Come Zinsou Koukpo

Background: Entomological surveillance for onchocerciasis is closely linked to riverine environments where black fly vectors breed. Relying solely on this xenomonitoring method may underestimate exposure risk in communities further away, as black flies can disperse over hundreds of kilometres. As endemic countries aim for elimination, understanding spatial heterogeneities in entomological indices is crucial. Methods: We therefore conducted spatially explicit entomological surveys around two historical breeding sites: Bétérou in Benin and Asubende in Ghana, during the 2023 and 2024 wet seasons. Using a radial sampling design, 40 settlements within 25 km around each site were selected. black flies were collected via Human Landing Catches at both breeding sites and settlements, and one Esperanza Window Trap at each settlement. Black flies were identified morphologically; biting rates were calculated relative to sampling effort; parity assessed by dissection; and parasite presence determined through O-150 PCR pool-screening and dissection. Generalised Additive Models described trends in entomological indices over distance. Results: A total of 76,050 black flies was collected over 656 sampling days, primarily in Bétérou. In Bétérou, black fly counts and biting rates generally declined with increasing distance from the breeding site, though notable spatial variation was observed. Positive O-150 PCR pools were found at 31 of 41 sites, with infective flies detected up to 19 km from the breeding site. Bétérou exceeded the WHO infectivity prevalence threshold for interrupted transmission in 2023 (0.11%) and 2024 (0.08%). Parous rates remained relatively constant across distance. In Asubende, similar trends were observed, but low catches limited trend analysis, and no infective flies were detected via PCR. Conclusions: These findings demonstrate that black fly dispersal and infectivity capable of sustaining transmission extend beyond breeding sites and first-line villages. This underscores the importance of broader, spatially extensive vector sampling to better evaluate transmission patterns and inform elimination efforts. To gain deeper understanding of the underlying spatial dynamics beyond descriptive trend analysis, comprehensive spatial–temporal modelling is required.

The Plasmodium transmission-blocking endosymbiont Microsporidia MB was previously identified in Anopheles gambiae s.l., but its association with the carria

In Benin, the resistance of An. gambiae s.l. mosquitoes to pyrethroids remains a major concern. The development and use of alternative insecticides seems to be the solution. Organophosphates (Actelic@300 CS) are currently the best candidates to meet this challenge. The present study aims to compare the genetic structure of vector populations of two species of the An. gambiae complex through the L1014F resistance allele of the Kdr gene during two periods marked by characteristic environments to better understand its impact on the dynamics and biology of these organisms. Eight localities divided into three agro-ecological zones, one of which is a control, were studied. Larval surveys were carried out during the rainy seasons from May to July 2016 for the pre-PID period and from May to November 2018 for the subsequent period. Larvae were reared at the insectarium of the Centre de Recherche Entomologique de Cotonou. The adult females obtained were identified morphologically and by molecular approaches. The genetic structure of the populations at various hierarchical levels could be determined using the L1014F and L1014L alleles of Kdr. Molecular analysis revealed three vector species of the An. gambiae complex both before and after IRS, two of which were in the majority. These were 171 An. coluzzii, 297 An. gambiae s.s., 11 An. arabiensis and 211 An. coluzzii, 256 An. gambiae s.s., 8 An. arabiensis respectively out of 479 sampled before treatment and 475 sampled after IRS. In both An. gambiae s.s. and An. coluzzii, the gene frequency of L1014F was found to increase significantly following treatment, approaching the limit of fixation in some populations. A deficit of heterozygosity is widespread with values of the indices, F_ST, F_SC and F_CT, expressing little or no differentiation within and between the defined populations. IRS didn’t adversely affect the selection and spread of the L1014F resistant allele of the Kdr gene. The apparent adaptation of An. coluzzii to polluted areas would be a factor in its proliferation to the detriment of its twin sister An. gambiae in IRS areas. The genetic structuring of the populations, whatever the species, remains almost the same despite the treatment.

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 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.

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. 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. 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). 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.

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. 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. 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). 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.

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. 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. 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. 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.