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Abstract Background Ethiopia continues to grapple with a persistent malaria burden, characterized by ongoing transmission and recurrent outbreaks. Human behavior influences both malaria exposure and the effectiveness of vector interventions, complicating malaria control efforts. Implementing tailored strategies that account for the complex interplay between human activities and vector behavior remains a challenge in both high- and low-transmission areas in Ethiopia, particularly for vulnerable highland populations and temporary labor migrants, due to lack of data. The aim of this study was to examine the spatiotemporal patterns of human—mosquito interactions and evaluate the effectiveness and suitability of long-lasting insecticidal nets (LLINs) in settings involving lowland resident populations, seasonal migrant workers and highland communities. Methods Concurrent human and vector behavior data were collected from high-transmission lowlands (residents and temporary migrant workers) and vulnerable highlands populations. Hourly human behavior observations (HBOs), which examined LLIN use, indoor versus outdoor human presence and sleeping patterns, were paired in a crossover design with mosquito sampling using US Centers for Disease Control light traps (CDC LT) as a proxy for mosquito biting behavior. The study was conducted during the peak (October–December 2022) and minor (March–May 2023) malaria transmission seasons (‘peak’ and ‘minor’) for a total of 368 nights. In the highlands, four villages consisting of eight households per village were selected for surveillance; in the lowlands, four villages consisting of two resident villages and two farm sites with migrant workers, with eight households/structures per village or farm, were used for data collection. CDC LT and HBO data were integrated to evaluate HBO-adjusted human biting rates (HBO-adjusted HBR) of Anopheles mosquitoes. Results In the highland villages, residents predominantly engaged in indoor activities, with their peak activity overlapping with the peak biting hours (1800-2200 hours). A substantial proportion of inhabitants slept indoors without LLINs in the peak and minor seasons (42.8% and 39.2%, respectively). Highland residents were significantly more exposed to malaria vectors indoors (88.4% peak, 88.6% minor) than outdoors during both transmission seasons. In lowland villages, both resident and seasonal migrant worker populations exhibited predominantly outdoor activity, particularly during peak biting hours (1800-2300 hours). Both residents and temporary migrants were significantly more exposed to Anopheles mosquitoes outdoors (resident: 65.0% peak, 67.1% minor; migrant: 70.5% peak, 80.0% minor) than indoors during both transmission seasons. LLIN usage was minimal and offered limited protection, with < 16.63% of person-time spent under nets by resident populations and 10.7% by migrant workers. Conclusions Malaria control in Ethiopia requires context-specific strategies tailored to diverse ecological settings that consider the impact of human behavior on exposure to Anopheles mosquitoes. Limited LLIN effectiveness, human activities coinciding with peak biting times and minimal LLIN usage create significant protection gaps. Comprehensive control necessitates supplemental tools addressing exposure in all locations and times. In the Ethiopian highlands, where indoor activities predominate, increased LLIN usage combined with targeted indoor residual spraying could reduce transmission. In lowland areas, both residents and seasonal migrant workers face relatively higher outdoor exposure risks, requiring additional measures, such as topical and spatial repellents. We recommend implementing data-driven, hyperlocal approaches based on specific human—vector interactions to enhance malaria control effectiveness across the Ethiopian highlands and lowlands. Graphical Abstract

Abstract Background The Greater Mekong Subregion (GMS) aims to eliminate all human malaria by 2030 and is making substantial progress toward this goal, with malaria increasingly confined to forest foci. These transmission foci are predominantly inhabited by ethnic minorities, local populations, and rural mobile and migrant populations working in mining and agriculture. The recommendations of the World Health Organization (WHO) on malaria elimination states that small population groups which constitute a large proportion of the malaria transmission reservoir should benefit from targeted strategies to reduce transmission overall. These population groups are exposed to malaria vector bites during the day due to Anopheles daytime biting, and during the night, due to low bed net use and open sleeping structures. Such characteristics limit the effectiveness of the WHO core vector control strategies [indoor residual spraying (IRS), insecticide-treated nets (ITNs)], which target indoor resting and indoor feeding mosquitoes. Interventions that target daytime and outdoor resting or biting mosquitoes, and which complement IRS and ITNs and drug strategies, may hasten a decline in the malaria burden. Methods This study evaluated two transfluthrin- and one metofluthrin-based volatile pyrethroid spatial repellents (VPSRs), and etofenprox insecticide-treated clothing (ITC) with and without a topical repellent in a semi-field system (SFS) at two research sites in Thailand, across two trial rounds. The study estimated the protective efficacies of the vector control tools against two pyrethroid-susceptible Anopheles minimus strains in the form of 15 interventions, including a combined VPSR and ITC intervention. The interventions’ modes of action were studied by measuring their impact on mosquito landing, and on key life history traits known to affect vectoral capacity (knockdown, post-exposure blood feeding, and 24-h mortality) using a block-randomized crossover design. The odds ratio (OR) for each intervention compared to the control on each outcome was estimated. Results All interventions substantially reduced An. minimus landings and prevented more than 50% mosquito landings when new (VPSRs) or unwashed (treated clothing). In addition to landing reduction, all interventions decreased post-exposure blood feeding, induced knockdown and increased mortality at 24 h. The VPSR interventions were generally more protective against landing than the treated clothing intervention. The combined intervention (VPSR + ITC) provided the greatest protection overall. Conclusion This SFS evaluation indicates an effect of these VPSR and ITC interventions in reducing An. minimus landing for the user, and indicates their potential for community protection by secondary modes of action. This study demonstrates the utility of SFS trials in the evaluation of bite prevention tools and emphasizes the need for multiple evaluations at different sites. It also highlights possible sources of biases observed, including the measuring of mosquito landing rather than biting, weather parameters, and low mosquito recapture.

Abstract Cambodia’s goal to eliminate malaria by 2025 is challenged by persistent transmission in forest and forest fringe areas, where people are exposed to Anopheles mosquito bites during the day and night. Volatile pyrethroid spatial repellents (VPSRs) and insecticide-treated clothing (ITC) could address these gaps. This study evaluated the outdoor application of one passive transfluthrin-based VPSR, four etofenprox-ITCs paired with a picaridin topical repellent, and a combination of VPSR and ITC against wild Anopheles landing in Cambodia. A 7 × 7 Latin-square study was conducted over 49 collection nights in temporary open structures in Mondulkiri Province. All interventions substantially reduced Anopheles landing, with protective efficacy ranging from 61 to 95%. Mathematical modeling showed significant reductions in vectoral capacity, especially with the combined ITC and VPSR and VPSR alone, albeit with decreased effectiveness over time. These interventions have the potential to reduce outdoor and daytime Anopheles biting, offering valuable contributions to malaria elimination efforts in Cambodia and the Greater Mekong Subregion, contingent upon achieving effective coverage and adherence.

Abstract Background Pyrethroid-based indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been employed as key vector control measures against malaria in Namibia. However, pyrethroid resistance in Anopheles mosquitoes may compromise the efficacy of these interventions. To address this challenge, the World Health Organization (WHO) recommends the use of piperonyl butoxide (PBO) LLINs in areas where pyrethroid resistance is confirmed to be mediated by mixed function oxidase (MFO). Methods This study assessed the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to WHO tube bioassays with 4% DDT and 0.05% deltamethrin insecticides. Additionally, the study explored the effect of piperonyl butoxide (PBO) synergist by sequentially exposing mosquitoes to deltamethrin (0.05%) alone, PBO (4%) + deltamethrin (0.05%), and PBO alone. The Anopheles mosquitoes were further identified morphologically and molecularly. Results The findings revealed that An. gambiae sensu stricto (s.s.) (62%) was more prevalent than Anopheles arabiensis (38%). The WHO tube bioassays confirmed resistance to deltamethrin 0.05% in the Oshikoto, Kunene, and Kavango West regions, with mortality rates of 79, 86, and 67%, respectively. In contrast, An. arabiensis displayed resistance to deltamethrin 0.05% in Oshikoto (82% mortality) and reduced susceptibility in Kavango West (96% mortality). Notably, there was reduced susceptibility to DDT 4% in both An. gambiae s.s. and An. arabiensis from the Kavango West region. Subsequently, a subsample from PBO synergist assays in 2020 demonstrated a high proportion of An. arabiensis in Oshana (84.4%) and Oshikoto (73.6%), and 0.42% of Anopheles quadriannulatus in Oshana. Non-amplifiers were also present (15.2% in Oshana; 26.4% in Oshikoto). Deltamethrin resistance with less than 95% mortality, was consistently observed in An. gambiae s.l. populations across all sites in both 2020 and 2021. Following pre-exposure to the PBO synergist, susceptibility to deltamethrin was fully restored with 100.0% mortality at all sites in 2020 and 2021. Conclusions Pyrethroid resistance has been identified in An. gambiae s.s. and An. arabiensis in the Kavango West, Kunene, and Oshikoto regions, indicating potential challenges for pyrethroid-based IRS and LLINs. Consequently, the data highlights the promise of pyrethroid-PBO LLINs in addressing resistance issues in the region.

Abstract Background The mosquito landing rate measured by human landing catches (HLC) is the conventional endpoint used to evaluate the impact of vector control interventions on human-vector exposure. Non-exposure based alternatives to the HLC are desirable to minimize the risk of accidental mosquito bites. One such alternative is the human-baited double net trap (HDN), but the estimated personal protection of interventions using the HDN has not been compared to the efficacy estimated using HLC. This semi-field study in Sai Yok District, Kanchanaburi Province, Thailand, evaluates the performance of the HLC and the HDN for estimating the effect on Anopheles minimus landing rates of two intervention types characterized by contrasting modes of action, a volatile pyrethroid spatial repellent (VSPR) and insecticide-treated clothing (ITC). Methods Two experiments to evaluate the protective efficacy of (1) a VPSR and (2) ITC, were performed. A block randomized cross-over design over 32 nights was carried out with both the HLC or HDN. Eight replicates per combination of collection method and intervention or control arm were conducted. For each replicate, 100 An. minimus were released and were collected for 6 h. The odds ratio (OR) of the released An. minimus mosquitoes landing in the intervention compared to the control arm was estimated using logistic regression, including collection method, treatment, and experimental day as fixed effects. Results For the VPSR, the protective efficacy was similar for the two methods: 99.3%, 95% CI (99.5–99.0) when measured by HLC, and 100% (100, Inf) when measured by HDN where no mosquitoes were caught (interaction test p = 0.99). For the ITC, the protective efficacy was 70% (60–77%) measured by HLC but there was no evidence of protection when measured by HDN [4% increase (15–27%)] (interaction test p

Abstract Background Although most of Panamá is free from malaria, localized foci of transmission persist, including in the Guna Yala region. Government-led entomological surveillance using an Entomological Surveillance Planning Tool (ESPT) sought to answer programmatically relevant questions on local entomological drivers of transmission and gaps in protection to guide local vector control decision-making. Methods The ESPT was used to design a sampling plan to answer priority programmatic questions about the appropriateness of Long Lasting Insecticidal Nets (LLINs) and spaces and times where humans remain exposed to Anopheles bites (gaps in protection) in the communities of Permé and Puerto Obaldía, Guna Yala. Adult Anopheles were sampled at three time points via human landing catches (HLCs) during the rainy and dry seasons (2018/2019). Human behaviour observations (HBOs) were conducted alongside HLCs to examine intervention use, indoor versus outdoor activity, and sleeping patterns. HLC and HBO data were integrated to evaluate HBO-adjusted human biting rate (HBR). Results A total of 7,431 adult Anopheles were collected across both sites. Of the 450 specimens molecularly confirmed to species-level, 75.5% (n = 340) were confirmed as Anopheles Nyssorhynchus albimanus, followed by Anopheles (Ny.) aquasalis. Anopheles host seeking activity was demonstrated to be primarily exophagic throughout all sampling periods and in both communities. When adjusted with HBOs, exposure to mosquito bites was predominantly indoors and overnight in Permé (Nov, Mar), compared to predominantly outdoors in Puerto Obaldía (Nov, Mar, Jul). Differences in site-specific human-vector exposure profiles were due to contrasting cultural and lifestyle practices between Permé and Puerto Obaldía (possibly partly influenced by the absence of electricity in Permé), and lower LLIN use in Permé. This evidence supported a previously planned LLIN campaign alongside a social behaviour change communication (SBCC) strategy in the Guna Yala Comarca (Jul 2019), which increased LLIN use. In turn, this led to a reduction of indoor exposure to mosquito bites, and a shift to predominant outdoor exposure to mosquito bites. Conclusion ESPT-based question-driven planning and the integration of HBOs, intervention, and HLC data generated evidence towards answering the programmatic questions. This evidence enabled the characterization of site-specific human-vector exposure profiles, and the quantification of remaining gaps in protection. These data also provide important insights into remaining gaps in protection that must be addressed to further reduce human exposure to mosquito bites at these sites.

RESUMEN Antecedentes Aunque la mayor parte de Panamá está libre de malaria, aun persisten focos de transmisión localizados, dentro de ellos la región de Guna Yala. La vigilancia entomológica liderada por el gobierno utilizando la Herramienta de Planificación de Vigilancia Entomológica (HPVE), tuvo como objetivo responder a preguntas programáticas relevantes para lograr una mejor comprensión de los factores entomológicos de transmisión locales y las brechas en la protección que, repercuten en la transmisión persistente de la malaria para guiar la toma de decisiones de control de vectores. Métodos En las localidades de Permé y Puerto Obaldía dentro de la región de Guna Yala, Panamá, se recolectaron Anopheles adultos en tres intervalos de tiempo fijos utilizando Capturas de Cebo Humano (CCH), Capturas con Trampas de Luz CDC (TL-CDC) y Capturas con Aplicación de Piretroides (CAP) durante las temporadas lluviosas y secas. Los mosquitos fueron identificados a nivel de especie mediante métodos moleculares. Se realizaron pruebas de susceptibilidad al fenitrotión en la especie principal del vector. Resultados En total, se recolectaron 7.537 adultos de Anopheles en ambos sitios. De los 493 especímenes con confirmación a nivel de especie, dos tercios (n = 340) fueron identificados como Nyssorhynchus albimanus, seguido de Anopheles aquasalis. En general, las tasas de picadura humanas (TPH)) de Anopheles fueron más altas en exteriores que en interiores, y fueron más altas en Permé que en Puerto Obaldía: la TPH nocturna en el exterior osciló entre 2,71 picaduras por persona por noche (ppn) (Puerto Obaldía) y 221,00 ppn (Permé), mientras que la TPN nocturna en el interior osciló entre 0,70 ppn (Puerto Obaldía) y 81,90 ppn (Permé). Generalmente, el punto más alto de picaduras se produjó durante las primeras horas de la noche. El rendimiento de las trampas de luz CDC fue significativamente más bajo, que el de las CCH. Por lo anterior, la TL CDC fue removida como método de captura después de la primera ronda de colectas. Las CAP dieron como resultado solo tres Anopheles en reposo en interiores recolectados. La resistencia de Ny. albimanus al fenitrotión fue confirmada, con solo 65,5% de mortalidad en el tiempo diagnóstico. Conclusión El comportamiento exofágico y vespertino del vector Anopheles, la ausencia de comportamiento de reposo en interiores y la presencia de resistencia al insecticida de intervención primaria, demuestran las limitaciones de la estrategia actual contra la malaria, incluyendo el Rociado Residual Intradomiciliario (RRI) y los Mosquiteros Impregnados con Insecticida de Larga Duración (MTILD) y marcan tanto las brechas en la protección como los factores que promueven la transmisión persistente de la malaria en Guna Yala. Estos hallazgos destacan la necesidad de una vigilancia entomológica continua y dirigida, basada en preguntas programáticas, que genere evidencia entomológica para informar una estrategia adaptativa de eliminación de la malaria.