The Anopheles coluzzii range extends into Kenya: detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

BACKGROUND: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. The current study sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well as describe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzii populations. METHODS: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing (WGS) data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Comparisons were made with An. coluzzii cohorts from West and Central Africa. RESULTS: This study reports the detection of An. coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points from which samples were analyzed and its presence confirmed through taxonomic analysis. Additionally, there was a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies up to 64%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. CONCLUSIONS: These findings emphasize the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.

The Anopheles coluzzii range extends into Kenya: Detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

Background: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. In the current study, we sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well asdescribe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzi populations. Methods: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Results: This study reports the detection of Anopheles coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points sampled and its presence confirmed through taxonomic analysis. Additionally, we found a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies of ~60%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. Conclusions: These findings emphasise the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

The Anopheles stephensi mosquito is an invasive malaria vector recently reported in Djibouti, Ethiopia, Sudan, Somalia, Nigeria, and Ghana. The World Health Organization has called on countries in Africa to increase surveillance efforts to detect and report this vector and institute appropriate and effective control mechanisms. In Kenya, the Division of National Malaria Program conducted entomological surveillance in counties at risk for An. stephensi mosquito invasion. In addition, the Kenya Medical Research Institute conducted molecular surveillance of all sampled Anopheles mosquitoes from other studies to identify An. stephensi mosquitoes. We report the detection and confirmation of An. stephensi mosquitoes in Marsabit and Turkana Counties by using endpoint PCR and morphological and sequence identification. We demonstrate the urgent need for intensified entomological surveillance in all areas at risk for An. stephensi mosquito invasion, to clarify its occurrence and distribution and develop tailored approaches to prevent further spread.

Towards an integrated vector management approach for sustainable control of schistosomiasis and malaria in Mwea, Kirinyaga County, Kenya: Baseline epidemiological and vector results.

Background: Vector control is an important approach in the control of most parasitic and vector-borne diseases including malaria, and schistosomiasis. Distribution of these two infections often overlaps and in such areas it's more economically viable to employ an integrated approach in the control of their vectors which largely shares the same breeding ecosystem. We carried out a baseline epidemiological and vector surveys for malaria and schistosomiasis in Mwea, Kirinyaga County, in preparation for the upscaling of integrated vector management (IVM) for the two diseases. Methods: This was a repeated cross sectional survey, where mosquito and snails were sampled during dry and wet seasons in three different ecological zones, Kiamaciri, Thiba and Murinduko to identify possible breeding sites. Mosquito larvae were collected using standard dippers, adults using CDC miniature light traps while snail vectors were sampled using standard snail scoops in different breeding habitats. A total of 1200 pupils from 12 primary schools were tested for malaria using rapid diagnostic tests (Malaria Pf/PAN Ag combo). Stool samples were processed using the Kato Katz technique for intestinal schistosomiasis. Results: The overall prevalence of intestinal schistosomiasis was 9.08 % (95 % CI: 07.00-11.00), with Kiamaciri zone recording the highest prevalence at 19 % (95%CI: 15.00-23.00) and Murinduko zone the least at 0.17 % (95%CI: 0.00-0.01). Majority of the infections were of light intensity 78.9 % (95%CI: 70.04-86.13). There was no positive malaria case detected in this study. Of the 3208 adult mosquitoes sampled during the dry season, 20.6 % (95 % CI: 19.25-22.08) were Anopheles gambiae s.l while 79.4 % (95 % CI: 77.92-80.75) were culicines. During the wet season, 3378 adult mosquitoes were collected, of which 14.7 % (95 % CI: 13.56-15.98) were Anopheles gambiae s.l and 85.3 % (95 % CI: 84.02-86.44) culicines. Overall, 4085 mosquito larvae were collected during the two seasons, of which, 57.3 % and 42.7 % were anopheles and culicine respectively. Majority of the larvae (85.1 % (95%CI: 84.01-86.10) were collected during the wet season, with only 14.9 % (95%CI: 14.10-16.00) being collected during the dry season. A total of 2292 fresh water vector snails were collected with a majority (69.6 % (95%CI: 68.00-71.10) being Biomphalaria pffeiferi responsible for transmission of intestinal schistosomiasis. Conclusion: This study demonstrates that intestinal schistosomiasis is prevalent in Kiamaciri and Thiba zones, and points to the possibility of active transmission of schistosomiasis in Murinduko zone. Malaria vectors were predominantly observed in all sites despite there being no malaria positive case. Culex quinquefaciatus responsible for the spread of several arboviruses was also observed. The presence of these vectors may lead to future disease outbreaks in the area if concerted control initiatives are not undertaken. The disease vectors shared the same breeding sites and thus its economical and feasible to adopt an integrated vector management approach in control efforts for these disease in the study area.

Vectorial capacity and TEP1 genotypes of Anopheles gambiae sensu lato mosquitoes on the Kenyan coast.

BACKGROUND: Malaria remains one of the most important infectious diseases in sub-Saharan Africa, responsible for approximately 228 million cases and 602,000 deaths in 2020. In this region, malaria transmission is driven mainly by mosquitoes of the Anopheles gambiae and, more recently, Anopheles funestus complex. The gains made in malaria control are threatened by insecticide resistance and behavioural plasticity among these vectors. This, therefore, calls for the development of alternative approaches such as malaria transmission-blocking vaccines or gene drive systems. The thioester-containing protein 1 (TEP1) gene, which mediates the killing of Plasmodium falciparum in the mosquito midgut, has recently been identified as a promising target for gene drive systems. Here we investigated the frequency and distribution of TEP1 alleles in wild-caught malaria vectors on the Kenyan coast. METHODS: Mosquitoes were collected using CDC light traps both indoors and outdoors from 20 houses in Garithe village, along the Kenyan coast. The mosquitoes were dissected, and the different parts were used to determine their species, blood meal source, and sporozoite status. The data were analysed and visualised using the R (v 4.0.1) and STATA (v 17.0). RESULTS: A total of 18,802 mosquitoes were collected, consisting of 77.8% (n = 14,631) Culex spp., 21.4% (n = 4026) An. gambiae sensu lato, 0.4% (n = 67) An. funestus, and 0.4% (n = 78) other Anopheles (An. coustani, An. pharoensis, and An. pretoriensis). Mosquitoes collected were predominantly exophilic, with the outdoor catches being higher across all the species: Culex spp. 93% (IRR = 11.6, 95% Cl [5.9-22.9] P < 0.001), An. gambiae s.l. 92% (IRR = 7.2, 95% Cl [3.6-14.5]; P < 0.001), An. funestus 91% (IRR = 10.3, 95% Cl [3.3-32.3]; P < 0.001). A subset of randomly selected An. gambiae s.l. (n = 518) was identified by polymerase chain reaction (PCR), among which 77.2% were An. merus, 22% were An. arabiensis, and the rest were not identified. We were also keen on identifying and describing the TEP1 genotypes of these mosquitoes, especially the *R3/R3 allele that was identified recently in the study area. We identified the following genotypes among An. merus: *R2/R2, *R3/R3, *R3/S2, *S1/S1, and *S2/S2. Among An. arabiensis, we identified *R2/R2, *S1/S1, and *S2/S2. Tests on haplotype diversity showed that the most diverse allele was TEP1*S1, followed by TEP1*R2. Tajima's D values were positive for TEP1*S1, indicating that there is a balancing selection, negative for TEP1*R2, indicating there is a recent selective sweep, and as for TEP1*R3, there was no evidence of selection. Phylogenetic analysis showed two distinct clades: refractory and susceptible alleles. CONCLUSIONS: We find that the malaria vectors An. gambiae s.l. and An. funestus are predominantly exophilic. TEP1 genotyping for An. merus revealed five allelic combinations, namely *R2/R2, *R3/R3, *R3/S2, *S1/S1 and *S2/S2, while in An. arabiensis we only identified three allelic combinations: *R2/R2, *S1/S1, and *S2/S2. The TEP1*R3 allele was restricted to only An. merus among these sympatric mosquito species, and we find that there is no evidence of recombination or selection in this allele.

Malaria vector bionomics in Taita-Taveta County, coastal Kenya.

BACKGROUND: Estimation of the composition and densities of mosquito species populations is crucial for monitoring the epidemiology of mosquito-borne diseases and provide information on local vectors to public health officials and policy-makers. The aim of this study was to evaluate malaria vector bionomics in ecologically distinct sites in Taita-Taveta County, Kenya. METHODS: Adult mosquitoes were collected using backpack aspirators and paired indoor/outdoor CDC light traps in 10 randomly selected households in six villages with distinct ecologies over a study period of 3 years. All Anopheles mosquitoes were morphotyped, and sibling species of Anopheles gambiae sensu lato (An. gambiae s.l.) were identified and separated by PCR analysis of extracted ribosomal DNA. All female anophelines were tested for sporozoite infectivity, with engorged females screened for blood-meal sources using the enzyme-linked immunosorbent assay technique. A subsample of those testing positive and those testing negative for Plasmodium in the ELISA were subjected to PCR assay. RESULTS: A total of eight different Anopheles species were collected both indoors and outdoors. Anopheles gambiae s.l. (82.6%, n = 5252) was the predominant species sensu lato, followed by Anopheles coustani sensu lato (An. coustani s.l.; (10.5%, n = 666) and Anopheles funestus sensu lato (An. funestus s.l.; 5.6%, n = 357). A subset of 683 mosquito samples representing An. gambiae s.l. (n = 580, approx. 11.0%) and An. funestus s.l. (n = 103, approx. 28.9%) were identified by molecular diagnostic assays into sibling species. The An. gambiae s.l. complex was composed of Anopheles arabiensis (62.5%, n = 363/580), An. gambiae sensu stricto (An. gambiae s.s.; 0.7%, n = 4/580), Anopheles merus (0.7%, n = 4/580) and Anopheles quadriannulatus (0.2%, n = 1/580), with the remaining samples (35.5%, n = 206/580) unamplified. Anopheles funestus s.l. was composed of An. rivulorum (14.6%, n = 15/103) and An. leesoni (11.6%, n = 12/103); the remaining samples were unamplified (73.8%, n = 76/103). A total of 981 samples were subjected to PCR analysis for malaria parasite detection; of these 16 (1.6%) were confirmed to be positive for Plasmodium falciparum. The overall human blood index was 0.13 (32/238). CONCLUSIONS: Anopheles gambiae, An. funestus and An. coustani are key malaria vectors in the Taveta region of Kenya, showing concurrent indoor and outdoor transmission. All of the vectors tested showed a higher propensity for bovine and goat blood than for human blood.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Rethinking human resources and capacity building needs for malaria control and elimination in Africa.

Despite considerable success in controlling malaria worldwide, progress toward achieving malaria elimination has largely stalled. In particular, strategies to overcome roadblocks in malaria control and elimination in Africa are critical to achieving worldwide malaria elimination goals-this continent carries 94% of the global malaria case burden. To identify key areas for targeted efforts, we combined a comprehensive review of current literature with direct feedback gathered from frontline malaria workers, leaders, and scholars from Africa. Our analysis identified deficiencies in human resources, training, and capacity building at all levels, from research and development to community involvement. Addressing these needs will require active and coordinated engagement of stakeholders as well as implementation of effective strategies, with malaria-endemic countries owning the relevant processes. This paper reports those valuable identified needs and their concomitant opportunities to accelerate progress toward the goals of the World Health Organization's Global Technical Strategy for Malaria 2016-2030. Ultimately, we underscore the critical need to re-think current approaches and expand concerted efforts toward increasing relevant human resources for health and capacity building at all levels if we are to develop the relevant competencies necessary to maintain current gains while accelerating momentum toward malaria control and elimination.

Genetic markers associated with insecticide resistance and resting behaviour in Anopheles gambiae mosquitoes in selected sites in Kenya.

BACKGROUND: Molecular diagnostic tools have been incorporated in insecticide resistance monitoring programmes to identify underlying genetic basis of resistance and develop early warning systems of vector control failure. Identifying genetic markers of insecticide resistance is crucial in enhancing the ability to mitigate potential effects of resistance. The knockdown resistance (kdr) mutation associated with resistance to DDT and pyrethroids, the acetylcholinesterase-1 (ace-1R) mutation associated with resistance to organophosphates and carbamates and 2La chromosomal inversion associated with indoor resting behaviour, were investigated in the present study. METHODS: Anopheles mosquitoes sampled from different sites in Kenya and collected within the context of malaria vector surveillance were analysed. Mosquitoes were collected indoors using light traps, pyrethrum spray and hand catches between August 2016 and November 2017. Mosquitoes were identified using morphological keys and Anopheles gambiae sensu lato (s.l.) mosquitoes further identified into sibling species by the polymerase chain reaction method following DNA extraction by alcohol precipitation. Anopheles gambiae and Anopheles arabiensis were analysed for the presence of the kdr and ace-1R mutations, while 2La inversion was only screened for in An. gambiae where it is polymorphic. Chi-square statistics were used to determine correlation between the 2La inversion karyotype and kdr-east mutation. RESULTS: The kdr-east mutation occurred at frequencies ranging from 0.5 to 65.6% between sites. The kdr-west mutation was only found in Migori at a total frequency of 5.3% (n = 124). No kdr mutants were detected in Tana River. The ace-1R mutation was absent in all populations. The 2La chromosomal inversion screened in An. gambiae occurred at frequencies of 87% (n = 30), 80% (n = 10) and 52% (n = 50) in Baringo, Tana River and Migori, respectively. A significant association between the 2La chromosomal inversion and the kdr-east mutation was found. CONCLUSION: The significant association between the 2La inversion karyotype and kdr-east mutation suggests that pyrethroid resistant An. gambiae continue to rest indoors regardless of the presence of treated bed nets and residual sprays, a persistence further substantiated by studies documenting continued mosquito abundance indoors. Behavioural resistance by which Anopheles vectors prefer not to rest indoors may, therefore, not be a factor of concern in this study's malaria vector populations.

Proceedings of an expert workshop on community agreement for gene drive research in Africa - Co-organised by KEMRI, PAMCA and Target Malaria.

Gene drive research is progressing towards future field evaluation of modified mosquitoes for malaria control in sub-Saharan Africa. While many literature sources and guidance point to the inadequacy of individual informed consent for any genetically modified mosquito release, including gene drive ones, (outside of epidemiological studies that might require blood samples) and at the need for a community-level decision, researchers often find themselves with no specific guidance on how that decision should be made, expressed and by whom. Target Malaria, the Kenya Medical Research Institute and the Pan African Mosquito Control Association co-organised a workshop with researchers and practitioners on this topic to question the model proposed by Target Malaria in its research so far that involved the release of genetically modified sterile male mosquitoes and how this could be adapted to future studies involving gene drive mosquito releases for them to offer reflections about potential best practices. This paper shares the outcomes of that workshop and highlights the remaining topics for discussion before a comprehensive model can be designed.

Insecticide resistance status in Anopheles gambiae (s.l.) in coastal Kenya.

BACKGROUND: The rapid and widespread evolution of insecticide resistance has emerged as one of the major challenges facing malaria control programs in sub-Saharan Africa. Understanding the insecticide resistance status of mosquito populations and the underlying mechanisms of insecticide resistance can inform the development of effective and site-specific strategies for resistance prevention and management. The aim of this study was to investigate the insecticide resistance status of Anopheles gambiae (s.l.) mosquitoes from coastal Kenya. METHODS: Anopheles gambiae (s.l.) larvae sampled from eight study sites were reared to adulthood in the insectary, and 3- to 5-day-old non-blood-fed females were tested for susceptibility to permethrin, deltamethrin, dichlorodiphenyltrichloroethane (DDT), fenitrothion and bendiocarb using the standard World Health Organization protocol. PCR amplification of rDNA intergenic spacers was used to identify sibling species of the An. gambiae complex. The An. gambiae (s.l.) females were further genotyped for the presence of the L1014S and L1014F knockdown resistance (kdr) mutations by real-time PCR. RESULTS: Anopheles arabiensis was the dominant species, accounting for 95.2% of the total collection, followed by An. gambiae (s.s.), accounting for 4.8%. Anopheles gambiae (s.l.) mosquitoes were resistant to deltamethrin, permethrin and fenitrothion but not to bendiocarb and DDT. The L1014S kdr point mutation was detected only in An. gambiae (s.s.), at a low allelic frequency of 3.33%, and the 1014F kdr mutation was not detected in either An. gambiae (s.s.) or An. arabiensis. CONCLUSION: The findings of this study demonstrate phenotypic resistance to pyrethroids and organophosphates and a low level of the L1014S kdr point mutation that may partly be responsible for resistance to pyrethroids. This knowledge may inform the development of insecticide resistance management strategies along the Kenyan Coast.

Entomological and Anthropological Factors Contributing to Persistent Malaria Transmission in Kenya, Ethiopia, and Cameroon.

INTRODUCTION: In order to improve our understanding of the fundamental limits of core interventions and guide efforts based on prioritization and identification of effective/novel interventions with great potentials to interrupt persistent malaria transmission in the context of high vector control coverage, the drivers of persistent disease transmission were investigated in three eco-epidemiological settings; forested areas in Cameroon, coastal area in Kenya and highland areas in Ethiopia. METHODS: Mosquitoes were sampled in three eco-epidemiological settings using different entomological sampling techniques and analysed for Plasmodium infection status and blood meal origin in blood-fed specimens. Human behavioural surveys were conducted to assess the knowledge and attitude of the population on malaria and preventive measures, their night activities, and sleeping pattern. The parasitological analysis was conducted to determine the prevalence of Plasmodium infection in the population using rapid diagnostic tests. RESULTS: Despite the diversity in the mosquito fauna, their biting behaviour was found to be closely associated to human behaviour in the three settings. People in Kenya and Ethiopia were found to be more exposed to mosquito bites during the early hours of the evening (18-21h) while it was in the early morning (4-6 am) in Cameroon. Malaria transmission was high in Cameroon compared to Kenya and Ethiopia with over 50% of the infected bites recorded outdoors. The non-users of LLINs were 2.5 to 3 times more likely to be exposed to the risk of acquiring malaria compared to LLINs users. Malaria prevalence was high (42%) in Cameroon, and more than half of the households visited had at least one individual infected with Plasmodium parasites. CONCLUSIONS: The study suggests high outdoor malaria transmission occurring in the three sites with however different determinants driving residual malaria transmission in these areas.

Bionomics and ecology of Anopheles merus along the East and Southern Africa coast.

Malaria transmission persists despite the scale-up of interventions such as long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). Understanding the entomological drivers of transmission is key for the design of effective and sustainable tools to address the challenge. Recent research findings indicate a shift in vector populations from the notorious Anopheles gambiae (s.s.) as a dominant vector to other species as one of the factors contributing to the persistence of malaria transmission. However, there are gaps in the literature regarding the minor vector species which are increasingly taking a lead role in malaria transmission. Currently, minor malaria vectors have behavioural plasticity, which allows their evasion of vector control tools currently in use. To address this, we have reviewed the role of Anopheles merus, a saltwater mosquito species that is becoming an important vector of malaria transmission along the East and Southern African coast. We performed a literature review from PubMed and Google Scholar and reviewed over 50 publications relating to An. merus's bionomics, taxonomy, spatial-temporal distribution and role in malaria transmission. We found that An. merus is an important vector of malaria and that it contributes to residual malaria transmission because of its exophilic tendencies, insecticide resistance and densities that peak during the dry seasons as the freshwater mosquitoes decline. Spatial and temporal studies have also shown that this species has increased its geographical range, densities and vectorial capacity over time. In this review, we highlight the resting behaviour and breeding habitats of this mosquito, which could be targeted for surveillance studies and control interventions.

Urban Ecology of Arboviral Mosquito Vectors Along the Kenyan Coast.

The purpose of this study was to determine the ecology of the common arboviral mosquito vectors in Mombasa, Kilifi and Malindi urban areas of coastal Kenya. Mosquito larvae were collected using standard dippers and pipettes. Egg survivorship in dry soil was evaluated by collecting soil samples from dry potential larval developmental sites, re-hydrating them for hatching and rearing of the eventual larvae to adults. Adult mosquitoes were collected with CDC light traps and BG-Sentinel traps. All blood-fed females were tested for bloodmeal origin. Mosquitoes were screened for arboviruses using RT-qPCR. Overall, the predominant species were Culex quinquefasciatus (Say) 72.4% (n = 2,364) and Aedes aegypti (L.), 25.7%, (n = 838). A total of 415 larval developmental sites were identified indoors (n = 317) and outdoors (n = 98). The most productive larval developmental sites, both indoors and outdoors, were assorted small containers, water tanks, drainages, drums, and jerricans. Overall, 62% (n = 18) of the soil samples collected were positive for larvae which were used as a proxy to measure the presence of eggs. The mosquitoes fed on humans (29.8%) and chickens (3.7%). Of 259 mosquitoes tested for viral infection, 11.6% were positive for Flavivirus only. The most productive larval developmental sites for arboviral vectors indoors were small containers, water tanks, jerricans, and drums whereas small containers, water tanks, drainage channels, buckets, tires, and water troughs were the productive larval developmental sites outdoors.

Evaluating the impact of larviciding with Bti and community education and mobilization as supplementary integrated vector management interventions for malaria control in Kenya and Ethiopia.

BACKGROUND: Malaria prevention in Africa is mainly through the use of long-lasting insecticide treated nets (LLINs). The objective of the study was to assess the effect of supplementing LLINs with either larviciding with Bacillus thuringiensis israelensis (Bti) or community education and mobilization (CEM), or with both interventions in the context of integrated vector management (IVM). METHODS: The study involved a factorial, cluster-randomized, controlled trial conducted in Malindi and Nyabondo sites in Kenya and Tolay site in Ethiopia, to assess the impact of the following four intervention options on mosquitoes and malaria prevalence: LLINs only (arm 1); LLINs and Bti (arm 2); LLINs and CEM (arm 3); and, LLINs combined with Bti and CEM (arm 4). Between January 2013 and December 2015, CDC light traps were used to sample adult mosquitoes during the second, third and fourth quarter of each year in 10 houses in each of 16 villages at each of the three study sites. Larvae were sampled once a fortnight from potential mosquito-breeding habitats using standard plastic dippers. Cross-sectional malaria parasite prevalence surveys were conducted involving a total of 11,846 primary school children during the 3-year period, including 4800 children in Tolay, 3000 in Malindi and 4046 in Nyabondo study sites. RESULTS: Baseline relative indoor anopheline density was 0.11, 0.05 and 0.02 mosquitoes per house per night in Malindi, Tolay and Nyabondo sites, respectively. Nyabondo had the highest recorded overall average malaria prevalence among school children at 32.4%, followed by Malindi with 5.7% and Tolay 1.7%. There was no significant reduction in adult anopheline density at each of the three sites, which could be attributed to adding of the supplementary interventions to the usage of LLINs. Malaria prevalence was significantly reduced by 50% in Tolay when using LLINs coupled with application of Bti, community education and mobilization. The two other sites did not reveal significant reduction of prevalence as a result of combining LLINs with any of the other supplementary interventions. CONCLUSION: Combining LLINs with larviciding with Bti and CEM further reduced malaria infection in a low prevalence setting in Ethiopia, but not at sites with relatively higher prevalence in Kenya. More research is necessary at the selected sites in Kenya to periodically determine the suite of vector control interventions and broader disease management strategies, which when integrated would further reduce adult anopheline populations and malaria prevalence beyond what is achieved with LLINs.

Ecological drivers of genetic connectivity for African malaria vectors Anopheles gambiae and An. arabiensis.

Anopheles gambiae and An. arabiensis are major malaria vectors in sub-Saharan Africa. Knowledge of how geographical factors drive the dispersal and gene flow of malaria vectors can help in combatting insecticide resistance spread and planning new vector control interventions. Here, we used a landscape genetics approach to investigate population relatedness and genetic connectivity of An. gambiae and An. arabiensis across Kenya and determined the changes in mosquito population genetic diversity after 20 years of intensive malaria control efforts. We found a significant reduction in genetic diversity in An. gambiae, but not in An. arabiensis as compared to prior to the 20-year period in western Kenya. Significant population structure among populations was found for both species. The most important ecological driver for dispersal and gene flow of An. gambiae and An. arabiensis was tree cover and cropland, respectively. These findings highlight that human induced environmental modifications may enhance genetic connectivity of malaria vectors.

Evaluating effectiveness of screening house eaves as a potential intervention for reducing indoor vector densities and malaria prevalence in Nyabondo, western Kenya.

BACKGROUND: Mosquito-proofing of houses using wire mesh screens is gaining greater recognition as a practical intervention for reducing exposure to malaria transmitting mosquitoes. Screening potentially protects all persons sleeping inside the house against transmission of mosquito-borne diseases indoors. The study assessed the effectiveness of house eaves screening in reducing indoor vector densities and malaria prevalence in Nyabondo, western Kenya. METHODS: 160 houses were selected for the study, with half of them randomly chosen for eaves screening with fibre-glass coated wire mesh (experimental group) and the other half left without screening (control group). Randomization was carried out by use of computer-generated list in permuted blocks of ten houses and 16 village blocks, with half of them allocated treatment in a ratio of 1:1. Cross-sectional baseline entomological and parasitological data were collected before eave screening. After baseline data collection, series of sampling of indoor adult mosquitoes were conducted once a month in each village using CDC light traps. Three cross-sectional malaria parasitological surveys were conducted at three month intervals after installation of the screens. The primary outcome measures were indoor Anopheles mosquito density and malaria parasite prevalence. RESULTS: A total of 15,286 mosquitoes were collected over the two year period using CDC light traps in 160 houses distributed over 16 study villages (mean mosquitoes = 4.35, SD = 11.48). Of all mosquitoes collected, 2,872 (18.8%) were anophelines (2,869 Anopheles gambiae sensu lato, 1 Anopheles funestus and 2 other Anopheles spp). Overall, among An. gambiae collected, 92.6% were non-blood fed, 3.57% were blood fed and the remaining 0.47% were composed of gravid and half gravid females. More indoor adult mosquitoes were collected in the control than experimental arms of the study. Results from cross-sectional parasitological surveys showed that screened houses recorded relatively low malaria parasite prevalence rates compared to the control houses. Overall, malaria prevalence was 5.6% (95% CI: 4.2-7.5) n = 1,918, with baseline prevalence rate of 6.1% (95% CI: 3.9-9.4), n = 481 and 3rd follow-up survey prevalence of 3.6% (95% CI: 2.0-6.8) n = 494. At all the three parasitological follow-up survey points, house screening significantly reduced the malaria prevalence by 100% (p < 0.001), 63.6% (p = 0.026), and 100% (p < 0.001) in the 1st, 2nd and 3rd follow-up surveys respectively. CONCLUSIONS: The study demonstrated that house eave screening has potential to reduce indoor vector densities and malaria prevalence in high transmission areas.

Surveillance of Culicine Mosquitoes in Six Villages of Taita-Taveta County, Kenya, With Host Determinations From Blood-Fed Females.

Culicine mosquitoes are vectors of human disease-causing pathogens like filarial worms and several arthropod-borne viruses (arboviruses). Currently, there has been an increase in emerging and re-emerging vector-borne diseases along coastal Kenya, which has been of major concern in public health. This study aimed at determining culicine mosquito species abundance, diversity and their host feeding preferences in Taita-Taveta County, Coastal Kenya. Entomological sampling was done during the long-wet season (March and May) and long dry season (June to October) 2016-2018. Mosquito sampling was done using CDC light traps and Backpack aspiration for indoor and outdoor environments. All culicine mosquitoes collected were identified morphologically and categorized according to their physiological status. Blood fed culicine mosquitoes were tested for bloodmeal sources using ELISA. In total, 3,278 culicine mosquitoes were collected, of which 738 (22.5 %) were found indoors and 2,540, (77.5 %) outdoors. The mosquitoes consisted of 18 species belonging to four genera: Aedes (7), Culex (8), Mansonia (2), and Coquillettidia (1). Overall, there was high mosquito species diversity (H) in outdoors (H = 2.4339) than in indoors (H = 2.2523), whereas even distribution (EH) was higher in indoors (EH = 0.9064) than outdoors (EH = 0.8266). Majorly the bloodmeals identified were from multiple host sources with (51.6%), single hosts (41.3%), and unidentified (7.2%). This study has demonstrated a high diversity of culicine mosquitoes with relaxed feeding tendencies. These mosquitoes are contributing to mosquito biting nuisance and the likelihood of exposure of populations to diseases of public health.

Species Composition, Phenotypic and Genotypic Resistance Levels in Major Malaria Vectors in Teso North and Teso South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Knockdown resistance (kdr) is strongly linked to pyrethroid insecticide resistance in Anopheles gambiae in Africa, which may have vital significance to the current increased use of pyrethroid-treated bed net programmes. The study is aimed at determining species composition, levels of insecticide resistance, and knockdown patterns in Anopheles gambiae sensu lato in areas with and areas without insecticide resistance in Teso North and Teso South subcounties, Western Kenya. MATERIALS AND METHODS: For WHO vulnerability tests, mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes (4944 at 100 mosquitoes per insecticide) which were exposed to 0.75% permethrin, 0.05% deltamethrin, and 0.1% bendiocarb using the WHO tube assay method. Species identification and kdr East gene PCRs were also performed on randomly selected mosquitoes from the collections; including adult mosquitoes (3448) sampled using standard collection methods. RESULTS: Anopheles gambiae sensu stricto were the majority in terms of species composition at 78.9%. Bendiocarb caused 100% mortality while deltamethrin had higher insecticidal effects (77%) on female mosquitoes than permethrin (71%). Susceptible Kengatunyi cluster had higher proportion of An. arabiensis (20.9%) than resistant Rwatama (10.7%). Kengatunyi mosquitoes exposed to deltamethrin had the highest KDT50 R of 8.2. Both Anopheles gambiae sensu stricto and Anopheles arabiensis had equal S allelic frequency of 0.84. Indoor resting mosquitoes had 100% mortality rate after 24 h since exposure. Overall SS genotypic frequency in Teso North and Teso South subcounties was 79.4% against 13.7% homozygous LL genotype and 6.9% heterozygous LS genotype. There was a significant difference (ρ < 0.05) in S allele frequencies between Kengatunyi (0.61) and Rwatama (0.95). Mosquito samples collected in 2013 had the highest S allelic frequency of 0.87. Discussion. Most likely, the higher the selection pressure exerted indoors by insecticidal nets, the higher were the resistance alleles. Use of pyrethroid impregnated nets and agrochemicals may have caused female mosquitoes to select for pyrethroid resistance. Different modes of action and chemical properties in different types of pyrethroids aggravated by a variety of edaphic and climatic factors may have caused different levels of susceptibility in both indoor and outdoor vectors to pyrethroids and carbamate. Species composition and populations in each collection method may have been influenced by insecticide resistance capacity in different species. Conclusions and Recommendations. Both phenotypic and genotypic insecticide resistance levels have been confirmed in Teso North and Teso South subcounties in Western Kenya. Insecticide resistance management practices in Kenya should be fast tracked and harmonized with agricultural sector agrochemical-based activities and legislation, and possibly switch to carbamate use in order to ease selection pressure on pyrethroids which are useable in insecticidal nets and indoor residual spray due to their low human toxicity. The implication of such high resistance levels in mosquitoes collected in Teso subcounties is that resistance is likely to persist and or even increase if monomolecules of permethrin and deltamethrin or both continue to be used in all net- and nonnet-based mosquito control purposes. Usage of mutually reinforcing piperonyl butoxide (PBO) that prohibits particular enzymes vital in metabolic activities inside mosquito systems and has been integrated into pyrethroid-LLINs to create pyrethroid-PBO nets is an extremely viable option.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. METHODS: Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. RESULTS: The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.