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.

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.

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.

Plasmodium cynomolgi (Pc) is one of the few parasite species that forms quiescent liver stage parasites known as hypnozoites and is therefore a suitable model for Plasmodium vivax. Very little is known about liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity. Here, we present the development of a Pc in vitro infection model using stem cell-derived hepatocytes from Macaca fascicularis. IPS cells were established on feeder free condition and differentiated into hepatocytes via inducible overexpression of key transcription factors. The generated hepatocytes were infected with Pc sporozoites and hypnozoite formation as well as schizont development were confirmed by immunofluorescence. This system is a promising tool to study the mechanisms underlying liver stage dormancy and facilitate drug discovery against hypnozoites.

BackgroundMalaria still is one of the most relevant infectious diseases in Brazil with 184,869 cases in 2019 and 62.8% of these cases occurred in only 6.2% of municipalities that had high transmission. The incidence of malaria is influenced by environmental, socioeconomic, demographic, and structural factors (organization and effectiveness of health services, land use, and infrastructure). MethodsWe use data from the Malaria Epidemiological Surveillance System aggregated by period and municipality, maintained by the Ministry of Health, from 2007 to 2019, and combine it with data from the National Institute for Space Researchs - Satellite Monitoring of Brazilian Amazon Forest Deforestation Project, 2010 Demographic Census Microdata and Registry of Health Establishments to analyze socioeconomic, demographic, environmental and health factors in two distinct periods. We use the Principal Component Analysis algorithm to create different principal components characteristics patterns in high-risk municipalities. ResultsThe Principal Component Analysis allowed the creation of three profiles of high-risk municipalities combining the contribution of different demographic, socioeconomic, environmental, and health characteristics with the contribution of the municipalities in each principal component. The first group with a vulnerability profile, that is, high-risk municipalities with cases more associated with vulnerability characteristics, the second group is one with a profile of occupation and working age, of high-risk municipalities with cases more related to socioeconomic and demographic variables linked with specific occupations and a working-age population and the third group with municipalities that has a mixed profile associated with different characteristics related to of P. falciparum in contrast of P. vivax cases, such as the municipality of Rio Preto da Eva, which for the cases of P. vivax, may be more related to children under 5 years and differently, P. falciparum in this municipality could be more related to gold panning, deforestation and the presence of large bodies of water. ConclusionsThere are particularities in socioeconomic, demographic, environmental, and health characteristics for infection by both types of Plasmodium and high-risk municipalities that become necessary to understand the differences in the profile of the population affected by malaria together with the environment in which they live, the weather, forest, hydrography and health conditions to assess the structure in these regions, in addition to analyzing the cases of P. falciparum separately from the cases of P. vivax, showing us that understand the socio-environmental determinants at the local level is essential for the success of malaria prevention and control strategies. Author SummaryLuciana C. Alves is an Associate Professor at the Institute of Philosophy and Human Sciences (IFCH) at the University of Campinas (Unicamp) and research scientist at the Population Studies Center Elza Berquo (Nepo) and Chair of the Department of Demography at the University of Campinas. Natalia M. Arruda is a doctoral student in Epidemiology at the National School of Public Health/Fiocruz. Vinicius S. Maia is a doctoral student at the Centre for Economic Demography at Lund University. Bianca C. Carlos is an assistant researcher at the Population Studies Center Elza Berquo (Nepo)/Unicamp. Carlos E. Beluzo is a Professor at the Federal Institute of Sao Paulo and a doctoral student at the Institute of Philosophy and Human Sciences (IFCH) at the University of Campinas (Unicamp).

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.

Ixodid ticks, such as Ixodes ovatus and Haemaphysalis flava, are important vectors of tick-borne diseases in Japan, such as Japanese spotted fever caused by Rickettsia japonica. This study describes the population genetic structure and gene flow of I. ovatus and H. flava and their Rickettsia infection rates along an altitudinal gradient. A total of 346 adult I. ovatus and 243 H. flava were analyzed for the presence of Rickettsia by nested PCR targeting the 17kDA, gltA, rOmpA, and rOmpB genes. Population genetic structure was performed using the mitochondrial cytochrome oxidase 1 (cox1) marker. The Rickettsia infection rates were 13.26% in I. ovatus and 6.17% in H. flava. For I. ovatus, the global FST value revealed significant genetic differentiation among the different populations, whereas H. flava showed non-significant genetic differentiation. The cox1 I. ovatus cluster dendrogram showed two cluster groups, while the haplotype network and phylogenetic tree showed three genetic groups. The two cluster groups and the three genetic groups found in I. ovatus showed a significant difference in their Rickettsia infection rates and mean altitude per group. No significant differences were found in the mean altitude or Rickettsia infection rates of H. flava. Our results suggest that low gene flow in the I. ovatus populations has caused spatially heterogenous Rickettsia infection rates along the altitudinal gradient. This information can be used in understanding the relationship between the tick vector, its pathogen, and environmental factors, such as altitude, and for the control of tick-borne diseases in Japan.

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.

Ixodid tick species such as Ixodes ovatus and Haemaphysalis flava are essential vectors of tick-borne diseases in Japan. In this study, we investigated the population genetic structures and gene flow of I. ovatus and H. flava as affected by the tick host mobility. We hypothesized that I. ovatus and H. flava may have differences in their genetic structure due to the low mobility of small rodent hosts of I. ovatus at the immature stage in contrast to the mediated dispersal of avian hosts for immature H. flava. We collected 307 adult I. ovatus and 220 adult H. flava from 29 and 17 locations across Niigata Prefecture, Japan. We investigated the genetic structure at two mitochondrial loci (cox1, 16S rRNA gene). For I. ovatus, pairwise FST and analysis of molecular variance (AMOVA) analyses of cox1 sequences indicated significant genetic variation among populations. Both cox1 and 16S rRNA markers showed non-significant genetic variation among locations for H. flava. The Bayesian tree and haplotype network of cox1 marker for I. ovatus samples in Niigata Prefecture found 3 genetic groups wherein most haplotypes in group 2 were distributed in low altitudinal areas. When we added cox1 sequences of I. ovatus from China to the phylogenetic analysis, three genetic groups (China 1, China 2, and Niigata and Hokkaido, Japan) were formed in the tree suggesting the potential for cryptic species in the genetic group in Japan. Our results support our hypothesis and suggest that the host preference of ticks at the immature stage may influence the genetic structure and gene flow of the ticks. This information is vital in understanding the tick-host interactions in the field to better understand the tick-borne disease transmission and in designing an effective tick control program.

Climate change is a complex problem that often disproportionately affects global health, including its influence on microbial ecosystems, which can lead to disease outbreaks such as Lyme disease (LD). However, the extent of climate variability and its influence on the spread of the most common vector-borne disease in countries neighboring the Baltic Sea and North Sea have not been fully quantified. Therefore, this study quantified the magnitude of the (LD) burden due to climate change in the most at-risk European countries. In this ecological study, the correlation between disease incidence over the years and climate change was tested using a Spearman correlation test, and the change in incidence of LD during the period 2000-2024 was assessed using Generalized Linear Models (GLMs) with a negative binomial distribution to handle overdispersion. The study found a strong positive relationship when accounting for the tick life cycle using lagged climate variables of two years to higher rates of LD in countries bordering the Baltic and North Seas. Most importantly, a unit increase in precipitation (mm/year), after adjusting for covariates, is associated with a higher disease rate (IRR Range= 1.15-1.24). Two years of delayed temperature effect has a similar relationship (IRR range = 1.11-1.27). These findings suggest that disproportionate climate change in the region influences the spread and burden of the disease in the northern European temperate climate, mostly observed after two years of delays. Compared to previous research, this study focused on the regional impact of climate change on microbial life and disease spillover, which causes public health problems. There is an urgent need for a collaborative and comprehensive program that includes environmental, human, animal and vector data factors for future research to aid in disease control.

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.

Central China has been reported to be one of the most important endemic areas of zoonotic infection by spotted fever group rickettsiae(SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV) and orthohantavirus hantanense(HTNV). Due to similar clinical symptoms, it is challenging to make a definite diagnosis rapidly and accurately in the absence of microbiological tests. In the present study, an all-in-one real-time PCR assay was developed for the simultaneous detection of nucleic acids from SFGR, SFTSV and HTNV. Three linear standard curves for determining SFGR-ompA, SFTSV-L and HTNV-L were obtained within the range of 101-106 copies/L, with the PCR amplification efficiencies ranging from 93.46% to 96.88% and the regression coefficients R2 of >0.99. The detection limit was 1.108 copies/L for SFGR-ompA, 1.075 copies/L for SFTSV-L and 1.006 copies/L for HTNV-L, respectively. Both the within-run and within-laboratory coefficients of variation on the cycle threshold (Ct) values were within the range of 0.53%-2.15%. It was also found there was no statistical difference in the Ct values between with and without other non-target bloodborne virus nucleic acids (PSFGR-ompA =0.186, PSFTSV-L=0.612, PHTNV-L=0.298). The sensitivity, specificity, positive and negative predictive value were all 100% for determining SFGR-ompA and SFTSV-L, 97%, 100%, 100% and 99.6% for HTNV-L, respectively. Therefore, the all-in-one real-time PCR assay appears to be a reliable, sensitive, rapid, high-throughput and low cost-effective method to diagnose the zoonotic infection by SFGR, SFTSV and HTNV. Author SummarySpotted fever, severe fever with thrombocytopenia syndrome (SFTS), and hemorrhagic fever with renal syndrome (HFRS) sporadically have outbreaks in central China. Due to the similarities in clinical symptoms and the absence of reliable diagnostic methods, clinical diagnosis and treatment frequently result in misdiagnosis or missed diagnosis. Thus, the development of a fast and accurate diagnostic method is crucial for prevention and precise treatment. In this study, we designed an all-in-one real-time PCR assay to differentiate spotted fever group rickettsiae(SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV) and orthohantavirus hantanense(HTNV). The gene ompA of SFGR, as well as the gene segment L of SFTSV and HTNV, were used as targets to design primers and probes for amplification. Through the verification of nucleic acid and clinical sample detection, the sensitivity of this detection method exceeded 97%, and its specificity was 100%. This new assay could be applied in epidemiology and clinical diagnosis, to control new outbreaks, reduce diagnostic and identification time, and improve test efficiency.

Recent reports from Thailand reveal a substantial surge in Plasmodium knowlesi cases over the past decade, with a more than eightfold increase in incidence by 2023 compared to 2018. This study investigates temporal changes in genetic polymorphism associated with the escalating transmission of P. knowlesi malaria in Thailand over the past two decades. Twenty-five P. knowlesi samples collected in 2018-2023 were sequenced for the 42-kDa region of pkmsp1 and compared with 24 samples collected in 2000-2009, focusing on nucleotide diversity, natural selection, recombination rate, and population differentiation. Seven unique haplotypes were identified in recent samples, compared to 15 in earlier samples. Nucleotide and haplotype diversities were lower in recent samples ({pi} = 0.016, Hd = 0.817) than in earlier samples ({pi} = 0.018, Hd = 0.942). Significantly higher synonymous substitution rates were observed in both sample sets (dS - dN = 2.77 and 2.43, p < 0.05), indicating purifying selection and reduced genetic diversity over time. Additionally, 8 out of 17 mutation points were located on B-cell epitopes, suggesting an adaptive response by the parasites to evade immune recognition. Population differentiation analysis using the fixation index (Fst) revealed high genetic differentiation between parasite populations in central and southern Thailand or Malaysia. Conversely, the relatively lower Fst value between southern Thailand and Malaysia suggests a closer genetic relationship, possibly reflecting historical gene flow. In conclusion, our findings highlight a decline in genetic diversity and evidence of purifying selection associated with the recently increased incidence of P. knowlesi malaria in Thailand. The minor genetic differentiation between P. knowlesi populations from southern Thailand and Malaysia suggests a shared recent ancestry of these parasites and underscores the need for coordinated efforts between the two countries for the elimination of P. knowlesi.

Anopheles barbirostris, a member of the Barbirostris Subgroup in the Anopheles genus, comprises a complex of species in South and Southeast Asia. This An. barbirostris complex includes eight species such as An. barbirostris sensu stricto (s.s.), An. campestris, An. dissidens, An. donaldi, An. saeungae, An. sarpangensis, An. vanderwulpi, and An. wejchoochotei. This study employed molecular markers, including ITS2 and COI genes, to investigate the phylogenetic relationships within An. barbirostris sensu lato (s.l.) populations from various locations in Indonesia. The analysis reveals the presence of nine distinct populations within this complex, including the first report of An. wejchoochotei in North Sulawesi and an unique Barbirostris population in Magelang, Central Java. This makes it a more complex metapopulation than previously thought. These findings provide critical insights into the diversity of malaria and lymphatic filariasis vectors in Indonesia. Understanding the complex structure of the An. barbirostris populations and their genetic diversity will be useful for effective vector control and disease elimination strategies.

Trypanosoma brucei is the etiologic agent of African trypanosomiasis, also known as sleeping sickness. In the mammalian host, T. brucei is present as replicative and non-replicative bloodstream forms (BSF) referred to as long slender and stumpy, respectively. In the insect vector, the developmental forms are procyclic (PCF), epimastigote, and metacyclic trypomastigote. It is well established that BSF use glucose as the main energy source, and PCF can use glucose but, in its absence, uses amino acids, mainly proline. Due to these facts, the metabolism of amino acids has been well studied in PCF, but less attention has been given to it in BSF. In the present work, we unveil the participation of glutamine (Gln), the most abundant amino acid in the human blood, in BSF proliferation. According to the data presented herein, in the absence of extracellular Gln, cell cycle progress, as well as kinetoplast and flagella distribution and function among daughter cells, are widely compromised, yielding polyploid multiflagellated cells with reduced motility. In addition, the Gln-poor medium resulted in cells with a massive alteration in the proteome glutamylation. These alterations were reverted when Gln was added to the culture media. An analysis of the glutamylproteome of cells incubated in Gln-poor medium, followed by a Gene Ontology (GO) analysis, suggested a relationship between glutamylation and biological processes such as gene expression and regulation, cell cycle, and bioenergetics metabolism, in addition to cytoskeleton dynamics in BSF of T. brucei.

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.

The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimise the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening. Author SummaryYearly, up two million people in poverty-stricken areas contract leishmaniasis, a disease caused by parasites of the genus Leishmania. When an infected sandfly takes a blood meal, Leishmania parasites enter the host where they are taken up by macrophages. Inside the macrophage, Leishmania parasites establish a niche where they can proliferate. Although this infection often leads to disability or death, the drugs currently available are lacking due to toxic side effects, high expenses or difficulties in usage. Drug screening assays that are currently used for compound screening often rely on mouse peritoneal macrophages. We have generated human induced pluripotent stem cell derived macrophages and used these as new host cells for Leishmania donovani in the testing of anti-leishmanial compounds. This model has many advantages. For one, it allows us to work with human cells, mimicking the natural infection more closely than possible with murine cells. Secondly, it allows to obtain bigger batches of uniform cells for screening campaigns. Finally, this approach aligns with the principle of 3R, replacing the use of animals for cultivation of Leishmania and drug screening purposes.

Profilins are actin binding proteins that play a central role in regulation of actin remodeling in all eukaryotic organisms. Leishmania, a family of protozoan parasites that cause leishmaniasis, express a single homolog of profilin, which differs from other eukaryotic profilins in that it contains an extra stretch of 20 amino acids (actin binding domain) through which it directly binds actin. We therefore considered it of interest to analyze the role of direct binding of profilin with actin in interactions and functions of profilin in L. donovani promastigotes. For this, we deleted the actin binding domain in L. donovani profilin (LdPfn), and then carried out comparative analysis of LdPfn and truncated LdPfn ({delta}LdPfn) interactomes by affinity pull-down and mass spectrometry. To assess the effect of deletion of the actin binding domain on the LdPfn functions, we expressed GFP conjugates of LdPfn and {delta}LdPfn in the wild type Leishmania cells and then carried out comparative analysis of their growth, cell division cycle and intracellular vesicle trafficking activity. Results revealed that expression of GFP-{delta}LdPfn in the wild type cells adversely affected their cell growth, intracellular vesicle trafficking and G1-to-S and S-to-G2/M phase transitions during their cell division cycle. Also, there was a complete loss of LdPfn binding to several cellular proteins including actin and mitochondrial outer membrane protein, porin (LdPorin) after deleting its actin binding domain. Further, to assess the effect of lack of LdPorin binding to {delta}LdPfn on the mitochondrial functions, we measured the cellular ATP levels in wild-type and transgenic promastigotes. The ATP levels were significantly increased by expressing GFP-{delta}LdPfn in wild-type cells. These results taken together strongly indicate that LdPfn-driven actin remodeling besides playing a pivotal role in regulation of intracellular vesicle trafficking and cell division cycle, it also plays an important role in regulation of Leishmania mitochondrial activity.

BackgroundAedes aegypti presence, human-vector contact rates, and Aedes-borne virus transmission are highly variable through time and space. The Lower Rio Grande Valley (LRGV), Texas, is one of the few regions in the U.S. where local transmission of Aedes-borne viruses occurs, presenting an opportunity to evaluate social, urbanistic, entomological, and mobility-based factors that modulate human exposure to Ae. aegypti. Methodology & Principal FindingsMosquitoes were collected using BG-Sentinel 2 traps during November 2021 as part of an intervention trial, with knowledge, attitudes, and practices (KAP) and housing quality surveys to gather environmental and demographic data. Human blood samples were taken from individuals and a Bitemark Assay (ELISA) was conducted to quantify human antibodies to the Ae. aegypti Nterm-34kDa salivary peptide as a measure of human exposure to bites. In total, 64 houses were surveyed with 142 blood samples collected. More than 80% of participants had knowledge of mosquito-borne diseases and believed mosquitoes to be a health risk in their community. Our best fit generalized linear mixed effects model found four fixed effects contributed significantly to explaining the variation in exposure to Ae. aegypti bites: higher annual household income, younger age, larger lot area, and higher female Ae. aegypti abundance per trap night averaged over 5 weeks prior to human blood sampling. ConclusionsMost surveyed residents recognized mosquitoes and the threat they pose to individual and public health. Urbanistic (i.e., lot size), social (i.e., income within a low-income community and age), and entomological (i.e., adult female Ae. aegypti abundance) factors modulate the risk of human exposure to Ae. aegypti bites. The use of serological biomarker assays, such as the Bitemark Assay, are valuable tools for surveillance and risk assessment of mosquito-borne disease, especially in areas like the LRGV where the transmission of target pathogens is low or intermittent. Author SummaryAedes aegypti is a mosquito vector with public health importance on the global scale as it transmits viruses such as dengue, chikungunya, and Zika. Although transmission rates of dengue and Zika are low in the U.S., there are a few regions, including south Texas, where local transmission has occurred. Our study aimed to evaluate the factors associated with risk of exposure to these viruses using a serological bioassay that measured antibody response to an Ae. aegypti salivary protein to assess human-vector contact. We collected mosquitoes, took human-blood samples, and conducted urbanistic and demographic surveys in November 2021 in eight communities in the Lower Rio Grande Valley, Texas. Our knowledge, attitude, and practices survey found that most residents recognized adult mosquitoes, though few individuals knew someone personally who been sick with a mosquito-borne disease. Outdoor adult female Ae. aegypti abundance was positively associated with exposure to mosquito bites. Household income, individual age, and lot area also significantly affected exposure levels. The Bitemark Assay we used in this study can be utilized as a tool for entomological risk assessment and could be used as an alternative to infection exposure in areas where mosquito-borne disease levels are low.