Understanding mosquito larval ecology is crucial for effective vector control strategies. While much is known about Anopheles larval habitat distribution, the impact of landscape (urban vs. rural) on Anopheles stephensi larval distribution and population dynamics in Ethiopia remains unclear. A longitudinal study was conducted from February 2023 to December 2024 in aquatic habitats in urban, peri-urban, and rural areas of Dire Dawa City Administration, eastern Ethiopia. Mosquito larvae and pupae were collected and reared in a field insectary until adult emergence. The female Anopheles were morphologically identified and further confirmed via polymerase chain reaction (PCR). The physicochemical parameters of the larval habitats were measured via a HANNA multiparameter water probe. Statistical analyses included general linear models, ANOVA, logistic regression, paired sample t tests, chi-square tests, principal component analysis and correlation analyses to assess larval presence, density, and distribution in relation to water physicochemical parameters across the three ecological settings. A total of 23,526 larvae and 1808 pupae of Anopheles mosquitoes were collected from 856 man-made habitats (bricks, plastic sheets, steel drums, tire tracks, canal ditches, and barrels) and 53 natural habitats (river edges, animal hoof prints, ponds, and swamps) in urban, peri-urban, and rural areas. Uncovered cemented cisterns were the main human-made larval habitats; river edges were the main natural habitats. Anopheles larvae were absent in the steel drums and plastic barrels at the rural sites. Anopheles larval density significantly differed across the different ecological settings (F = 7.8, df = 1, 908, p = 0.005) and among the different larval habitat types (F = 326.2, df = 1, 907, p < 0.001). Among the 2934 reared adults, 74.8

Background:Understanding mosquito larval ecology is essential for planning and implementations of vector control strategies. The biotic and abiotic factors affect larval occurrence , density, survival and morphogenesis of mosquitoes. Artificial containers are very suitable larval habitats for some species of Anopheles and Aedes mosquitoes in urban and peri-urban settings. Therefore, this study we identified, mapped and characterized larval habitats, estimated larval density and indices larval habitats. In addition, we determined the species composition of Anopheles mosquitoes and species evenness in urban, peri-urban and rural areas of Dire Dawa city adminstration. Methods:Larval habitats were surveyed and identified monthly for a period of 16 months from February 2023 to December 2024 in urban, peri-urban and rural areas of Dire Dawa. Mosquito larvae and pupae were collected and those larvae identified as Anopheles were fed on fish-food. Emerged adults were provided with 10% sucrose solution and kept under standard conditions in field insectary. Females Anopheles was identified morphologically and further species-specific PCR assay was employed to identify members of An.gambiae s.l. In addition, real time PCRassay was performed to identify An.stephensi and An.arabiensis. Water samples were taken from the larval habitats and the physico-chemical parameters were measured using HANNA Multi-parameter (H198194). Larval habitat diversity, larval abundance and distribution were assessed across the three ecological settings (urban, peri-urban and rural). Results:A total of 23, 526 larvae and 1,808 pupae of Anopheles mosquitoes were collected from 909 man-made(uncovered cemented cisterns (Brick), plastic sheets, steel drums, Tire tracks, Canal ditch, plastic tanker/Barrel) and natural habitats (River edges, ponds, animal hoof prints and swaps) in urban, peri-urban areas of Dire Dawa. The highest mean larval density (51 larvae per dip) of Anopheles mosquitoes was recorded from peri-urban sites in uncovered water tanker (brick) followed by urban site in brick (46 larvae per dip). Anopheles larvae were not found in steel drum and plastic barrels in rural sites.A total of 2,934 adult Anopheles mosquitoes were emerged from immatures collected from all sites, of which 75% (2194/2934) were An. stephensi, and 22 % (636 / 2934) were An.arabiensis. The remaining 3.0% were An. pharoensis, An. coustani, An .amharicus, and An. pretoriensis. Anopheles stephensi, An. arabiensis and An. amharicus shared the same habitats across the three ecologies. Larval density was positively correlated with availability of brick making, proximity to houses, urban setting, presence of competitors /predators, vegetation cover, shade cover and substrate type. But larval presence was not correlated with presence/absence of intervention. Larva/pupa presence were positively correlated with pH (r=0.264, p=0.01) and water pressure (r=0.21, p<0.05).There was positive correlation among temperature, electrical conductivity (EC), total dissolved solids (TDS), salinity and dissolved oxygen (DO) and negative correlation among temperature with resistivity, pH with mvPH. Larval presence was positively correlated with water salinity and pH. Conclusion:Anopheles stephensi was the predominant species found in the study area, followed by An.arabiensis, An.amharicu, An.pharoensis, An.coustani and An.pretoriensis. Uncovered water tankers (Bricks) were the most prolific artificial habitats in urban and peri-urban sites followed by plastic sheets while natural habitats such as hoof prints and river basins were the most efficient habitat types in rural and urban settings, respectively. Anopheles stephensi was found in natural habitats of Butuji and legehare rivers from urban site, and in rural sites from man-made habitat of plastic sheet.Anopheles amharicus larvae was found in plastic sheet, an artificial habitat common in urban, peri-urban and rural areas.We report here the occurrence of An.stephensi in rural areas, breeding in natural habitats, and co-existing with An. gambiae s.l complex.Availability of brick making, shorter distance from living houses being in urban ecology were directly correlated with larval density. Habitat abundance and positivity of uncovered water tankers (cemented cisterns or bricks) in urban and pre-urban sites could indicate for feasibility and proper implementation of larval source management.

BackgroundLong-lasting insecticidal nets (LLINs) are central to malaria control and designed to protect for up to three years; however, increasing pyrethroid resistance undermines their effectiveness. Piperonyl butoxide (PBO) LLINs, designed to enhance pyrethroid efficacy, are recommended in high-resistance areas, yet their long-term operational bio-efficacy remains unclear. This study evaluated the killing efficacy of PBO LLINs over three years of field use compared to standard LLINs and monitored insecticide resistance in local Anopheles gambiae s.l. populations.MethodsDuring a trial in Muhoroni, western Kenya (2021–2024), standard and PBO LLINs were collected at 6, 18, and 36 months of use. Residual bio-efficacy was assessed using WHO cone bioassays against a susceptible Anopheles gambiae s.s. Kisumu strain and field An. gambiae s.l. populations. WHO tube and bottle assays determined insecticide resistance, while synergist assay assessed metabolic resistance. Quantitative polymerase chain reaction detected target-site mutations (kdr-1014 F/S and Ace-1 G119S).ResultsMolecular identification confirmed all An. gambiae s.l. were Anopheles arabiensis, showing increasing pyrethroid resistance, with deltamethrin mortality declining from 96.3% (2021) to 22.7% (2024) while remaining susceptible to pirimiphos-methyl and clothianidin. PBO pre-exposure restored deltamethrin mortality from 22.7% to 98.9%. The frequency of 1014F increased from 0.09 to 0.17 and 1014S from 0.04 to 0.06, with no Ace-1 mutations detected. Standard LLINs retained >80% efficacy against the susceptible strain for 18 months but were below threshold against field mosquitoes even when new. PBO LLINs were effective at baseline against field populations but declined sharply, with mortality dropping to 24% by 6 months. Overall, both net types exhibited a marked decline in killing efficacy over time against field mosquitoes, with mortality falling < 20% within six months.ConclusionAnopheles arabiensis showed increasing pyrethroid resistance, driven largely by metabolic mechanisms. Standard LLINs showed suboptimal killing efficacy against field populations, while PBO LLINs achieved high baseline efficacy but declined significantly by six months. These findings indicate that PBO LLINs can improve protection against resistant vectors but may be insufficient in high-resistance areas, underscoring the need for alternative non-pyrethroid interventions, strategic deployment, and revised LLIN re-distribution cycles aligned with their functional lifespan.

BACKGROUND:Surveillance of molecular markers associated with antimalarial resistance in Plasmodium falciparum is critical for tracking the emergence, evolution, and spread of resistant malaria parasites in the population for timely and effective interventions. As shifting use of sulfadoxine-pyrimethamine (SP) in Kenya constitutes a differential selection pressure, this study compared resistance genotypes and haplotypes in P. falciparum isolates from endemic and epidemic regions of western Kenya. METHODS:A cross-sectional design was employed to collect blood samples from febrile patients residing in Ahero in Kisumu County, an endemic region, and Marani in Kisii County, an epidemic region. Molecular markers for antifolate resistance, dihydrofolate reductase (Pfdhfr) and dihydrofolate synthetase (Pfdhps), were genotyped for selected samples (N = 112) from Kisumu (n = 60) and Kisii (n = 52). Subsequent analysis was conducted for sequence polymorphisms, mutation frequency and haplotype prevalence. RESULTS:Genotyping of SP resistance markers identified 436H (28.8%), 437G (99%), and 540E (97.1%) in Pfdhps and 51I (100%), 59R (97.3%), and 108N (100%) in Pfdhfr as mutations that presented high frequency, with low multiplicity of infection (MOI) in both Kisumu (0.3196) and Kisii (0.2738). The double mutant SGEAA (70.18% in Kisumu vs. 51.06% in Kisii) and triple mutant HGEAA (26.31% vs. 44.68%) in Pfdhps, along with the triple mutant IRNI (86.67% vs. 98.08%) in Pfdhfr, exhibited significant regional differences in prevalence (p < 0.05). The Pfdhps-Pfdhfr haplotype analysis revealed a high prevalence of the quintuple mutant SGEAA-IRNI (57.89% in Kisumu vs. 48.94% in Kisii; p > 0.05) and a significantly higher prevalence of the sextuple mutant HGEAA-IRNI in Kisii compared to Kisumu (44.68% vs. 16.31%; p < 0.05). Comparatively, given that Pfdhps-516F and Pfdhfr-164L were the dominant alleles in Kisumu, while the Pfdhps-436H allele was dominant in Kisii, along with HGEAA and HGEAA-IRNI haplotypes (p < 0.05), they highlight regional variation in SP resistance genotypes and haplotypes. CONCLUSIONS:This study demonstrates that the fully resistant double Pfdhps-SGEAA and triple Pfdhfr-IRNI haplotypes have approached fixation in both endemic and epidemic regions, while the dominance of the 164L allele in the endemic region signals the emergence of super-resistance. These findings suggest a review of the therapeutic efficacy of SP and continuous surveillance of resistance due to the presence of fully resistant haplotype (SGEAA-IRNI) and super-resistant haplotype (F/HGEAA-IRNL) in the population of P. falciparum strains in western Kenya.

Malaria remains a leading cause of mortality in Kenya, despite concerted efforts in malaria vector control. Reducing outdoor transmission is a key factor in addressing residual malaria. Outdoor latrines are characterized as semi-sheltered structures with humid environments, which may provide an ideal resting site for Anopheles species to feed on human blood and subsequently rest. This study aimed to quantify the abundance of adult mosquitoes in houses and outdoor latrines, as well as explore the environmental factors associated with mosquito abundance. Monthly mosquito sampling was conducted in 50 houses and their corresponding outdoor latrines using Prokopack aspirators from July 2023 to April 2024. Household interviews were conducted concurrently to collect data on the number of individuals sleeping in the houses and the quantity of bednets used within the households. In addition, blood meal sources were identified through polymerase chain reaction (PCR) analysis of blood-fed mosquitoes collected from December 2023 to April 2024. Among anopheline species, the An. funestus group was the most common, followed by An. gambiae s.l. in both houses and latrines. In anophelines, the human blood index was 50.0

Malaria treatment interventions play a central role in the prevention and control of Plasmodium falciparum infections worldwide. However, the emergence and spread of resistant P. falciparum parasites pose a serious public health challenge, as they reduce the effectiveness of antimalarial drugs and contribute to an increased global malaria burden. This study conducted a comparative molecular surveillance of antimalarial drug resistance in P. falciparum isolates from the Lake endemic and highland epidemic-prone regions of western Kenya. A cross-sectional study design was employed to screen patients presenting with malaria-associated symptoms among residents of the lake-endemic region of Nyando sub-County in Kisumu County and the highland epidemic-prone region of Marani sub-County in Kisii County. Single-nucleotide polymorphisms (SNPs) were genotyped in the chloroquine resistance transporter (Pfcrt), multidrug resistance 1 (Pfmdr1), and Kelch 13 (Pfk13) genes to identify validated molecular markers associated with resistance to chloroquine, artemisinin derivatives, and artemisinin partner drugs. A comparative analysis was conducted to assess mutation frequencies, genetic diversity, and selection pressure on molecular markers associated with antimalarial drug resistance between the Lake-endemic region and the highland epidemic-prone region. Genotyping of molecular markers detected M74I, N75E, and K76T mutations each at a frequency of 5.4

Background Increasing urbanization in tropical Africa may create new niches for malaria vectors, potentially leading to higher disease transmission rates. Vector control efforts remain largely targeted at ecologically rural bio-complexities with multiple hosts. Understanding mosquito species composition, ecology, host diversity and biting behavior in urban areas is crucial for planning effective control. This study assessed mosquito species diversity, abundance, behavioral patterns, and Plasmodium sporozoite infection rates of Anopheles vectors along an urban-rural transect in Kisumu city, western Kenya. Methods Indoor and outdoor host-seeking and resting adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and mechanical aspirators (Prokopack) along an urban-rural transect. Females Anopheles mosquitoes collected were identified using morphological taxonomic keys to species level. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further processed using polymerase chain reaction (PCR) to identify members of each complex/group. Subsequently, sporozoite infection rates of the anopheline mosquitoes were determined using a multiplexed real-time quantitative PCR (qPCR) assay. Result A total of 3,394 female Anopheles mosquitoes were collected and identified. These comprised of An. gambiae s.l. (68%), An. funestus group (19.8%), An. coustani (7.8%), An. pharoensis (2.6%), An. maculipalipis (1.6%), and An. leesoni (0.2%). All six species were found in urban zone, but only three were found in peri-urban and rural sites. Overall, urban collections accounted for the majority of these collections (55.5%) of mosquitoes collected, followed by those from peri-urban (30%) and rural sites (14.5%). Species distribution across the three ecotypes showed Anopheles arabiensis was the dominant species in urban (84.3%) and peri-urban (89%) sites, while An. gambiae s.s. was predominantly found in the rural zone (60.2%) alongside An. arabiensis (39.7%). Anopheles funestus was the predominant species in peri-urban (98.4%) and rural (85.7%) areas, with An. leesoni accounted for 1.6% and 14.3%, respectively. In urban areas, all samples from the An. funestus group were identified as An. funestus s.s.. Majority (55.5%) of Anopheles mosquitoes were collected indoors, while secondary vectors were primarily caught outdoors. Overall, sporozoite rates were higher outdoors 3.5% compared to indoors 1.45% in rural areas. Specifically, sporozoite infectivity rates for An. funestus, An. gambiae s.s and An. arabiensis collected indoors in the rural zone was 2.5%, 1.4% and 1% respectively. Outdoors in rural areas, An. gambiae had a sporozoite rate of 5.3%, while An. arabiensis had a rate of 2.1%. In peri-urban areas An. gambiae had a sporozoite rate of 2.3%. No sporozoites were detected in samples from urban sites. Conclusion The study highlights a shift of diversity of Anopheles species towards urban areas with increased outdoor activity, and significant outdoor malaria transmission in rural and peri-urban areas, emphasizing the need for tools targeting outdoor-biting mosquitoes. The presence of An. funestus in urban settings is of interest and highlights the critical importance of sustained entomological surveillance to inform integrated vector control and prevent future transmission risks.

Anti-malaria interventions typically reduce the intensity of Plasmodium transmission, but the effects of reduced transmission on P. falciparum population biology remain unclear. Highly polymorphic microsatellite markers in P. falciparum were used to investigate genetic diversity, polyclonality and genetic structure among populations in areas of varying malaria transmission intensity across Kenya. We also assessed relationships between metrics derived from genetic data, transmission intensity estimates and bioclimatic variables. Despite an overall reduction in transmission intensity across Kenya from 2005 to 2014, we found that parasite populations maintained high genetic diversity and that genetic diversity correlated more closely with past transmission intensity estimates in the year 2000 as compared to contemporary estimates in 2014. In contrast, we found genetic structuring to be significant, consistent with our observation of shifting parasite migration patterns in western Kenya. Both genetic diversity and polyclonality increased with higher precipitation in the dry season, revealing the potential impacts of changing climate patterns on parasite population dynamics. Whereas fragmentation of P. falciparum populations increases opportunities for spatially targeted interventions in Kenya, the high genetic diversity of isolates in our study signals enhanced adaptability of parasites.

BACKGROUND:Long-lasting insecticidal nets (LLINs) are vital for malaria control in sub-Saharan Africa, but their durability is challenged by fabric decay and pyrethroid resistance. This study assessed the physical integrity and bioefficacy of piperonyl butoxide-LLINs (PBO-LLINs) and pyrethroid-only LLINs (pyrethroid-LLINs) after 1.5 years of use in western Kenya, where resistance is widespread. METHODS:A survey on net integrity and insecticide efficacy was conducted in randomly selected households (101-107 per group per visit) from three villages per net type group in Muhoroni Sub-County, Kisumu County. Physical integrity surveys were done after every six months while residual bio-efficacy was after every three months for 18 months. Physical integrity and residual bio-efficacy studies were conducted following WHO guidelines. RESULTS:PBO-LLINs exhibited higher physical integrity than pyrethroid-LLINs over time. At 18 months, 45.2% (61/135) of pyrethroid-LLINs and 21.8% (31/142) of PBO-LLINs were torn, with pHI values of 2494.1 ± 1696.4 and 1618.6 ± 1056.7, respectively. Net type, net age and house wall structures significantly influenced net integrity (p < 0.05). Torn nets were significantly more common in pyrethroid-LLIN households with mud-unplastered [OR=5.323 (95% CI = 1.685-16.816), p = 0.004] and corrugated iron walls [OR=6.31 (95% CI = 2.10-18.93), p < 0.001] and in PBO-LLIN households with mud-unplastered walls [OR=9.823 (95% CI = 1.487-64.898), p = 0.018]. Against the Kisumu susceptible Anopheles gambiae s.s, both net types decreased in mortality at baseline (when new) from 97.6% to 18.4% and 98.6% to 18.5% for pyrethroid and PBO-LLINs respectively at 18 months. Against a Bungoma pyrethroid-resistant Anopheles gambiae s.s, mosquito mortality with pyrethroid-LLINs declined from 36.9% when new to 6.8% at 18 months, while PBO-LLINs dropped from 55.6% to 11.8%. CONCLUSION:Both physical integrity and bioefficacy of LLINs declined significantly within 18 months. The findings demonstrate that not all nets in the field offer maximum protection by this time point, calling for net care education and further evaluation of PBO-LLINs especially in pyrethroid-resistant regions.

BACKGROUND:Pyrethroid-treated nets have reduced malaria in endemic areas, but insecticide resistance has hindered progress, prompting WHO to recommend piperonyl butoxide (PBO) based long-lasting insecticidal nets (LLINs). Kenya adopted PBO nets, but their usage is not well documented. This study aims to assess the uptake and effect of PBO nets versus pyrethroid-only LLINs (pyrethroid-LLINs) on malaria transmission in Western Kenya. METHODS:A cross-sectional study was conducted in Muhoroni Sub-County, Kisumu County, one year after net distribution between November and December 2023. Twelve villages were randomly selected, comprising 380 households and divided into two intervention arms of six villages. Group 1, with 181 households, received pyrethroid-LLIN, while Group 2, with 199 households, received PBO-LLINs. Data on net ownership and usage was collected using a standardized semi-structured questionnaire. Finger prick blood smears were collected on slides for microscopic examination, while dry blood spots (DBS) on filter paper were collected for real-time PCR (RT-PCR) diagnosis of Plasmodium infection in both intervention groups. Logistic regression was used to identify factors influencing net ownership and malaria prevalence, while a generalized linear model assessed factors affecting net usage. The χ² test was used to evaluate differences in demographic characteristics between the intervention arms. RESULTS:Overall, higher net ownership and usage was recorded in PBO-LLINs group compared to the pyrethroid-LLIN group after one year of net distribution. Among households, 89% in the pyrethroid-LLIN group and 96% in the PBO-LLIN group owned at least one net. Net usage was 80.2% in the pyrethroid-LLIN group and 87.6% in the PBO-LLIN group. Net attrition rate was 17.9% in pyrethroid-LLIN and 7.6% for PBO-LLIN group. Households with nets were 1.3 times more likely to use them [aOR=1.338 (95% CI = 1.224-1.462), p < 0.001). Bed ownership was 50.8% in the pyrethroid-LLIN group and 55.3% in the PBO-LLIN group. Not owning a bed decreased the likelihood of net usage by 13.3% [aOR=0.867 (95% CI = 0.816-0.920), p < 0.001]. Between the two groups, 75.7% of households in the pyrethroid-LLIN group and 66.8% in the PBO-LLIN group reported bedbug infestations in their homes. Bedbug infestation significantly affected net ownership (p = 0.018). Malaria parasite prevalence was 34.7% by qPCR in the pyrethroid-LLIN group and 29.0% in the PBO-LLIN group, with a prevalence ratio of 0.84 (95% CI: 0.65-1.03). CONCLUSION:The study observed higher bednet ownership, usage, and lower malaria prevalence in the PBO-LLIN clusters compared to the pyrethroid-LLIN clusters. However, bedbug infestations and insufficient sleeping beds hindered net ownership and usage, limiting their overall effectiveness. These findings highlight the need for National Malaria Control Programs (NMCPs) to enhance community education on proper net use and maintenance through targeted initiatives, such as household visits and local workshops. Furthermore, incorporating bedbug control strategies and regular surveillance will improve compliance and net usage. Addressing these challenges will maximize the impact of PBO-LLINs and other next-generation nets, strengthening malaria control and elimination efforts in both urban and rural settings.

After several decades in development, two malaria vaccines based on the same antigen and with very similar constructs and adjuvants, RTS,S/AS01 (RTS,S) and R21/Matrix-M (R21), were recommended by the WHO for widespread vaccination of children. These vaccines are much-needed additions to malaria control programs that, when used in conjunction with other control measures, will help to accelerate reductions in malaria morbidity and mortality. Although R21 is not yet available, RTS,S is currently being integrated into routine vaccine schedules in some areas. However, the efficacy of RTS,S is partial, short-lived, and varies widely according to age and geographic location. It is not clear why RTS,S induces protection in some individuals and not others, what the immune mechanisms are that favor protective immunity with RTS,S, and how immune mechanisms are influenced by host and environmental factors. Several studies suggest that higher levels of previous malaria exposure negatively impact RTS,S clinical efficacy. In this article, we summarize data suggesting that previous malaria exposures negatively impact the efficacy of RTS,S and other malaria vaccine candidates. We highlight recent evidence suggesting that increasing malaria exposure impairs the generation of functional antibody responses to RTS,S. Finally, we discuss how investigation of clinical and immune factors associated with suboptimal responses to RTS,S can be used to develop strategies to optimize RTS,S, which will remain relevant to R21 and next-generation vaccines.

BACKGROUND:The rise of insecticide resistance poses a growing challenge to the effectiveness of vector control tools, particularly in rural areas. However, the urban setting has received comparatively less focus despite its significance in attracting rural to urban migration. Unplanned urbanization, often overlooked, exacerbates insecticide resistance as Anopheles mosquitoes adapt to the polluted environments of rapidly expanding cities. This study aimed to assess the insecticide susceptibility status of malaria vectors and identify potential underlying mechanisms across three distinct ecological settings characterized by differing levels of urbanization in Kisumu County, Kenya. METHODS:The study was conducted in 2022-2023 in Kisumu County, western Kenya. Field-derived An. gambiae (s.l.) larvae collected from a long stretch of urban-to-rural continuum were phenotyped as either resistant or susceptible to six different insecticides using the World Health Organization (WHO) susceptibility test. Polymerase chain reaction (PCR) techniques were used to identify the species of the An. gambiae complex and screened for mutations at voltage-gated sodium channels (Vgsc-1014F, Vgsc-1014S, Vgsc-1575Y) and acetylcholinesterase (Ace1) target site mutation 119S. Metabolic enzyme activities (non-specific β-esterases and monooxygenases) were evaluated in mosquitoes not exposed to insecticides using microplate assays. Additionally, during larval sampling, a retrospective questionnaire survey was conducted to determine pesticide usage by the local inhabitants. RESULTS:Anopheles arabiensis dominated in urban (96.2%) and peri-urban (96.8%) areas, while An. gambiae (s.s.) was abundant in rural settings (82.7%). Urban mosquito populations showed high resistance intensity to deltamethrin (Mortality rate: 85.2% at 10x) and suspected resistance to Pirimiphos-methyl and bendiocarb while peri-urban and rural populations exhibited moderate resistance intensity to deltamethrin (mortality rate >98% at 10x). Preexposure of mosquitoes to a synergist piperonyl butoxide (PBO) significantly increased mortality rates: from 40.7% to 88.5% in urban, 51.9% to 90.3% in peri-urban, and 55.4% to 87.6% in rural populations for deltamethrin, and from 41.4% to 78.8% in urban, 43.7% to 90.7% in peri-urban, and 35% to 84.2% in rural populations for permethrin. In contrast, 100% mortality to chlorfenapyr and clothianidin was observed in all the populations tested. The prevalence of L1014F mutation was notably higher in urban An. arabiensis (0.22) unlike the peri-urban (0.11) and rural (0.14) populations while the L1014S mutation was more prevalent in rural An. gambiae (0.93). Additionally, urban An. arabiensis exhibited elevated levels of mixed function oxidases (0.8/mg protein) and non-specific esterases (2.12/mg protein) compared to peri-urban (0.57/mg protein and 1.5/mg protein, respectively) and rural populations (0.6/mg protein and 1.8/mg protein, respectively). Pyrethroids, apart from their use in public health through LLINs, were being highly used for agricultural purposes across all ecological settings (urban 38%, peri-urban 36% and rural 37%) followed by amidine group, with organophosphates, neonicotinoids and carbamates being of secondary importance. CONCLUSION:These findings show high resistance of An. arabiensis to insecticides commonly used for vector control, linked with increased levels of detoxification enzymes. The observed intensity of resistance underscores the pressing issue of insecticide resistance in urban areas, potentially compromising the effectiveness of vector control measures, especially pyrethroid-treated LLINs. Given the species' unique behavior and ecology compared to An. gambiae, tailored vector control strategies are needed to address this concern in urban settings.

Background Understanding of malaria ecology is a prerequisite for designing locally adapted control strategies in resource-limited settings. The aim of this study was to utilize the spatial heterogeneity in malaria transmission for the designing of adaptive interventions. Methods Field collections of clinical malaria incidence, asymptomatic Plasmodium infection, and malaria vector data were conducted from 108 randomly selected clusters which covered different landscape settings including irrigated farming, seasonal flooding area, lowland dryland farming, and highlands in western Kenya. Spatial heterogeneity of malaria was analyzed and classified into different eco-epidemiological zones. Results There was strong heterogeneity and detected hot/cold spots in clinical malaria incidence, Plasmodium prevalence, and vector abundance. The study area was classified into four zones based on clinical malaria incidence, parasite prevalence, vector density, and altitude. The two irrigated zones have either the highest malaria incidence, parasite prevalence, or the highest malaria vector density; the highlands have the lowest vector density and parasite prevalence; and the dryland and flooding area have the average clinical malaria incidence, parasite prevalence and vector density. Different zones have different vector species, species compositions and predominant species. Both indoor and outdoor transmission may have contributed to the malaria transmission in the area. Anopheles gambiae sensu stricto ( s.s. ), Anopheles arabiensis , Anopheles funestus s.s. , and Anopheles leesoni had similar human blood index and malaria parasite sporozoite rate. Conclusion The multi-transmission-indicator-based eco-epidemiological zone classifications will be helpful for making decisions on locally adapted malaria interventions.

Background Timely molecular surveillance of Plasmodium falciparum kelch 13 ( k13 ) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022. Methods Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected. Results Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li’s F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%). Conclusions This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.

Background In western Kenya, not all malaria cases are reported as stipulated in the community case management of malaria (CCMm) strategy. This underreporting affects the equity distribution of malaria commodities and the evaluation of interventions. The current study aimed to evaluate the effectiveness of community health volunteers’ active case detection and management of malaria in western Kenya. Methods Cross-sectional active case detection (ACD) of malaria survey was carried out between May and August 2021 in three eco-epidemiologically distinct zones in Kisumu, western Kenya: Kano Plains, Lowland lakeshore and Highland Plateau. The CHVs conducted biweekly ACD of malaria household visits to interview and examine residents for febrile illness. The Community Health Volunteers (CHVs) performance during the ACD of malaria was observed and interviews done using structured questionnaires. Results Of the total 28,800 surveyed, 2597 (9%) had fever and associated malaria symptoms. Eco-epidemiological zones, gender, age group, axillary body temperature, bed net use, travel history, and survey month all had a significant association with malaria febrile illness (p < 0.05). The qualification of the CHV had a significant influence on the quality of their service. The number of health trainings received by the CHVs was significantly related to the correctness of using job aid (χ 2 = 6.261, df = 1, p = 0.012) and safety procedures during the ACD activity (χ 2 = 4.114, df = 1, p = 0.043). Male CHVs were more likely than female CHVs to correctly refer RDT-negative febrile residents to a health facility for further treatment (OR = 3.94, 95% CI = 1.85–5.44, p < 0.0001). Most of RDT-negative febrile residents who were correctly referred to the health facility came from the clusters with a CHV having 10 years of experience or more (OR = 1.29, 95% CI = 1.05–1.57, p = 0.016). Febrile residents in clusters managed by CHVs with more than 10 years of experience (OR = 1.82, 95% CI = 1.43–2.31, p < 0.0001), who had a secondary education (OR = 1.53, 95% CI = 1.27–1.85, p < 0.0001), and were over the age of 50 (OR = 1.44, 95% CI = 1.18–1.76, p < 0.0001), were more likely to seek malaria treatment in public hospitals. All RDT positive febrile residents were given anti-malarial by the CHVs, and RDT negatives were referred to the nearest health facility for further treatment. Conclusions The CHV’s years of experience, education level, and age had a significant influence on their service quality. Understanding the qualifications of CHVs can assist healthcare systems and policymakers in designing effective interventions that assist CHVs in providing high-quality services to their communities.

Background The rise of insecticide resistance against malaria vectors in sub-Saharan Africa has resulted in the need to consider other methods of vector control. The potential use of biological methods, including larvivorous fish, Bacillus thuringiensis israelensis (Bti) and plant shading, is sustainable and environmentally friendly options. This study examined the survivorship of Anopheles arabiensis and Anopheles funestus larvae and habitat productivity in four permanent habitat types in Homa Bay county, western Kenya. Methods Predator densities were studied in a laboratory setup while habitat productivity and larval survivorship was studied in field setup. Results Fish were observed as the most efficient predator (75.8% larval reduction rate) followed by water boatman (69%), and dragonfly nymph (69.5%) in predation rates. Lower predation rates were observed in backswimmers (31%), water beetles (14.9%), water spiders (12.2%), mayflies (7.3%), and tadpoles (6.9%). Increase in predator density in the field setup resulted in decreased Culex larval density. Larval and pupa age–specific distribution was determined and their survivorship curves constructed. Combined larvae (Stage I–IV) to pupa mortality was over 97% for An. arabiensis and 100% for An. funestus . The highest larval stage survival rate was from larval stages I to II and the lowest from larval stage IV to pupa. Stage-specific life tables indicated high mortality rates at every developmental stage, especially at the larval stage II and III. Conclusion Determination of the efficiency of various larval predators and habitat productivity will help with the correct identification of productive habitats and selection of complementary vector control methods through environmental management and/or predator introduction (for instance fish) in the habitats.

Abstract Background Despite the substantial reduction of under-five mortality (U5M) across the world, about 15,000 under-fives (U5s) die daily. Majority occurs in developing countries with Sub-Saharan African countries like Kenya accounting for over 50%. In Kenya, the former Nyanza Province in western region exhibits the highest U5MR of 82 deaths/1000 live births. Karemo Sub-County in Nyanza Province displays twice as much U5MR as Nyanza Province despite having relatively good access to private and public health facilities. Moreover, the influence of maternal risk factors on U5M remains unknown. Methodology: The current cross-sectional study assessed the influence of maternal risk factors associated with U5M in Karemo Sub-County in Siaya County in 299 households of women of reproductive age (15–49 years). Data was collected using pre-tested structured questionnaire on a face-to-face interview. Differences between proportions were determined using Chi-square test while binary logistic regression was used to determine the association between maternal factors and U5M. Results Results show that children of older mothers (35–49 years) had increased mortality relative to those of younger mothers (< 20 years) (OR = 2.484; 95%CI: 1.249–4.940; P = 0.009). Short preceding birth interval of (< 2 years) was associated with increased risk of U5M relative to longer preceding birth intervals (> 2 years) (OR = 2.079; 95%CI: 1.240–3.485; P = 0.005). Relative to primary education level, children born to mothers who had attained tertiary level had reduced risk of U5M (OR = 0.408; 95%CI: 0.208-0.800; P = 0.009). Higher birth orders of 4 and above were five times more associated withU5M compared to lower birth orders (OR = 5.442; 95%CI: 2.789–10.620; P < 0.0001). Polygamy was associated with increased risk of U5M (OR = 3.370; 95%CI: 1.922–5.910; P < 0.0001). Similarly, lower death rates of about 61% were observed among children of married mothers compared to those of single mothers (OR = 0.382; 95%CI: 0.218–0.669; P = 0.001). Employment, subsistent farmers and small-scale business mothers showed increased risk to U5M (OR = 3.505, 95%CI: 1.657–7.414; P = 0.001), 2.1 (OR = 2.196; 95%CI: 1.190–4.053; P = 0.012) and 5.6 (OR = 5.639; 95%CI: 2.871–11.077; P < 0.0001), respectively. Conclusion Strategies targeting promotion of girl-child education above primary levels, women economic empowerment and acceptance of family planning are considered fundamental interventions and should be emphasized in reducing U5M in this region.

Gravid Anopheles malaria vectors depend on both chemical and physical (including microbial) cues for selection of preferred habitats for oviposition. This study focused on assessing the effects of bacterial composition and habitat metabolites on malaria vector larval availability in irrigated and non-irrigated potential larval sources. Water samples were collected from larval positive and negative habitats in the irrigated and non- irrigated areas of Homa Bay county. Bacteria cultured from the water samples were subjected to Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for species identification. DNA was extracted from the colonies and polymerase chain reaction (PCR) and sequencing done. Finally, the metabolite composition of larval positive and negative habitats was determined. MALDI-TOF MS results revealed that Bacillus was the only genera identified from larval sources in the non-irrigated zone. In the irrigated area, Shigella was the dominant genera (47%) while Escherichia coli was the abundant species (13/51). Of the sequenced isolates, 65% were Bacillus. Larvicidal isolates Brevibacillus brevis, Bacillus subtilis, and Exiguobacterium profundum were isolated and grouped with Bacillus mojavensis, Bacillus tequilensis, Bacillus stercoris, and Brevibacillus agri. Irrigated areas with larvae had reduced crude fat (0.01%) and protein content (0.13%) in comparison to those without larvae. In irrigated and non- irrigated areas, larval presence was evident in habitats with high total chlorophyll content (1.12 μg/g vs 0.81μg/g and 3.37 μg/g vs 0.82). Aquatic habitats with larvae in both irrigated and non-irrigated areas exhibited higher sugar concentration than habitats without larvae; however, when compared, non-irrigated areas with larvae had higher sugar concentration than similar habitats in irrigated areas. In addition, substantial concentrations of Manganese, Calcium, and Copper were found in aquatic habitats containing larvae in both irrigated and non-irrigated areas. These results allow for prospective examination as potential larvicidal or adulticidal agents and could be considered when designing potential vector control interventions.

Knowledge of insect dispersal is relevant to the control of agricultural pests, vector-borne transmission of human and veterinary pathogens, and insect biodiversity. Previous studies in a malaria endemic area of the Sahel region in West Africa revealed high-altitude, long-distance migration of insects and various mosquito species. The objective of the current study was to assess whether similar behavior is exhibited by mosquitoes and other insects around the Lake Victoria basin region of Kenya in East Africa. Insects were sampled monthly from dusk to dawn over 1 year using sticky nets suspended on a tethered helium-filled balloon. A total of 17,883 insects were caught on nets tethered at 90, 120, and 160 m above ground level; 818 insects were caught in control nets. Small insects (<0.5 cm, n = 15,250) were predominant regardless of height compared with large insects (>0.5 cm, n = 2,334) and mosquitoes (n = 299). Seven orders were identified; dipteran was the most common. Barcoding molecular assays of 184 mosquitoes identified 7 genera, with Culex being the most common (65.8%) and Anopheles being the least common (5.4%). The survival rate of mosquitoes, experimentally exposed to high-altitude overnight, was significantly lower than controls maintained in the laboratory (19% vs. 85%). There were no significant differences in mosquito survival and oviposition rate according to capture height. These data suggest that windborne dispersal activity of mosquito vectors of malaria and other diseases occurs on a broad scale in sub-Saharan Africa.

Several sub-Saharan African countries rely on irrigation for food production. This study examined the impact of environmental modifications resulting from irrigation on the ecology of aquatic stages of malaria vectors in a semi-arid region of western Kenya. Mosquito larvae were collected from irrigated and non-irrigated ecosystems during seasonal cross-sectional and monthly longitudinal studies to assess habitat availability, stability, and productivity of anophelines in temporary, semipermanent, and permanent habitats during the dry and wet seasons. The duration of habitat stability was also compared between selected habitats. Emergence traps were used to determine the daily production of female adult mosquitoes from different habitat types. Malaria vectors were morphologically identified and sibling species subjected to molecular analysis. Data was statistically compared between the two ecosystems. After aggregating the data, the overall malaria vector productivity for habitats in the two ecosystems was estimated. Immatures of the malaria vector (Anopheles arabiensis) Patton (Diptera: Culicidae) comprised 98.3% of the Anopheles in both the irrigated and non-irrigated habitats. The irrigated ecosystem had the most habitats, higher larval densities, and produced 85.8% of emerged adult females. These results showed that irrigation provided conditions that increased habitat availability, stability, and diversity, consequently increasing the An. arabiensis production and potential risk of malaria transmission throughout the year. The irrigated ecosystems increased the number of habitats suitable for Anopheles breeding by about 3-fold compared to non-irrigated ecosystems. These results suggest that water management in the irrigation systems of western Kenya would serve as an effective method for malaria vector control.