Acta Tropica

Leishmaniasis is a neglected tropical disease. Parasite strategies and the evaluation of drugs and vaccines are inferred from studies carried out using mouse models and macrophages. The development of model organisms with no ethical restrictions will contribute to our knowledge of leishmaniasis. Acanthamoeba castellanii, a free-living protozoan, is known to interact with various microorganisms. In this study, the interaction between the amoeba A. castellanii and the trypanosomatid Leishmania amazonensis was investigated by combining quantitative kinetics analysis, optical, fluorescence, electronic, confocal, and live video microcopy. We sought to standardize protocols for the co-culture; the optimal experimental conditions were: RPMI medium + 10% SFB at 26{degrees}C. L. amazonensis invades A. castellanii through its acanthopods, and the promastigotes interact with the trophozoites via their flagellum, which also occurs when parasites infect mammalian macrophages. The forms of L. amazonensis inside the amoeba become rounded and lose their flagellum; they are similar to amastigotes. These round forms were isolated from trophozoites after 3 h of co-culture and differentiated into promastigotes, demonstrating their viability inside amoeba. The percentage of amoebas with L. amazonensis was reduced overtime. Thus, considering that A. castellanii can clear Leishmania, this interaction could serve as an effective model of cellular leishmanicidal mechanisms.

The enforcement of COVID-19 interventions by diverse governmental bodies, coupled with the indirect impact of COVID-19 on short-term environmental changes (e.g. plant shutdowns lead to lower greenhouse gas emissions), influences the dengue vector. This provides a unique opportunity to investigate the impact of COVID-19 on dengue transmission and generate insights to guide more targeted prevention measures. We aim to compare dengue transmission patterns and the exposure-response relationship of environmental variables and dengue incidence in the pre- and during-COVID-19 to identify variations and assess the impact of COVID-19 on dengue transmission. We initially visualized the overall trend of dengue transmission from 2012-2022, then conducted two quantitative analyses to compare dengue transmission pre-COVID-19 (2017-2019) and during-COVID-19 (2020-2022). These analyses included time series analysis to assess dengue seasonality, and a Distributed Lag Non-linear Model (DLNM) to quantify the exposure-response relationship between environmental variables and dengue incidence. We observed that all subregions in Thailand exhibited remarkable synchrony with a similar annual trend except 2021. Cyclic and seasonal patterns of dengue remained consistent pre- and during-COVID-19. Monthly dengue incidence in three countries varied significantly. Singapore witnessed a notable surge during-COVID-19, particularly from May to August, with cases multiplying several times compared to pre-COVID-19, while seasonality of Malaysia weakened. Exposure-response relationships of dengue and environmental variables show varying degrees of change, notably in Northern Thailand, where the peak relative risk for the maximum temperature-dengue relationship rose from about 3 to 17, and the max RR of overall cumulative association 0-3 months of relative humidity increased from around 5 to 55. Our study is the first to compare dengue transmission patterns and their relationship with environmental variables before and during COVID-19, showing that COVID-19 has affected dengue transmission at both the national and regional level, and has altered the exposure-response relationship between dengue and the environment. Author SummaryDengue fever is a typical tropical disease transmitted via mosquito bites. COVID-19 lockdowns have altered human-mosquito contact patterns that impacted dengue transmission. Additionally, lockdowns caused short-term environmental changes that affected dengue vector breeding. In fact, during the COVID-19 period, the normal prevention and treatment of dengue in many dengue-endemic countries was negatively affected due to the sweep of COVID-19, such as strained allocation of medical resources and misreporting of cases. Therefore, this offers a unique chance to study the impact of COVID-19 on dengue transmission, guiding targeted and reasonable prevention measures. We used a series of analytical approaches including time series analysis, space-time scan statistics, and distributed lag non-linear model to compare the differences in dengue transmission patterns and its exposure-response relationships with four environmental variables (average monthly precipitation, average monthly relative humidity, monthly maximum temperature, and monthly minimum temperature) before and during COVID-19 in three Southeast Asian countries: Malaysia, Singapore and Thailand at the province scale. We found that the dengue transmission pattern and its relationship with the environmental variables changed differently. For instance, seasonality and infections heightened in Singapore during COVID-19 and peak relative risk between max temperature and dengue has rose significantly in Northern Thailand.

Vector control programs worldwide are facing the challenge of mosquitoes becoming resistant to available insecticides. Larviciding is a crucial preventative measure for dengue control but data on insecticide resistance of larval Ae. aegypti in the Middle Eastern Region are limited. This study assesses the susceptibility status of Ae. aegypti collected from the two most important dengue foci in Saudi Arabia, Jeddah and Makkah, to important chemical and biological larvicides; the organophosphate temephos and Bacillus thuringiensis israelensis, Bti). Whilst worldwide, and particularly in Latin America, high-level resistance to temephos is common, Jeddah and Makkah populations exhibited full susceptibility to both temephos and Bti. These data suggest each can be considered by vector control programs for preventative dengue control in the region, as part of temporal rotations or spatial mosaics to manage insecticide resistance.

Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor(R) G2 (IG2), a first-in-class dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insects mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2s public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. Therefore, we tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with established mixed-function oxidases and Vgsc-L995F knockdown resistance alleles were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 hours overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI: 0.23-0.46) and 0.43 (95% CI: 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI: 0.22-0.41). We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes. Author summaryMalaria remains a serious problem in many tropical and sub-tropical areas, affecting the welfare and health of many individuals. Since 2016, malaria has increased and the emergence of mosquitoes that are resistant to different classes of insecticides used in vector control tools may have contributed to some of this increase. Therefore, insecticides with a different mode of action are required to manage vector resistance to insecticides used for public health vector control. One of the main resistance mechanisms is metabolic resistance where mosquitoes upregulate detoxification enzymes to break down insecticides. Chlorfenapyr is a pyrrole-pro-insecticide that is metabolised by these detoxification enzymes from chlorfenapyr to tralopyril that disrupts mitochondrial function in mosquitoes. We therefore hypothesized that the metabolites of chlorfenapyr may also have an effect on Plasmodia since they, too possess mitochondria and this could reduce the development of Plasmodium in mosquitoes that survived a sub-lethal dose of chlorfenapyr. In this study we established and evaluated a modified WHO tunnel assay to investigate the effect of chlorfenapyr in Plasmodium-infected Anopheles mosquitoes. In this bioassay, we found that chlorfenapyr substantially reduces the proportion of Plasmodium-infected mosquitoes at doses sub-lethal to mosquitoes. Our findings demonstrate that chlorfenapyr provides additional benefits beyond mosquito killing although the mechanism of action requires further elucidation.

Despite declining malaria cases in Thailand, surveillance in endemic areas is crucial. Retrospectively analyzing samples from Tak province, Thailand, we found prevalent drugresistant Plasmodium falciparum, particularly to mefloquine and sulfadoxine/pyrimethamine. Notably, mutations indicating resistance to artemisinin were detected at low frequencies, suggesting evolving resistance. These findings stress the need for continuous surveillance to guide control strategies and prevent outbreaks, even with decreasing cases, to sustain malaria elimination efforts.

Sphingosine-1-phosphate (S1P) a bioactive lipid is produced in its primary reservoir, erythrocytes by an enzyme Sphingosine kinase-1 (SphK-1). The activation of such kinases and the subsequent S1P generation and secretion in the blood serum represent a major regulator of many cellular signaling cascades. Orthologue of sphingosine kinases 1 and 2 (SphK-1 and 2) that catalyze the phosphorylation of sphingosine generating S1P are not present in malaria parasite. The malaria parasite, Plasmodium falciparum, is an intracellular obligatory organism that reside in the human erythrocyte during its blood stage life cycle and orchestrates many metabolic interactions with host for its survival. Given the regulatory role of S1P, we targeted host SphK-1 by a generic pharmacological inhibitor N,N-Dimethyl-sphingosine (DMS) and analyzed growth of intra-erythrocytic parasite. We found that reducing S1P levels by inhibiting host SphK-1 activity led to halted parasite growth and ultimately cell death. Reduced intracellular S1P levels were attributed to decreased glycolysis marked by the low uptake of glucose by parasite and by less production of lactate, a byproduct of glycolysis. Reduced glycolysis was mediated by decrease translocation of the glycolytic enzyme, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to the cytosol of infected erythrocytes and cell death. Knocking down of erythrocyte SphK-1 is not lethal to the host and being a host encoded enzyme, targeting it with safe and specific drugs will not lead to the problem of resistance; thus, SphK-1 represents a potent target for the development of therapeutics against intra-erythrocytic P. falciparum.\n\nAuthor SummaryErythrocytes membrane enzyme Sphingosine kinase-1 (SphK-1) produces Sphingosine-1-phosphate (S1P) a bioactive lipid by phosphorylation of Sphingosine (Sph). S1P generated by activation of SphK is prosurvival signal and regulate cell growth. The malaria parasite, Plasmodium falciparum, is an intracellular obligatory pathogen that reside in erythrocyte during its blood stage life cycle and orchestrates many metabolic interactions with its host erythrocytes for survival. Orthologue of SphK-1/ 2 are not present in malaria parasite, therefore treatment with SphK inhibitor targeted host SphK-1 and led to reduced S1P level. The reduction in host S1P led to halted parasite growth and cell death. Furthermore, reduced erythrocyte S1P levels led to decreased glycolysis marked by the low uptake of glucose by parasite and by less production of lactate. Erythrocyte SphK-1 being a host encoded enzyme, is resistance safe and represents a potent target for the development of therapeutics against intra-erythrocytic P. falciparum.

Plasmodium vivax uses Duffy binding protein (PvDBP1) to bind to the Duffy Antigen-Chemokine Receptor (DARC) to invade human erythrocytes. Individuals who lack DARC expression (Duffy-negative) are thought to be resistance to P. vivax. In recent years, P. vivax malaria is becoming more prevalent in Africa with a portion of these cases detected in Duffy-negatives. Apart from DBP1, members of the reticulocyte binding protein (RBP) and tryptophan-rich antigen (TRAg) families may also play a role in erythrocyte invasion. While the transcriptomes of the Southeast Asian and South American P. vivax are well documented, the gene expression profile of P. vivax in Africa and more specifically the expression level of several erythrocyte binding gene candidates as compared to DBP1 are largely unknown. This paper characterized the first P. vivax transcriptome in Africa and compared with those from the Southeast Asian and South American isolates. The expression of 4,404 gene transcripts belong to 12 functional groups including 43 specific erythrocyte binding gene candidates were examined. Overall, there were 10-26% differences in the gene expression profile amongst the geographical isolates, with the Ethiopian and Cambodian P. vivax being most similar. Majority of the gene transcripts involved in protein transportation, housekeeping, and host interaction were highly transcribed in the Ethiopian P. vivax. Erythrocyte binding genes including PvRBP2a and PvRBP3 expressed six-fold higher than PvDBP1and 60-fold higher than PvEBP/DBP2. Other genes including PvRBP1a, PvMSP3.8, PvMSP3.9, PvTRAG2, PvTRAG14, and PvTRAG22 also showed relatively high expression. Differential expression was observed among geographical isolates, e.g., PvDBP1 and PvEBP/DBP2 were highly expressed in the Cambodian but not the Brazilian and Ethiopian isolates, whereas PvRBP2a and PvRBP2b showed higher expression in the Ethiopian and Cambodian than the Brazilian isolates. Compared to Pvs25, the standard biomarker for detecting female gametocytes, PvAP2-G (PVP01_1440800), GAP (PVP01_1403000), and Pvs47 (PVP01_1208000) were highly expressed across geographical samples. These findings provide an important baseline for future comparisons of P. vivax transcriptomes from Duffy-negative infections and highlight potential biomarkers for improved gametocyte detection.

Uncontrolled urbanization led to a specific environment, promoting the spread of Aedes mosquitoes in many African cities. The objective of the study was to assess human exposure to Aedes mosquito bites in the urban area of Abidjan, Cote dIvoire, by using antibody-based biomarkers. A cross-sectional study was undertaken during the short rainy season in 3 different neighborhoods (Bromakote, Anoumabo, and Petit-Bassam) presenting various socio-environmental and entomological characteristics. The specific IgG responses to Aedes Nterm-34kDa salivary peptide, previously validated as a relevant individual biomarker of exposure to mosquito bites, were assessed in children and analyzed according to neighborhoods and age classes. The specific IgG level was significantly different according to the 3 neighborhoods and higher in Bromakote compared to Anoumabo and Petit-Bassam. No significant difference in specific IgG level was observed according to age. We also noticed an association between the level of specific IgG responses and Ae. aegypti densities or various socio-environmental factors. This study indicated that Human exposure to Aedes vector appeared to be dependent on neighborhoods within the same city, which could be related to some socio-environmental factors. Antibody-based biomarkers of human exposure to Aedes bites could be a helpful tool for assessing the heterogeneity of urban exposure to arbovirus vectors in the framework of monitoring the risk of arbovirus infections, and for evaluating the effectiveness of vector control strategies in national control programs.

BackgroundEchinococcosis is a zoonotic disease caused by cestodes of the genus Echinococcus. Alveolar echinococcosis (AE), caused by E. multilocularis, primarily affects the liver and shows infiltrative, tumor-like growth of the metacestode stage. If untreated, AE is lethal. AE remains a neglected disease with current treatments based on albendazole or mebendazole that are parasitostatic, and not curative, underscoring the need for more effective therapies. Niclosamide is a chlorinated salicylanilide derivative with proven activities against intestinal helminths but is inactive against tissue-dwelling helminths due to poor absorption and limited bioavailability. In this study, we repurposed niclosamide ethanolamine (NEN), a formulation with improved systemic exposure, for the treatment of E. multilocularis infection in vitro and in vivo. Methodology/Principal FindingsWe assessed the in vitro efficacy of niclosamide and NEN against E. multilocularis metacestode vesicles (IC50<0.2 {micro}M) and primary parasite cells (IC50<0.3 {micro}M), with active concentrations largely corresponding to NEN levels reachable in the liver. Metabolic analysis suggested that NEN acts as a mitochondrial uncoupler. Electron microscopy showed that NEN-treatments induced profound structural damage in the metacestode vesicle tissue, but mitochondrial ultrastructure was not notably affected. In mice intraperitoneally infected with E. multilocularis, NEN was orally administered during 9 weeks either alone, or in combination with albendazole. Pharmacokinetic analyses showed that NEN reached blood level concentrations above 1 {micro}M. However, the parasite burden in NEN-treated mice was not significantly reduced. Conclusions/SignificanceAlthough niclosamide and NEN demonstrated potent activity against E. multilocularis in vitro, this efficacy did not translate in the mouse model. The lack of in vivo activity could be attributed to several factors such as infection model, limited drug uptake by the parasite in the animal, or the rapid metabolization of the compound. Future studies should explore novel niclosamide derivatives and formulations to enhance efficacy against AE in vivo. Author SummaryAlveolar echinococcosis (AE) is a severe disease caused by the larval stage of the fox tapeworm Echinococcus multilocularis. The parasite forms tumor-like lesions in the liver and can spread to other organs. The currently licensed drugs for the treatment of AE are not always effective, require long-term use, and can cause side effects that frequently require treatment interruption. Therefore, safer and more efficacious treatment options are urgently needed. Niclosamide is frequently applied for the treatment of adult tapeworm infections in the intestine, but its limited uptake and low biodistribution renders the compound unsuitable for systemic treatment. In this study, we tested a non-toxic salt formulation, niclosamide ethanolamine (NEN), exhibiting improved absorption. In vitro, NEN was highly effective against E. multilocularis metacestode vesicles. It induced profound structural alterations in metacestode vesicles and impaired the mitochondrial membrane potential, and thus interfering in energy production. However, NEN was not effective against AE in experimentally infected mice. Our results suggest that NEN treatment appears promising in vitro, but to translate to the in vivo situation, new formulations and delivery strategies should be developed to increase absorption, bioavailability and metabolic stability of the compound for an effective treatment for AE.

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in millions of dollars production losses to the livestock industry. To understand the dispersal origin and the spread patterns of F. gigantica is important for preventing the disease. A total of 53 Fasciola flukes collected from buffalo and goat in the Punjab province of Pakistan, were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffalo and goats in Pakistan was indicated by the genetic analysis of two distinct mitochondrial markers [NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1)]. Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes were clustered in haplogroup A. Fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, or possibly come from neighbouring Middle East countries. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica regarding the development of sustainable parasite control strategies.

Malaria is the worldwide infectious disease causing significant losses of human lives every year. The deployment of artemisinin-based combination therapies (ACTs) has helped to reduce the losses caused by malaria. However, resistance to ACTs evolved especially by Plasmodium falciparum occurs and becomes more and more threats for human lives. Up to now, there is no effective way to solve this drug-resistance problem. Here we propose a new ACT, which contains traditional ACT plus iron supplement, so-called ferrous ACT (FACT). The FACT is promising to become a safe and effective new formulation to combat ACT resistance malaria, helping WHO reach its 2030s Malaria Control Goal.

In June 2022, a pool of five mosquitoes that were morphologically classified as Anopheles gambiae and caught in Kisumu (Kenya) were tested for blood-meal analysis. Of the 19.6% (11/56) amplicon sequence variants assigned to mosquito species using basic local alignment search tool (BLAST), one had 15 hits matching Anopheles stephensi.

A field trial was conducted using 10% lambda-cyhalothrin microcapsule suspension to provide a method for killing ticks and preventing diseases in outdoor gatherings of people or temporary resettlement places after disasters. In this study, three field experimental sites were selected, and each experimental site was set up with a test area and a control area. Before pesticide application, the tick density in three test areas and three control areas was surveyed using the flagging method. Subsequently, two methods were used for pesticide spraying: motorized fogging and electric constant-volume spraying (with the pesticide diluted 300 times). The relative density decline rate of ticks was calculated in three test sites on days 1, 7, 14, 21, and 28 after spraying, and all experimental areas achieved good tick-killing effects. Even without prohibiting wild animals, grazing sheep, and dogs (which are often infested with ticks and not treated) from entering the trial sites, spraying 10% lambda-cyhalothrin microcapsule suspension could maintain a tick-free (low-density) state for approximately 3-4 weeks. Our study provides an idea for controlling epidemics through tick elimination during the high incidence period of tick-borne diseases.

Chikungunya, an arboviral disease transmitted by Aedes mosquitoes, shares clinical similarities with dengue, but is distinguished by prolonged joint pain. Following a major outbreak in Bangladesh in 2017, chikungunya nearly vanished from its territory. The study participants were recruited prospectively following specific inclusion criteria and obtained written informed consent. Out of 1280 febrile individuals screened, 569 met the criteria of fever onset within 2-5 days, accompanied by symptoms such as headache, myalgia, bone and joint pain, rash, nausea, vomiting, or diarrhea. Of these, 474 underwent real-time RT-PCR testing. Among the samples tested, 213 were PCR-positive for at least one arbovirus. Chikungunya cases totaled 55, including 7 coinfections (6 with DENV, and the first documented CHIKV-ZIKV coinfection in Bangladesh). No infections were reported from January to August, with a peak in October and November. Most CHIKV infections (72.7%) had moderate to high viral loads, with common symptoms of joint pain, myalgia, and headaches. The resurgence of Chikungunya in late 2024 underscores the potential for a major outbreak in 2025, necessitating proactive measures to mitigate public health impact and ensure a robust response to this re-emerging threat.

Diabetes and malaria are major morbidities that impair hepatic and renal functions. This study investigated the effects of the hydromethanol extract of Ocimum basilicum (OCB) on parasitemia, biochemical markers, and coagulation parameters in diabetic BALB/C mice infected with Plasmodium berghei. Fifty-six male mice were divided into eight groups (n=7): normal control, diabetes only, malaria only, diabetes + malaria, malaria + OCB, diabetes + OCB, diabetes + malaria + OCB, and diabetes + malaria + metformin. Diabetes was induced by streptozotocin (40 mg/kg, i.p.) for 5 consecutive days, while Plasmodium berghei was inoculated in the malaria groups, and infection was confirmed by Giemsa-stained thin smears. Treatments consisted of OCB (100 mg/kg) or metformin (250 mg/kg) orally for 7 days; controls received phosphate-buffered saline. OCB significantly (p<0.05) reduced parasitemia in infected groups compared with untreated controls. In diabetic and malaria-induced mice, elevated fasting blood glucose, creatinine, and urea were markedly reduced by OCB, with decreases of 33.22%, 70.58%, and 26.32%, respectively, in the malaria + diabetes + OCB group relative to the untreated group. Serum alanine and aspartate aminotransferases were also lowered by OCB more effectively than metformin, indicating hepatoprotective activity. Coagulation profiles showed no significant differences in activated partial thromboplastin time and prothrombin time between OCB-treated and control groups, although prothrombin time decreased in the diabetes + OCB group. These findings demonstrate that O. basilicum possesses anti-plasmodial, antihyperglycemic, and organ-protective effects, highlighting its potential as a source of phytopharmaceutical agents for the treatment and management of malaria and diabetes.

BackgroundUrbanization and climate change exacerbate mosquito-borne disease transmission by expanding mosquito habitats in densely populated areas. Traditional mosquito control methods, such as eliminating breeding sites or using insecticides, face challenges in urban settings due to practicality and resistance issues. This study introduces Human-Controlled Breeding Sites (HCBS), a novel, low-cost, and environmentally friendly mosquito control method tailored for urban and suburban households. HCBS provides controlled oviposition sites to attract gravid female mosquitoes, disrupting their reproductive cycle without chemical deterrents. MethodsThe HCBS method involves deploying devices designed to mimic natural breeding sites, encouraging mosquitoes to lay eggs in a controlled environment. After 10 days, eggs, larvae, and pupae are collected and destroyed. The HCBS device, fabricated using 3D-printed ABS material in five colors (white, black, red, green, blue), was tested in a semi-controlled laboratory in La Molina, Peru. The experiment evaluated oviposition preferences of Aedes aegypti by counting larvae and pupae across five 10-day cycles, with device colors randomized to minimize bias. ResultsHCBS devices successfully attracted gravid Aedes aegypti for oviposition, with green devices showing the highest initial preference (2-17 larvae/pupae per cycle), though larvae were later observed in all colors, indicating color is not essential. The method achieved 100% effectiveness in disrupting the mosquito life cycle by eliminating eggs, larvae, and pupae within the 10-day timeframe. The low-cost design and use of household materials enhance its scalability and community engagement potential. ConclusionsHCBS offers a sustainable alternative to conventional mosquito control by targeting early life stages, reducing populations with minimal environmental impact. Its adaptability to household settings and cost-effectiveness make it suitable for urban and suburban areas. Further research is needed to optimize device design, assess long-term efficacy, and integrate HCBS into disaster risk management systems for broader public health applications, particularly in regions like Peru with documented insecticide resistance.

Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown that Leishmania modulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy in Leishmania is inconclusive because of the limited tools available to study the Leishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy in Leishmania major. Transmission electron microscopy (TEM) allowed us to visualize Leishmania autophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments in Leishmania major by detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation in Leishmania parasites that could provide insights into developing improved therapeutic targets against leishmaniasis.

Understanding the susceptibility status of mosquitoes to insecticides is critical for effective decision making regarding the use or rotation of insecticides in control programs. In this study, we demonstrated the use of amino acid profiling for the detection of deltamethrin-resistant Aedes albopictus (L.). Mosquitoes collected in the field were first tested with WHO adulticide bioassay kits, and the amino acid profiles of the resistant mosquitoes were compared with the susceptible strain of Ae. albopictus. Samples were lyophilized and derived by silylation and then analyzed by gas chromatography-mass spectrometry (GC-MS). Using standardized, known concentrations of amino acids, we quantified the amino acids in both resistant and susceptible strains. An independent t-test was performed to compare the concentrations of each amino acid between strains. Logistic regression was then performed to assess the relationship between amino acid concentrations and susceptibility status of the mosquitoes. Our results showed that the amino acids in resistant mosquitoes differed significantly from those in susceptible mosquitoes, with the exception of serine. Further regression analysis showed that seven amino acids significantly predicted susceptibility, suggesting that they are suitable as biological indicators for rapid assessment of resistance status in field mosquitoes. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/604257v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@7c3c91org.highwire.dtl.DTLVardef@6c7ad1org.highwire.dtl.DTLVardef@1f36900org.highwire.dtl.DTLVardef@172b13d_HPS_FORMAT_FIGEXP M_FIG C_FIG Identification of deltamethrin-resistant mosquitoes based on differences in the amino acid profile: Deltamethrin-susceptible and -resistant mosquito strains were lyophilised and converted into esters by silylation, which were then analysed using a gas chromatography-mass spectrometer (GC-MS). The difference between susceptible and resistant could be classified by developing a classification model with logistic regression.

Clonorchiasis is one of the major parasitic diseases in South Korea. Spatially explicit estimates of the infection risk are important for control and intervention. We did a systematic review of collected prevalence data on Clonorchis sinensis infection in South Korea. Data of potential influencing factors (e.g., environmental and social-economic factors) was obtained from open-access databases. Bivariate Bayesian geostatistical joint modeling approaches were applied to analyze disease data, within a logit regression in combination of potential influencing factors and spatial-temporal random effects. We identified surveys of C. sinensis infection done at 1362 unique locations, and presented the first spatial-temporal risk maps at high spatial resolution (5x5km) in South Korea. High infection risk areas shrunk significantly from 1970 to 2017. The overall risk decreased since the start of the national deworming program in 1969, and then slightly increased since the year 1995 when the program suspended, and maintained stable since 2005 when the Clonorchiasis Eradication Program begun. The population-weighted prevalence was estimated as 3.87% (95% BCI: 3.04-4.82%) in 2017, accounting to 1.92 (95% BCI: 1.51-2.40) million infected people. Although the prevalence over the country has been low, C. sinensis infection was still endemic in areas of eastern and southern regions, particularly the five major river basins. We also defined factors significantly correlated, such as, distance to the nearest open water bodies, annual precipitation, and land surface temperature at night. All findings above provide important information on spatial-targeting control and preventive strategies of C. sinensis infection in South Korea.

Despite increasing reports and concerns about resistance development to public-health insecticides in malaria-vectors, significant steps have been put into the quest for novel strategies to disrupt the disease transmission cycle by targeting insect-vectors hence sustaining vector management. This study evaluates the toxicity potential of oils of insecticidal plants shortlisted in an ethnobotanical survey on the larvae and adult stages of Anopheles gambiae. Oils from leaves of Hyptis suaveolens, Ocimum gratissimum, Nicotiana tabacum, Ageratum conyzoides and fruit-peel of Citrus sinensis were extracted by steam-distillation using a Clevenger apparatus. Larvae and female adults of deltamethrin susceptible Anopheles gambiae were gotten from an already established colony in the Entomological Research Laboratory, University of Ilorin. Twenty-five third instar stage larvae were used for larvicidal assays while twenty 2-5 days old adults were used for the adulticidal assays in five replicates. After which, coupled gas chromatography-mass spectrometry analysis (GC-MS) was performed to determine the major chemical-constituents of plant oils. A. gambiae exposed to H. suaveolens and C. sinensis demonstrated significantly higher larval toxicity (94.7-100%) after 24 hours. At 48 hours, the mortality induced by the oils of the four plants peaked at 100%. N. tabacum (0.50 mg/ml) induced the highest percentage adult mortality (100%) on A. gambiae which compared favourably with the positive control Deltamethrin (0.05%). The lowest KdT50 was observed with 0.25 mg/ml of N. tabacum (20.3 minutes) while the lowest KdT95 was observed with 0.10mg/ml of A. conyzoides (35.97 mins) against adult A. gambiae. D-limonene is the key chemical-constituent in oils from C. sinensis and A. conyzoides. The significant larval and adult mortality rates, lower lethal concentration and knockdown times demonstrated by the evaluated plant oils showed promising outcomes that can be further developed for vector control management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/503910v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@35404corg.highwire.dtl.DTLVardef@105440org.highwire.dtl.DTLVardef@b64f8org.highwire.dtl.DTLVardef@11f079_HPS_FORMAT_FIGEXP M_FIG C_FIG