study team and the local leaders as described by Paré Toé . The 31% reduction of LLINs use during the dry season in Benin is comparable to that observed in most of the West African countries (Figure 3C) [24, 41, 43]. When populations were not bothered by the mosquitoes, they did not use the treated nets [47, 59]. In the present longitudinal study, neither asymptomatic infection nor malaria attack was affected by the use of LLINs. The impact of LLINs was lower than expected since the correct use gave a 26% of individual protective effect only against infection without influencing malaria morbidity. Moreover, both curves of use and correct use of LLINs varied in the same way through the surveys (Figure 3C).
In conclusion, the health district of Ouidah-Kpomassè-Tori Bossito is a mesoendemic area characterized by a moderate level of pyrethroid resistance of vectors and a high heterogeneity of malaria infection between villages. Malaria infection and disease did not vary through the year. The used LLINs rate was high and only the correct use of LLINs was found to reduce malaria infection without influencing malaria morbidity.
This research was realized in the context of the Project FSP/REFS N° 2006-22 supported by the Ministère Français des Affaires Etrangères et Européennes. A financial contribution was also given by the President Malaria Initiative of the US Government. We thank the nurses, the microscopists and the entomological technicians who collected the data. We thank also the administration of the health district of Ouidah-Kpomassè-Tori Bossito for their strong collaboration. We are grateful for all the inhabitants of the health district of Ouidah-Kpomassè-Tori Bossito who took part in the surveys and participated actively in the data collection. We thank Pr Robin Bailey from London School of Hygiene & Tropical Medicine, Department of Infectious and Tropical Diseases (London, England) for his linguistic correction.
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