Effect of Bendiocarb (Ficam ® 80% WP) on Entomological Indices of Malaria Transmission by Indoor Residual Spraying in Burkina Faso, West Africa

Context: The vector control is essential in malaria prevention strategies in several endemic countries in Africa including Burkina Faso. The high transmission of malaria occurs during the period of high vector abundance (Au-gust to October) in Burkina Faso. Therefore, a vector control strategy based on the use of indoor residual spraying targeting this period should provide effective protection against malaria. This study aimed to evaluate the effect of bendiocarb applied in indoor residual spraying on entomological parameters of malaria transmission in a pyrethroid resistance area in southwestern, Burkina Faso. Methods: CDC light trap and early morning collections by pyrethrum spray catches were performed monthly to determine the change in entomological parameter within malaria vector in sprayed (Diebougou) and unsprayed sites (Dano). The female’s malaria vectors collected by both methods were used to determine their blood feeding pattern, biting and sporozoites rates as well as the malaria transmission risk estimated by entomological inoculation rate. Results: A total of 26,276 mosquitoes (13,555 anopheline and 12,721 other culicines) were collected using both CDC light trap (9158 addition, mean human biting rate of An. gambiae s.l. and An. funestus s.l. were significantly lower in sprayed areas compared to unsprayed areas (P < 0.05). Overall, malaria vector transmission risk was significant four-fold lower in villages which received IRS (P = 0.0001) whatever the malaria vectors species (An. gambiae s.l. and An. An. funestus s.l.). Conclusions: The results showed that in the sprayed area (Diebougou), vector densities, human biting rates and malaria transmission risks were very lower than unsprayed areas (Dano). The findings also showed a change in vector behavior especially within An. funestus s.l. which became more zoophagic following IRS. The indoor residual spraying could be promoted as a control tool in areas where malaria transmission occurs during a given period of year.


Introduction
Vector control is one of the key elements of malaria control strategies [1]. In Africa, vector control relies mainly on two effective and complementary tools: long-lasting insecticidal mosquito nets (LLINs) and indoor residual spraying (IRS) [2]. Several studies have demonstrated the effectiveness of both tools in reducing the incidence of malaria-related morbidity and mortality [3] [4] in Africa [5] [6] [7] [8] [9]. However, these tools, in particular LLINs, are impregnated with insecticides from the pyrethroid family. Unfortunately, the recent evolution and the expansion of resistance to this class of insecticide in West Africa in Anopheles gambiae ss is a major problem for sustainability in malaria prevention in Africa [10] [11]. For this reason, the search for alternative tools using a non-pyrethroid insecticide has become a necessity [12]. Indeed, since 2006, the World Health Organization (WHO) encouraged a scale-up of IRS for vector-borne disease control, using one of several classes of insecticide that are suitable for IRS [13]. In 2008, of 108 malaria-endemic burdened countries, 44 reported the use of IRS [14]. The outcomes of indoor residual spraying towards curtailing malaria transmission are firstly to decrease the life span of vector mosquitoes and also to reduce the malaria vectors density [15]. There are several insecticide formulations currently prequalified by WHO for IRS: namely organophosphates, carbamates, pyrethroids and neonicotinoids. Moreover, the effectiveness of the IRS depends on many factors, such as the residual efficacy of the insecticide in formulations for the IRS, the feeding behavior of malaria vectors resting inside houses and susceptibility of vectors local populations to insecticide used for the IRS [16]. Residual  160 Advances in Entomology mosquitoes to insecticides is very important to the understanding of how chemicals work in the control of disease transmission [17]. Indeed, in northern Nigeria [18], IRS led to a high decrease in the total vector population and showed also a reduction in the incidence of malaria among children, the malaria parasites rate and fever, and an apparent effect on mortality of 1 -4-year-old children. In Kenya, IRS with fenitrothion in Kisumu town [19] and the LLINs use in the south coast of Kenya [20], showed a decrease in An. funestus s.l and An. gambiae s.l. populations due to the IRS, while the high bed net coverage was followed by a much reduced human biting rate and a diminishing role of An. gambiae s.s.
in malaria transmission.
In Burkina Faso, malaria was the most common cause of outpatient consulta-  areas (peripheral, central, sub-urban, presence of water source, etc) and had the same type of walls such as "banco" (a mixture of mud and water) and cement.

Mosquito Sampling and Identification
Following IRS, the researche institute (Institut de Recherche en Sciences de la Indoor CDC LT were installed about 1.5 m above the ground next to an occu-

Laboratory Processing
All anophelines were separate and assigned to species based on morphological characters using standard identification keys [35]. Legs of each An. gambiae s.l.
female collected using CDC light trap and PSC methods were tested by PCR for molecular identification of An. gambiae complex species [36]. Aliquots of DNA extracted from PCR positive specimens of An. gambiae s.s. were subjected to PCR assays for identification of the molecular "M" and "S" forms [37] that are currently An. gambiae and An. coluzzii. The heads and thoraces of host-seeking females were tested by enzyme-linked immunosorbent assay-circumsporozoite protein (ELISA-CSP) for Plasmodium falciparum detection using the protocol of Wirtz et al., [38]. The blood meals source from freshly fed females collected using early morning collections (PSC method) were used to assess An. gambiae s.l.
host preference. Therefore, a random selection of 30 specimens per month and per district were tested by a direct ELISA bloodmeal source detection [39] using anti-host immunoglobulin G (IgG) conjugated against human, bovine, pig, donkey and sheep blood. Unfortunately, parity rates could not be assessed because females died while in traps and were too dry for dissection. All the mosquito samples collected were stored individually in numbered vials with desiccant.

Data Analysis
The mean human biting rate (HBR) was calculated for each specie collected by CDC LT by dividing the total number of captured specimens by the total person-nights for the collection period. The mean indoor resting density (IRD) was defined as the total number of mosquitoes (per species) collected by PSC divided by the total number of rooms sampled. The circumsporozoite (CSP) rate was calculated as the proportion of mosquitoes infected with P. falciparum sporozoites. The malaria vectors anthropophilic rate was calculated as the proportion of female mosquitoes with human blood out of the total tested for blood-meal source. The entomological inoculation rate (EIR) was calculated by multiplying the HBR indoor/outdoor and the CSP rate.
All the measured parameters were computed and analysed using the free software GraphPad 5.0 version. Data were compared with the Pearson chi 2 or Fisher exact tests and odds ratio were calculated to determine the impact of IRS in study sites. All calculations were expressed with the statistically significant threshold set at P ≤ 0.05.  (Table 1) (Figure 3(a)). However, in Diebougou An. gambiae s.l. human biting rates of by indoor CDC light trap were found to be lower at 2 and 5 b/p/n indoors in June and July respectively. The An. funestus s.l.

Density and Species Composition of Malaria Vectors
indoor human biting rate was less than 1 b/p/n (Figure 4(a)). A similar trend was recorded for indoor resting densities, with Dano having approximately double the catch size of Diebougou ( Figure 5(a) & Figure 5(b)). The highest resting densities by indoor PSC collection with a mean value in July reaching 49 An. gambiae s.l. per house per night in Dano ( Figure 5(a)). The catch size was generally low in outdoor CDC light trap collections in both sites (Figure 3(b) & Figure 4(b)).

Post-Spraying Data
A summary of mean biting rates is presented in Figure 3 for An. gambiae s.l. and Figure 4 for An. funestus s.l. In addition, the number of mosquitoes collected by month and by site is summarized in Table 2 & Table 3.
In the period post-IRS (August to December) the mean indoor biting rate per person per night (b/p/n) was significantly highest in the unsprayed sites (mean =  biting rates were particularly low in both sites, with a mean of <3 bites per person per night (Figure 3(b)). But, the exposure to mosquito bites outdoors was slightly, but more increased in Diebougou (sprayed area) after treatment compared to Dano, the unsprayed area but the difference was not significant (P > 0.05).
Similar results were observed in An. gambiae s.l. and An. funestus s.l. biting   (Table 3).
The results of CSP-ELISA assays and entomological inoculation rate of An. gambiae s.l. and An. funestus s.l. are presented in Table 2 and Table 3

An. gambiae s.l. and An. funestus s.l. Blood Meal Sources
The results presented in Figure 6 included data from indoor PSC collections, for An. gambiae s.l. and An. funestus s.l. from the two areas. Irrespective of the sampling month, the proportion of An. gambiae s.l. blood-fed on human was highest, reaching more than 80% of the total of 335 females analysed, both in the sprayed and unsprayed areas. No female was recorded blood-fed only on animals. For An. funestus s.l. the feeding patterns were quite different, especially in Figure 6. Proportion of An. gambiae s.l. and An. funestus s.l. blood-fed on humans, animals or mixed from sprayed area (Diebougou) and unsprayed area (Dano).

Discussion
The IRS carried out in the Diébougou district of had a positive impact because it was able to protect 115,639 inhabitants including 23,118 children under 5 years and 2188 pregnant women who are the most vulnerable in thr country. The study showed that two species An. gambiae s.l. and An. funestus s.l. were predominant vectors of malaria transmission in study areas [28] collected using CDC LT and pyrethrum spray catches. Our findings confirmed again the An gambiae specie predominance among An. gambiae complex members in this part of Burkina Faso [29]. In addition, the results also indicated that the An. gambiae s.l. entomological inoculation rate was 4-fold lower in sprayed area compared to the unsprayed area, after the implementation of the IRS primarily due to a lower indoor biting rate and a significant decrease of malaria vectors sporozoite rates. However, the indoor resting density of vectors declined in the sprayed houses following IRS, but increased slightly in October, probably due to the relatively short residual duration of bendiocarb indicated in a separate manuscript. This drastic drop could be also due to the lethal effect of bendiocarb on the anophelines resistant to pyrethroids [40]. The biting rates observed outdoors were slightly higher in intervention areas compared to control areas and may be an early sign of biting behavior change. In conclusion IRS did not reduce the endophily behaviour from that of the baseline but had significantly reduced the density of mosquitoes resting indoors in sprayed area compared to the unsprayed area. In addition, EIR might have been impacted by the observed outdoor biting behaviour, and the reduced residual efficacy of the insecticide after  [40] even though such efficacy did not last more than three months (discussed in a separate manuscript). The biting rates observed in outdoors were higher in intervention areas compared to control areas assuming a less pronounced behavior change of vectors.
In conclusion IRS did not reduce the endophily behavior from that of the baseline though it had significantly reduced the density of mosquitoes resting indoors compared to the control area. In addition, the findings have also shown An. gambiae s.l. were particularly anthropophilic in the two areas with few cases of mixed blood meals and no pure animal blood meals identified. This feeding pattern was the inverse for An. funestus s.l. in the intervention area where more females showed a large plasticity of the host range (zoo-anthropophilic). The results have shown that An. gambiae specie (former S-form) was the major malaria vector species biting in the southwestern region in Burkina Faso. Indeed, this corroborates previous reports [27] [28] of the Anopheline distribution in Burkina Faso, which explained the abundance of An. gambiae species by the ecological characteristics in this area.

Conclusions
The pilot study of IRS with bendiocarb appeared to have a significant impact on malaria transmission in the sprayed areas, as measured by EIR. Indeed, the results illustrated that IRS was strong enough to reduce mosquito abundance, sporozoite rate and EIR in pyrethroids resistance areas. However, the baseline period indicated intrinsic differences in biting rates between Dano and Diebougou before IRS.
The findings also showed a change in vector behavior, with An. funestus s.l.
becoming more zoophagic after IRS. Furthermore, the residual efficacy of IRS did not last more than three months. In areas of high transmission, other insec-

Ethics Approval and Consent to Participate
Ethical approval for this study was granted by the Ethical Committee of the Ministry of Health in Burkina Faso. The mosquito collectors gave prior informed consent and they were vaccinated against yellow fever. They were also subjected to regular medical check-ups with preventive treatments of malaria.