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Background: Cambodia is targeting the elimination of malaria by 2025. The last remaining pockets of malaria in Cambodia are concentrated among populations exposed to forested areas, but the size of these populations is not well understood. To plan for the procurement and distribution of vector-control tools, chemoprophylaxis, and other commodities for malaria prevention and surveillance, robust estimates of the population at greatest risk are required., Objective: This study aims to estimate the number of forest-exposed individuals residing in Cambodia's highest-burden operational districts (ODs) in 2 provinces with active malaria transmission., Methods: In April 2023, a multistage, in-person survey was conducted among residents in the 2 ODs in Cambodia with the highest malaria burden: Sen Monorom in Mondulkiri province and Phnom Srouch in Kampong Speu province. In each OD, 10 villages were randomly selected, and 35 households were randomly selected from each village. To estimate the number of individuals at high risk of malaria-defined as residing within 1 km of a forest or traveling at least once per week to the forest-respondents were asked about the distance from their household to the nearest forested area, and their travel patterns to forested areas. To account for mobility (ie, to avoid double-counting), respondents also provided information on overnight stays at other households in the selected villages in the past month. In the 4 selected villages in Sen Monorom OD where Project BITE forest packs (an intervention in the larger research program) had been distributed prior to the survey, respondents were also asked questions to determine if they had received such a pack, to develop smaller scale "multiplier method" estimates of at-risk individuals in each of those villages., Results: In Sen Monorom, 138 households and 872 individuals were enrolled in the survey, and in Phnom Srouch, 163 households and 844 individuals were enrolled. The estimated percentage of female householders was 49.7% (852/1716) across both ODs; the median age was 22 (IQR 12-37) years in Sen Monorom and 24.5 (IQR 16.0-40.5) years in Phnom Srouch (total age range 3-86). Based on mobility-adjusted survey estimates alone, 32% (280/706; 95% CI 19.9-47.2) of residents in Sen Monorom (an estimated 12,133-20,135 individuals) and 36% (68/198; 95% CI 24.5-45.5) of residents in Phnom Srouch (an estimated 1717-2203 individuals), met risk criteria for forest exposure. Between 125 and 186 individuals were estimated to be at risk in each of the 4 villages where the multiplier method could be applied., Conclusions: This study provides estimates of the number of individuals potentially at high risk for malaria infection due to forest exposure in 2 ODs in Cambodia. These estimates can support planning for malaria control and elimination efforts. The straightforward methods of household surveys and multipliers should be feasible for many national malaria control programs., (©Jerry O Jacobson, Dyna Doum, Neil F Lobo, Siv Sovannaroth, Allison Tatarsky, David J McIver. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org), 27.09.2024.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Malaria in eastern Indonesia remains high despite significant reduction and elimination in other parts of the country. A rapid entomological assessment was conducted in eight high malaria endemic regencies of Papua Province, Indonesia, to expedite malaria elimination efforts in this region. This study aims to characterize specific, actionable endpoints toward understanding where and when malaria transmission is happening, where interventions may function best, and identify gaps in protection that result in continued transmission. The entomological assessment included identifying potential vectors through human landing catch (HLC), indoor morning and night resting collections, identification of larval sites through surveillance of water bodies, and vector incrimination toward understanding exposure to malaria transmission. Human landing catches (HLCs) and larval collections identified 10 Anopheles species, namely Anopheles koliensis, Anopheles punctulatus, Anopheles farauti, Anopheles hinesorum, Anopheles longirostris, Anopheles peditaeniatus, Anopheles tesselatus, Anopheles vagus, Anopheles subpictus and Anopheles kochi. The most common and abundant species found overall were An. koliensis and An. punctulatus, while An. farauti was found in large numbers in the coastal areas of Mimika and Sarmi Regencies. Vector incrimination on Anopheles collected from HLCs and night indoor resting demonstrated that An. koliensis and An. punctulatus carried Plasmodium in Keerom, Jayapura, and Sarmi Regencies. Analysis of HLCs for the most common species revealed that the An. koliensis and An. punctulatus, bite indoors and outdoors at equal rates, while An. farauti predominantly bite outdoors. Larval surveillance demonstrated that most water bodies in and surrounding residential areas contained Anopheles larvae. This study demonstrated indoor and outdoor exposure to mosquito bites and gaps in protection, enabling exposure to infectious bites in all regencies. This explains why current malaria control efforts focusing on indoor protection have failed to substantially reduce malaria incidence in the region. Optimization of insecticide-treated bed nets (ITNs), as well as installment of mosquito screens in houses, may further reduce indoor transmission. For outdoor transmission, the use of community-centric approaches to reduce or eliminate larval sources within and surrounding the village through the guidance of locally stationed entomologists, along with Social and Behavior Change mediated health education towards the local adoption of mosquito protection tools during outdoor activities, may reduce malaria transmission., (© 2024. The Author(s).)

Entomological evaluations of vector control tools often use human landing catches (HLCs) as a standard measure of a direct human-vector contact. However, some tools have additional characteristics, such as mortality, and HLCS are not sensitive for measuring other effects beyond landing inhibition. Therefore, additional measures may need to be considered when evaluating these tools for public health use. This study has two main aims (1) the evaluate the accuracy of HLCs as a proxy for feeding and (2) to compare the predicted reduction in vectorial capacity when we do and do not consider these additional characteristics. To achieve this, we analyse previously published semi-field data from an experiment which used HLCs and another where mosquitoes were allowed to feed in the presence of different dosages of the volatile pyrethroid spatial repellent, transfluthrin. We compare results for two mathematical models: one which only considers the reduction in feeding effect and one which also considers mortality before and after feeding (using data gathered by the aspiration of mosquitoes after the semi-field feeding/landing period and 24 h survival monitoring). These Bayesian hierarchical models are parameterised using Bayesian inference. We observe that, for susceptible mosquitoes, reduction in landing is underestimated by HLCs. For knockdown resistant mosquitoes the relationship is less clear; with HLCs sometimes appearing to overestimate this characteristic. We find HLCs tend to under-predict the relative reduction in vectorial capacity in susceptible mosquitoes while over-predicting this impact in knockdown-resistant mosquitoes. Models without secondary effects have lower predicted relative reductions in vectorial capacities. Overall, this study highlights the importance of considering additional characteristics to reduction in biting of volatile pyrethroid spatial repellents. We recommend that these are considered when evaluating novel vector control tools., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

The invasive Anopheles stephensi mosquito has rapidly expanded in range in Africa over the past decade. Consistent with World Health Organization guidelines, routine entomologic surveillance of malaria vectors in Accra, Ghana, now includes morphologic and molecular surveillance of An. stephensi mosquitoes. We report detection of An. stephensi mosquitoes in Ghana.

Background: The emergence of insecticide resistance and outdoor transmission in malaria-endemic areas underlines the urgent need to develop innovative tools, such as spatial repellents (SR), that may circumvent this residual transmission. With limited options for effective insecticides, regular resistance monitoring is warranted for selecting and using appropriate tools. This study evaluates the pyrethroid knockdown resistance (kdr) allele before and after implementing a transfluthrin-based spatial repellent (SR) intervention in placebo-treated clusters., Methods: This study looks at the frequency distribution of the kdr allele in Sumba Island from June 2015 to August 2018. Insecticide susceptibility tests were carried out on female Anopheles sp. aged 3-5 days against permethrin 21.5 μg/ml, deltamethrin 12.5 μg/ml, and transfluthrin 10 μg/ml using CDC bottle assay. PCR sequencing of representative samples from adult mosquito collections and insecticide tests revealed the presence of kdr mutations (L1014F and L1014S) in the VGSC gene., Results: A total of 12 Anopheles species, Anopheles tesselatus, Anopheles. aconitus, Anopheles barbirostris, Anopheles kochi, Anopheles annularis, Anopheles maculatus, Anopheles sundaicus, Anopheles flavirostris, Anopheles balabacensis, Anopheles indefinitus, Anopheles subpictus, and Anopheles vagus were analysed. Anopheles vagus and An. sundaicus predominated in the larval populations. Susceptibility assays for all insecticides identified fully susceptible phenotypes in all species examined. Anopheles increasing frequency of kdr mutant alleles during the 3 year SR deployment was observed in both SR-treated and placebo areas, a statistically significant increase occurred in each arm. However, it is unclear how significant SR is in causing the increase in mutant alleles. The L1014S, knockdown resistance east type (kdr-e) allele was detected for the first time among the mosquito samples in this study. The L1014F, knockdown resistance west type (kdr-w) allele and heteroduplex form (wild-type-mutant) were found in almost all Anopheles species examined, including An. vagus, An. aconitus, An. subpictus, An. tesselatus, An. annularis, An. flavirostris and An. sundaicus., Conclusion: The presence of fully susceptible phenotypes over time, along with an increase in the frequency distribution of the L1014F/S mutations post-intervention, suggest drivers of resistance external to the study, including pyrethroid use in agriculture and long-lasting insecticidal nets (LLINs). However, this does not negate possible SR impacts that support resistance. More studies that enable the comprehension of possible SR-based drivers of resistance in mosquitoes need to be conducted., (© 2024. The Author(s).)

Background: Different anopheline species (even within a species group/complex) can differ in their feeding and resting behaviours, which impact both malaria transmission patterns as well as the efficacy of vector control interventions. While morphological identification of sampled specimens is an important first step towards understanding species diversity and abundance, misidentification can result in the implementation of less effective vector control measures, and consequently smaller reductions in the number of local malaria cases. Focusing on southern Mozambique, a malaria pre-elimination area where malaria remains persistent, the aims of this preliminary study were to use molecular identification (CO1 and ITS2 barcoding) to (1) validate the results from the morphological identification (with a particular focus on Anopheles pharoensis and Anopheles squamosus), and (2) have a closer look at the Anopheles coustani group (which includes Anopheles tenebrosus and Anopheles ziemanni)., Methods: Female anopheline mosquitoes (n = 81) were identified morphologically and subsequently sequenced at the ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or cytochrome oxidase subunit 1 (CO1) loci towards species determination., Results: Out of the 62 specimens that were identified morphologically to species, 4 (6.5%) were misidentified. Regarding the An. coustani group, morphological identification showed that several members are present in southern Mozambique, including An. coustani sensu lato (s.l.), An. ziemanni and An. tenebrosus. However, based on both ITS2 and CO1 sequences, the exact species remains unknown for the latter two members until voucher sequences are available for comparison., Conclusion: The reason(s) for morphological misidentification of anopheline mosquitoes need to be mitigated. This is usually related to both the capacity (i.e. training) of the microscopist to identify anopheline species, and the information provided in the dichotomous identification key. As the An. coustani complex contributes to (residual) malaria transmission in sub-Saharan Africa, it may play a role in the observed persistent malaria in southern Mozambique. A better baseline characterizing of the local anophelines species diversity and behaviours will allow us to improve entomological surveillance strategies, better understand the impact of vector control on each local vector species, and identify new approaches to target those vector species., (© 2024. The Author(s).)

The effectiveness of vector-control tools is often assessed by experiments as a reduction in mosquito landings using human landing catches (HLCs). However, HLCs alone only quantify a single characteristic and therefore do not provide information on the overall impacts of the intervention product. Using data from a recent semi-field study which used time-stratified HLCs, aspiration of non-landing mosquitoes, and blood feeding, we suggest a Bayesian inference approach for fitting such data to a stochastic model. This model considers both personal protection, through a reduction in biting, and community protection, from mosquito mortality and disarming (prolonged inhibition of blood feeding). Parameter estimates are then used to predict the reduction of vectorial capacity induced by etofenpox-treated clothing, picaridin topical repellents, transfluthrin spatial repellents and metofluthrin spatial repellents, as well as combined interventions for Plasmodium falciparum malaria in Anopleles minimus. Overall, all interventions had both personal and community effects, preventing biting and killing or disarming mosquitoes. This led to large estimated reductions in the vectorial capacity, with substantial impact even at low coverage. As the interventions aged, fewer mosquitoes were killed; however the impact of some interventions changed from killing to disarming mosquitoes. Overall, this inference method allows for additional modes of action, rather than just reduction in biting, to be parameterised and highlights the tools assessed as promising malaria interventions., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: Declaration of interests The authors declare no competing interests.

Bangladesh has dramatically reduced malaria by 93% from 2008 to 2020. The strategy has been district-wise, phased elimination; however, the last districts targeted for elimination include remote, forested regions which present several challenges for prevention, detection, and treatment of malaria. These districts border Myanmar which harbors Plasmodium falciparum malaria parasites resistant to artemisinins, key drugs used in artemisinin-based combination therapies (ACTs) that have been vital for control programs. Challenges in monitoring emergence of artemisinin resistance (AR), tracking parasite reservoirs, changes in vector behavior and responses to insecticides, as well as other environmental and host factors (including the migration of Forcibly Displaced Myanmar Nationals; FDMNs) may pose added hazards in the final phase of eliminating malaria in Bangladesh., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Mosquito-borne diseases are major global health problems that threaten nearly half of the world's population. Conflicting resources and infrastructure required by the coronavirus disease 2019 (COVID-19) global pandemic have resulted in the vector control process being more demanding than ever. Although novel vector control paradigms may have been more applicable and efficacious in these challenging settings, there were virtually no reports of novel strategies being developed or implemented during COVID-19 pandemic. Evidence shows that the COVID-19 pandemic has dramatically impacted the implementation of conventional mosquito vector measures. Varying degrees of disruptions in malaria control and insecticide-treated nets (ITNs) and indoor residual spray (IRS) distributions worldwide from 2020 to 2021 were reported. Control measures such as mosquito net distribution and community education were significantly reduced in sub-Saharan countries. The COVID-19 pandemic has provided an opportunity for innovative vector control technologies currently being developed. Releasing sterile or lethal gene-carrying male mosquitoes and novel biopesticides may have advantages that are not matched by traditional vector measures in the current context. Here, we review the effects of COVID-19 pandemic on current vector control measures from 2020 to 2021 and discuss the future direction of vector control, taking into account probable evolving conditions of the COVID-19 pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lu, Sui, Lobo, Fouque, Gao, Lu, Lv, Deng and Wang.)

Background: Namibia's focus on the elimination of malaria requires an evidence-based strategy directed at understanding and targeting the entomological drivers of malaria transmission. In 2018 and 2019, the Namibia National Vector-borne Diseases Control Program (NVDCP) implemented baseline entomological surveillance based on a question-based approach outlined in the Entomological Surveillance Planning Tool (ESPT). In the present study, we report on the findings of the ESPT-based NVDCP on baseline vector species composition and bionomic traits in malaria endemic regions in northern Namibia, which has the aim of generating an evidence base for programmatic decision-making., Methods: Nine representative sentinel sites were included in the 2018 entomological surveillance program (Kunene, Omusati, Oshana, Ohangwena, Oshikoto, Otjozondjupa, Kavango West, Kavango East and Zambezi); the number was reduced to four sites in 2019 due to limited funding (Ohangwena, Kavango West, Kavango East, and Zambezi). In the 2018 baseline collections, multiple sampling methods (human landing catches, pyrethroid spray catches, U.S. Centers for Disease Control and Prevention light traps [CDC-LTs], resting boxes [RBs] and larval sampling) were utilized to evaluate indoor/outdoor human biting rates, resting behaviors and insecticide resistance (IR). CDC-LTs and RBs were not used in 2019 due to low and non-representative sampling efficacies., Results: Overall, molecular evidence demonstrated the presence of three primary mosquito vectors, namely Anopheles arabiensis, rediscovered Anopheles gambiae sensu stricto and Anopheles funestus sensu stricto, alongside Anopheles squamosus and members of the Anopheles coustani complex. Vectors were found to bite throughout the night (1800 hours 0600 hours) both indoors and outdoors, with An. arabiensis having the highest biting rates outdoors. Low numbers of indoor resting Anopheles point to possible low indoor residual spraying (IRS) efficacy-with An. arabiensis found to be the major vector species resting indoors. The IR tests demonstrated varying country-wide resistance levels to the insecticide deltamethrin, with the resistance levels confirmed to have increased in 2019, evidence that impacts national programmatic decision-making. Vectors demonstrated susceptibility to the insecticides dichlorodiphenyltrichloroethane, bendiocarb and Actellic 300CS in 2018, with mosquitoes from only one site (Kavango West) demonstrating possible resistance to DDT. Targeted and question-based entomological surveillance enabled a rapid and focused evidence base to be built, showing where and when humans were being bitten and providing entomological data on long-lasting insecticidal nets, IRS efficacy and insecticide resistance, which the Ministry of Health and Social Services-Namibia can use to further build a monitoring and evaluation framework for understanding the drivers of transmission., Conclusion: Identification and characterization of species-specific bionomic traits allows for an understanding of where and when vector human contact may occur as well as the potential impact of interventions. Low indoor resting rates as well as the presence of insecticide resistance (and the increase in its frequency) point to the need for mosquito-behavior-directed and appropriate interventions as well as the requirement for a resistance mitigation strategy. The ESPT-based question- and minimal essential indicator-based operational research strategy provides programs with directed and focused data for facilitating decision-making while requiring limited funding and capacity., (© 2023. The Author(s).)

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 < 0.001)., Conclusions: Interactions between mosquitoes, bite prevention tools and the sampling method may impact the estimated intervention protective efficacy. Consequently, the sampling method must be considered when evaluating these interventions. The HDN is a valid alternative trapping method (relative to the HLC) for evaluating the impact of bite prevention methods that affect mosquito behaviour at a distance (e.g. VPSR), but not for interventions that operate through tarsal contact (e.g., ITC)., (© 2023. The Author(s).)

Background: Malaria is a mosquito-borne disease that is one of the most serious public health issues globally and a leading cause of mortality in many developing countries worldwide. Knowing the prevalence of both symptomatic and asymptomatic malaria on a subnational scale allows for the estimation of the burden of parasitaemia present in the transmission system, enabling targeting and tailoring of resources towards greater impact and better use of available capacity. This study aimed to determine the PCR-based point prevalence of malaria infection, by parasite species, among three high-risk populations in Mondulkiri province, Cambodia: forest rangers, forest dwellers, and forest goers., Methods: A cross-sectional survey was performed during the transmission season in November and December 2021. Blood samples collected on filter paper from participants (n = 1301) from all target groups were screened for Plasmodium spp using PCR., Results: Malaria prevalence among all study participants was 6.7% for any Plasmodium species. Malaria prevalence in the forest ranger group was 8.1%, was 6.8% in forest goers, and 6.4% in forest dwellers; all infections were asymptomatic. Plasmodium vivax was detected in all participant groups, while the few Plasmodium falciparum infections were found in goers and dwellers. 81% of all infections were due to P. vivax, 9% were due to P. falciparum, 3% due to Plasmodium cynomolgi, and the rest (7%) remained undefined. Gender was associated with malaria infection prevalence, with male participants having higher odds of malaria infection than female participants (OR = 1.69, 95% CI 1.08-2.64). Passively collected malaria incidence data from the Cambodian government were also investigated. Health facility-reported malaria cases, based on rapid diagnostic tests, for the period Jan-Dec 2021 were 521 Plasmodium vivax (0.89% prevalence), 34 P. falciparum (0.06%) and four P. falciparum + mixed (0.01%)-a total of 559 cases (0.95%) for all of Mondulkiri., Conclusion: This reservoir of asymptomatic parasitaemia may be perpetuating low levels of transmission, and thus, new strategies are required to realize the goal of eliminating malaria in Cambodia by 2025., (© 2023. The Author(s).)

Background: Joint efforts by government and non-government organizations have helped to reduce malaria in Bangladesh and set the country on a clear path to eventual malaria elimination. However, achieving that goal would be challenging without a comprehensive understanding of vector bionomics., Methods: Targeted capturing of Anopheles mosquitoes over a rainy season, utilizing specific sampling methods, including human landing catches (HLCs), CDC-light traps (CDC-LTs), and pyrethrum spray catches (PSCs) were aimed to characterize entomological drivers of transmission in four sites of Bandarban, Bangladesh., Results: Molecular characterization of a subset of 4637 mosquitoes has demonstrated the presence of at least 17 species whose capture rates were representative of the rainy season. Species compositions and bionomic traits did not vary between sites with Anopheles maculatus having the highest landing rate by HLCs and Anopheles vagus having the highest capture rate with CDC-LTs. Interestingly, Anopheles species compositions and capture rates varied significantly (p < 0.05) for An. vagus, between HLCs and its often-used proxy-CDC-LTs- suggesting impacts on downstream analysis. CDC-LTs capture rates demonstrated differing compositions with indoor and outdoor biting rates. For example, Anopheles nigerrimus and Anopheles nivipes were more endophagic by HLCs and more exophagic by CDC-LTs. The use of a cow-baited CDC-LT also demonstrated significantly different results when compared to a human-baited CDC-LT considering the high degree of anthropophily in these species. The exception to both zoophily and indoor resting was An. vagus, which demonstrated both anthropophily and high resting rates indoors-pointing to this species being a possible primary vector at this site., Conclusion: A diverse Anopheles fauna in Bandarban has been confirmed through molecular methods, highlighting the potential impact of sampling techniques. Given the complexity of the local ecosystem, a better understanding of mosquito behaviour and ecology is required to achieve the goal of malaria elimination in Bangladesh., (© 2023. The Author(s).)

Presently, the most common malaria control tools-i.e., long lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS)-are limited to targeting indoor biting and resting behaviors of Anopheles mosquito species. Few interventions are targeted towards malaria control in areas where transmission is driven or persists due to outdoor biting behaviors. This study investigated a volatile pyrethroid-based spatial repellent (VPSR) designed to bridge this gap and provide protection from mosquito bites in outdoor spaces. Southern Province, Zambia, is one such environment where outdoor biting is suspected to contribute to malaria transmission, where people are active in the evening in open-walled outdoor kitchens. This study assessed the VPSR in replica kitchens within a controlled semi-field environment. Endpoints included effects on mosquito host seeking, immediate and delayed mortality, deterrence, blood feeding inhibition, and fertility. Host-seeking was reduced by approximately 40% over the course of nightly releases in chambers containing VPSR devices. Mosquito behavior was not uniform throughout the night, and the modeled effect of the intervention was considerably higher when hourly catch rates were considered. These two observations highlight a limitation of this overnight semi-field design and consideration of mosquito circadian rhythms is recommended for future semi-field studies. Additionally, deterrence and immediate mortality were both observed in treatment chambers, with evidence of delayed mortality and a dose related response. These results demonstrate a primarily personal protective mode of action with possible positive and negative community effects. Further investigation into this primary mode of action will be conducted through a field trial of the same product in nearby communities., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Burton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Artemisinin-based combination therapy (ACT) is the most effective treatment for malaria, and has significantly reduced morbimortality. Polymorphisms associated with the Plasmodium falciparum Kelch gene (Pfkelch13) have been associated with delayed parasite clearance even with ACT treatment., Methods: The Pfkelch13 gene was sequenced from P. falciparum infected patients (n = 159) with uncomplicated malaria in Niger. An adequate clinical and parasitological response (ACPR) was reported in 155 patients. Four (n = 4) patients had treatment failure (TF) that were not reinfections-two of which had late parasitological failures (LPF) and two had late clinical failures (LCF)., Results: Thirteen single nucleotide polymorphisms (SNPs) were identified of which seven were non-synonymous (C469R, T508S, R515T, A578S, I465V, I437V, F506L,), and three were synonymous (P443P, P715P, L514L). Three SNP (C469R, F506L, P715P) were present before ACT treatment, while seven mutations (C469R, T508S, R515T, L514L, P443P, I437V, I465V) were selected by artemether/lumefantrine (AL)-five of which were non-synonymous (C469R, T508S, R515T, I437V, I465V). Artesunate/amodiaquine (ASAQ) has selected any mutation. One sample presented three cumulatively non-synonymous SNPs-C469R, T508S, R515T., Conclusions: This study demonstrates intra-host selection of Pfkelch13 gene by AL. The study highlights the importance of LCF and LPF parasites in the selection of resistance to ACT. Further studies using gene editing are required to confirm the potential implication of resistance to ACT with the most common R515T and T508S mutations. It would also be important to elucidate the role of cumulative mutations., (© 2023. The Author(s).)

Progress in reducing both malaria cases and deaths has stalled with regression seen in many geographies. While significant attention is given to the contributing challenges of drug and insecticide resistance, 'residual' malaria is often diminished to transmission resulting from outdoor-biting or zoophagic/opportunistic mosquito vectors. These specific vector bionomic traits are only part of the problem, as residual transmission may be driven by (a combination of) (1) sub-optimal intervention coverage, quality, acceptance, and/or usage, (2) drug resistance, (3) insecticide resistance, (4) refractory, resistant and adaptive vector and human behaviours that lower intervention effectiveness, (5) lack of, limited access to, and/or willingness to use healthcare systems, (6) diagnostic sensitivity along with the parallel issue of hrp2/3 mutations, (7) (inter)national policy, (8) the research and development pipeline, and (9) external factors such as natural disasters and conflict zones. Towards combating the minimization of this extensive and multipronged issue among the scientific community, funding agencies, and public health officials responsible for guiding or developing malaria programmes, an alternative way of describing this transmission is proposed by focusing in on the causative 'gaps in protection'. Defining and wording it as such zeros in on the drivers that result in the observed remaining (or increasing) transmission, allowing the malaria community to focus on solutions by identifying the actual causes. Outlining, defining and quantifying the gaps in protection for a given system is of utmost importance to understand what needs to be done, differentiating what can be done versus what cannot be tackled at that moment, along with delineating the technical and financial capacity required., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Background: Human landing catches (HLC) are an entomological collection technique in which humans are used as attractants to capture medically relevant host-seeking mosquitoes. The use of this method has been a topic of extensive debate for decades mainly due to ethical concerns. Many alternatives to HLC have been proposed; however, no quantitative review and meta-analysis comparing HLC to outdoor alternative trapping methods has been conducted., Methods: A total of 58 comparisons across 12 countries were identified. We conducted a meta-analysis comparing the standardized mean difference of Anopheles captured by HLC and alternative traps. To explain heterogeneity, three moderators were chosen for analysis: trap type, location of study, and species captured. A meta-regression was fit to understand how the linear combination of moderators helped in explaining heterogeneity. The possibility of biased results due to publication bias was also explored., Results: Random-effects meta-analysis showed no statistically significant difference in the mean difference of Anopheles collected. Moderator analysis was conducted to determine the effects of trap type, geographical location of study, and the species of Anopheles captured. On average, tent-based traps captured significantly more Anopheles than outdoor HLC (95% CI: [- .9065, - 0.0544]), alternative traps in Africa captured on average more mosquitoes than outdoor HLC (95% CI: [- 2.8750, - 0.0294]), and alternative traps overall captured significantly more Anopheles gambiae s.l. than outdoor HLC (95% CI: [- 4.4613, - 0.2473]) on average. Meta-regression showed that up to 55.77% of the total heterogeneity found can be explained by a linear combination of the three moderators and the interaction between trap type and species. Subset analysis on An. gambiae s.l. showed that light traps specifically captured on average more of this species than HLC (95% CI: [- 18.3751, - 1.0629]). Publication bias likely exists. With 59.65% of studies reporting p-values less than 0.025, we believe there is an over representation in the literature of results indicating that alternative traps are superior to outdoor HLC., Conclusions: Currently, there is no consensus on a single "magic bullet" alternative to outdoor HLC. The diversity of many alternative trap comparisons restricts potential metrics for comparisons to outdoor HLC. Further standardization and specific question-driven trap evaluations that consider target vector species and the vector control landscape are needed to allow for robust meta-analyses with less heterogeneity and to develop data-driven decision-making tools for malaria vector surveillance and control., (© 2022. The Author(s).)

Background: Bangladesh has reduced malaria incidence and mortality by over 75% between 2010 and 2020. Widespread long-lasting insecticidal net (LLIN) distribution and use is one of the measures responsible for this success. Recalcitrant malaria hotspots within the Chittagong Hill Tracts districts suggest important drivers of malaria risk may remain uncharacterized., Methods: Towards understanding how household-level human behaviour impacts exposure to mosquitoes, parallel human landing catches and human behavioural observations were conducted in four households for 6 months (May-October) over the rainy season in the Bandarban District. Analysis quantifies spatiotemporal human behaviour-adjusted exposure to Anopheles with and without LLINs., Results: This small-scale operational study demonstrates that human spatial and temporal presence along with LLIN use drives exposure to Anopheles. Though the four households had both outdoor and indoor exposure, especially in the evening (1800-2000 h) and early morning (0400-0500 h), data points to household-based heterogeneity in malaria exposure even with similar LLIN access., Conclusion: Incorporating human behaviour into exposure estimates can be used to understand the efficacy and limitations of local vector control strategies and identify gaps in protection, as well as where present intervention strategies may be optimized., (© 2022. The Author(s).)

Background: Although the Republic of Namibia has significantly reduced malaria transmission, regular outbreaks and persistent transmission impede progress towards elimination. Towards an understanding of the protective efficacy, as well as gaps in protection, associated with long-lasting insecticidal nets (LLINs), human and Anopheles behaviors were evaluated in parallel in three malaria endemic regions, Kavango East, Ohangwena and Zambezi, using the Entomological Surveillance Planning Tool to answer the question: where and when are humans being exposed to bites of Anopheles mosquitoes?, Methods: Surveillance activities were conducted during the malaria transmission season in March 2018 for eight consecutive nights. Four sentinel structures per site were selected, and human landing catches and human behavior observations were consented to for a total of 32 collection nights per site. The selected structures were representative of local constructions (with respect to building materials and size) and were at least 100 m from each other. For each house where human landing catches were undertaken, a two-person team collected mosquitoes from 1800 to 0600 hours., Results: Surveillance revealed the presence of the primary vectors Anopheles arabiensis, Anopheles gambiae sensu stricto (s.s.) and Anopheles funestus s.s., along with secondary vectors (Anopheles coustani sensu lato and Anopheles squamosus), with both indoor and outdoor biting behaviors based on the site. Site-specific human behaviors considerably increased human exposure to vector biting. The interaction between local human behaviors (spatial and temporal presence alongside LLIN use) and vector behaviors (spatial and temporal host seeking), and also species composition, dictated where and when exposure to infectious bites occurred, and showed that exposure was primarily indoors in Kavango East (78.6%) and outdoors in Ohangwena (66.7%) and Zambezi (81.4%). Human behavior-adjusted exposure was significantly different from raw vector biting rate., Conclusions: Increased LLIN use may significantly increase protection and reduce exposure to malaria, but may not be enough to eliminate the disease, as gaps in protection will remain both indoors (when people are awake and not using LLINs) and outdoors. Alternative interventions are required to address these exposure gaps. Focused and question-based operational entomological surveillance together with human behavioral observations may considerably improve our understanding of transmission dynamics as well as intervention efficacy and gaps in protection., (© 2022. The Author(s).)

Malaria vector control interventions in Sumba, Indonesia, have not been able to eliminate malaria. Human drivers of exposure to Anopheles bites were investigated as part of a larger clinical trial evaluating the impact of a spatial repellent product on malaria incidence. Human behavioral observations (HBOs) evaluating temporal and spatial presence, sleeping behaviors, and insecticide treated net (ITN) use, were collected parallel to entomological collections-indoor and outdoor human landing catches (HLCs), and house hold surveys. Data demonstrates that mosquito access to humans, enabled by structurally open houses, is evident by the similar entomological landing rates both inside and outside households. The presence of animals inside houses was associated with increased mosquito entry-however, the number of humans present inside houses was not related to increased mosquito landing. Analyzing mosquito landing rates with human behavior data enables the spatial and temporal estimation of exposure to Anopheles bites, accounting for intervention (ITN) presence and usage. Human behavior adjusted exposure to Anopheles bites was found to be highest in the early in the evening, but continued at lower levels throughout the night. Over the night, most exposure (53%) occurred when people were indoors and not under the protection of nets (asleep or awake) followed by exposure outside (44%). Characterized gaps in protection are outdoor exposure as well as exposure indoors-when awake, and when asleep and not using ITNs. Interestingly, in the primary trial, even though there was not a significant impact of the spatial repellent on vector biting rates by themselves (16%), when factoring in human behavior, there was approximately 28% less exposure in the intervention arm than in the placebo arm. The treated arm had less human behavior adjusted bites in all spaces evaluated though there was proportionally higher exposure indoors. This analysis points to the importance of using HBOs both towards understanding gaps in protection as well as how interventions are evaluated. To mitigate ongoing transmission, understanding context specific spatial and temporal exposure based on the interactions of vectors, humans and interventions would be vital for a directed evidence-based control or elimination strategy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Rozi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Vector-borne diseases are among the most burdensome infectious diseases worldwide with high burden to health systems in developing regions in the tropics. For many of these diseases, vector control to reduce human biting rates or arthropod populations remains the primary strategy for prevention. New vector control interventions intended to be marketed through public health channels must be assessed by the World Health Organization for public health value using data generated from large-scale trials integrating epidemiological endpoints of human health impact. Such phase III trials typically follow large numbers of study subjects to meet necessary power requirements for detecting significant differences between treatment arms, thereby generating substantive and complex datasets. Data is often gathered directly in the field, in resource-poor settings, leading to challenges in efficient data reporting and/or quality assurance. With advancing technology, mobile data collection (MDC) systems have been implemented in many studies to overcome these challenges. Here we describe the development and implementation of a MDC system during a randomized-cluster, placebo-controlled clinical trial evaluating the protective efficacy of a spatial repellent intervention in reducing human infection with Aedes-borne viruses (ABV) in the urban setting of Iquitos, Peru, as well as the data management system that supported it. We discuss the benefits, remaining capacity gaps and the key lessons learned from using a MDC system in this context in detail., (© 2022. The Author(s).)

The effectiveness of artemisinin-based combination therapies (ACTs) depends not only on that of artemisinin but also on that of partner molecules. This study aims to evaluate the prevalence of mutations in the Pfdhfr , Pfdhps, and Pfmdr1 genes from isolates collected during a clinical study. Plasmodium genomic DNA samples extracted from symptomatic malaria patients from Dogondoutchi, Niger, were sequenced by the Sanger method to determine mutations in the Pfdhfr (codons 51, 59, 108, and 164), Pfdhps (codons 436, 437, 540, 581, and 613), and Pfmdr1 (codons 86, 184, 1034, and 1246) genes. One hundred fifty-five (155) pre-treatment samples were sequenced for the Pfdhfr, Pfdhps, and Pfmdr1 genes. A high prevalence of mutations in the Pfdhfr gene was observed at the level of the N51I (84.97%), C59R (92.62%), and S108N (97.39%) codons. The key K540E mutation in the Pfdhps gene was not observed. Only one isolate was found to harbor a mutation at codon I431V. The most common mutation on the Pfmdr1 gene was Y184F in 71.43% of the mutations found, followed by N86Y in 10.20%. The triple-mutant haplotype N51I/C59R/S108N (IRN) was detected in 97% of the samples. Single-mutant (ICS and NCN) and double-mutant (IRS, NRN, and ICN) haplotypes were prevalent at 97% and 95%, respectively. Double-mutant haplotypes of the Pfdhps (581 and 613) and Pfmdr (86 and 184) were found in 3% and 25.45% of the isolates studied, respectively. The study focused on the molecular analysis of the sequencing of the Pfdhfr , Pfdhps, and Pfmdr1 genes. Although a high prevalence of mutations in the Pfdhfr gene have been observed, there is a lack of sulfadoxine pyrimethamine resistance. There is a high prevalence of mutation in the Pfmdr184 codon associated with resistance to amodiaquine. These data will be used by Niger's National Malaria Control Program to better monitor the resistance of Plasmodium to partner molecules in artemisinin-based combination therapies.

Background: To eliminate malaria in southern Mozambique, the National Malaria Control Programme and its partners are scaling up indoor residual spraying (IRS) activities in two provinces, Gaza and Inhambane. An entomological surveillance planning tool (ESPT) was used to answer the programmatic question of whether IRS would be effective in target geographies, given limited information on local vector bionomics., Methods: Entomological intelligence was collected in six sentinel sites at the end of the rainy season (April-May 2018) and the beginning of the dry season (June-July 2018). The primary objective was to provide an 'entomological snapshot' by collecting question-based, timely and high-quality data within one single week in each location. Host-seeking behaviour (both indoors and outdoors) was monitored by human-baited tent traps. Indoor resting behaviour was quantified by pyrethrum spray catches and window exit traps., Results: Five different species or species groups were identified: Anopheles funestus sensu lato (s.l.) (66.0%), Anopheles gambiae s.l. (14.0%), Anopheles pharoensis (1.4%), Anopheles tenebrosus (14.1%) and Anopheles ziemanni (4.5%). Anopheles funestus sensu stricto (s.s.) was the major vector among its sibling species, and 1.9% were positive for Plasmodium falciparum infections. Anopheles arabiensis was the most abundant vector species within the An. gambiae complex, but none tested positive for P. falciparum infections. Some An. tenebrosus were positive for P. falciparum (1.3%). When evaluating behaviours that impact IRS efficacy, i.e. endophily, the known primary vector An. funestus s.s., was found to rest indoors-demonstrating at least part of its population will be impacted by the intervention if insecticides are selected to which this vector is susceptible. However, other vector species, including An. gambiae s.l., An. tenebrosus, An. pharoensis and An. ziemanni, showed exophilic and exophagic behaviours in several of the districts surveilled., Conclusion: The targeted approach to entomological surveillance was successful in collecting question-based entomological intelligence to inform decision-making about the use of IRS in specific districts. Endophilic An. funestus s.s. was documented as being the most prevalent and primary malaria vector suggesting that IRS can reduce malaria transmission, but the presence of other vector species both indoors and outdoors suggests that alternative vector control interventions that target these gaps in protection may increase the impact of vector control in southern Mozambique., (© 2022. The Author(s).)

Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti , thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes -borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Background: The East Nusa Tenggara province, Indonesia, contributed to 5% of malaria cases nationally in 2020, with other mosquito-borne diseases, such as dengue and filariasis also being endemic. Monitoring of spatial and temporal vector species compositions and bionomic traits is an efficient method for generating evidence towards intervention strategy optimization and meeting disease elimination goals., Methods: The impact of a spatial repellent (SR) on human biting mosquitoes was evaluated as part of a parent cluster-randomized, double-blinded, placebo-controlled trial, in Sumba, East Nusa Tenggara. A 10-month (June 2015-March 2016) baseline study was followed by a 24-month intervention period (April 2016 to April 2018)-where half the clusters were randomly assigned either a passive transfluthrin emanator or a placebo control., Results: Human-landing mosquito catches documented a reduction in landing rates related to the SR. Overall, there was a 16.4% reduction (21% indoors, and 11.3% outdoors) in human biting rates (HBR) for Anopheles. For Aedes, there was a 44.3% HBR reduction indoors and a 35.6% reduction outdoors. This reduction was 38.3% indoors and 39.1% outdoors for Armigeres, and 36.0% indoors and 32.3% outdoors for Culex species. Intervention impacts on the HBRs were not significant and are attributed to large inter-household and inter cluster variation. Anopheles flavirostris, Anopheles balabacensis and Anopheles maculatus individually impacted the overall malaria infections hazard rate with statistically significance. Though there was SR-based protection against malaria for all Anopheles species (except Anopheles sundaicus), only five (Anopheles aconitus, Anopheles kochi, Anopheles tessellatus, An. maculatus and An. sundaicus) demonstrated statistical significance. The SR numerically reduced Anopheles parity rates indoors and outdoors when compared to the placebo., Conclusion: Evidence demonstrating that Anopheles vectors bite both indoors and outdoors indicates that currently implemented indoor-based vector control tools may not be sufficient to eliminate malaria. The documented impact of the SR intervention on Aedes, Armigeres and Culex species points to its importance in combatting other vector borne diseases. Studies to determine the impact of spatial repellents on other mosquito-borne diseases is recommended., (© 2022. The Author(s).)

Mosquitoes are important vectors that transmit pathogens to human and other vertebrates. Each mosquito species has specific ecological requirements and bionomic traits that impact human exposure to mosquito bites, and hence disease transmission and vector control. A study of human biting mosquitoes and their bionomic characteristics was conducted in West Sumba and Southwest Sumba Districts, Nusa Tenggara Timur Province, Indonesia from May 2015 to April 2018. Biweekly human landing catches (HLC) of night biting mosquitoes both indoors and outdoors caught a total of 73,507 mosquito specimens (59.7% non-Anopheles, 40.3% Anopheles). A minimum of 22 Culicinae species belonging to four genera (Aedes, Armigeres, Culex, Mansonia), and 13 Anophelinae species were identified. Culex quinquefasciatus was the dominant Culicinae species, Anopheles aconitus was the principal Anopheles species inland, while An. sundaicus was dominant closer to the coast. The overall human biting rate (HBR) was 10.548 bites per person per night (bpn) indoors and 10.551 bpn outdoors. Mosquitoes biting rates were slightly higher indoors for all genera with the exception of Anopheles, where biting rates were slightly higher outdoors. Diurnal and crepuscular Aedes and Armigeres demonstrated declining biting rates throughout the night while Culex and Anopheles biting rates peaked before midnight and then declined. Both anopheline and non-anopheline populations did not have a significant association with temperature (p = 0.3 and 0.88 respectively), or rainfall (p = 0.13 and 0.57 respectively). The point distribution of HBR and seasonal variables did not have a linear correlation. Data demonstrated similar mosquito-human interactions occurring outdoors and indoors and during early parts of the night implying both indoor and outdoor disease transmission potential in the area-pointing to the need for interventions in both spaces. Integrated vector analysis frameworks may enable better surveillance, monitoring and evaluation strategies for multiple diseases., Competing Interests: The authors have declared that no competing interest exist. Author Michael J. Bangs was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

As gene drive mosquito projects advance from contained laboratory testing to semi-field testing and small-scale field trials, there is a need to assess monitoring requirements to: i) assist with the effective introduction of the gene drive system at field sites, and ii) detect unintended spread of gene drive mosquitoes beyond trial sites, or resistance mechanisms and non-functional effector genes that spread within trial and intervention sites. This is of particular importance for non-localized gene drive projects, as the potential scale of intervention means that monitoring is expected to be more costly than research, development and deployment. Regarding monitoring needs for population replacement systems, lessons may be learned from experiences with Wolbachia -infected mosquitoes, and for population suppression systems, from experiences with releases of genetically sterile male mosquitoes. For population suppression systems, assessing monitoring requirements for tracking population size and detecting rare resistant alleles are priorities, while for population replacement systems, allele frequencies must be tracked, and pressing concerns include detection of gene drive alleles with non-functional effector genes, and resistance of pathogens to functional effector genes. For spread to unintended areas, open questions relate to the optimal density and placement of traps and frequency of sampling in order to detect gene drive alleles, drive-resistant alleles or non-functional effector genes while they can still be effectively managed. Invasive species management programs face similar questions, and lessons may be learned from these experiences. We explore these monitoring needs for gene drive mosquito projects progressing through the phases of pre-release, release and post-release., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rašić, Lobo, Jeffrey Gutiérrez, Sánchez C. and Marshall.)

Background: Though 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 that would enhance the understanding of both local entomological drivers of transmission and gaps in protection that result in persisting malaria transmission to guide local vector control decision-making., Methods: The ESPT was used to design a sampling plan centered around the collection of minimum essential indicators to investigate the relevance of LLINs and IRS in the communities of Permé and Puerto Obaldía, Guna Yala, as well as to pinpoint any remaining spaces and times where humans are exposed to Anopheles bites (gaps in protection). Adult Anopheles were collected at three time points via human landing catches (HLCs), CDC Light Traps (LT), and pyrethrum spray catches (PSCs) during the rainy and dry seasons. Mosquitoes were identified to species via molecular methods. Insecticide susceptibility testing of the main vector species to fenitrothion was conducted., Results: In total, 7537 adult Anopheles were collected from both sites. Of the 493 specimens molecularly confirmed to species, two thirds (n = 340) were identified as Nyssorhynchus albimanus, followed by Anopheles aquasalis. Overall Anopheles human biting rates (HBRs) were higher outdoors than indoors, and were higher in Permé than in Puerto Obaldía: nightly outdoor HBR ranged from 2.71 bites per person per night (bpn) (Puerto Obaldía), to 221.00 bpn (Permé), whereas indoor nightly HBR ranged from 0.70 bpn (Puerto Obaldía) to 81.90 bpn (Permé). Generally, peak biting occurred during the early evening. The CDC LT trap yields were significantly lower than that of HLCs and this collection method was dropped after the first collection. Pyrethrum spray catches resulted in only three indoor resting Anopheles collected. Insecticide resistance (IR) of Ny. albimanus to fenitrothion was confirmed, with only 65.5% mortality at the diagnostic time., Conclusion: The early evening exophagic behaviour of Anopheles vectors, the absence of indoor resting behaviours, and the presence of resistance to the primary intervention insecticide demonstrate limitations of the current malaria strategy, including indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs), and point to both gaps in protection and to the drivers of persisting malaria transmission in Guna Yala. These findings highlight the need for continued and directed entomological surveillance, based on programmatic questions, that generates entomological evidence to inform an adaptive malaria elimination strategy., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

Anopheles hinesorum is a mosquito species with variable host preference. Throughout New Guinea and northern Australia, An .  hinesorum feeds on humans (it is opportunistically anthropophagic) while in the south-west Pacific's Solomon Archipelago, the species is abundant but has rarely been found biting humans (it is exclusively zoophagic in most populations). There are at least two divergent zoophagic (nonhuman biting) mitochondrial lineages of An .  hinesorum in the Solomon Archipelago representing two independent dispersals. Since zoophagy is a derived (nonancestral) trait in this species, this leads to the question: has zoophagy evolved independently in these two populations? Or conversely: has nuclear gene flow or connectivity resulted in the transfer of zoophagy? Although we cannot conclusively answer this, we find close nuclear relationships between Solomon Archipelago populations indicating that recent nuclear gene flow has occurred between zoophagic populations from the divergent mitochondrial lineages. Recent work on isolated islands of the Western Province (Solomon Archipelago) has also revealed an anomalous, anthropophagic island population of An .  hinesorum . We find a common shared mitochondrial haplotype between this Solomon Island population and another anthropophagic population from New Guinea. This finding suggests that there has been recent migration from New Guinea into the only known anthropophagic population from the Solomon Islands. Although currently localized to a few islands in the Western Province of the Solomon Archipelago, if anthropophagy presents a selective advantage, we may see An .  hinesorum emerge as a new malaria vector in a region that is now working on malaria elimination., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The host decoy trap (HDT) is a surveillance trap that presents a combination of heat, visual and odour stimuli to attract bloodmeal-seeking mosquitoes. Here we employed a semi-field study to demonstrate the role of the visual attributes present on the HDT on the effectiveness of Aedes aegypti capture. Our results show that the HDT is an effective means of capturing Ae. aegypti mosquitoes in semi-field conditions, with a per trial capture rate of up to 69% across four visually distinct HDTs. The solid black coloured HDT captured more mosquitoes than HDTs with black-white stripes, black-white checkerboard patches or solid white colour by a factor of 1.9, 1.7 and 1.5, respectively. In all cases, mosquito capture was not evenly distributed on the HDT surface, with captures on the HDT's outer downwind half, away from the odour delivery, exceeding captures on the inner upwind half. We conclude that the solid black surface of the original HDT design is more effective than the other surfaces (white or black/white patterns) for the capture of Ae. aegypti. Our results demonstrate that mosquito attraction to the thermal and odorant cues of the HDT is modulated by visual information., (© 2020 The Royal Entomological Society.)

Background: Malaria vector control has been implemented chiefly through indoor interventions targeting primary vectors resulting in population declines-pointing to a possible greater proportional contribution to transmission by secondary malaria vectors with their predominant exophagic and exophilic traits. With a historical focus on primary vectors, there is paucity of data on secondary malaria vectors in many countries in Africa. This study sought to determine the species compositions and bionomic traits, including proportions infected with Plasmodium falciparum and phenotypic insecticide resistance, of secondary vectors in three sites with high malaria transmission in Kisumu County, western Kenya., Methods: Cross-sectional sampling of adult Anopheles was conducted using indoor and outdoor CDC light traps (CDC-LT) and animal-baited traps (ABTs) in Kakola-Ombaka and Kisian, while larvae were sampled in Ahero. Secondary vectors captured were exposed to permethrin using WHO bioassays and then analyzed by ELISA to test for proportions infected with P. falciparum sporozoites. All Anopheles were identified to species using morphological keys with a subset being molecularly identified using ITS2 and CO1 sequencing for species identification., Results: Two morphologically identified secondary vectors captured-An. coustani and An. pharoensis-were determined to consist of four species molecularly. These included An. christyi, An. sp. 15 BSL-2014, an unidentified member of the An. coustani complex (An. cf. coustani) and a species similar to that of An. pharoensis and An. squamosus (An. cf. pharoensis). Standardized (Anopheles per trap per night) capture rates demonstrate higher proportions of secondary vectors across most trapping methods-with overall indoor and outdoor CDC-LTs and ABT captures composed of 52.2% (n = 93), 78.9% (n = 221) and 58.1% (n = 573) secondary vectors respectively. Secondary vectors were primarily caught outdoors. The overall proportion of secondary vectors with P. falciparum sporozoite was 0.63% (n = 5), with the unidentified species An. cf. pharoensis, determined to carry Plasmodium. Overall secondary vectors were susceptible to permethrin with a > 99% mortality rate., Conclusions: Given their high densities, endophily equivalent to primary vectors, higher exophily and Plasmodium-positive proportions, secondary vectors may contribute substantially to malaria transmission. Unidentified species demonstrate the need for further morphological and molecular identification studies towards further characterization. Continued monitoring is essential for understanding their temporal contributions to transmission, the possible elevation of some to primary vectors and the development of insecticide resistance.

Background: A goal of malaria epidemiological interventions is the detection and treatment of parasite reservoirs in endemic areas-an activity that is expected to reduce local transmission. Since the gametocyte is the only transmissible stage from human host to mosquito vector, this study evaluated the pre and post presence of gametocytes during a mass screening and treatment (MST) intervention conducted during 2013 in East Nusa Tenggara, Indonesia., Methods: RT-qPCR targeting pfs25 and pvs25 transcripts-gametocyte molecular markers for Plasmodium falciparum and Plasmodium vivax, respectively, was performed to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax-infected subjects over the course of the MST study. The presence of both asexual and sexual parasites in microscopic and submicroscopic infections was compared from the start and end of the MST, using proportion tests as well as parametric and non-parametric tests., Results: Parasite prevalence remained unchanged for P. falciparum (6% = 52/811 versus 7% = 50/740, p = 0.838), and decreased slightly for P. vivax (24% = 192/811 versus 19% = 142/740, p = 0.035) between the MST baseline and endpoint. No significant difference was observed in gametocyte prevalence for either P. falciparum (2% = 19/803 versus 3% = 23/729, p = 0.353, OR = 1.34, 95%CI = 0.69-2.63), or P. vivax (7% = 49/744 versus 5% = 39/704, p = 0.442, OR = 0.83, 95%CI = 0.52-1.31). Even though there was an insignificant difference between the two time points, the majority of parasite positive subjects at the endpoint had been negative at baseline (P. falciparum: 66% = 29/44, P. vivax: 60% = 80/134). This was similarly demonstrated for the transmissible stage-where the majority of gametocyte positive subjects at the endpoint were negative at baseline (P. falciparum: 95% = 20/21, P. vivax: 94% = 30/32). These results were independent of treatment provided during MST activities. No difference was demonstrated in parasite and gametocyte density between both time points either in P. falciparum or P. vivax., Conclusion: In this study area, similar prevalence rates of P. falciparum and P. vivax parasites and gametocytes before and after MST, although in different individuals, points to a negligible impact on the parasite reservoir. Treatment administration based on parasite positivity as implemented in the MST should be reevaluated for the elimination strategy in the community. Trial registration Clinical trials registration NCT01878357. Registered 14 June 2013, https://www.clinicaltrials.gov/ct2/show/NCT01878357.

Advances in genomics have led to an appreciation that introgression is common, but its evolutionary consequences are poorly understood. In recent species radiations the sharing of genetic variation across porous species boundaries can facilitate adaptation to new environments and generate novel phenotypes, which may contribute to further diversification. Most Anopheles mosquito species that are of major importance as human malaria vectors have evolved within recent and rapid radiations of largely nonvector species. Here, we focus on one of the most medically important yet understudied anopheline radiations, the Afrotropical Anopheles funestus complex (AFC), to investigate the role of introgression in its diversification and the possible link between introgression and vector potential. The AFC comprises at least seven morphologically similar species, yet only An. funestus sensu stricto is a highly efficient malaria vector with a pan-African distribution. Based on de novo genome assemblies and additional whole-genome resequencing, we use phylogenomic and population genomic analyses to establish species relationships. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification. The most recent introgression event involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus , an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: Understanding local anopheline vector species and their bionomic traits, as well as related human factors, can help combat gaps in protection., Methods: In San José de Chamanga, Esmeraldas, at the Ecuadorian Pacific coast, anopheline mosquitoes were sampled by both human landing collections (HLCs) and indoor-resting aspirations (IAs) and identified using both morphological and molecular methods. Human behaviour observations (HBOs) (including temporal location and bed net use) were documented during HLCs as well as through community surveys to determine exposure to mosquito bites. A cross-sectional evaluation of Plasmodium falciparum and Plasmodium vivax infections was conducted alongside a malaria questionnaire., Results: Among 222 anopheline specimens captured, based on molecular analysis, 218 were Nyssorhynchus albimanus, 3 Anopheles calderoni (n = 3), and one remains unidentified. Anopheline mean human-biting rate (HBR) outdoors was (13.69), and indoors (3.38) (p = 0.006). No anophelines were documented resting on walls during IAs. HBO-adjusted human landing rates suggested that the highest risk of being bitten was outdoors between 18.00 and 20.00 h. Human behaviour-adjusted biting rates suggest that overall, long-lasting insecticidal bed nets (LLINs) only protected against 13.2% of exposure to bites, with 86.8% of exposure during the night spent outside of bed net protection. The malaria survey found 2/398 individuals positive for asymptomatic P. falciparum infections. The questionnaire reported high (73.4%) bed net use, with low knowledge of malaria., Conclusion: The exophagic feeding of anopheline vectors in San Jose de Chamanga, when analysed in conjunction with human behaviour, indicates a clear gap in protection even with high LLIN coverage. The lack of indoor-resting anophelines suggests that indoor residual spraying (IRS) may have limited effect. The presence of asymptomatic infections implies the presence of a human reservoir that may maintain transmission.

According to the WHO, unmanaged insecticide resistance may lead to increases in malaria-related mortality and morbidity. Bangladesh, having made significant progress in malaria control efforts, has recently seen an upswing in malaria cases-58% of which occurred in Bandarban district. Toward identifying entomological drivers of increased malaria, an entomological survey including Anopheles susceptibility to the insecticides in use was conducted in Bandarban. Anopheles vagus , the primary vector of malaria, was found to be resistant to both permethrin and deltamethrin-with only 29% and 55% mortality at 30 minutes, respectively. Intervention strategies in this area-all based on pyrethroids, may need to be reevaluated toward closing this gap in protection and increasing intervention efficacy.

Background: Understanding local Anopheles species compositions and bionomic traits are vital for an effective malaria vector intervention strategy. Though eight malaria vectors, including species complexes, have been documented across the island of Sulawesi, Indonesia, a comprehensive survey linking morphological and molecular species identification has not been conducted in this global hotspot of biodiversity., Results: Eighteen distinct species of Anopheles were molecularly identified in a 1 km 2 area in Karama village, West Mamuju Province, Sulawesi. Known species included An. aconitus, An. karwari, An. peditaeniatus, An. vagus, An. barbirostris, An. tessellatus, An. nigerrimus, An. crawfordi, An. maculatus, An. flavirostris and An. kochi. Of the 18 distinct sequence groups identified through both ribosomal DNA internal transcribed spacer region 2, and mitochondrial DNA cytochrome c oxidase subunit 1 loci, 8 could not be identified to species through comparison to published sequences. The comparison of morphological and molecular identities determined that interpretations of local species compositions for primary and expected species in Karama (An. barbirostris and An. vagus) had the highest rate of accuracy (92.1% and 87.6%, respectively) when compared to molecular analysis. However, the remaining distinct sequences molecularly identified to species were identified correctly by morphological methods less frequently, from 0 to 83%., Conclusions: Karama, Indonesia has a high diversity of Anopheles spp. The unexpected high number of Anopheles species in a small area points to possible complex transmission dynamics and limitations with vector control based on possible varying behaviors and interactions with both humans and interventions. Morphological identification of Anopheles spp. in this study was more accurate for primary and expected species than secondary or unexpected species. Finally, the inability to identify seven sequence groups to species with consensus sequences implies that future studies employing sequencing are required to clarify species compositions in the Nigerrimus Subgroup, among others, as well as their distribution and vector status. Use of molecular methods in conjunction with morphological investigations for analysis of species composition, population dynamics and bionomic characteristics is directly implicated in understanding drivers of malaria transmission, intervention effectiveness, and the pursuit of malaria elimination.