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Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found in 75.3% of water sources surveyed and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa.

Background Anopheles stephensi , an invasive malaria vector, was first detected in Africa nearly 10 years ago. After the initial finding in Djibouti, it has subsequently been found in Ethiopia, Sudan and Somalia. To better inform policies and vector control decisions, it is important to understand the distribution, bionomics, insecticide susceptibility, and transmission potential of An. stephensi . These aspects were studied as part of routine entomological monitoring in Ethiopia between 2018 and 2020. Methods Adult mosquitoes were collected using human landing collections, pyrethrum spray catches, CDC light traps, animal-baited tent traps, resting boxes, and manual aspiration from animal shelters. Larvae were collected using hand-held dippers. The source of blood in blood-fed mosquitoes and the presence of sporozoites was assessed through enzyme-linked immunosorbent assays (ELISA). Insecticide susceptibility was assessed for pyrethroids, organophosphates and carbamates. Results Adult An. stephensi were collected with aspiration, black resting boxes, and animal-baited traps collecting the highest numbers of mosquitoes. Although sampling efforts were geographically widespread, An. stephensi larvae were collected in urban and rural sites in eastern Ethiopia, but An. stephensi larvae were not found in western Ethiopian sites. Blood-meal analysis revealed a high proportion of blood meals that were taken from goats, and only a small proportion from humans. Plasmodium vivax was detected in wild-collected An. stephensi . High levels of insecticide resistance were detected to pyrethroids, carbamates and organophosphates. Pre-exposure to piperonyl butoxide increased susceptibility to pyrethroids. Larvae were found to be susceptible to temephos. Conclusions Understanding the bionomics, insecticide susceptibility and distribution of An. stephensi will improve the quality of a national response in Ethiopia and provide additional information on populations of this invasive species in Africa. Further work is needed to understand the role that An. stephensi will have in Plasmodium transmission and malaria case incidence. While additional data are being collected, national programmes can use the available data to formulate and operationalize national strategies against the threat of An. stephensi .

Targeted amplicon sequencing is a powerful and efficient tool for interrogating the Plasmodium falciparum genome, generating actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive, and reproducible. We developed, characterized, and implemented MAD 4 HatTeR, an amplicon sequencing panel based on Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing, along with a bioinformatic pipeline for data analysis. Additionally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. Laboratory control and field samples were used to demonstrate the panel’s high sensitivity and robustness. MAD 4 HatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes, key markers for drug and diagnostic resistance (including duplications and deletions), and CSP and potential vaccine targets. The panel can also detect non- falciparum Plasmodium species. MAD 4 HatTeR successfully generated data from low-parasite-density dried blood spot and mosquito midgut samples and detected minor alleles at within-sample allele frequencies as low as 1% with high specificity in high-parasite-density dried blood spot samples. Gene deletions and duplications were reliably detected in mono- and polyclonal controls. Data generated by MAD 4 HatTeR were highly reproducible across multiple laboratories. The successful implementation of MAD 4 HatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MAD 4 HatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.

Background Plant-based mosquito control methods may use as a supplementary malaria vector control strategy. This study aimed to evaluate the effect of smoking ethno-medicinal plants on indoor density and feeding activity of malaria vectors at early hours of the night and its residual effect after midnight in southern Ethiopia. Methods Both field and tent trials were conducted to evaluate the impact of smoking Juniperus procera leaves , Eucalyptus globulus seeds and Olea europaea leaves in Kolla Shara Village from July 2016 to February 2017. For the field trial, five grass-thatched traditional huts (three for ethno-medicinal plants and two as control [only charcoal smoking and non-charcoal smoking]) were used. Indoor host-seeking mosquitoes were collected by CDC light traps. A Latin square design was employed to minimize the bias due to the variation in house location and different sampling nights. For the tent experiment, 25 3–5-day-old starved wild female Anopheles mosquitoes reared from the larvae were released into the tents where a calf was tethered at the mid-point of each tent. Results A total of 614 Anopheles mosquitoes belonging to 5 species were collected from 5 huts, of which 93.4% was An. arabiensis ; O. europaea, E. globulus and J. procera reduced the indoor density of An. arabiensis , with the mean percentage drop of 80%, 73% and 70%, respectively. In the tent trial, smoking of these plants had significant knockdown effects and inhibited feeding on the calves ( F  = 383.5, DF = 3, P  < 0.01). The mean knockdown effect due to O. europaea was relatively high (17.7 ± 0.54; 95% CI 16.8–18.6), while it was only 0.9 ± 0.1 (95% CI 0.29–1.52) in the control tents. All the test plants used in the tent trial caused significantly inhibited feeding activity of An. arabiensis on the host ( F  = 383.5, DF = 3, P  < 0.01). About 94.5%, 89.5% and 86% of mosquitoes were unfed because of the smoking effect of O. europaea , E. globulus and J. procera , respectively, whereas only 19.5% were unfed in the control tent. Conclusions Smoking ethno-medicinal plant materials reduced indoor density of malaria vectors and inhibited feeding on calves inside the tents. Thus, plant-based mosquito control methods may play a vital role in reducing mosquito bites in the early hours of the night and thereby reduce residual malaria transmission. Graphical 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

Background Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. Methods Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households’ exposure to An. stephensi . Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. Results Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75–100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi , ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9–100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi , and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis . None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax ). Conclusions This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission. Graphical Abstract

Seasonal movement of less-immune people from low- to high- transmission regions increase malaria risk and may introduce parasite strains to both areas. This study examined Plasmodium falciparum genetic diversity and connectivity between low-transmission highlands and endemic lowlands in Ethiopia to assess the contribution of seasonal agricultural migration in sustaining transmission. P. falciparum qPCR-positive dried blood spots collected from highland health facilities and lowland agricultural worksites were sequenced using multiplexed amplicon sequencing. Complexity of infection (COI) and infection pairwise relatedness were estimated and used for clustering analysis. Lowland populations (seasonal workers and residents) had higher COI and polyclonal infection rates (mean COI 2.62, 62%, n = 581) than highland residents (mean COI 2.00, 40%, n = 599). Similar expected heterozygosity (He ≈ 0.4) was observed, and P. falciparum infections from worksites showed high genetic connectivity between highland and lowland populations, with extensive parasite sharing, including 27 related clusters in highland cases and 10 in seasonal workers. Integrating parasite genomic data with epidemiological information revealed strong connectivity and low genetic differentiation between these regions linked by seasonal migration. These findings highlight how agricultural mobility likely drives parasite diversity and gene flow, implicating its role in sustaining malaria transmission.

Background In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s mitochondrial (mt) cytochrome oxidase I ( COX-I ) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods A PCR-based method targeting the Plasmodium mt COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection time points (days 0.5, 1, 6, 9, 12, 15). The head and thorax and the abdomen for each specimen were tested separately with each method. Agreement between methods at each infection stage was measured using Cohen’s kappa measure of test association. Results Infection status of mosquitoes was assessed in approximately 90 head/thorax and 90 abdomen segments at each time point; in total, 538 head/thorax and 534 abdomen segments were tested. In mosquitoes bisected after 0.5, 1, and 6 days post-infection (dpi), the mt COX-I PCR detected Plasmodium DNA in both the abdomen (88, 78, and 67%, respectively) and head/thorax segments (69, 60, and 44%, respectively), whilst CSP ELISA detected sporozoites in only one abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9, 12, and 15 dpi in both the head and thorax and abdomen. There was fair agreement between methods for time points 9–15 dpi (κ = 0.312, 95% CI: 0.230–0.394). Conclusions The mt COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the mt COX-I PCR is a poor candidate for identifying infectious mosquitoes. Graphical Abstract

Background Ethiopia has shown notable progress in reducing the burden of malaria over the past two decades. Because of this progress, the country has shifted efforts from control to elimination of malaria. This study was conducted to analyse the malaria epidemiology and stratification of incidence in the malaria elimination setting in eastern Ethiopia. Methods A retrospective study was conducted to analyse the epidemiology of malaria by reviewing the district health office data from 2013 to 2019 in Harari Region. In addition, three years of sub-district level malaria data were used to stratify the malaria transmission intensity. Malaria interventions (Long-lasting insecticidal nets [LLIN] and indoor residual spraying [IRS]) employed were reviewed to analyse the intervention coverage at the Regional level. Descriptive statistics were used to show the malaria transmission in terms of years, season and species of the malaria parasite. Incidence rate per 1000 population and death rate per 1 000 000 population at risk were computed using the total population of each year. Results In the Harari Region, malaria incidence showed a more pronounced declining trend from 2017 to 2019. Plasmodium falciparum , P. vivax and mixed infections accounted for 69.2%, 30.6% and 0.2% of the cases, respectively. There was an increment in malaria intervention coverage and improved malaria diagnosis. In the year 2019 the coverage of LLIN and IRS in the Region were 93.4% and 85.1% respectively. The annual malaria incidence rate dropped from 42.9 cases per 1000 population in 2013 to 6.7 cases per 1000 population in 2019. Malaria-related deaths decreased from 4.7 deaths per 1 000 000 people annually in 2013 to zero, and there have been no deaths reported since 2015. The malaria risk appears to be heterogeneous and varies between districts. A higher number of malaria cases were recorded in Erer and Jenella districts, which constitute 62% of the cases in the Region. According to the sub-district level malaria stratification, there was shrinkage in the malaria transmission map and about 70% of the sub-districts have achieved elimination targets. Conclusions In the Harari Region, malaria morbidity and mortality have been significantly declined. Thus, if this achievement is sustained and scaling-up of the existing malaria prevention and control strategies by focusing on those populations living in the higher malaria transmission districts and sub-districts, planning of malaria elimination from the study area might be feasible.

Background The protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands—resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands. Methods Hourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1). Results In the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) ( n  = 1970, 41.9%), followed by An. demeilloni ( n  = 1133, 24.1%) and An. cinereus ( n  = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. ( n  = 1190, 36.9%), An. pretoriensis ( n  = 899, 27.9%), and An. demeilloni ( n  = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations. Conclusions The high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations. Graphical Abstract