The frequency of kdr and ace-1 alleles in Anopheles gambiae s.l. before and during indoor residual spraying (IRS) implementation and four years after IRS withdrawal in three districts in Atacora, Benin.

BACKGROUND: Indoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l. METHOD: Identification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy-Weinberg equilibrium and genetic differentiation within and between populations were assessed. RESULTS: Prevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of "An. gambiae s.s. and An. coluzzii") populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations. CONCLUSIONS: This study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations.

Field performance of three mosquito collection methods for assessing the entomological efficacy of dual-active ingredient long-lasting insecticidal nets.

Selection of mosquito collection methods is of crucial importance to evaluate the impact of vector control tools on entomological outcomes. During a cluster randomised control trial evaluating the relative efficacy of two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs, we assessed the performance of different mosquito collection methods: Human landing catches (HLC), Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSC). Anopheles mosquitoes were collected using three collection methods in 4 houses, in each of the 60 trial clusters at baseline and every quarter for 24 months using PSCs and HLCs, while CDC light traps were performed during two quarters only. Mean density of vectors collected per method per night was the highest with HLCs (15.9), followed by CDC light traps (6.8); with PSCs (1.1) collecting 10 times less mosquitoes than HLCs. All three collection methods collected fewer mosquitoes in the Interceptor G2® dual a.i. arm, compared to the other trial arms, although only HLCs and PSCs demonstrated strong evidence of this due to a greater number of collection rounds undertaken, than CDC light traps. The broadly similar results regarding the differential impact of the two dual a.i. LLINs showed by the three collection methods suggest that the more ethically acceptable, cheaper, and logistically simpler methods such as CDC light traps could be prioritised for use in large community trials for measuring the efficacy of vector control tools.

Evidence of Transmission of Plasmodium vivax 210 and Plasmodium vivax 247 by Anopheles gambiae and An. coluzzii, Major Malaria Vectors in Benin/West Africa.

Current diagnostic and surveillance systems in Benin are not designed to accurately identify or report non-Plasmodium falciparum (Pf) human malaria infections. This study aims to assess and compare the prevalence of circumsporozoite protein (CSP) antibodies of Pf and P. vivax (Pv) in Anopheles gambiae s.l. in Benin. For that, mosquito collections were performed through human landing catches (HLC) and pyrethrum spray catches (PSC). The collected mosquitoes were morphologically identified, and Pf, Pv 210, and Pv 247 CSP antibodies were sought in An. gambiae s.l. through the ELISA and polymerase chain reaction (PCR) techniques. Of the 32,773 collected mosquitoes, 20.9% were An. gambiae s.l., 3.9% An. funestus gr., and 0.6% An. nili gr. In An. gambiae s.l., the sporozoite rate was 2.6% (95% CI: 2.1-3.1) for Pf, against 0.30% (95% CI: 0.1-0.5) and 0.2% (95% CI: 0.1-0.4), respectively, for Pv 210 and Pv 247. P. falciparum sporozoite positive mosquitoes were mostly An. gambiae (64.35%), followed by An. coluzzii (34.78%) and An. arabiensis (0.86%). At the opposite, for the Pv 210 sporozoite-positive mosquitoes, An. coluzzii and An. gambiae accounted for 76.92% and 23.08%, respectively. Overall, the present study shows that P. falciparum is not the only Plasmodium species involved in malaria cases in Benin.

Entomological Characteristics of Malaria Transmission across Benin: An Essential Element for Improved Deployment of Vector Control Interventions.

Entomological surveillance in Benin has historically been limited to zones where indoor residual spraying was performed or where long-standing sentinel surveillance sites existed. However, there are significant country-wide gaps in entomological knowledge. The National Malaria Control Program (NMCP) assessed population dynamics of Anopheles vectors and malaria transmission in each of Benin's 12 departments to create an entomological risk profile. Two communes per department (24/77 communes) were chosen to reflect diverse geographies, ecologies and malaria prevalence. Two villages per commune were selected from which four households (HH) per village were used for human landing catches (HLCs). In each HH, an indoor and outdoor HLC occurred between 7 p.m. and 7 a.m. on two consecutive nights between July−September 2017. Captured Anopheles were identified, and ovaries were dissected to determine parous rate. Heads and thoraces were tested for Plasmodium falciparum sporozoites by ELISA. The Entomological Inoculation Rate (EIR) was calculated as the product of mosquito bite rate and sporozoite index. Bite rates from An. gambiae s.l., the primary vector species complex, differed considerably between communes; average sporozoite infection index was 3.5%. The EIR ranged from 0.02 infectious bites (ib) per human per night in the departments of Ouémé and Plateau to 1.66 ib/human/night in Collines. Based on transmission risk scales, Avrankou, Sakété and Nikki are areas of low transmission (0 < EIR < 3 ib/human/year), Adjarra, Adja Ouèrè, Zè, Toffo, Bopa, Pehunco, Pèrèrè and Kandi are of medium transmission (3 < EIR < 30 ib/human/year), and the other remaining districts are high transmission (EIR > 30 ib/human/year). The heterogeneous and diverse nature of malaria transmission in Benin was not readily apparent when only assessing entomological surveillance from sentinel sites. Prospectively, the NMCP will use study results to stratify and deploy targeted vector control interventions in districts with high EIRs to better protect populations most at-risk.

Assessing Insecticide Susceptibility and Resistance Intensity of Anopheles gambiae s.l. Populations From Some Districts of Benin Republic, West Africa.

Pyrethroid resistance is widespread in sub-Saharan Africa. The objective of this study was to assess the insecticide resistance intensity in Anopheles gambiae s.l. (Diptera: Culicidae) in four districts of Benin in order to better understand how pyrethroid-only nets are likely to be effective. Thus, adult females of An. gambiae s.l., reared from field-collected larvae were used for assessing resistance intensity to permethrin and deltamethrin. They were tested at 1×, 5×, and 10× the diagnostic dose, using both WHO susceptibility tube testing and CDC bottle bioassays. Identification of molecular species, as well as of L1014F Kdr and Ace-1R mutations was performed using the PCR. The level of expression of biochemical enzymes was also evaluated. Overall, moderate to high resistance intensity to permethrin and deltamethrin was observed, irrespective of the testing method. While the L1014F Kdr frequency was high (>75%), Ace-1R was low (≤6%) in An. gambiaes.s. and Anopheles coluzzii, the two predominant species [52% (95% CI: 44.8-59.1) and 45% (95% CI: 38.0-52.2), respectively]. Anopheles arabiensis was found at very low frequency (3%, 95%CI: 1.1-6.4). For Biochemical analyses, α and ß-esterases were over-expressed in all four districts, while mixed-function oxidases (MFOs) were over-expressed in only one. Overall, the two testing methods led to comparable conclusions, though there were a few inconsistencies between them. The moderate-high resistance intensity observed in the study area suggests that dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) may provide better control of insecticide-resistant mosquitoes.

Pyrethroid Resistance Intensity in Anopheles gambiae s.l. from Different Agricultural Production Zones in Benin, West Africa.

Agricultural production activities usually occur in Benin with the use of a huge amount of insecticides including pyrethroids for pest control. It is therefore important to regularly monitor pyrethroid resistance intensity in Anopheles gambiae s.l., the main malaria vector. This study was conducted in cereal, cotton, rice growing, and urban market gardening areas throughout the country in 2018 and 2019. Females An. gambiae s.l. field-collected as larvae were exposed to deltamethrin 1 × (0.05%), 2 × (0.1%), 5 × (0.25%), and 10 × (0.5%) and permethrin 1 × (0.75%), 2 × (1.5%), 5 × (3.75%), and 10 × (7.5%). Synergist assays were also performed using World Health Organization articles combining piperonyl butoxide (PBO) (4%) + deltamethrin 1 × and, PBO (4%) + Permethrin 1 × . Molecular species and L1014F kdr mutation were identified using PCR. Expression of metabolic enzymes was also assessed through biochemical tests. After exposure to permethrin and deltamethrin 10 × , An. gambiae s.l. displayed mortality rates <98%. Synergist assays induced significantly higher mortality rates than pyrethroids alone (p < 0.05). An. gambiae s.l. complex was composed of An. gambiae s.s., Anopheles coluzzii, and Anopheles arabiensis, with mean frequency of the L1014F kdr mutation >75%. Overexpression of nonspecific α and ß esterases was observed in the cereal, cotton, and urban market gardening areas, while an overexpression of mixed function oxidases was observed in the cotton and rice growing areas. Overall, An. gambiae s.l. showed high resistance intensity to both deltamethrin and permethrin. The synergist and biochemical tests performed suggest that PBO long-lasting insecticidal nets may provide a greater control of pyrethroid-resistant mosquitoes.

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.

Involvement of Anopheles nili in Plasmodium falciparum transmission in North Benin.

BACKGROUND: Several studies carried out in Benin have shown the involvement of Anopheles gambiae sensu lato (s.l.), the Anopheles funestus group in malaria transmission, but none of them reported the contribution of the Anopheles nili group to the transmission of this disease. The current study investigated the question through an entomological cross-sectional survey performed in Northern Benin. METHODS: Mosquito samplings were performed in September and October 2017 in 4 villages located in two districts: Bambaba and Wodara (Kérou district) and, Péhunco 2 and Béké (Péhunco district). The collections were carried out indoors and outdoors using human landing catches (HLC) to assess the human biting rate (HBR) and pyrethrum spray catches (PSC) to evaluate the blood feeding rate and the blood meal origin using the ELISA test. All collected mosquitoes were morphologically identified and, the polymerase chain reaction (PCR) technique was used for molecular identification of sibling species of An. gambiae s.l., An. funestus group and An. nili group sporozoite index (SI) was also assessed by the ELISA test. RESULTS: Overall, An. gambiae s.l., An. funestus group and An. nili group were the three vectors found in the study area. A significantly higher human biting rate (HBR) was recorded in An. nili group (5 bites/human/night) compared to An. funestus group (0.656 bites/human/night) in the Kérou district (p < 0.0001). Anopheles gambiae s.l. displayed the highest HBR (26.19 bites/human/night) in the same district. The entomological inoculation rate (EIR) was 1.875 infected bites/human/month in An. nili group against 13.05 infected bites/human/month in An. gambiae s.l. and 0.938 infected bites/human/month in An. funestus group in Kérou. In Péhunco, the EIR was 1.02 infected bites/human/month in An. gambiae s.l. PCR results showed that An. nili sensu stricto (s.s.) and An. funestus s.s. were the only species of the An. nili and An. funestus groups, respectively. The anthropophagic character of An. gambiae s.l. was also highlighted. CONCLUSION: This study provides useful information on the contribution of An. nili group as secondary vector to malaria transmission in northern Benin. Broader studies must also be carried out in a larger study area to assess the involvement of other Anopheles species to malaria transmission. This will aid to better plan malaria vector control interventions.

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.

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.

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.