A transfer learning approach to identify Plasmodium in microscopic images.

Plasmodium parasites cause Malaria disease, which remains a significant threat to global health, affecting 200 million people and causing 400,000 deaths yearly. Plasmodium falciparum and Plasmodium vivax remain the two main malaria species affecting humans. Identifying the malaria disease in blood smears requires years of expertise, even for highly trained specialists. Literature studies have been coping with the automatic identification and classification of malaria. However, several points must be addressed and investigated so these automatic methods can be used clinically in a Computer-aided Diagnosis (CAD) scenario. In this work, we assess the transfer learning approach by using well-known pre-trained deep learning architectures. We considered a database with 6222 Region of Interest (ROI), of which 6002 are from the Broad Bioimage Benchmark Collection (BBBC), and 220 were acquired locally by us at Fundação Oswaldo Cruz (FIOCRUZ) in Porto Velho Velho, Rondônia-Brazil, which is part of the legal Amazon. We exhaustively cross-validated the dataset using 100 distinct partitions with 80% train and 20% test for each considering circular ROIs (rough segmentation). Our experimental results show that DenseNet201 has a potential to identify Plasmodium parasites in ROIs (infected or uninfected) of microscopic images, achieving 99.41% AUC with a fast processing time. We further validated our results, showing that DenseNet201 was significantly better (99% confidence interval) than the other networks considered in the experiment. Our results support claiming that transfer learning with texture features potentially differentiates subjects with malaria, spotting those with Plasmodium even in Leukocytes images, which is a challenge. In Future work, we intend scale our approach by adding more data and developing a friendly user interface for CAD use. We aim at aiding the worldwide population and our local natives living nearby the legal Amazon's rivers.

Melatonin as a Circadian Marker for Plasmodium Rhythms.

Plasmodium, a digenetic parasite, requires a host and a vector for its life cycle completion. Most Plasmodium species display circadian rhythmicity during their intraerythrocytic cycle within the host, aiding in immune evasion. This rhythmicity, however, diminishes in in vitro cultures, highlighting the importance of host-derived signals for synchronizing the parasite's asexual cycle. Studies indicate a species-specific internal clock in Plasmodium, dependent on these host signals. Melatonin, a hormone the pineal gland produces under circadian regulation, impacts various physiological functions and is extensively reviewed as the primary circadian marker affecting parasite rhythms. Research suggests that melatonin facilitates synchronization through the PLC-IP3 signaling pathway, activating phospholipase C, which triggers intracellular calcium release and gene expression modulation. This evidence strongly supports the role of melatonin as a key circadian marker for parasite synchronization, presenting new possibilities for targeting the melatonin pathway when developing novel therapeutic approaches.

Host metabolomic responses in recurrent P. vivax malaria.

Malaria is the leading parasitic disease worldwide, with P. vivax being a major challenge for its control. Several studies have indicated metabolomics as a promising tool for combating the disease. The study evaluated plasma metabolomic profiles of patients with recurrent and non-recurrent P. vivax malaria in the Brazilian Amazon. Metabolites extracted from the plasma of P. vivax-infected patients were subjected to LC-MS analysis. Untargeted metabolomics was applied to investigate the metabolic profile of the plasma in the two groups. Overall, 51 recurrent and 59 non-recurrent patients were included in the study. Longitudinal metabolomic analysis revealed 52 and 37 significant metabolite features from the recurrent and non-recurrent participants, respectively. Recurrence was associated with disturbances in eicosanoid metabolism. Comparison between groups suggest alterations in vitamin B6 (pyridoxine) metabolism, tyrosine metabolism, 3-oxo-10-octadecatrienoate ß-oxidation, and alkaloid biosynthesis II. Integrative network analysis revealed enrichment of other metabolic pathways for the recurrent phenotype, including the butanoate metabolism, aspartate and asparagine metabolism, and N-glycan biosynthesis. The metabolites and metabolic pathways predicted in our study suggest potential biomarkers of recurrence and provide insights into targets for antimalarial development against P. vivax.

In Vitro and in Vivo Antimalarial Activity, Cytotoxicity and Phytochemical HRMS2 Profile of Plants from the Western Pará State, Brazilian Amazonia.

Ethnopharmacology and botanical taxonomy are valid criteria used to selecting plants for antimalarial bioprospection purposes. Based on these two criteria, ethanol extracts of 11 plants from Santarém City vicinities, Western Pará State, Brazilian Amazonia, had their in vitro antiplasmodial activity against chloroquine-resistant Plasmodium falciparum (W2 clone) assessed by the PfLDH method, whereas their cytotoxicity to HepG2-A16 cells was assessed through MTT assay. Acmella oleracea, Siparuna krukovii and Trema micrantha extracts disclosed the highest rate of parasite growth inhibition (90 %) in screening tests. In vivo antimalarial assays were conducted with these extracts against Plasmodium berghei (NK 65 strain) infected mice. Inhibition rate of parasite multiplication ranged from 41.4 % to 60.9 % at the lowest extract dose (25 mg/kg). HPLC-ESI-HRMS2 analyses allowed the putative identification of alkylamides, fatty acids, flavonoid glycosides and alkaloids in ethanol extracts deriving from these three plant species. Results pointed towards A. oleracea flowers ethanol extract as the most promising potential candidate to preclinical studies aiming the development of antimalarial phytomedicine.

Unravelling the genome of the brackish water malaria vector Anopheles aquasalis.

Malaria is a severe public health problem in several developing tropical and subtropical countries. Anopheles aquasalis is the primary coastal malaria vector in Central and South America and the Caribbean Islands, and it has the peculiar feature of living in water with large changes in salinity. Recent research has recognised An. aquasalis as an important model for studying the interactions of murine and human Plasmodium parasites. This study presents the complete genome of An. aquasalis and offers insights into its evolution and physiology. The genome is similar in size and gene content to other Neotropical anophelines, with 162 Mb and 12,446 protein-coding genes. There are 1387 single-copy orthologs at the Diptera level (eg. An. gambiae, An. darlingi and Drosophila melanogaster). An. aquasalis diverged from An. darlingi, the primary malaria vector in inland South America, nearly 20 million years ago. Proteins related to ion transport and metabolism belong to the most abundant gene families with 660 genes. We identified gene families relevant to osmosis control (e.g., aquaporins, vacuolar-ATPases, Na+/K+-ATPases, and carbonic anhydrases). Evolutionary analysis suggests that all osmotic regulation genes are under strong purifying selection. We also observed low copy number variation in insecticide resistance and immunity-related genes for all known classical pathways. The data provided by this study offers candidate genes for further studies of parasite-vector interactions and for studies on how anophelines of brackish water deal with the high fluctuation in water salinity. We also established data and insights supporting An. aquasalis as an emerging Neotropical malaria vector model for genetic and molecular studies.

A new Plasmodium vivax reference genome for South American isolates.

BACKGROUND: Plasmodium vivax is the second most important cause of human malaria worldwide, and accounts for the majority of malaria cases in South America. A high-quality reference genome exists for Papua Indonesia (PvP01) and Thailand (PvW1), but is lacking for South America. A reference genome specifically for South America would be beneficial though, as P. vivax is a genetically diverse parasite with geographical clustering. RESULTS: This study presents a new high-quality assembly of a South American P. vivax isolate, referred to as PvPAM (P. vivax Peruvian AMazon). The genome was obtained from a low input patient sample from the Peruvian Amazon and sequenced using PacBio technology, resulting in a highly complete assembly with 6497 functional genes. Telomeric ends were present in 17 out of 28 chromosomal ends, and additional (sub)telomeric regions are present in 12 unassigned contigs. A comparison of multigene families between PvPAM and the PvP01 genome revealed remarkable variation in vir genes, and the presence of merozoite surface proteins (MSP) 3.6 and 3.7. Three dhfr and dhps drug resistance associated mutations are present in PvPAM, similar to those found in other Peruvian isolates. Mapping of publicly available South American whole genome sequencing (WGS) data to PvPAM resulted in significantly fewer variants and truncated reads compared to the use of PvP01 or PvW1 as reference genomes. To minimize the number of core genome variants in non-South American samples, PvW1 is most suited for Southeast Asian isolates, both PvPAM and PvW1 are suited for South Asian isolates, and PvPAM is recommended for African isolates. Interestingly, non-South American samples still contained the least subtelomeric variants when mapped to PvPAM, indicating high quality of the PvPAM subtelomeric regions. CONCLUSIONS: Our findings show that the PvPAM reference genome more accurately represents South American P. vivax isolates in comparison to PvP01 and PvW1. In addition, PvPAM has a high level of completeness, and contains a similar number of annotated genes as PvP01 or PvW1. The PvPAM genome therefore will be a valuable resource to improve future genomic analyses on P. vivax isolates from the South American continent.

Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

In Silico Studies of Four Compounds of Cecropia obtusifolia against Malaria Parasite.

Malaria is a disease that affects many people in the world. In Mexico, malaria remains an active disease in certain regions, particularly in the states of Chiapas and Chihuahua. While antimalarial effects have been attributed to some species of Cecropia in various countries, no such studies have been conducted in Mexico. Therefore, the objective of this study was to evaluate the in silico antimalarial activity of some active compounds identified according to the literature in the species of Cecropia obtusifolia, belonging to the Cecropiaceae family, such as ursolic acid, α-amyrin, chrysin, and isoorientin. These compounds were evaluated with specific molecular docking and molecular dynamics (MD) studies using three different malarial targets with the PDB codes 1CET, 2BL9, and 4ZL4 as well as the prediction of their pharmacokinetic (Pk) properties. Docking analysis revealed the following best binding energies (kcal/mol): isoorientin-1CET (-9.1), isoorientin-2BL9 (-8.8), and chrysin-4ZL4 (-9.6). MD simulation validated the stability of the complexes. Pharmacokinetics analysis suggested that the compounds would generally perform well if administered. Therefore, these results suggest that these compounds may be used as potential drugs for the treatment of malaria.

Interplay Between the Immune and Nervous Cognitive Systems in Homeostasis and in Malaria.

The immune and nervous systems can be thought of as cognitive and plastic systems, since they are both involved in cognition/recognition processes and can be architecturally and functionally modified by experience, and such changes can influence each other's functioning. The immune system can affect nervous system function depending on the nature of the immune stimuli and the pro/anti-inflammatory responses they generate. Here we consider interactions between the immune and nervous systems in homeostasis and disease, including the beneficial and deleterious effects of immune stimuli on brain function and the impact of severe and non-severe malaria parasite infections on neurocognitive and behavioral parameters in human and experimental murine malaria. We also discuss the effect of immunization on the reversal of cognitive deficits associated with experimental non-severe malaria in a model susceptible to the development of the cerebral form of the illness. Finally, we consider the possibility of using human vaccines, largely exploited as immune-prophylactics for infectious diseases, as therapeutic tools to prevent or mitigate the expression of cognitive deficits in infectious and chronic degenerative diseases.

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest.

BACKGROUND: Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. METHODS: Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. RESULTS: Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. CONCLUSIONS: For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

The first complete genome of the simian malaria parasite Plasmodium brasilianum.

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species.

Testosterone induces sexual dimorphism during infection with Plasmodium berghei ANKA.

Malaria is the most lethal parasitic disease worldwide; men exhibit higher mortality and more severe symptomatology than women; however, in most studies of immune response in malaria, sex is not considered a variable. Sex hormones 17ß-oestradiol and testosterone are responsible for the main physiological differences between sexes. When interacting with their receptors on different immune cells, they modify the expression of genes that modulate cell proliferation, differentiation, and synthesis of cytokines. The immunosuppressive activity of testosterone is well accepted; however, its participation in the sexual dimorphism of the immune response to malaria has not been studied. In this work, we analysed whether altering the concentration of testosterone, through increasing the concentration of this hormone for exogenous administration for three weeks, or gonadectomy before infection with Plasmodium berghei ANKA affects different cells of the immune response necessary for parasite clearance. We also assessed the concentration of pro-and anti-inflammatory cytokines in male and female CBA/Ca mice infected or not with the parasite. Our results show that testosterone changes affect females more than males, resulting in sex-associated patterns. Testosterone administration increased parasitaemia in intact males while reducing it in intact females leading to a dimorphic pattern. In addition, gonadectomy increased parasitaemia in both sexes. Moreover, testosterone administration prevented both weight loss caused by the infection in females and hypothermia in gonadectomized mice of both sexes. Boosting testosterone concentration increased CD3+ and CD8+ populations but decreased the B220+ cells exclusively in females. Additionally, testosterone reduced IFN-γ concentration and increased IL-6 levels only in females, while in males, testosterone increased the number of NK cells. Finally, gonadectomy decreased TNF-α concentration in both sexes. Our results demonstrate that testosterone induces different patterns depending on sex and testosterone concentration. The results of this work contribute to understanding the impact of modifying testosterone concentration on the immune response specific against Plasmodium and the participation of this hormone in sexual dimorphism in malaria.

Antimalarial Pyrido[1,2-a]benzimidazoles Exert Strong Parasiticidal Effects by Achieving High Cellular Uptake and Suppressing Heme Detoxification.

Pyrido[1,2-a]benzimidazoles (PBIs) are synthetic antiplasmodium agents with potent activity and are structurally differentiated from benchmark antimalarials. To study the cellular uptake of PBIs and understand the underlying phenotype of their antiplasmodium activity, their antiparasitic activities were examined in chloroquine (CQ)-susceptible and CQ-resistant Plasmodium falciparumin vitro. Moreover, drug uptake and heme detoxification suppression were examined in Plasmodium berghei-infected mice. The in vitro potency of PBIs is comparable to most 4-aminoquinolines. They have a speed of action in vitro that is superior to that of atovaquone and an ability to kill rings and trophozoites. The antiparasitic effects observed for the PBIs in cell culture and in infected mice are similar in terms of potency and efficacy and are comparable to CQ but with the added advantage of demonstrating equipotency against both CQ susceptible and resistant parasite strains. PBIs have a high rate of uptake by parasite cells and, conversely, a limited rate of uptake by host cells. The mechanism of cellular uptake of the PBIs differs from the ion-trap mechanism typically observed for 4-aminoquinolines, although they share key structural features. The high cellular uptake, attractive parasiticidal profile, and susceptibility of resistant strains to PBIs are desirable characteristics for new antimalarial agents.

CCR6 expression reduces mouse survival upon malarial challenge with Plasmodium berghei NK65 strain.

BACKGROUND: It has been demonstrated that proteins expressed by liver-stage Plasmodium parasites can inhibit the translocation of transcription factors to the nucleus of different cells. This process would hinder the expression of immune genes, such as the CCL20 chemokine. OBJECTIVE: Since CCR6 is the only cognate receptor for CCL20, we investigated the importance of this chemokine-receptor axis against rodent malaria. METHODS: CCR6-deficient (KO) and wild-type (WT) C57BL/6 mice were challenged with Plasmodium berghei (Pb) NK65 sporozoites or infected red blood cells (iRBCs). Liver parasitic cDNA, parasitemia and serum cytokine concentrations were respectively evaluated through reverse transcription-polymerase chain reaction (RT-PCR), staining thin-blood smears with Giemsa solution, and enzyme-linked immunosorbent assay (ELISA). FINDINGS: Although the sporozoite challenges yielded similar liver parasitic cDNA and parasitemia, KO mice presented a prolonged survival than WT mice. After iRBC challenges, KO mice kept displaying higher survival rates as well as a decreased IL-12 p70 concentration in the serum than WT mice. CONCLUSION: Our data suggest that malaria triggered by PbNK65 liver- or blood-stage forms elicit a pro-inflammatory environment that culminates with a decreased survival of infected C57BL/6 mice.

Proteomic analysis of Plasmodium berghei in the ring phase during in vivo antiparasitic treatment with kramecyne.

Malaria is a parasitic disease of global importance due to its high annual death toll. The treatment for this infection is difficult for the increase in the populations of parasites resistant to the existing medicines, the development of new antimalarials is urgent needed. Several products developed for the control of malaria from herbalist have had a profound impact, for example, quinine obtained from the bark of the cinchona tree and recently those derived from artemisinin, whose discovery was the reason for the awarding of the 2015 Nobel Prize. The aim of the present study was to evaluate a compound named kramecyne extracted of "chayotillo" (Krameria cystisoides) plant used by the antiparasitic effect against some blood and intestinal protozoa (Giardia duodenalis y Trypanosoma cruzi). In addition is using for the treatment of inflammatory diseases. Measuring parasitaemia at different times, it was observed that in mice treated with kramecyne, it reached only 14% of parasitaemia at 7 days with a dose of 15 mg/kg, using chloroquine as a control drug, because it has not been demonstrated that parasites that infect rodents have developed resistance against this drug. Our results showed that kramecyne decreases the expression of parasite proteins that participate in biological processes, such as invasion, cytoadherence, pathogenicity and energy metabolism. With these results, it is proposed that this compound has repercussions on the metabolism of the parasite and could be useful for use as an antimalarial.

The primate malaria parasites Plasmodium malariae, Plasmodium brasilianum and Plasmodium ovale spp.: genomic insights into distribution, dispersal and host transitions.

During the twentieth century, there was an explosion in understanding of the malaria parasites infecting humans and wild primates. This was built on three main data sources: from detailed descriptive morphology, from observational histories of induced infections in captive primates, syphilis patients, prison inmates and volunteers, and from clinical and epidemiological studies in the field. All three were wholly dependent on parasitological information from blood-film microscopy, and The Primate Malarias" by Coatney and colleagues (1971) provides an overview of this knowledge available at that time. Here, 50 years on, a perspective from the third decade of the twenty-first century is presented on two pairs of primate malaria parasite species. Included is a near-exhaustive summary of the recent and current geographical distribution for each of these four species, and of the underlying molecular and genomic evidence for each. The important role of host transitions in the radiation of Plasmodium spp. is discussed, as are any implications for the desired elimination of all malaria species in human populations. Two important questions are posed, requiring further work on these often ignored taxa. Is Plasmodium brasilianum, circulating among wild simian hosts in the Americas, a distinct species from Plasmodium malariae? Can new insights into the genomic differences between Plasmodium ovale curtisi and Plasmodium ovale wallikeri be linked to any important differences in parasite morphology, cell biology or clinical and epidemiological features?

Larval habitats, species composition and distribution of malaria vectors in regions with autochthonous and imported malaria in Roraima state, Brazil.

BACKGROUND: Malaria control requires local action. Assessing the vector diversity and abundance provides information on the local malariogenic potential or risk of transmission. This study aimed to determine the Anopheles species composition, habitats, seasonal occurrence, and distribution in areas with autochthonous and imported malaria cases in Roraima State. METHODS: A longitudinal study was conducted from January 2017 to October 2018, sampling larvae and adult mosquitoes in three municipalities of Roraima State: Boa Vista, Pacaraima and São João da Baliza. These areas have different risks of malaria importation. Four to six mosquito larval habitats were selected for larval sampling at each municipality, along with two additional sites for adult mosquito collection. All larval habitats were surveyed every two months using a standardized larval sampling methodology and MosqTent for adult mosquitoes. RESULTS: A total of 544 Anopheles larvae and 1488 adult mosquitoes were collected from the three municipalities studied. Although the species abundance differed between municipalities, the larvae of Anopheles albitarsis s.l., Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were collected from all larval habitats studied while Anopheles darlingi were collected only from Boa Vista and São João da Baliza. Adults of 11 species of the genus Anopheles were collected, and the predominant species in Boa Vista was An. albitarsis (88.2%) followed by An. darlingi (6.9%), while in São João da Baliza, An. darlingi (85.6%) was the most predominant species followed by An. albitarsis s.l. (9.2%). In contrast, the most abundant species in Pacaraima was Anopheles braziliensis (62%), followed by Anopheles peryassui (18%). Overall, the majority of anophelines exhibited greater extradomicile than peridomicile-biting preference. Anopheles darlingi was the only species found indoors. Variability in biting times was observed among species and municipalities. CONCLUSION: This study revealed the composition of anopheline species and habitats in Boa Vista, Pacaraima and São João da Baliza. The species sampled differed in their behaviour with only An. darlingi being found indoors. Anopheles darlingi appeared to be the most important vector in São João da Baliza, an area of autochthonous malaria, and An. albitarsis s.l. and An. braziliensis in areas of low transmission, although there were increasing reports of imported malaria. Understanding the diversity of vector species and their ecology is essential for designing effective vector control strategies for these municipalities.

Presence and potential distribution of malaria-infected New World primates of Costa Rica.

BACKGROUND: In South and Central America, Plasmodium malariae/Plasmodium brasilianum, Plasmodium vivax, Plasmodium simium, and Plasmodium falciparum has been reported in New World primates (NWP). Specifically in Costa Rica, the presence of monkeys positive to P. malariae/P brasilianum has been identified in both captivity and in the wild. The aim of the present study was to determine the presence of P. brasilianum, P. falciparum, and P. vivax, and the potential distribution of these parasites-infecting NWP from Costa Rica. METHODS: The locations with PCR (Polymerase Chain Reaction) positive results and bioclimatic predictors were used to construct ecological niche models based on a modelling environment that uses the Maxent algorithm, named kuenm, capable to manage diverse settings to better estimate the potential distributions and uncertainty indices of the potential distribution. RESULTS: PCR analysis for the Plasmodium presence was conducted in 384 samples of four primates (Howler monkey [n = 130], White-face monkey [n = 132], Squirrel monkey [n = 50], and red spider monkey [n = 72]), from across Costa Rica. Three Plasmodium species were detected in all primate species (P. falciparum, P. malariae/P. brasilianum, and P. vivax). Overall, the infection prevalence was 8.9%, but each Plasmodium species ranged 2.1-3.4%. The niche model approach showed that the Pacific and the Atlantic coastal regions of Costa Rica presented suitable climatic conditions for parasite infections. However, the central pacific coast has a more trustable prediction for malaria in primates. CONCLUSIONS: The results indicate that the regions with higher suitability for Plasmodium transmission in NWP coincide with regions where most human cases have been reported. These regions were also previously identified as areas with high suitability for vector species, suggesting that enzootic and epizootic cycles occur.

Spatial cluster analysis of Plasmodium vivax and P. malariae exposure using serological data among Haitian school children sampled between 2014 and 2016.

BACKGROUND: Estimation of malaria prevalence in very low transmission settings is difficult by even the most advanced diagnostic tests. Antibodies against malaria antigens provide an indicator of active or past exposure to these parasites. The prominent malaria species within Haiti is Plasmodium falciparum, but P. vivax and P. malariae infections are also known to be endemic. METHODOLOGY/PRINCIPAL FINDINGS: From 2014-2016, 28,681 Haitian children were enrolled in school-based serosurveys and were asked to provide a blood sample for detection of antibodies against multiple infectious diseases. IgG against the P. falciparum, P. vivax, and P. malariae merozoite surface protein 19kD subunit (MSP119) antigens was detected by a multiplex bead assay (MBA). A subset of samples was also tested for Plasmodium DNA by PCR assays, and for Plasmodium antigens by a multiplex antigen detection assay. Geospatial clustering of high seroprevalence areas for P. vivax and P. malariae antigens was assessed by both Ripley's K-function and Kulldorff's spatial scan statistic. Of 21,719 children enrolled in 680 schools in Haiti who provided samples to assay for IgG against PmMSP119, 278 (1.27%) were seropositive. Of 24,559 children enrolled in 788 schools providing samples for PvMSP119 serology, 113 (0.46%) were seropositive. Two significant clusters of seropositivity were identified throughout the country for P. malariae exposure, and two identified for P. vivax. No samples were found to be positive for Plasmodium DNA or antigens. CONCLUSIONS/SIGNIFICANCE: From school-based surveys conducted from 2014 to 2016, very few Haitian children had evidence of exposure to P. vivax or P. malariae, with no children testing positive for active infection. Spatial scan statistics identified non-overlapping areas of the country with higher seroprevalence for these two malarias. Serological data provides useful information of exposure to very low endemic malaria species in a population that is unlikely to present to clinics with symptomatic infections.

Susceptibility of Field-Collected Nyssorhynchus darlingi to Plasmodium spp. in Western Amazonian Brazil.

Mosquito susceptibility to Plasmodium spp. infection is of paramount importance for malaria occurrence and sustainable transmission. Therefore, understanding the genetic features underlying the mechanisms of susceptibility traits is pivotal to assessing malaria transmission dynamics in endemic areas. The aim of this study was to investigate the susceptibility of Nyssorhynchus darlingi-the dominant malaria vector in Brazil-to Plasmodium spp. using a reduced representation genome-sequencing protocol. The investigation was performed using a genome-wide association study (GWAS) to identify mosquito genes that are predicted to modulate the susceptibility of natural populations of the mosquito to Plasmodium infection. After applying the sequence alignment protocol, we generated the variant panel and filtered variants; leading to the detection of 202,837 SNPs in all specimens analyzed. The resulting panel was used to perform GWAS by comparing the pool of SNP variants present in Ny. darlingi infected with Plasmodium spp. with the pool obtained in field-collected mosquitoes with no evidence of infection by the parasite (all mosquitoes were tested separately using RT-PCR). The GWAS results for infection status showed two statistically significant variants adjacent to important genes that can be associated with susceptibility to Plasmodium infection: Cytochrome P450 (cyp450) and chitinase. This study provides relevant knowledge on malaria transmission dynamics by using a genomic approach to identify mosquito genes associated with susceptibility to Plasmodium infection in Ny. darlingi in western Amazonian Brazil.