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The paper presents an innovative computational framework for predictive solutions for simulating the spread of malaria. The structure incorporates sophisticated computing methods to improve the reliability of predicting malaria outbreaks. The study strives to provide a strong and effective tool for forecasting the propagation of malaria via the use of an AI-based recurrent neural network (RNN). The model is classified into two groups, consisting of humans and mosquitoes. To develop the model, the traditional Ross-Macdonald model is expanded upon, allowing for a more comprehensive analysis of the intricate dynamics at play. To gain a deeper understanding of the extended Ross model, we employ RNN, treating it as an initial value problem involving a system of first-order ordinary differential equations, each representing one of the seven profiles. This method enables us to obtain valuable insights and elucidate the complexities inherent in the propagation of malaria. Mosquitoes and humans constitute the two cohorts encompassed within the exposition of the mathematical dynamical model. Human dynamics are comprised of individuals who are susceptible, exposed, infectious, and in recovery. The mosquito population, on the other hand, is divided into three categories: susceptible, exposed, and infected. For RNN, we used the input of 0 to 300 days with an interval length of 3 days. The evaluation of the precision and accuracy of the methodology is conducted by superimposing the estimated solution onto the numerical solution. In addition, the outcomes obtained from the RNN are examined, including regression analysis, assessment of error autocorrelation, examination of time series response plots, mean square error, error histogram, and absolute error. A reduced mean square error signifies that the model's estimates are more accurate. The result is consistent with acquiring an approximate absolute error close to zero, revealing the efficacy of the suggested strategy. This research presents a novel approach to solving the malaria propagation model using recurrent neural networks. Additionally, it examines the behavior of various profiles under varying initial conditions of the malaria propagation model, which consists of a system of ordinary differential equations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nisar 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: The Magude Project assessed the feasibility of eliminating malaria in Magude district, a low transmission setting in southern Mozambique, using a package of interventions, including long-lasting insecticidal nets (LLINs). As the efficacy of LLINs depends in part on their physical integrity, this metric was quantified for Olyset® Nets post mass-distribution, in addition to net use, care and handling practices and other risk factors associated with net physical integrity., Methods: Nets were collected during a cross-sectional net evaluation, nine months after the Magude project commenced, which was 2 years after the nets were distributed by the National Malaria Control Programme (NMCP). The physical integrity of the nets was assessed by counting and sizing the holes at different positions on each net. A structured questionnaire was administered to assess how the selected net was used and treated (care, wash and repair). Net bio-efficacy was assessed following the standard World Health Organization (WHO) cone bioassay procedures., Results: Out of the 170 Olyset® Nets included in the analysis, 63.5% had been used the night before. The main reason for not using a net was the notion that there were no mosquitoes present. The average number of people using each net was 1.79. Two thirds of the nets had only been washed once or twice since distribution. Most nets (80.9%) were holed and 18% were torn, but none of the risk factors were significantly associated with net integrity, except for presence of mice in the household. Less than half of the participants noticed holes in holed nets, and of those only 38.6% attempted to repair those. None of the six nets that were tested for bio-efficacy passed the WHO threshold of 80% mosquito mortality., Conclusion: Overall the majority of Olyset® Nets were in serviceable condition two years post-distribution, but their insecticidal effect may have been lost. This study-together with previous evidence on suboptimal access to and use of LLINs in Magude district-highlights that LLINs as an intervention could have been optimized during the Magude project to achieve maximum intervention impact., (© 2024. The Author(s).)

Background: Malaria is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Human-to-human transmission depends on a mosquito vector; thus, the interruption of parasite transmission from humans to mosquitoes is an important approach in the fight against malaria. The parasite stages infectious to mosquitoes are the gametocytes, sexual stages that are ingested by the vector during a blood meal and transform into male and female gametes in the midgut. Immunity against sexual stage antigens expressed by gametocytes, gametes, and the zygote formed after fertilisation can interrupt the parasite sexual cycle in the mosquito. This transmission blocking immunity is mediated by specific antibodies ingested during the mosquito blood feed, inhibiting the parasite development in the midgut. Merozoite thrombospondin related anonymous protein (MTRAP) is a merozoite and gametocyte surface protein essential for gamete egress from erythrocytes and for parasite transmission to mosquitoes., Objectives: Here, we evaluated the potential of the P. berghei MTRAP to elicit antibodies with the ability to inhibit gamete fertilisation in vitro., Methods: We expressed a soluble recombinant PbMTRAP and used it to immunise BALB/c mice. The transmission blocking activity of the anti-rPbMTRAP antibodies was tested through in vivo challenge experiments followed by in vitro conversion assays., Findings: Immunisations with the rPbMTRAP induced a strong antibody response and the antibodies recognised the native protein by Western Blot and IFA. Anti-rPbMTRAP present in the blood stream of immunised mice partially inhibited gamete conversion into ookinetes., Conclusion: Our results indicate that antibodies to PbMTRAP may reduce but are not sufficient to completely block transmission.

Background: The malaria incidence data from a malaria prevention study from the Rift Valley, Central Ethiopia, were reanalysed. The objective was to investigate whether including an administrative structure within the society, which may have required consideration in the protocol or previous analysis, would provide divergent outcomes on the effect measures of the interventions., Methods: A cluster-randomized controlled trial lasting 121 weeks with 176 clusters in four groups with 6071 households with 34,548 persons was done: interventions combining indoor residual spraying (IRS) and insecticide-treated nets (ITNs), IRS alone, ITNs alone and routine use. The primary outcome was malaria incidence. A multilevel negative binomial regression model was employed to examine the impact of the kebele (smallest administrative unit) and the proximity of homes to the primary mosquito breeding sites as potential residual confounders (levels). The study also assessed whether these factors influenced the effect measures of the interventions., Results: The study's initial findings revealed 1183 malaria episodes among 1059 persons, with comparable effects observed across the four intervention groups. In the reanalysis, the results showed that both ITN + IRS (incidence rate ratio [IRR] 0.63, P < 0.001) and ITN alone (IRR 0.78, P = 0.011) were associated with a greater reduction in malaria cases compared to IRS (IRR 0.90; P = 0.28) or the control (reference) group. The combined usage of IRS with ITN yields better outcomes compared to the standalone use of ITN and surpasses the effectiveness of IRS in isolation., Conclusion: The findings indicate that implementing a combination of IRS and ITN and also ITN alone decrease malaria incidence. Furthermore, there was an observed synergistic impact when ITN and IRS were used in combination. Considering relevant social structures as potential residual confounders is of paramount importance., Trial Registration: PACTR201411000882128 (08 September 2014)., (© 2024. The Author(s).)

Background: By 2022, the Government of Indonesia had successfully eliminated malaria in 389 out of 514 districts but continues to face a challenge in Eastern Indonesia where 95% of the total 2021 malaria cases were reported from Papua, West Papua and Nusa Tenggara Timur provinces. There is an increased recognition that malaria elimination will require a better understanding of the human behavioural factors hindering malaria prevention and treatment, informed by local context and local practice., Methods: This research used a light-touch immersion research approach. Field researchers lived in communities over several days to gather data through informal conversations, group-based discussions using visual tools, participant observation and direct experience. The study was conducted in four high malaria endemic areas in Papua, West Papua, and Sumba Islands in Nusa Tenggara Timur., Results: The research highlights how people's perception of malaria has changed since the introduction of effective treatment which, in turn, has contributed to a casual attitude towards early testing and adherence to malaria treatment. It also confirms that people rarely accept there is a link between mosquitoes and malaria based on their experience but nevertheless take precautions against the annoyance of mosquitoes. There is widespread recognition that babies and small children, elderly and incomers are more likely to be seriously affected by malaria and separately, more troubled by mosquitoes than indigenous adult populations. This is primarily explained by acclimatization and strong immune systems among the latter., Conclusions: Using immersion research enabled behaviour research within a naturalistic setting, which in turn enabled experiential-led analysis of findings and revealed previously unrecognized insights into attitudes towards malaria in Eastern Indonesia. The research provides explanations of people's lack of motivation to consistently use bed nets, seek early diagnosis or complete courses of treatment. The felt concern for the wellbeing of vulnerable populations highlighted during light touch immersion provides an entry point for future social behaviour change communication interventions. Rather than trying to explain transmission to people who deny this connection, the research concludes that it may be better to focus separately on the two problems of malaria and mosquitoes (especially for vulnerable groups) thereby resonating with local people's own experience and felt concerns., (© 2024. The Author(s).)

Introduction: with imported malaria cases in a given population, the question arises as to what extent the local cases are a consequence of the imports or not. We perform a modeling analysis for a specific area, in a region aspiring for malaria-free status., Methods: data on malaria cases over ten years is subjected to a compartmental model which is assumed to be operating close to the equilibrium state. Two of the parameters of the model are fitted to the decadal data. The other parameters in the model are sourced from the literature. The model is utilized to simulate the malaria prevalence with or without imported cases., Results: in any given year the annual average of 460 imported cases, resulted in an end-of-year season malaria prevalence of 257 local active infectious cases, whereas without the imports the malaria prevalence at the end of the season would have been fewer than 10 active infectious cases. We calculate the numerical value of the basic reproduction number for the model, which reveals the extent to which the disease is being eliminated from the population or not., Conclusion: without the imported cases, over the ten seasons of malaria, 2008-2018, the KwaZulu-Natal province would have been malaria-free over at least the last 7 years of the decade indicated. This simple methodology works well even in situations where data is limited., Competing Interests: The authors declare no competing interests., (Copyright: Peter Joseph Witbooi et al.)

The successful completion of gamete fertilization is essential for malaria parasite transmission, and this process can be targeted by intervention strategies. In this study, we identified a conserved gene (PBANKA&#95;0813300) in the rodent malaria parasite Plasmodium berghei, which encodes a protein of 54 kDa (designated as Pbs54). Localization studies indicated that Pbs54 is associated with the plasma membranes of gametes and ookinetes. Functional studies by gene disruption showed that the Δpbs54 parasites had no defect in asexual proliferation, gametocyte development, or gametogenesis. However, the interactions between male and female gametes were significantly decreased compared with wild-type parasites. The Δpbs54 lines did not show a further reduction in zygote and ookinete numbers during in vitro culture, indicating that the defects were probably restricted to gamete fertilization. Consistent with this finding, mosquitoes fed on Δpbs54-infected mice showed a 30.1% reduction in infection prevalence and a 74.7% reduction in oocyst intensity. Cross-fertilization assay indicated that both male and female gametes were impaired in the Δpbs54 parasites. To evaluate its transmission-blocking potential, we obtained polyclonal antibodies from mice immunized with the recombinant Pbs54 (rPbs54) protein. In vitro assays showed that anti-rPbs54 sera inhibited ookinete formation by 42.7%. Our experiments identified Pbs54 as a fertility factor required for mosquito transmission and a novel candidate for a malaria transmission-blocking vaccine., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Gene drive modified mosquitoes (GDMMs) have the potential to address Africa's persistent malaria problem, but are still in early stages of development and testing. Continuous engagement of African stakeholders is crucial for successful evaluation and implementation of these technologies. The aim of this multi-country study was, therefore, to explore the insights and recommendations of key stakeholders across Africa on the potential of GDMMs for malaria control and elimination in the continent., Methods: A concurrent mixed-methods study design was used, involving a structured survey administered to 180 stakeholders in 25 countries in sub-Saharan Africa, followed by 18 in-depth discussions with selected groups and individuals. Stakeholders were drawn from academia, research and regulatory institutions, government ministries of health and environment, media and advocacy groups. Thematic content analysis was used to identify key topics from the in-depth discussions, and descriptive analysis was done to summarize information from the survey data., Results: Despite high levels of awareness of GDMMs among the stakeholders (76.7%), there was a relatively low-level of understanding of their key attributes and potential for malaria control (28.3%). When more information about GDMMs was provided to the stakeholders, they readily discussed their insights and concerns, and offered several recommendations to ensure successful research and implementation of the technology. These included: (i) increasing relevant technical expertise within Africa, (ii) generating local evidence on safety, applicability, and effectiveness of GDMMs, and (iii) developing country-specific regulations for safe and effective governance of GDMMs. A majority of the respondents (92.9%) stated that they would support field trials or implementation of GDMMs in their respective countries. This study also identified significant misconceptions regarding the phase of GDMM testing in Africa, as several participants incorrectly asserted that GDMMs were already present in Africa, either within laboratories or released into the field., Conclusion: Incorporating views and recommendations of African stakeholders in the ongoing research and development of GDMMs is crucial for instilling stakeholder confidence on their potential application. These findings will enable improved planning for GDMMs in Africa as well as improved target product profiles for the technologies to maximize their potential for solving Africa's enduring malaria challenge., (© 2023. The Author(s).)

There is an increasing awareness of the association between tourism activity and risks of emerging mosquito-borne diseases (MBDs) worldwide. In previous studies we showed that hotels in Zanzibar may play an important role in maintaining residual foci of mosquito vectors populations of public health concern. These findings indicated larval sources removal (LSR) interventions may have a significant negative impact on vector communities. However, a thorough analysis of the response vector species to potential LSM strategies must be evaluated prior to implementation of a large-scale area-wide control campaign. Here we propose a protocol for evaluation of the impact of LSR against mosquito vectors at hotel settings in Zanzibar. This protocol is set to determine the efficacy of LSR in a randomized control partial cross-over experimental design with four hotel compounds representing the unit of randomization for allocation of interventions. However, the protocol can be applied to evaluate the impact of LRS in more than four sites. Proposed interventions are active removal of disposed containers, and installation of water dispenser to replace single use discarded plastic water bottles, which were identified as the most important source of mosquitoes studied hotels. The ideal time for allocating intervention to the intervention arms the dry season, when the mosquito abundance is predictably lower. The possible impact of interventions on mosquito occurrence and abundance risks is then evaluated throughout subsequent rainy and dry seasons. If an appreciable reduction in mosquito abundance and occurrence risks is observed during the trial period, intervention could be extended to the control arm to determine whether any potential reduction of mosquito density is reproducible. A rigorous evaluation of the proposed LRS interventions will inspire large scale trials and provide support for evidence-based mosquito management at hotel facilities in Zanzibar and similar settings., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kampango 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: Irrigated rice cultivation in sub-Saharan Africa not only brings more malaria vectors to nearby communities, but also greater malaria risk. To aid the implementation of mosquito control in rice-growing communities, it is necessary to understand how farmers understand, view and manage their responsibility in mosquito generation and whether they are interested in coordinating to minimize it., Methods: Qualitative methods (observation grids, semi-structured in-depth interviews and focus group discussions) were used to reveal the perceptions of mosquitoes and their control in two irrigated rice farming communities in central Côte d'Ivoire near the M'bé and Lokapli irrigation schemes., Results: All rice farmers viewed mosquitoes as severe nuisances, and most acknowledged that they caused djèkouadjo (malaria) and were less numerous during harmattan (dry season). Many study participants believed that mosquitoes originated from grasses and stagnant water around villages. Only those living closer in proximity (~ 1 km) to the paddies believed that mosquitoes came from the bas-fonds (irrigated lowlands). However, they did not associate mosquito production with rice cultivation. Some farmers believed that there were more mosquitoes in recent years than historically because of the dam construction, but remarked on the importance of the dam (and bas-fonds) for their livelihood. Many farmers were not convinced that mosquito control could occur at farm-level., Conclusions: To enhance accountability amongst rice farmers, there is a need for greater awareness on the rice-mosquito link, and emphasis that the link does not imply a trade-off between food production and health. Training should not only be directed towards farming communities, but also agricultural and health extension workers. Future riceland mosquito control methods must focus on improving crop productivity and address collective action problems that may occur., (© 2023. The Author(s).)

Studies on the applications of infrared (IR) spectroscopy and machine learning (ML) in public health have increased greatly in recent years. These technologies show enormous potential for measuring key parameters of malaria, a disease that still causes about 250 million cases and 620,000 deaths, annually. Multiple studies have demonstrated that the combination of IR spectroscopy and machine learning (ML) can yield accurate predictions of epidemiologically relevant parameters of malaria in both laboratory and field surveys. Proven applications now include determining the age, species, and blood-feeding histories of mosquito vectors as well as detecting malaria parasite infections in both humans and mosquitoes. As the World Health Organization encourages malaria-endemic countries to improve their surveillance-response strategies, it is crucial to consider whether IR and ML techniques are likely to meet the relevant feasibility and cost-effectiveness requirements-and how best they can be deployed. This paper reviews current applications of IR spectroscopy and ML approaches for investigating malaria indicators in both field surveys and laboratory settings, and identifies key research gaps relevant to these applications. Additionally, the article suggests initial target product profiles (TPPs) that should be considered when developing or testing these technologies for use in low-income settings., (© 2023. The Author(s).)

Preventing parasite transmission from humans to mosquitoes is recognised to be critical for achieving elimination and eradication of malaria. Consequently developing new antimalarial drugs with transmission-blocking properties is a priority. Large screening campaigns have identified many new transmission-blocking molecules, however little is known about how they target the mosquito-transmissible Plasmodium falciparum stage V gametocytes, or how they affect their underlying cell biology. To respond to this knowledge gap, we have developed a machine learning image analysis pipeline to characterise and compare the cellular phenotypes generated by transmission-blocking molecules during male gametogenesis. Using this approach, we studied 40 molecules, categorising their activity based upon timing of action and visual effects on the organisation of tubulin and DNA within the cell. Our data both proposes new modes of action and corroborates existing modes of action of identified transmission-blocking molecules. Furthermore, the characterised molecules provide a new armoury of tool compounds to probe gametocyte cell biology and the generated imaging dataset provides a new reference for researchers to correlate molecular target or gene deletion to specific cellular phenotype. Our analysis pipeline is not optimised for a specific organism and could be applied to any fluorescence microscopy dataset containing cells delineated by bounding boxes, and so is potentially extendible to any disease model., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tsebriy 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.)

Studies on the timing of anopheline host-seeking are important for understanding mosquito ecology, behaviors, and possible role in disease transmission. To study the evening crepuscular host-seeking activity of anophelines and the effects of moonlight, anopheline mosquitoes were collected with light traps settled in a livestock area in the Brazilian Cerrado region. Traps (Silva traps) were placed next to animal enclosures at 1.5 m asl. The research was divided in 2 experiments: the first experiment (12 nights) comprised 2 trapping periods, one from 6 PM to 7 PM and one from 7 PM to 6 AM, and the second experiment (16 evenings) divided in three 20-min intervals based on the 3 twilight phases (civil: 6-6:20 PM; nautical: 6:20-6:40 PM; and astronomical: 6:40-7 PM). A total of 2,815 anopheline mosquitoes and 9 species were found. The main species were Anopheles triannulatus s.l., An. argyritarsis, An. goeldii, and An. evanse. Host-seeking mosquitoes were more frequently active during the first hour after sunset, with a significant peak in the second 20-min interval. After that, a decrease in the number of individuals was observed from the astronomical twilight onwards. Moonlight did not affect the evening flight activity of anophelines. By using LED-based passive light traps, the evening arrival time of anophelines at blood-feeding sites was demonstrated, and it may be a key time window for malaria vector control interventions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although larval diet quality may affect adult mosquito fitness, its impact on parasite development is scarce. Plant pollen from Zea mays, Typha latifolia, and Prosopis juliflora was ultraviolet-sterilized and examined for effects on larval development, pupation rate, adult mosquito longevity, survival and infectivity. The control larvae were fed Tetramin fish food as a comparator food. Four treatment and two control groups were used for each pollen diet, and each experimental tray had 25 larvae. Female An. arabiensis were starved overnight and exposed to infectious blood using a membrane-feeding system. The Kaplan-Meier curves and log-rank test were used for analysis. The Z. mays pollen diet increased malaria mosquito survival and pupation rate (91.3%) and adult emergence (85%). Zea mays and Tetramin fish food had comparable adulthood development times. Adults who emerged from larvae fed Z. mays pollen had the longest average wing length (3.72 mm) and were more permissive to P. vivax (45%) and P. falciparum (27.5%). They also survived longer after feeding on infectious blood and had the highest number of P. vivax oocysts. Zea mays pollen improved larval development, adult mosquito longevity, survival and infectivity to Plasmodium. Our findings suggest that malaria transmission in Z. mays growing villages should be monitored., (© 2023. Springer Nature Limited.)

Mosquito-borne diseases like dengue and malaria cause a significant global health burden. Unfortunately, current insecticides and environmental control strategies aimed at the vectors of these diseases are only moderately effective in decreasing disease burden. Understanding and manipulating the interaction between the mosquito holobiont (i.e., mosquitoes and their resident microbiota) and the pathogens transmitted by these mosquitoes to humans and animals could help in developing new disease control strategies. Different microorganisms found in the mosquito's microbiota affect traits related to mosquito survival, development, and reproduction. Here, we review the physiological effects of essential microbes on their mosquito hosts; the interactions between the mosquito holobiont and mosquito-borne pathogen (MBP) infections, including microbiota-induced host immune activation and Wolbachia-mediated pathogen blocking (PB); and the effects of environmental factors and host regulation on the composition of the microbiota. Finally, we briefly overview future directions in holobiont studies, and how these may lead to new effective control strategies against mosquitoes and their transmitted diseases., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Malaria-transmitting mosquitoes are skilled human hunters, selectively choosing their prey based on a complex array of sensory cues. A new study unveils a distinct pattern of preference for human-associated olfactory cues that underlies the selective behavior of these mosquitoes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen. IMPORTANCE Blocking human-to-vector transmission is an essential step toward malaria elimination. Gametocytes are solely responsible for achieving this transmission and represent an opportunity for therapeutic intervention. While these falciform-shaped parasite stages were first discovered in the 1880s, our understanding of the genetic determinants responsible for their formation and molecular mechanisms that drive their development is limited. In this work, we developed a scalable screening methodology with piggyBac mutants to identify genes that influence the development of gametocytes in the most lethal human malaria parasite, P. falciparum. By doing so, we lay the foundation for large-scale functional genomic studies specifically designed to address remaining questions about sexual commitment, maturation, and mosquito infection in P. falciparum. Such functional genetic screens will serve to expedite the identification of essential pathways and processes for the development of novel transmission-blocking agents., Competing Interests: The authors declare no conflict of interest.

Plasmodium parasites are the etiological agents of malaria, a disease responsible for over half a million deaths annually. Successful completion of the parasite's life cycle in the vertebrate host and transmission to a mosquito vector is contingent upon the ability of the parasite to evade the host's defenses. The extracellular stages of the parasite, including gametes and sporozoites, must evade complement attack in both the mammalian host and in the blood ingested by the mosquito vector. Here, we show that Plasmodium falciparum gametes and sporozoites acquire mammalian plasminogen and activate it into the serine protease plasmin to evade complement attack by degrading C3b. Complement-mediated permeabilization of gametes and sporozoites was higher in plasminogen-depleted plasma, suggesting that plasminogen is important for complement evasion. Plasmin also facilitates gamete exflagellation through complement evasion. Furthermore, supplementing serum with plasmin significantly increased parasite infectivity to mosquitoes and lowered the transmission-blocking activity of antibodies to Pfs230, a potent vaccine candidate currently in clinical trials. Finally, we show that human factor H, previously shown to facilitate complement evasion by gametes, also facilitates complement evasion by sporozoites. Plasmin and factor H simultaneously cooperate to enhance complement evasion by gametes and sporozoites. Taken together, our data show that Plasmodium falciparum gametes and sporozoites hijack the mammalian serine protease plasmin to evade complement attack by degrading C3b. Understanding of the mechanisms of complement evasion by the parasite is key to the development of novel effective therapeutics. IMPORTANCE Current approaches to control malaria are complicated by the development of antimalarial-resistant parasites and insecticide-resistant vectors. Vaccines that block transmission to mosquitoes and humans are a plausible alternative to overcome these setbacks. To inform the development of efficacious vaccines, it is imperative to understand how the parasite interacts with the host immune response. In this report, we show that the parasite can co-opt host plasmin, a mammalian fibrinolytic protein to evade host complement attack. Our results highlight a potential mechanism that may reduce efficacy of potent vaccine candidates. Taken together, our results will inform future studies in developing novel antimalarial therapeutics., Competing Interests: The authors declare no conflict of interest.

Sierra Leone is vulnerable to a wide range of vector-borne diseases transmitted by mosquitoes, tsetse flies, black flies, and other vectors. Malaria, lymphatic filariasis, and onchocerciasis have posed the greatest threat and have received the most attention in terms of vector control and capacity for diagnosis. However, malaria infection rates remain high, and there is evidence of circulation of other vector-borne diseases, such as chikungunya and dengue, which may go undiagnosed and unreported. The limited understanding of the prevalence and transmission of these diseases restricts the capacity for predicting outbreaks, and impedes the planning of appropriate responses. We review the available literature and gather expert opinions from those working in the country to report on the status of vector-borne disease transmission and control in Sierra Leone, and present an assessment of the threats of these diseases. Our discussions highlight an absence of entomological testing for disease agents and the need for more investment in surveillance and capacity strengthening.

Some parasitic diseases, such as malaria, require two hosts to complete their lifecycle: a human and an insect vector. Although most malaria research has focused on parasite development in the human host, the life cycle within the vector is critical for the propagation of the disease. The mosquito stage of the Plasmodium lifecycle represents a major demographic bottleneck, crucial for transmission blocking strategies. Furthermore, it is in the vector, where sexual recombination occurs generating " de novo " genetic diversity, which can favor the spread of drug resistance and hinder effective vaccine development. However, understanding of vector-parasite interactions is hampered by the lack of experimental systems that mimic the natural environment while allowing to control and standardize the complexity of the interactions. The breakthrough in stem cell technologies has provided new insights into human-pathogen interactions, but these advances have not been translated into insect models. Here, we review in vivo and in vitro systems that have been used so far to study malaria in the mosquito. We also highlight the relevance of single-cell technologies to progress understanding of these interactions with higher resolution and depth. Finally, we emphasize the necessity to develop robust and accessible ex vivo systems (tissues and organs) to enable investigation of the molecular mechanisms of parasite-vector interactions providing new targets for malaria control., 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 Parres-Mercader, Pance and Gómez-Díaz.)

Alphonse Laveran (Nobel Prize 1907) played a pioneering role in discovering the causative agent of malaria, a disease that has existed since time immemorial, and long emblematic of the miasma theory until the end of the 19 th century. In 1880, this unknown military doctor discovered the role of a hematazoan in malaria, designated Plasmodium. This was the first protozoan to be discovered in an infectious disease, at a time when bacteria were mainly suspected. This major discovery led to the identification of the role of mosquitoes in the spread of malaria by Ronald Ross (Nobel Prize 1902) and Battista Grassi. The recurrence of malaria attacks over many years was for a long time an enigma only solved after the Second World War by the discovery of the exo-erythrocytic cycle of Plasmodium. Progress was then made in treatment, from cinchona bark, quinine and chloroquine, to the recent discovery of artemisinin in 1972 by the Chinese researcher Tu Youyou (Nobel Prize 2015)., (Copyright © 2023 SFMTSI.)

Safe and effective malaria transmission-blocking chemotherapeutics would allow a community-level approach to malaria control and eradication efforts by targeting the mosquito sexual stage of the parasite life cycle. However, only a single drug, primaquine, is currently approved for use in reducing transmission, and drug toxicity limits its widespread implementation. To address this limitation in antimalarial chemotherapeutics, we used a recently developed transgenic Plasmodium berghei line, Ookluc, to perform a series of high-throughput in vitro screens for compounds that inhibit parasite fertilization, the initial step of parasite development within the mosquito. Screens of antimalarial compounds, approved drug collections, and drug-like molecule libraries identified 185 compounds that inhibit parasite maturation to the zygote form. Seven compounds were further characterized to block gametocyte activation or to be cytotoxic to formed zygotes. These were further validated in mosquito membrane-feeding assays using Plasmodium falciparum and P. vivax. This work demonstrates that high-throughput screens using the Ookluc line can identify compounds that are active against the two most relevant human Plasmodium species and provides a list of compounds that can be explored for the development of new antimalarials to block transmission.

Background: Once a mainstay of malaria elimination operations, larval source management (LSM)-namely, the treatment of mosquito breeding habitats-has been marginalized in Africa in favour of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of new technologies, and mosquitoes' growing resistance to insecticides used in LLINs and IRS raise renewed interest in LSM., Methods: A digitally managed larviciding (DML) operation in three of the seven districts of São Tomé and Príncipe (STP) was launched by the Ministry of Health (MOH) and ZzappMalaria LTD. The operation was guided by the Zzapp system, consisting of a designated GPS-based mobile application and an online dashboard, which facilitates the detection, sampling and treatment of mosquito breeding sites. During the operation, quality assurance (QA) procedures and field management methods were developed and implemented., Results: 12,788 water bodies were located and treated a total of 128,864 times. The reduction impact on mosquito population and on malaria incidence was 74.90% and 52.5%, respectively. The overall cost per person protected (PPP) was US$ 0.86. The cost varied between areas: US$ 0.44 PPP in the urban area, and US$ 1.41 PPP in the rural area. The main cost drivers were labour, transportation and larvicide material., Conclusion: DML can yield highly cost-effective results, especially in urban areas. Digital tools facilitate standardization of operations, implementation of QA procedures and monitoring of fieldworkers' performance. Digitally generated spatial data also have the potential to assist integrated vector management (IVM) operations. A randomized controlled trial (RCT) with a larger sample is needed to further substantiate findings., (© 2023. The Author(s).)

Background: Since 2004, malaria transmission on Bioko Island has declined significantly as a result of the scaling-up of control interventions. The aim of eliminating malaria from the Island remains elusive, however, underscoring the need to adapt control to the local context. Understanding the factors driving the risk of malaria infection is critical to inform optimal suits of interventions in this adaptive approach., Methods: This study used individual and household-level data from the 2015 and 2018 annual malaria indicator surveys on Bioko Island, as well as remotely-sensed environmental data in multilevel logistic regression models to quantify the odds of malaria infection. The analyses were stratified by urban and rural settings and by survey year., Results: Malaria prevalence was higher in 10-14-year-old children and similar between female and male individuals. After adjusting for demographic factors and other covariates, many of the variables investigated showed no significant association with malaria infection. The factor most strongly associated was history of travel to mainland Equatorial Guinea (mEG), which increased the odds significantly both in urban and rural settings (people who travelled had 4 times the odds of infection). Sleeping under a long-lasting insecticidal net decreased significantly the odds of malaria across urban and rural settings and survey years (net users had around 30% less odds of infection), highlighting their contribution to malaria control on the Island. Improved housing conditions indicated some protection, though this was not consistent across settings and survey year., Conclusions: Malaria risk on Bioko Island is heterogeneous and determined by a combination of factors interacting with local mosquito ecology. These interactions grant further investigation in order to better adapt control according to need. The single most important risk factor identified was travel to mEG, in line with previous investigations, and represents a great challenge for the success of malaria control on the Island., (© 2023. The Author(s).)

Background: Malaria disproportionately affects low-income households in rural communities where poor housing is common. Despite evidence that well-constructed and mosquito-proofed houses can reduce malaria risk, housing improvement is rarely included in malaria control toolboxes. This study assessed the need, magnitude, and opportunities for housing improvement to control malaria in rural Tanzania., Methods: A mixed-methods study was conducted in 19 villages across four district councils in southern Tanzania. A structured survey was administered to 1292 community members to assess need, perceptions, and opportunities for housing improvement for malaria control. Direct observations of 802 houses and surrounding environments were done to identify the actual needs and opportunities, and to validate the survey findings. A market survey was done to assess availability and cost of resources and services necessary for mosquito-proofing homes. Focus group discussions were conducted with key stakeholders to explore insights on the potential and challenges of housing improvement as a malaria intervention., Results: Compared to other methods for malaria control, housing improvement was among the best understood and most preferred by community members. Of the 735 survey respondents who needed housing improvements, a majority needed window screening (91.1%), repairs of holes in walls (79.4%), door covers (41.6%), closing of eave spaces (31.2%) and better roofs (19.0%). Community members invested significant efforts to improve their own homes against malaria and other dangers, but these efforts were often slow and delayed due to high costs and limited household incomes. Study participants suggested several mechanisms of support to improve their homes, including government loans and subsidies., Conclusion: Addressing the need for housing improvement is a critical component of malaria control efforts in southern Tanzania. In this study, a majority of the community members surveyed needed modest modifications and had plans to work on those modifications. Without additional support, their efforts were however generally slow; households would take years to sufficiently mosquito-proof their houses. It is, therefore, crucial to bring together the key players across sectors to reduce barriers in malaria-proofing housing in endemic settings. These may include government subsidies or partnerships with businesses to make housing improvement more accessible and affordable to residents., (© 2023. The Author(s).)

Female mosquitoes of most species require a blood meal for egg development. When biting a human host to collect this blood meal, they can spread dangerous diseases such as malaria, yellow fever, or dengue. Researchers use videography to study many aspects of mosquito behavior, including in-flight host-seeking, takeoff, and landing behaviors, as well as probing and blood feeding, and more. Here, we introduce protocols on how to use videography to capture and analyze mosquito movements at high spatial and temporal resolution, in two and three dimensions., (© 2023 Cold Spring Harbor Laboratory Press.)

Malaria is an infectious disease transmitted by mosquitos, whose control is hampered by drug resistance evolution in the causing agent, protist parasites of the genus Plasmodium , as well as by the resistance of the mosquito to insecticides. New approaches to fight this disease are, therefore, needed. Research into targeted drug delivery is expanding as this strategy increases treatment efficacies. Alternatively, targeting the parasite in humans, here we use single-chain polymer nanoparticles (SCNPs) to target the parasite at the ookinete stage, which is one of the stages in the mosquito. This nanocarrier system provides uniquely sized and monodispersed particles of 5-20 nm, via thiol-Michael addition. The conjugation of succinic anhydride to the SCNP surface provides negative surface charges that have been shown to increase the targeting ability of SCNPs to Plasmodium berghei ookinetes. The biodistribution of SCNPs in mosquitos was studied, showing the presence of SCNPs in mosquito midguts. The presented results demonstrate the potential of anionic SCNPs for the targeting of malaria parasites in mosquitos and may lead to progress in the fight against malaria.

Antigens expressed during the sexual development of malaria parasites are transmission-blocking vaccine (TBV) targets. Pb22, a protein expressed and localized to the plasma membrane of gametes and ookinetes in Plasmodium berghei, is an excellent TBV candidate. Here, we evaluated the TB potential of the Plasmodium vivax ortholog Pv22 using a transgenic P. berghei parasite line and P. vivax clinical isolates. The full-length recombinant Pv22 (rPv22) protein was produced and used to immunize mice and rabbits to obtain antibodies. We generated a transgenic P. berghei line (TrPv22Pb) by inserting the pv22 gene into the pb22 locus and showed that Pv22 expression completely rescued the defects in male gametogenesis of the pb22 deletion parasite. Since Pv22 in the transgenic parasite showed similar expression and localization patterns to Pb22, we used the TrPv22Pb parasite as a surrogate to evaluate the TB potential of Pv22. In mosquito feeding assays, mosquitoes feeding on rPv22-immunized mice infected with TrPv22Pb parasites showed a 49.3-53.3 % reduction in the oocyst density compared to the control group. In vitro assays showed that the rPv22 immune sera significantly inhibited exflagellation and ookinete formation of the TrPv22Pb parasites. In a direct membrane feeding assay using three clinical P. vivax isolates, the rabbit anti-rPv22 antibodies also significantly decreased the oocyst density by 53.7, 30.2, and 26.2 %, respectively. This study demonstrated the feasibility of using transgenic P. berghei parasites expressing P. vivax antigens as a potential tool to evaluate TBV candidates. However, the much weaker TB activity of Pv22 obtained from two complementary assays suggest that Pv22 may not be a promising TBV candidate for P. vivax., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This article explores the extent to which the emergence of networked conceptions of etiology and network-oriented approaches to the organization of medical practice were historically congruent. Focusing on interwar malariology, it contextualizes the development of ecological approaches to infection management and control in terms of mosquito-borne malariotherapeutic practice. In Britain, mosquito breeding programs directed toward the therapeutic infection of mental hospital patients prompted malariologists to modify and refine existing environmental approaches to malaria. Breeding mosquitoes, attending to patients, and maintaining sources of malarial blood modified malariologists' etiological presumptions, contributing to a wider breakdown of associations between race, place, and disease. Simultaneously, the emergence of an international network of malariotherapy-devoted institutions helped transform malariological practice. Examination of a collaboration between British and Romanian malariologists shows one way in which this network contributed to the transformation of malariology from a formal League of Nations-focused endeavor to one distributed along common lines of research and prevention.

The mosquito-borne disease, malaria, continues to impose a devastating health and economic burden worldwide. In malaria-endemic areas, insecticide-treated nets (ITNs) have been useful in curtailing the burden of the disease. However, mosquito resistance to insecticides, decay in ITN efficacy, net attrition, etc., undermine the effectiveness of ITNs in combatting malaria. In this study, mathematical models that account for asymptomatic infectious humans (through a partially immune class or a separate asymptomatic infectious class), insecticide resistance, and decay in ITN efficacy are proposed and analyzed. Analytical and numerical results of the models when ITN efficacy is constant show that there are parameter regimes for which a backward bifurcation occurs. Local and global sensitivity analyses are performed to identify parameters (some of which are potential targets for disease control) with the most significant influence on the control reproduction (R c ) and disease prevalence. These influential parameters include the maximum biting rate of resistant mosquitoes, ITN coverage, initial ITN efficacy against sensitive mosquitoes, the probability that an infectious mosquito (human) infects a susceptible human (mosquito), and the rate at which adult mosquitoes develop (lose) resistance to insecticides. Simulations of the models show that accounting for asymptomatic infectious humans through a separate class, or not accounting for the decay in ITN efficacy leads to an underestimation of disease burden. In particular, if the initial efficacy of ITNs against sensitive and resistance mosquitoes is 96%, the minimum ITN coverage required to reduce R c below one (and hence, contain malaria) is approximately 11% (27%) lower when ITN efficacy is averaged (constant) for a model with a separate asymptomatic class. For the model with a partially immune class and decaying ITN efficacy, reducing R c below one is impossible even if the entire populace uses ITNs. The study shows that replacing ITNs before their prescribed lifespans, or designing ITNs with longer lifespans is important for malaria control. Furthermore, the study shows that piperonyl butoxide (PBO) ITNs (which inhibit or reverse insecticide resistance) outperform regular ITNs in malaria control. Hence, prospects for effectively controlling malaria are enhanced by widespread use of high quality ITNs (e.g. PBO ITNs), especially if the useful lifespans of the ITNs are long enough and the ITNs are replaced before the end of their useful lifespans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.

Morphogenesis of many protozoans depends on a polarized establishment of cortical cytoskeleton containing the subpellicular microtubules (SPMTs), which are apically nucleated and anchored by the apical polar ring (APR). In malaria parasite Plasmodium, APR emerges in the host-invading stages, including the ookinete for mosquito infection. So far, the fine structure and molecular components of APR as well as the underlying mechanism of APR-mediated apical positioning of SPMTs are largely unknown. Here, we resolve an unprecedented APR structure composed of a top ring plus approximate 60 radiating spines. We report an APR-localizing and SPMT-binding protein APR2. APR2 disruption impairs ookinete morphogenesis and gliding motility, leading to Plasmodium transmission failure in mosquitoes. The APR2-deficient ookinetes display defective apical anchorage of APR and SPMT due to the impaired integrity of APR. Using protein proximity labeling, we obtain a Plasmodium ookinete APR proteome and validate ten undescribed APR proteins. Among them, APRp2 and APRp4 directly interact with APR2 and also mediate the apical anchorage of SPMTs. This study sheds light on the molecular basis of APR in the organization of Plasmodium ookinete SPMTs., (© 2022. The Author(s).)

Background: The use of insecticide-treated bed nets has been proven to be effective in reducing malaria transmission in highly endemic areas. Use of long-lasting insecticidal nets (LLINs) has been embraced by many malaria endemic countries. LLINs are up to 95% effective in inhibiting blood feeding, when used consistently even after 7 years. The challenge, however, is enhancing their consistent use, especially by the most vulnerable groups (children under 5 years and pregnant women). The study established factors associated with consistent use of bed nets for malaria control among children under 5 years in Soroti district., Methods: The study employed a cross-sectional design, with multi-stage sampling of households. A total of 400 households (HH) were sampled and the HH head in each household interviewed. Key informant interviews (KIIs) were conducted with 7 key informants who were knowledgeable on the subject matter. Data analysis was done using SPSS 17.0 at Univariate, Bivariate and Multivariable levels; after entry and cleaning. Key informants' data were summarized manually; verbatim quotes and text used to reinforce quantitative data in line with objectives., Results: Only 56.8% of the 690 children under 5 years used bed nets consistently. The factors affecting consistent bed net use were age of the child, their use of bed nets the previous night, occupation of caretaker, respondents' perceived susceptibility, perceived risk of getting malaria, size and shape of the bed nets. Rectangular nets were difficult to hang daily in huts according to most key informants., Conclusion: Consistent bed net use among under fives is still below the RBM target of 85% by 2015 and can be enhanced by providing conical bed nets and setting aside a health education programme to emphasize the effectiveness of even one mosquito in spreading malaria at night to the entire household and ability of bed nets to stop transmission better than other methods., (© 2022. The Author(s).)

Background: Cambodia has made significant progress towards achieving malaria elimination by 2025. Cases continue to decrease and are primarily concentrated in forested areas. Forest-goers are most at risk of malaria due to their proximity to the forest, poor sleeping conditions, frequent mobility, and distance from health services. Consistent use of long-lasting insecticidal nets or hammock nets (LLINs/LLIHNs), early diagnosis and treatment of cases are central to reducing disease burden. The aim of this study was to understand forest-goers' knowledge, attitudes, and practices related to malaria prevention and care-seeking, and to identify key behavioural determinants of LLIN/LLIHN use and prompt care-seeking within 24 h of developing a fever., Methods: A mixed-methods study design consisting of a cross-sectional survey and qualitative in-depth interviews was implemented in two Cambodian provinces. Survey participants (N = 654) were recruited using respondent driven sampling. Interview participants (N = 28) were selected using purposive sampling. Findings from the survey were analysed using univariate and bivariate analysis and multivariate weighted logistic regression. Interviews were coded and analysed using thematic content analysis., Results: All study participants had heard of malaria and 98% knew that malaria was transmitted by mosquitoes. LLIN/LLIHN ownership was high (94%). Although 99% of participants perceived LLIN/LLIHN use as an important malaria prevention measure, only 76% reported using one during their last visit to the forest. Only 39% of survey participants who reported seeking care did so within the recommended 24 h from fever onset during their last febrile illness. Among all study participants, 43% did not seek any healthcare during their last febrile episode. In controlled regression models, perceived community social norms were significantly associated with LLIN/LLIHN use (OR: 2.7, 96% CI 1.99-2.64) and care-seeking within 24 h of fever onset (OR: 1.7, 95% CI 1.00-2.88). Social support from other forest-goers was also significantly associated with LLIN/LLIHN use (OR: 4.9, 95% CI 1.32-18.12)., Conclusions: Study findings are consistent with other studies on LLIN/LLIHN use and care-seeking behaviours. While rates of LLIN/LLIHN ownership were high among the study population, rates of use were not as high. More concerning were the delayed care-seeking behaviours. Social behaviour change activities should incorporate social norms and social support as mechanisms for behaviour change given the identified positive correlations with LLIN/LLIHN use and prompt care-seeking., (© 2022. The Author(s).)

Differentiation of asexually replicating parasites into gametocytes is critical for successful completion of the sexual phase of the malaria parasite life cycle. Gametes generated from gametocytes fuse to form a zygote which differentiates into ookinetes and oocysts. The sporozoites are formed inside oocysts which migrate to the salivary glands for next cycle of human infection. These morphologically and functionally distinct stages require stage-specific gene expression via specific transcriptional regulators. The capacity of high mobility group box (HMGB) proteins to interact with DNA in a sequence independent manner enables them to regulate higher order chromosome organization and regulation of gene expression. Plasmodium falciparum HMGB2 ( Pf HMGB2) shows a typical L- shaped predicted structure which is similar to mammalian HMG box proteins and shows very high protein sequence similarity to Py HMGB2 and Pb HMGB2. Functional characterization of Pf HMGB2 by gene deletion ( Pfhmgb2¯ ) showed that knockout parasites develop normally as asexual stages and undergo gametocytogenesis. Transmission experiments revealed that Pfhmgb2¯ can infect mosquitoes and develop as oocyst stages. However, transmission was reduced compared to wild type (WT) parasites and as a consequence, the salivary gland sporozoites were reduced in number. In summary, we demonstrate that PfHMGB2 has no role in asexual growth and a modest role in sexual phase development and parasite transmission to the mosquito., 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 Kumar and Kappe.)

Background: Larval source management (LSM) may help reduce Plasmodium parasite transmission in malaria-endemic areas. LSM approaches include habitat modification (permanently or temporarily reducing mosquito breeding aquatic habitats); habitat manipulation (temporary or recurrent change to environment); or use of chemical (e.g. larviciding) or biological agents (e.g. natural predators) to breeding sites. We examined the effectiveness of habitat modification or manipulation (or both), with and without larviciding. This is an update of a review published in 2013., Objectives: 1. To describe and summarize the interventions on mosquito aquatic habitat modification or mosquito aquatic habitat manipulation, or both, on malaria control. 2. To evaluate the beneficial and harmful effects of mosquito aquatic habitat modification or mosquito aquatic habitat manipulation, or both, on malaria control., Search Methods: We used standard, extensive Cochrane search methods. The latest search was from January 2012 to 30 November 2021., Selection Criteria: Randomized controlled trials (RCT) and non-randomized intervention studies comparing mosquito aquatic habitat modification or manipulation (or both) to no treatment or another active intervention. We also included uncontrolled before-after (BA) studies, but only described and summarized the interventions from studies with these designs. Primary outcomes were clinical malaria incidence, malaria parasite prevalence, and malaria parasitaemia incidence., Data Collection and Analysis: We used standard Cochrane methods. We assessed risk of bias using the Cochrane RoB 2 tool for RCTs and the ROBINS-I tool for non-randomized intervention studies. We used a narrative synthesis approach to systematically describe and summarize all the interventions included within the review, categorized by the type of intervention (habitat modification, habitat manipulation, combination of habitat modification and manipulation). Our primary outcomes were 1. clinical malaria incidence; 2. malaria parasite prevalence; and 3. malaria parasitaemia incidence. Our secondary outcomes were 1. incidence of severe malaria; 2. anaemia prevalence; 3. mean haemoglobin levels; 4. mortality rate due to malaria; 5. hospital admissions for malaria; 6. density of immature mosquitoes; 7. density of adult mosquitoes; 8. sporozoite rate; 9. entomological inoculation rate; and 10., Harms: We used the GRADE approach to assess the certainty of the evidence for each type of intervention., Main Results: Sixteen studies met the inclusion criteria. Six used an RCT design, six used a controlled before-after (CBA) study design, three used a non-randomized controlled design, and one used an uncontrolled BA study design. Eleven studies were conducted in Africa and five in Asia. Five studies reported epidemiological outcomes and 15 studies reported entomological outcomes. None of the included studies reported on the environmental impacts associated with the intervention. For risk of bias, all trials had some concerns and other designs ranging from moderate to critical. Ten studies assessed habitat manipulation (temporary change to the environment). This included water management (spillways across streams; floodgates; intermittent flooding; different drawdown rates of water; different flooding and draining regimens), shading management (shading of drainage channels with different plants), other/combined management approaches (minimal tillage; disturbance of aquatic habitats with grass clearing and water replenishment), which showed mixed results for entomological outcomes. Spillways across streams, faster drawdown rates of water, shading drainage canals with Napier grass, and using minimal tillage may reduce the density of immature mosquitoes (range of effects from 95% reduction to 1.7 times increase; low-certainty evidence), and spillways across streams may reduce densities of adult mosquitoes compared to no intervention (low-certainty evidence). However, the effect of habitat manipulation on malaria parasite prevalence and clinical malaria incidence is uncertain (very low-certainty evidence). Two studies assessed habitat manipulation with larviciding. This included reducing or removal of habitat sites; and drain cleaning, grass cutting, and minor repairs. It is uncertain whether drain cleaning, grass cutting, and minor repairs reduces malaria parasite prevalence compared to no intervention (odds ratio 0.59, 95% confidence interval (CI) 0.42 to 0.83; very low-certainty evidence). Two studies assessed combination of habitat manipulation and permanent change (habitat modification). This included drainage canals, filling, and planting of papyrus and other reeds for shading near dams; and drainage of canals, removal of debris, land levelling, and filling ditches. Studies did not report on epidemiological outcomes, but entomological outcomes suggest that such activities may reduce the density of adult mosquitoes compared to no intervention (relative risk reduction 0.49, 95% CI 0.47 to 0.50; low-certainty evidence), and preventing water stagnating using drainage of canals, removal of debris, land levelling, and filling ditches may reduce the density of immature mosquitoes compared to no intervention (ranged from 10% to 55% reductions; low-certainty evidence). Three studies assessed combining manipulation and modification with larviciding. This included filling or drainage of water bodies; filling, draining, or elimination of rain pools and puddles at water supply points and stream bed pools; and shoreline work, improvement and maintenance to drainage, clearing vegetation and undergrowth, and filling pools. There were mixed effect sizes for the reduction of entomological outcomes (moderate-certainty evidence). However, filling or draining water bodies probably makes little or no difference to malaria parasite prevalence, haemoglobin levels, or entomological inoculation rate when delivered with larviciding compared to no intervention (moderate-certainty evidence)., Authors' Conclusions: Habitat modification and manipulation interventions for preventing malaria has some indication of benefit in both epidemiological and entomological outcomes. While the data are quite mixed and further studies could help improve the knowledge base, these varied approaches may be useful in some circumstances., (Copyright © 2022 The Authors. Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.)

Cyclin-dependent kinases (CDKs) and cyclins are critical cell cycle regulators in eukaryotes. In this study, we functionally characterized a CDK-related kinase (CRK5) of the human malaria parasite Plasmodium falciparum. P. falciparum CRK5 ( Pf CRK5) was expressed in asexual blood stages and sexual gametocyte stages, but showed male gametocyte- specific expression. In contrast to previous findings, we showed that gene deletion Pfcrk5 - parasites grew normally as asexual stages and underwent normal gametocytogenesis to stage V gametocytes. However, Pfcrk5 - parasites showed a severe defect in male gametogenesis, which was evident by a significant reduction in the emergence of male gametes (exflagellation). This defect caused a severe reduction of parasite transmission to the mosquito. Genetic crosses performed using sex-specific sterile transgenic parasites revealed that Pfcrk5 - parasites suffered a defect in male fertility but female gametes were fertile. Taken together, these results demonstrate that Pf CRK5 is a critical sexual stage kinase which regulates male gametogenesis and transmission to the mosquito. IMPORTANCE Gametocytes are parasite sexual stages which differentiate from asexually replicating parasites. These stages are necessary for the completion of sexual phase of the parasite life cycle. Inside the mosquito midgut, gametocytes rapidly get activated to form fertilization competent gametes. These stages present a bottleneck in the parasite life cycle. In this study, we demonstrate that P f CRK5 is important for male gametogenesis and therefore regulates parasite transmission to the mosquito. Our study identifies P f CRK5 as a potential target for the development of drugs to block malaria transmission.

Background: Malaria remains an important public health problem. Research in 1900 suggested house modifications may reduce malaria transmission. A previous version of this review concluded that house screening may be effective in reducing malaria. This update includes data from five new studies., Objectives: To assess the effects of house modifications that aim to reduce exposure to mosquitoes on malaria disease and transmission., Search Methods: We searched the Cochrane Infectious Diseases Group Specialized Register; Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE (PubMed); Embase (OVID); Centre for Agriculture and Bioscience International (CAB) Abstracts (Web of Science); and the Latin American and Caribbean Health Science Information database (LILACS) up to 25 May 2022. We also searched the World Health Organization International Clinical Trials Registry Platform, ClinicalTrials.gov, and the ISRCTN registry to identify ongoing trials up to 25 May 2022., Selection Criteria: Randomized controlled trials, including cluster-randomized controlled trials (cRCTs), cross-over studies, and stepped-wedge designs were eligible, as were quasi-experimental trials, including controlled before-and-after studies, controlled interrupted time series, and non-randomized cross-over studies. We sought studies investigating primary construction and house modifications to existing homes reporting epidemiological outcomes (malaria case incidence, malaria infection incidence or parasite prevalence). We extracted any entomological outcomes that were also reported in these studies., Data Collection and Analysis: Two review authors independently selected eligible studies, extracted data, and assessed the risk of bias. We used risk ratios (RR) to compare the effect of the intervention with the control for dichotomous data. For continuous data, we presented the mean difference; and for count and rate data, we used rate ratios. We presented all results with 95% confidence intervals (CIs). We assessed the certainty of evidence using the GRADE approach., Main Results: One RCT and six cRCTs met our inclusion criteria, with an additional six ongoing RCTs. We did not identify any eligible non-randomized studies. All included trials were conducted in sub-Saharan Africa since 2009; two randomized by household and four at the block or village level. All trials assessed screening of windows, doors, eaves, ceilings, or any combination of these; this was either alone, or in combination with roof modification or eave tube installation (an insecticidal "lure and kill" device that reduces mosquito entry whilst maintaining some airflow). In one trial, the screening material was treated with 2% permethrin insecticide. In five trials, the researchers implemented the interventions. A community-based approach was adopted in the other trial. Overall, the implementation of house modifications probably reduced malaria parasite prevalence (RR 0.68, 95% CI 0.57 to 0.82; 5 trials, 5183 participants; moderate-certainty evidence), although an inconsistent effect was observed in a subpopulation of children in one study. House modifications reduced moderate to severe anaemia prevalence (RR 0.70, 95% CI 0.55 to 0.89; 3 trials, 3643 participants; high-certainty evidence). There was no consistent effect on clinical malaria incidence, with rate ratios ranging from 0.38 to 1.62 (3 trials, 3365 participants, 4126.6 person-years). House modifications may reduce indoor mosquito density (rate ratio 0.63, 95% CI 0.30 to 1.30; 4 trials, 9894 household-nights; low-certainty evidence), although two studies showed little effect on this parameter., Authors' Conclusions: House modifications - largely screening, sometimes combined with insecticide and lure and kill devices - were associated with a reduction in malaria parasite prevalence and a reduction in people with anaemia. Findings on malaria incidence were mixed. Modifications were also associated with lower indoor adult mosquito density, but this effect was not present in some studies., (Copyright © 2022 The Authors. Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.)

Insect vectors are responsible for spreading many infectious diseases, yet interactions between pathogens/parasites and insect vectors remain poorly understood. Filling this knowledge gap matters because vectors are evolving in response to the deployment of vector control tools (VCTs). Yet, whilst the evolutionary responses of vectors to VCTs are being carefully monitored, the knock-on consequences for parasite evolution have been overlooked. By examining how mosquito responses to VCTs impact upon malaria parasite ecology, we derive a framework for predicting parasite responses. Understanding how VCTs affect the selection pressures imposed on parasites could help to mitigate against parasite evolution that leads to unfavourable epidemiological outcomes. Furthermore, anticipating parasite evolution will inform monitoring strategies for VCT programmes as well as uncovering novel VCT strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Genetic engineering of mosquitoes represents a promising tactic for reducing human suffering from malaria. Gene-drive techniques being developed that suppress or modify populations of Anopheles gambiae have the potential to be used with, or even possibly obviate, microbial and synthetic insecticides. However, these techniques are new and therefore there is attendant concern and uncertainty from regulators, policymakers, and the public about their environmental risks. Therefore, there is a need to assist decision-makers and public health stewards by assessing the risks associated with these newer mosquito management tactics so the risks can be compared as a basis for informed decision making. Previously, the effect of gene-drive mosquitoes on water quality in Africa was identified as a concern by stakeholders. Here, we use a comparative risk assessment approach for the effect of gene-drive mosquitoes on water quality in Africa. We compare the use of existing larvicides and the proposed genetic techniques in aquatic environments. Based on our analysis, we conclude that the tactic of gene-drive Anopheles for malaria management is unlikely to result in risks to aquatic environments that exceed current tactics for larval mosquitoes. As such, these new techniques would likely comply with currently recommended safety standards., (© 2022. The Author(s).)

Background: Malaria is one of the prime reasons for medical consultation and the major cause of morbidity and mortality in Mali. To assess and understand the dynamics of social representations of malaria, the anthropological research was conducted in the Wayerema II neighbourhood of the health district of Sikasso, southern Mali., Methods: This was an ethnographic study conducted qualitatively in 2011 and 2016 through informal conversations, 70 semi-structured interviews, and participant observations with key actors. The observations, conversations, and interviews investigated local people's perceptions and knowledge about malaria, and how and to what extent the cultural and popular representations of the disease can have an impact on therapeutic routes., Results: Mosquitoes are the principal agent of the transmission of malaria. However, the ubiquitous yet casually-claimed aetiological agents, causative, nosographic entities differ from-although sometimes integrated into-the biomedical dimension. For example, some communities perceive Kono, a complicated and pernicious form of malaria that often occurs among children, to originate from a supernatural force. "Bird disease" is another term used for Kono in Mali and other West African countries. Thus, overall, Kono is defined through the entanglements with cultural factors, namely the idiosyncratic habits, customs, and beliefs of the population of Wayerema II neighbourhood in the health district of Sikasso, Southern Mali. Wayerema II residents particularly tend to link therapeutic recourse amongst the afflicted not only to biomedical models but to sociocultural and popular perceptions and representations of malaria., Conclusion: In the findings, self-medication through both traditional and modern medical techniques was the most frequent therapeutic modality. Hence, the integration of local popular knowledge with the biomedical register can contribute to a comprehensive understanding of social representations and perceptions of malaria, and qualitative improvements in the malaria control programme., (© 2022. The Author(s).)

Background: Plasmodium parasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genus Culex. Culex quinquefasciatus, the mosquito vector of avian malaria in Hawai'i, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied., Methods: To evaluate the gene expression response of mosquitoes exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i, offspring of wild-collected Hawaiian Cx. quinquefasciatus were fed on a domestic canary infected with a fresh isolate of Plasmodium relictum GRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands., Results: Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as a Plasmodium agonist in C. quinquefasciatus. Overall, the differential gene expression response of Hawaiian Culex exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i was low, but more pronounced during ookinete invasion., Conclusions: This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response of Culex infected with a bird Plasmodium and those reported in Anopheles infected with human Plasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges of Culex with avian Plasmodium., (© 2022. The Author(s).)

Malaria-induced bacteremia has been shown to result from intestinal mast cell (MC) activation. The appearance of MCs in the ileum and increased intestinal permeability to enteric bacteria are preceded by an early Th2-biased host immune response to infection, characterized by the appearance of IL-4, IL-10, mast cell protease (Mcpt)1 and Mcpt4, and increased circulating basophils and eosinophils. Given the functional similarities of basophils and MCs in the context of allergic inflammation and the capacity of basophils to produce large amounts of IL-4, we sought to define the role of basophils in increased intestinal permeability, in MC influx, and in the development of bacteremia in the context of malaria. Upon infection with nonlethal Plasmodium yoelii yoelii 17XNL, Basoph8 × ROSA-DTα mice or baso (-) mice that lack basophils exhibited increased intestinal permeability and increased ileal MC numbers, without any increase in bacterial 16S ribosomal DNA copy numbers in the blood, relative to baso (+) mice. Analysis of cytokines, chemokines, and MC-associated factors in the ileum revealed significantly increased TNF-α and IL-13 at day 6 postinfection in baso (-) mice compared with baso (+) mice. Moreover, network analysis of significantly correlated host immune factors revealed profound differences between baso (-) and baso (+) mice following infection in both systemic and ileal responses to parasites and translocated bacteria. Finally, basophil depletion was associated with significantly increased gametocytemia and parasite transmission to Anopheles mosquitoes, suggesting that basophils play a previously undescribed role in controlling gametocytemia and, in turn, mammalian host-to-mosquito parasite transmission., (Copyright © 2022 The Authors.)

A deterministic differential equation model for the dynamics of terrestrial forms of mosquito populations is studied. The model assesses the impact of multiple probing attempts by mosquitoes that quest for blood within human populations by including a waiting class for mosquitoes that failed a blood feeding attempt. The equations are derived based on the idea that the reproductive cycle of the mosquito can be viewed as a set of alternating egg laying and blood feeding outcomes realised on a directed path called the gonotrophic cycle pathway. There exists a threshold parameter, the basic offspring number for mosquitoes, whose nature is affected by the way we interpret the transitions involving the different classes on the gonotrophic cycle path. The trivial steady state for the system, which always exists, can be globally asymptomatically stable whenever the threshold parameter is less than unity. The non-trivial steady state, when it exists, is stable for a range of values of the threshold parameter but can also be driven to instability via a Hopf bifurcation. The model's output reveals that the waiting class mosquitoes do contribute positively to sustain mosquito populations as well as increase their interactions with humans via increased frequency and initial amplitude of oscillations. We conclude that to understand human-mosquito interactions, it is informative to consider multiple probing attempts; known to occur when mosquitoes quest for blood meals within human populations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Background: With the emergence of resistance to front-line antimalarials, there is an urgent need to develop new medicines, including those targeting sexual development. This study aimed to assess the activity of a panel of phosphatase inhibitors against the sexual development of Plasmodium berghei and evaluate their potential as transmission-blocking agents., Methods: Twenty-five compounds were screened for transmission-blocking activity in vitro using the P. berghei ookinete culture assay. The inhibitory effects on male gametogenesis, gamete-ookinete, and zygote-ookinete formation were evaluated. The transmission-blocking activity of two compounds was evaluated using an in vivo mosquito feeding assay. Their cytotoxic effects were assessed on the human cell line HepG2., Results: Twelve compounds inhibited P. berghei ookinete formation with an IC 50  < 10 μM. Two compounds, BVT-948 and alexidine dihydrochloride, significantly inhibited different developmental stages from gametogenesis through ookinete maturation. They also showed a substantial in vivo transmission-blocking activity by the mosquito feeding assay., Conclusions: Some phosphatase inhibitors effectively inhibited Plasmodium sexual development and exhibited evident transmission-blocking activity, suggesting that phosphatases are valid targets for antimalarial development., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria., Competing Interests: The authors have declared that no competing interests exist.

Background: Gene drives are a genetic engineering method where a suite of genes is inherited at higher than Mendelian rates and has been proposed as a promising new vector control strategy to reinvigorate the fight against malaria in sub-Saharan Africa., Methods: Using an agent-based model of malaria transmission with vector genetics, the impacts of releasing population-replacement gene drive mosquitoes on malaria transmission are examined and the population replacement gene drive system parameters required to achieve local elimination within a spatially-resolved, seasonal Sahelian setting are quantified. The performance of two different gene drive systems-"classic" and "integral"-are evaluated. Various transmission regimes (low, moderate, and high-corresponding to annual entomological inoculation rates of 10, 30, and 80 infectious bites per person) and other simultaneous interventions, including deployment of insecticide-treated nets (ITNs) and passive healthcare-seeking, are also simulated., Results: Local elimination probabilities decreased with pre-existing population target site resistance frequency, increased with transmission-blocking effectiveness of the introduced antiparasitic gene and drive efficiency, and were context dependent with respect to fitness costs associated with the introduced gene. Of the four parameters, transmission-blocking effectiveness may be the most important to focus on for improvements to future gene drive strains because a single release of classic gene drive mosquitoes is likely to locally eliminate malaria in low to moderate transmission settings only when transmission-blocking effectiveness is very high (above ~ 80-90%). However, simultaneously deploying ITNs and releasing integral rather than classic gene drive mosquitoes significantly boosts elimination probabilities, such that elimination remains highly likely in low to moderate transmission regimes down to transmission-blocking effectiveness values as low as ~ 50% and in high transmission regimes with transmission-blocking effectiveness values above ~ 80-90%., Conclusion: A single release of currently achievable population replacement gene drive mosquitoes, in combination with traditional forms of vector control, can likely locally eliminate malaria in low to moderate transmission regimes within the Sahel. In a high transmission regime, higher levels of transmission-blocking effectiveness than are currently available may be required., (© 2022. The Author(s).)

Gene drive mosquitoes are increasingly considered a potential transformational tool for vector control of malaria mosquitoes. As part of efforts to promote responsible research in this field, a number of guidance documents have been published by the World Health Organization, National Academies and expert groups. While virtually all recent guidance documents on gene drive research stress the importance of stakeholder engagement activities, no specific guidelines on implementing them have been established. Target Malaria, a not-for-profit research consortium developing a vector-control gene drive approach to eliminate malaria, has reflected on how its stakeholder engagement strategy translates engagement guidance documents into practice. The project analysed and addressed the tension between the context specificities and the international recommendations. The engagement strategy combines published recommendations for responsible gene drive research, information collected from the local context where the project operates and a set of principles guiding the choices made. This strategy was first developed during the early phases of the project's research, years ahead of any activities with gene drive mosquitoes in those countries of operations. These earlier activities, and their related engagement, allow the project to develop and adapt an engagement strategy appropriate for potential gene drive research in its field site countries. This paper offers a description of a stakeholder engagement strategy operationalization based on (1) adaptation to stakeholder preferences, (2) inclusiveness and (3) empowerment and accountability. The authors hope to offer concrete examples to support other projects with the development and implementation of their engagement strategies with particular attention to the co-development principle., (© 2022. The Author(s).)