Pesticides and the evolution of the genetic structure of Anopheles coluzzii populations in some localities in Benin (West Africa).

BACKGROUND: Changes in the natural habitats of insect groups are determined the genetic polymorphisms between individuals. The objective of this study was to establish the genetic structure of the Anopheles coluzzii populations in four localities of Benin. METHODS: Insecticide surveys and larval sampling were conducted on 4 study localities, including Cotonou, Ketou, Zagnanado, and Sô-Ava. Molecular characterizations were performed on the Anopheles mosquitoes collected with the allelic and genotypic frequencies of kdr gene determined. The multiple comparison Chi square test for proportions was performed with R version 3.3.3. Next, the observed heterozygosity, expected heterozygosity, and indices of fixation, and genetic differentiation were estimated. Finally, the Hardy-Weinberg equilibrium (EHW) was determined to assess whether panmixia exists in the different populations of mosquitoes of the agroecological zones under study. RESULTS: Carbamates, pyrethroids, organophosphorus and organochlorines use have been reported in all localities except Sô-Ava. Anopheles coluzzii was strongly represented across all study localities. The L1014F allele was observed in the localities of Kétou, Cotonou and Zagnanado. Likewise, insecticide selection pressure of homozygous resistant individuals (L1014F/L1014F) was significantly higher in Kétou, Cotonou and Zagnanado (p value < 0.05). Surprisingly in Sô-Ava, a relatively high frequency of the L1014F allele despite the reported absence of pesticide use was observed. All mosquito populations were found to be deficient in heterozygosity across the study sites (FIS< 0). No genetic differentiation (FST< 0) was observed in the localities of Zagnanado and Kétou. CONCLUSION: The survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides.

The current distribution and characterization of the L1014F resistance allele of the kdr gene in three malaria vectors (Anopheles gambiae, Anopheles coluzzii, Anopheles arabiensis) in Benin (West Africa).

BACKGROUND: The fight against malaria faces various biological obstacles, including the resistance of parasites to anti-malarial drugs and the resistance of mosquito vectors to insecticides. The resistance of Anopheles gambiae sensu lato (s.l.) to pyrethroids, the only class of insecticides used to impregnate mosquito nets, is known in Benin; the expansion of this resistance is influenced by the existence of gene flow between species, otherwise by the presence or absence of the kdr mutation in them. The objective of this study is to determine the spatial distribution of An. gambiae and the level of expression of the pyrethroid resistance kdr gene in seven agro-ecological zones of Benin. METHODS: The study was conducted in 18 localities belonging to seven agro-ecological zones where environmental parameters varied. The sites represent the main areas of eco-epidemiological malaria in Benin. Anopheles gambiae larvae were collected in natural breeding sites using ladles and dipping method and reared under standard conditions. These larvae were reared under standard conditions of temperature and humidity (26 to 30 °C and 60 to 90%) at the insectarium of the Centre de Recherche Entomologique de Cotonou (CREC). Adult female mosquitoes having emerged are morphologically and molecularly identified. Homozygous resistant (1014F/1014F), homozygous sensitive (1014L/1014L) and heterozygous (1014F/1014L) genotypes of the L1014F kdr gene mutation are determined by PCR. RESULTS: A total of 677 An. gambiae was subjected at the PCR. The results revealed the presence of three vector species of the An. gambiae complex, of which 409 Anopheles coluzzii, 259 An. gambiae, 5 hybrids (An. coluzzii/An. gambiae) and 4 Anopheles arabiensis in the different agro-ecological zones. The four An. arabiensis were only found in Dassa, a locality in the cotton zone of central Benin. The frequency of distribution of the L1014F allele of the kdr gene varies from 84.48 to 100% in An. gambiae, from 80 to 100% in An. coluzzii and from 0 to 75% in An. arabiensis in the different agro-ecological zones. Moreover, a significant difference is generally observed in the distribution of the L1014F allele (P < 0.05). By comparing in pairs the distribution frequencies of this allele in the two species by agro-ecological zone, only a significant difference is noted in the central cotton and fishery zones (P = 0.0496). CONCLUSION: In summary, even if the data are in small portions, the An. Arabiensis species was found only in central Benin and the L1014F allele of the kdr gene is widespread and seems to fix in all the species recorded in the different agro-ecological zones. This situation amplifies the problem of resistance, which could eventually be a significant obstacle for the malaria vectors control. Similarly, a study of their genetic structure via the L1014F allele is necessary in order to put in place strategies to manage this resistance. These strategies will take into account both the ecology and the genetic diversity of the organisms involved to preserve the effectiveness of pyrethroids, the only insecticides used for the impregnation of mosquito nets.

Population Dynamics of Anopheles gambiae s.l. and Culex quinquefasciatus in Rural and Urban Settings Before an Indoor Residual Spraying Campaign in Northern Benin.

Background: The purpose of this report is to provide information on Culicidae diversity; biting behavior and spatio-seasonal variation of abundance of Anopheles gambiae s.l. and Culex quinquefasciatus in rural and urban settings of the Alibori and Donga regions, Northern Benin, where an indoor residual spraying (IRS) campaign to control malaria is planned. Methods: Both human landing catches, associated with pyrethrum spray catches were used to monitor the mosquito populations in 12 sites with 1 urban and 1 rural located in each of the 6 districts randomly selected in the two targeted regions. After morphological identification of all mosquito specimens, biting behavior and density of An. gambiae s.l. and Cx quinquefasciatus were studied. PCR was also performed on An. gambiae s.l., to identify sibling species and its seasonal variation. Results: A total of 10,367 mosquitoes were captured, related to 14 species of the genera, Anopheles, Aedes, Culex and Mansonia. Of the total species collection, 40.39% were An. gambiae s.l. and 56.85% were Cx. quinquefasciatus. An. gambiae s.l. was more abundant in Donga (2521 specimens) compared with Alibori (1666 specimens). The opposite trend was observed with Cx. quinquefasciatus (2162 specimens in Donga against 4028 in Alibori). An. gambiae s.l. was predominant and displayed a higher blood feeding rate in rural areas, whereas Cx. quinquefasciatus was in majority in urban areas. An. gambiae s.l. was more endophagic, whereas Cx. quinquefasciatus showed similar indoor and outdoor biting behavior. An. gambiae s.l. was composed of An. coluzzii found in majority in the drought, and An. gambiae, which was predominant in the rainy season. Conclusion: The predominance of the malaria vector, An. gambiae s.l. and their higher blood feeding rate and their significantly high endophagy in rural areas indicate that these areas should be primarily targeted with the IRS operations to have a substantial impact on malaria transmission. Endophagy, characteristic of An. gambiae s.l. in our study area, suggests that IRS will have a positive impact on vector control if implemented 1 week before June that is the onset of the rainy season.

Genetic structure of Anopheles gambiae s.s populations following the use of insecticides on several consecutive years in southern Benin.

BACKGROUND: Several studies have reported the strong resistance of Anopheles gambiae s.l. complex species to pyrethroids. The voltage-dependent sodium channel (Vgsc) gene is the main target of pyrethroids and DDT. In Benin, the frequency of the resistant allele (L1014F) of this gene varies along the north-south transect. Monitoring the evolution of resistance is necessary to better appreciate the genetic structure of vector populations in localities subject to the intensive use of chemicals associated with other control initiatives. The purpose of this study was to map the distribution of pyrethroid insecticide resistance alleles of the Kdr gene in malaria vectors in different regions and ecological facies in order to identify the evolutionary forces that might be the basis of anopheline population dynamics. METHODS: The characterization of Anopheles gambiae s.l. populations and resistance mechanisms were performed using adult mosquitoes obtained from larvae collected in the four agroecological zones in southern Benin. Genomic DNA extraction was performed on whole mosquitoes.The extracted genomic DNA from them were used for the molecular identification of species in Anopheles gambiae s.l. complex and the identification of genotypes related to pyrethroid resistance as the Kdr gene amino acid position 1014 in sodium channel. Molecular speciation and genotyping of Kdr resistant alleles (1014) were done using PCR.Genepop software version 4.2 was used to calculate allelic and genotypic frequencies in each agroecological zone. The p value of the allelic frequency was determined using the binomial test function in R version 3.3.3. The Hardy-Weinberg equilibrium was checked for each population with Genetics software version 1.3.8.1. The observed heterozygosity and the expected heterozygosity as well as the fixation index and genetic differentiation index within and between populations were calculated using Genepop software version 4.2. RESULTS: During the study period, Anopheles coluzzii was the major species in all agroecological zones while Anopheles gambiae was scarcely represented. Regardless of the species, resistant homozygote individuals (L1014F/L1014F) were dominant in all agroecological zones, showing a strong selection of the resistant allele (L1014F). All populations showed a deficit of heterozygosity. No genetic differentiation was observed between the different populations of the two species. For Anopheles coluzzii, there was a small differentiation among the populations of the central cotton and bar-lands zones. The genetic differentiation was modest among the population of the fisheries zone (Fst = 0.1295). The genetic differentiation was very high in the population of Anopheles gambiae of the bar-lands zone (Fst = 0.2408). CONCLUSION: This study revealed that the use of insecticides in Benin for years has altered the genetic structure of Anopheles gambiae s.s. populations in all agroecological zones of southern Benin. It would be desirable to orientate vector control efforts towards the use of insecticides other than pyrethroids and DDT or combinations of insecticides with different modes of action.

Small-scale field evaluation of the efficacy and residual effect of Fludora® Fusion (mixture of clothianidin and deltamethrin) against susceptible and resistant Anopheles gambiae populations from Benin, West Africa.

BACKGROUND: In recognition of the threat of insecticide resistance in vectors of malaria, the WHO Global Malaria Programme recommends the development of an appropriate and comprehensive response to insecticide resistance. In principle, good resistance management practice requires the application of multiple insecticides of different modes of action, for example, in rotations and mixtures. Insecticides recommended by the World Health Organization for indoor residual spraying and long-lasting insecticide nets are limited. It is, therefore, judicious to prevent the rapid spread of insecticide resistance by evaluating new insecticides formulations with different modes of action and long residual effect. METHODS: Fludora® Fusion, a new neonicotinoid IRS formulation (a mixture of 500 g/kg clothianidin and 62.5 g/kg deltamethrin applied 200 mg ai/sqm + 25 mg ai/sqm, respectively) was tested. Small scale field evaluation of this product was conducted in the district of Dangbo in Benin, to compare its efficacy and residual effect on cement and mud walls against those of clothianidin 200 mg ai/sqm (WG 70) alone, and of deltamethrin 25 mg ai/sqm (WG 250) alone. WHO wall cone bioassays were conducted monthly with laboratory susceptible Anopheles "Kisumu" and wild Anopheles gambiae sensu stricto (s.s.) population from Dangbo. The induced mortality by each treatment per wall substrate for 24 h, 48 h, and 72 h post exposure were recorded every month and analysed. RESULTS: Fludora® Fusion and clothianidin WG 70 showed mortality rates over 80% WHO bio-efficacy threshold on cement walls either with susceptible or resistant An. gambiae s.s. over a period of 10 and 9 months, respectively. Treatment with Fludora® Fusion and clothianidin WG 70 on the mud walls showed residual effect for 6 months and 5 months respectively against both susceptible and resistant mosquitoes. During the whole evaluation period, deltamethrin WG 250 showed mortality rates below 80% against resistant Anopheles population. Furthermore, the knock down rates observed with the Fludora® Fusion combination were significantly higher (p < 5%) than those induced by Clothiandin WG 70. CONCLUSION: Both the Fludora® Fusion combination and clothianidin alone showed very good and lasting efficacy for IRS against resistant Anopheles with some residual benefit provided by the combination. The residual efficacy of the Fludora® Fusion combination evaluated at 10 months shows this product is a good candidate for IRS interventions.

Blood feeding behaviour comparison and contribution of Anopheles coluzzii and Anopheles gambiae, two sibling species living in sympatry, to malaria transmission in Alibori and Donga region, northern Benin, West Africa.

BACKGROUND: The main goal of this study was to assess the blood feeding behaviour and the contribution Anopheles coluzzii and Anopheles gambiae, 2 sibling species of An. gambiae sensu stricto. present and living in sympatry in 2 regions of northern Benin targeted for indoor residual spraying (IRS). METHODS: The study was carried out in 6 districts of 2 regions of Benin (Alibori and Donga). Human landing catches (HLC) performed inside and outside of the households and pyrethrum spray captures (PSC) carried out in bedrooms were used to sample vector populations (An. gambiae and An. coluzzii). Collected mosquitoes were analysed to estimate the human biting rate indoors and outdoors, the circumsporozoite antigen positivity, and the anthropophagic index using ELISA methodology. Polymerase chain reaction was used to estimate the frequency of the knockdown resistance (kdr) L1014F and the ace-1 mutations, 2 markers associated respectively with pyrethroids and carbamate/organophosphate insecticide resistance. RESULTS: A higher blood feeding rate was observed in An. gambiae compared to An. coluzzii as well as, a non-pronounced outdoor biting behavior in both species. The latter showed similar anthropophagic and sporozoite rates. However the analysis indicates a seasonal difference in the contribution of each species to malaria transmission associated with shifts in resting behaviour. Anopheles coluzzii females accounted for most of the detected infections: 86% in Alibori and 79% in Donga, during the dry season versus 14.4% and 21.2%, respectively for An. gambiae during the same period. This relationship was reversed in Donga during the rainy season (66% for An. gambiae against 34% for An. coluzzii). Results also indicated lower frequencies of kdr L1014F and ace-1 in An. coluzzii versus An. gambiae. CONCLUSION: Despite similarity in some parameters related to malaria transmission in both surveyed species, An. coluzzii is potentially a more important malaria vector because of high density in the region. It is also characterized by lower frequencies of the ace-1 mutation than is An. gambiae. The ongoing use of pirimiphos methyl (organophosphate) for IRS should continue to show a good impact in Alibori and Donga because of the very low level of the ace-1 mutation in both species.

Efficacy of a novel mode of action of an indoor residual spraying product, SumiShield® 50WG against susceptible and resistant populations of Anopheles gambiae (s.l.) in Benin, West Africa.

BACKGROUND: Scale-up of the distribution of long-lasting insecticide-treated bed nets and indoor residual spraying with insecticides over the last decade have contributed to the considerable decrease of malaria morbidity and mortality in sub-Saharan Africa. Due to the increasing pyrethroid resistance intensity and the spread of carbamate resistance in Anopheles gambiae (s.s.) mosquitoes and the limited number of insecticides recommended by the WHO for vector control, alternative insecticide formulations for IRS with long-lasting residual activity are required to sustain the gains obtained in most malaria-endemic countries. METHODS: SumiShield 50WG (clothianidin 300 mg ai/m2) developed by Sumitomo Chemical was evaluated alongside deltamethrin 25 mg ai/m2 (K-Othrine 250 WG) against a pyrethroid resistant Anopheles gambiae (s.l.) population in experimental huts in Covè, Benin. Residual activity was also tested in cone bioassays with the susceptible An. gambiae "Kisumu" strain and the local wild resistant population. RESULTS: The results showed very low toxicity from deltamethrin (mortality rates ranged between 1-40%) against host-seeking resistant Anopheles populations. SumiShield in contrast gave an overall mean mortality of 91.7% at the 120 h observation across the eight- month observation period following spraying. The residual activity measured using cone tests was over the 80% WHO threshold for 24 weeks for resistant wild Anopheles population and 32 weeks for the susceptible strain "Kisumu" after the spraying. CONCLUSIONS: SumiShield is a good candidate for IRS in areas of permanent malaria transmission and where Anopheles populations are resistant to other conventional insecticides such as pyrethroids. It would be interesting to complete experimental huts studies by assessing the efficacy and residual effect of SumiShield 50WG at community level (small-scale field testing) in an area where vectors are highly resistant to insecticides.

Impact of Insecticide Resistance on the Effectiveness of Pyrethroid-Based Malaria Vectors Control Tools in Benin: Decreased Toxicity and Repellent Effect.

Since the first evidence of pyrethroids resistance in 1999 in Benin, mutations have rapidly increased in mosquitoes and it is now difficult to design a study including a control area where malaria vectors are fully susceptible. Few studies have assessed the after effect of resistance on the success of pyrethroid based prevention methods in mosquito populations. We therefore assessed the impact of resistance on the effectiveness of pyrethroids based indoor residual spraying (IRS) in semi-field conditions and long lasting insecticidal nets (LLINs) in laboratory conditions. The results observed showed low repulsion and low toxicity of pyrethroids compounds in the test populations. The toxicity of pyrethroids used in IRS was significantly low with An. gambiae s.l (< 46%) but high for other predominant species such as Mansonia africana (93% to 97%). There were significant differences in terms of the repellent effect expressed as exophily and deterrence compared to the untreated huts (P<0.001). Furthermore, mortality was 23.71% for OlyseNet® and 39.06% for PermaNet®. However, with laboratory susceptible "Kisumu", mortality was 100% for both nets suggesting a resistance within the wild mosquito populations. Thus treatment with pyrethroids at World Health Organization recommended dose will not be effective at reducing malaria in the coming years. Therefore it is necessary to study how insecticide resistance decreases the efficacy of particular pyrethroids used in pyrethroid-based vector control so that a targeted approach can be adopted.

Six years of experience in entomological surveillance of indoor residual spraying against malaria transmission in Benin: lessons learned, challenges and outlooks.

BACKGROUND: From 2008 to 2013, a prevention intervention against malaria based on indoor residual spraying (IRS) was implemented in Benin. From 2008 to 2012, Ficam M(®), a bendiocarb-containing product was used for house spraying, in association with pirimiphos methyl EC (Actellic EC) in 2013. This operation aimed to strengthen the effectiveness of treated nets so as to expedite the achievement of Millennium Development Goals (MDGs): the reduction of morbidity and mortality due to malaria by 75 % from 2000 to 2015. METHODS: Monitoring and evaluation (M&E) was implemented in order to evaluate the impact of IRS intervention on malaria transmission. Anopheles gambiae s.l. populations were sampled by human landing catch. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behaviour of Anopheles vectors the main malaria vector in the treated areas. The residual activity of insecticide in the treated walls was also assessed using WHO bioassay test. RESULTS: The purpose of this project was to draw attention to new challenges and future prospects for the success of IRS in Benin. The main strength of the intervention was a large-scale operation in which more than 80 % of the houses were treated due to the strong adhesion of population. In addition, a significant reduction of the EIR in areas under IRS were observed. However, there were many challenges including the high cost of IRS implementation and the identification of suitable areas to implement IRS. This was because of the low and short residual effect of the insecticides recommended for IRS and the management strategy for vector resistance to insecticides. This indicated that challenges are accompanied by suggested solutions. For the cost of IRS to be accessible to states, then local organizations need to be created in partnership with the National Malaria Control Programme (NMCP) in order to ensure relevant planning and implementation of IRS. CONCLUSION: As an anticipatory measure against vector resistance, this paper proposes various methods, such as periodic IRS based on a combination of two or three insecticides of different classes used in rotation every two or three years.

Evidence of multiple mechanisms providing carbamate and organophosphate resistance in field An. gambiae population from Atacora in Benin.

BACKGROUND: Insecticide resistance in Anopheles gambiae s.l is a major concern to malaria vector control programmes. In West Africa, resistance is mainly due to target-site insensitivity arising from a single point mutation. Metabolic-based resistance mechanisms have also been implicated and are currently being investigated in west Africa. The aim of this study is to better understand the origins of carbamate and organophosphate resistance in An. gambiae population from Atacora, Benin in West Africa. METHODS: Anopheles mosquitoes were reared from larvae collected in two districts (Kouandé and Tanguiéta) of the Atacora department. Mosquitoes were then exposed to WHO impregnated papers. Four impregnated papers were used: carbamates (0.1% bendiocarb, 0.1% propoxur) and organophosphates (0.25% pirimiphos methyl, 1% fenitrothion). PCR assays were run to determine the members of the An. gambiae complex, as well as phenotypes for insensitive acetylcholinesterase (AChE1). Biochemical assays were also carried out to detect any increase in the activity of enzyme typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transferase). RESULTS: 769 female of An. gambiae mosquitoes from Kouandé and Tanguiéta were exposed to bendiocarb, propoxur, pirimiphos methyl and fenitrothion. Bioassays showed resistance with low mortality to bendiocarb (78.57% to 80.17%), propoxur (77.21% to 89.77%), and fenitrothion (89.74% to 92.02%). On the other hand, the same populations of An. gambiae from Kouandé and Tanguiéta showed high susceptibility to pirimiphos methyl with recorded mortality of 99.02% and 100% respectively. The low rate of ace-1R allele frequency (3.75% among survivors and 0.48% among dead) added to the high proportion of homozygous susceptible specimens which survived the WHO bioassays (8/28), suggest that the ace-1 mutation could not entirely explain Anopheles gambiae resistance to carbamate and organophosphate. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GST, MFO and NSE compared to a susceptible strain An. gambiae Kisumu. CONCLUSIONS: Anopheles gambiae populations resistance from Atacora is multifactorial and includes target-site mutation and metabolic mechanism. The co-implication of both resistance mechanisms in An. gambiae s.l may be a serious obstacle for the future success of malaria control operations based on LLINs and IRS.

Good performances but short lasting efficacy of Actellic 50 EC Indoor Residual Spraying (IRS) on malaria transmission in Benin, West Africa.

BACKGROUND: The National Malaria Control Program (NMCP) has been using pirimiphos methyl for the first time for indoor residual spraying (IRS) in Benin. The first round was a success with a significant decrease of entomological indicators of malaria transmission in the treated districts. We present the results of the entomological impact on malaria transmission. Entomologic parameters in the control area were compared with those in intervention sites. METHODS: Mosquito collections were carried out in three districts in the Atacora-Dongo region of which two were treated with pirimiphos methyl (Actellic 50EC) (Tanguiéta and Kouandé) and the untreated (Copargo) served as control. Anopheles gambiae s.l. populations were sampled monthly by human landing catch. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behavior of Anopheles vectors. In the three districts, mosquito collections were organized to follow the impact of pirimiphos methyl IRS on malaria transmission and possible changes in the behavior of mosquitoes. The residual activity of pirimiphos methyl in the treated walls was also assessed using WHO bioassay test. RESULTS: A significant reduction (94.25%) in human biting rate was recorded in treated districts where an inhabitant received less than 1 bite of An. gambiae per night. During this same time, the entomological inoculation rate (EIR) dramatically declined in the treated area (99.24% reduction). We also noted a significant reduction in longevity of the vectors and an increase in exophily induced by pirimiphos methyl on An. gambiae. However, no significant impact was found on the blood feeding rate. Otherwise, the low residual activity of Actellic 50 EC, which is three months, is a disadvantage. CONCLUSION: Pirimiphos methyl was found to be effective for IRS in Benin. However, because of the low persistence of Actellic 50EC used in this study on the treated walls, the recourse to another more residual formulation of pirimiphos methyl is required.

Laboratory and field evaluation of the impact of washings on the effectiveness of LifeNet®, Olyset® and PermaNet® 2.0 in two areas, where there is a high level of resistance of Anopheles gambiae to pyrethroids, Benin, West Africa.

BACKGROUND: An investigation carried out in Benin has shown that, in some areas close to rivers where density of mosquitoes is high, long-lasting, insecticidal bed nets (LLINs) are permanently used. In such areas, LLINs are washed every month. Based on this situation, the 20-wash minimum efficacy advised by the manufacturers would be inadequate. The main goal of this study was to evaluate the effectiveness of LifeNet®, Olyset® and Permanet® 2.0 washed several times against Anopheles gambiae sensu stricto (s.s.) populations, which have developed high resistance to pyrethroids. METHODS: Efficacy of LifeNet®, Olyset® and PermaNet® 2.0 washed 30 and 40 times was expressed in terms of blood-feeding inhibition rate, deterrence, induced exophily and mortality rates. This WHOPES phase II evaluation, conducted in experimental huts in Akron (southern Benin) and in Malanville (northern Benin), was accompanied by WHOPES Phase I evaluation. RESULTS: Over 40 successive washes, LifeNet® induced a mortality rate over 80% in phase I. However, beyond 10 washes, Permanet® 2.0 and Olyset induced dramatically reduced mortality rates, respectively 12.5 and 2.5%. With regard to Phase II results, unwashed LifeNet®, LifeNet® and Olyset® washed 30 and 40 times induced a similar exophily rate per study site (at least 58% in Malanville and at least 71% in Akron). Regarding blood feeding inhibition, LifeNet® and Olyset® washed 30 and 40 times significantly reduced wild An. gambiae s.s. blood feeding showing a similar personal protection as unwashed LifeNet®. LifeNet® washed 30 and 40 times induced mortality rates significantly higher than those induced by Olyset® and Permanet® 2.0 (P < 0,05). CONCLUSION: LifeNet®, followed by Olyset®, have shown good efficacy against host-seeking resistant An. gambiae s.s. population in experimental huts in Benin. Lifenet® have shown to be an effective and promising vector control tool to prevent malaria in areas where repeated washings is a common practice in the community.

The residual life of bendiocarb on different substrates under laboratory and field conditions in Benin, Western Africa.

BACKGROUND: The efficacy of bendiocarb against pyrethroid resistant An. gambiae and the residual life of this insecticide on different substrates were evaluated under laboratory and field conditions. METHODS: Bioassays according to the WHO (World Health Organization) standard protocol were carried out on different substrates impregnated with bendiocarb. Data were analyzed using a binomial regression model with R software. RESULTS: A good efficacy of the bendiocarb against pyrethroid resistant An. gambiae and a high variation of its residual life according to the surfaces treated was observed. The probability that a female mosquito died after exposure to a treated substrate was below 80% after 13 weeks for the teak wood; 7 weeks for the wall made with a mixture of sand and cement and 6 weeks for walls made with red clay and those made with a mixture of the red clay and cement. CONCLUSIONS: Considering the residual life of bendiocarb on walls made with red clay, the main substrates treated during IRS campaigns in rural areas in Benin, more than 2 treatments rounds per year would be necessary to achieve a long term efficacy of IRS using bendiocarb in these areas. Financial and logistical resources required to achieve such levels of coverage need more political will from leaders of African endemic countries. While waiting for innovative malaria control tool, alternative insecticides or combinations of insecticides have to be used for insecticide resistance management in Benin.

A shift from indoor residual spraying (IRS) with bendiocarb to long-lasting insecticidal (mosquito) nets (LLINs) associated with changes in malaria transmission indicators in pyrethroid resistance areas in Benin.

BACKGROUND: Indoor residual spraying (IRS) was implemented in the department of Ouémé-Plateau, southern Benin, in 2008 and withdrawn in 2011, when long lasting insecticidal nets (LLINs) were distributed to the communities that were previously targeted by IRS. Did the LLIN strategy provide a better level of protection against malaria transmission than IRS? METHODS: Entomological surveillance was carried out to assess indicators of transmission risk during the last year of IRS and the first year after the LLIN intervention was put in place (2010-2011). Mosquito biting density was sampled by human landing collection (HLC). Females of Anopheles gambiae s.l. were dissected to estimate the parity rates and the blood meal index. A subsample of the An. gambiae s.l. collection was tested for presence of Plasmodium falciparum sporozoites. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behavior of Anopheles vectors. RESULTS: There were significant increases in all the indicators following withdrawal of IRS. Vector biting density (p<0.001) and longevity (OR=3.81[3.01-4.82] 95% CI; p<0.001) of the An. gambiae s.l. increased significantly; so too did the blood meal index (OR=1.48 [1.1-1.99] 95% CI; p<0.001). Entomological inoculation rate, after IRS withdrawal at one surveillance site, Adjohoun, rose two fold (9.0 infected bites/person/9 months (Apr-Dec 2011) versus 3.66 infective bites/person during the 9 months preceding IRS (Apr-Dec 2010). A second site, Missérété, experienced a six-fold increase after IRS cessation (15.1 infective bites/person/9 months versus 2.41 during IRS). Exophily after IRS cessation decreased significantly in all areas (p<0.001) suggesting that mosquitoes were more likely to rest in houses with LLINs, than in houses subjected to IRS. CONCLUSION: LLINs did not impact on indicators of transmission to the same levels as did IRS after IRS withdrawal.

Decreased proportions of indoor feeding and endophily in Anopheles gambiae s.l. populations following the indoor residual spraying and insecticide-treated net interventions in Benin (West Africa).

BACKGROUND: In many parts of Africa as in Benin, the main strategies of vector control are based on the scaling-up of Long Lasting Insecticide Treated Nets (LLITNs) and indoor residual spraying (IRS). The need to understand the biological implications of IRS in large scale and full coverage of LLITNs is paramount. It is in this context that the present study was conducted. It aims to evaluate the effect of a large scale IRS using a non-pyrethroid insecticide and full coverage of deltamethrin treated nets on the behavior of An. gambiae s.l. in the intervention areas compared to untreated areas used as controls. METHODS: Mosquitoes were collected using human landing catches, pyrethrum spray catches and window exit traps to assess reduction of entry rate, endophily rate, endophagy rate and overall mortality rate in natural populations of An. gambiae s.l. before IRS and LLITNs intervention (2007) and after in 2008 and 2010. RESULTS: In the IRS arm, endophily rate was 67.13% before intervention and 4.5% after intervention, whereas in the control arm it was stable at 51.67% (P > 0 .05). In the LLITN arm endophily rates also decreased after intervention. After the IRS, no gravid mosquitoes were collected from all treated localities, but LLITN performance was not that spectacular. The proportion of mosquitoes biting indoors in the IRS arm decreased from 67.09% before intervention to 42.85% after intervention, compared to a low but significant decrease (71.31% to 57. 46%) in the LLITN arm.The use of vector control tools and behavior of the host would be the main factors that modify the behavior of taking a human blood meal observed on An. gambiae s.l. inside human dwellings. CONCLUSION: The impact on the behavior of An. gambiae s.l. observed with the bendiocarb used in IRS was highly effective compared with the free distribution of LLITNs in terms of mortality and the decrease of proportions of indoor feeding. Despite this efficacy, there is a need for complementary tools and research of alternative strategy oriented on effective health education, and the use of powerful tools such as IRS, LLITNs, larviciding and repellents.

Impact of three years of large scale Indoor Residual Spraying (IRS) and Insecticide Treated Nets (ITNs) interventions on insecticide resistance in Anopheles gambiae s.l. in Benin.

BACKGROUND: In Benin, Indoor Residual Spraying (IRS) and long-lasting insecticidal nets (LLINs) are the cornerstones of malaria prevention. In the context of high resistance of Anopheles gambiae to pyrethroids, The National Malaria Control Program (NMCP) has undertaken a full coverage of IRS in a no-flood zone in the Oueme region, coupled with the distribution of LLINs in a flood zone. We assessed the impact of this campaign on phenotypic resistance, kdr (knock-down resistance) and ace-1(R) (insensitive acetylcholinesterase) mutations. METHODS: Insecticides used for malaria vector control interventions were bendiocarb WP (0.4 g/m²) and deltamethrin (55 mg/m²), respectively for IRS and LLINs. Susceptibility status of An. gambiae was assessed using World Health Organization bioassay tests to DDT, permethrin, deltamethrin and bendiocarb in the Oueme region before intervention (2007) and after interventions in 2008 and 2010. An. gambiae specimens were screened for identification of species, molecular M and S forms and for the detection of the West African kdr (L1014F) as well as ace-1(R) mutations using PCR techniques. RESULTS: The univariate logistic regression performed showed that kdr frequency has increased significantly during the three years in the intervention area and in the control area. Several factors (LLINs, IRS, mosquito coils, aerosols, use of pesticides for crop protection) could explain the selection of individual resistant An. gambiae. The Kdr resistance gene could not be the only mechanism of resistance observed in the Oueme region. The high susceptibility to bendiocarb is in agreement with a previous study conducted in Benin. However, the occurrence of ace-1(R) heterozygous individuals even on sites far from IRS areas, suggests other factors may contribute to the selection of resistance other than those exerted by the vector control program. CONCLUSION: The results of this study have confirmed that An.gambiae have maintained and developed the resistance to pyrethroids, but are still susceptible to bendiocarb. Our data clearly shows that selection of resistant individuals was caused by other insecticides than those used by the IRS and LLINs.