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Klebsiella pneumoniae (Kp) is currently a top priority for the development of alternative therapeutic strategies, according to the World Health Organisation, due to serious concerns regarding infections caused by multiple drug resistant (MDR) bacteria. The convergence of hypervirulence and MDR represents a major threat to public health worldwide. Kp displays an extensive genomic diversity, reflecting a variable repertoire of resistance and virulence factors. Despite this heterogeneity, the type VI secretion system (T6SS) is encoded in most Kp-genomes and plays important roles in competition and pathogenesis. The T6SS is a large macromolecular complex assembled in the cytoplasm that spans the inner and outer membranes. Upon activation, it undergoes conformational changes allowing the delivery of effectors into the extracellular milieu or target cells. Herein, we summarise the current knowledge on the Kp-T6SS, presenting a chronological overview of published studies, discussing the mechanisms, signals, and regulators involved in T6SS expression. Next, we detail both predicted and characterised effector proteins, including Tle1, Pld1 and VgrG4. Finally, we discuss the presence of Kp-T6SS in mobile genetic elements or in clinical samples, highlighting the importance of genomic vigilance and Kp-T6SS detection in high-risk cases. This review integrates available evidence, identifies knowledge gaps, and outlines future directions.

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BACKGROUND Anopheles darlingi is the primary vector of malaria in the Americas, particularly in the Amazon, where it thrives in forest margins. This species exhibits considerable flexibility in feeding behaviour, adapting to environmental conditions and host availability. Previous studies on its attraction to human odour have relied mainly on baited traps, with limited research using vertical olfactometry to explore host-seeking behaviour. OBJECTIVES This study aimed to assess the feasibility of using vertical and horizontal olfactometry to investigate the behavioural responses of both wild and laboratory-reared (F1) An. darlingi females to human odours. The odours were presented through direct (hands and feet) and indirect (sweat-impregnated synthetic substrates) stimuli. METHODS Wild mosquitoes were collected from Porto Velho and Candeias do Jamari, Brazil, and laboratory-reared (F1) mosquitoes were bred under controlled conditions. A vertical olfactometer was employed to assess short-range attraction, and a dual-choice horizontal olfactometer evaluated host-seeking behaviour. Human odours were obtained from volunteers’ hands, feet, and worn socks. Mosquito responses were analysed for attraction, activity, and inactivity, with statistical analysis performed using two-factor analysis of variance (ANOVA). FINDINGS Wild An. darlingi mosquitoes showed significantly higher attraction to human odours compared to F1 mosquitoes in both the vertical and horizontal olfactometers. Wild mosquitoes were more attracted to feet and worn socks than F1 mosquitoes, which exhibited low attraction to both stimuli. The preference index (PI) was higher in wild mosquitoes for both hand and sock odours, indicating a stronger attraction to human odours. MAIN CONCLUSIONS Wild An. darlingi mosquitoes exhibit a stronger behavioural response to human odours compared to F1 mosquitoes. The use of olfactometry, particularly vertical and horizontal methods, proved effective in studying An. darlingi mosquito’s host-seeking behaviour and can be applied to further research on vector behaviour and potential control strategies.

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Plague, caused by Yersinia pestis, remains a historically significant and reemerging zoonotic threat worldwide. Often erroneously considered a medieval relic, the disease persists in natural foci, including Brazil, where it was introduced in 1899 via maritime trade. Over the past 125 years, the country has experienced cyclical outbreaks concentrated in the northeast, where ecological conditions support enzootic transmission among wild rodents and their fleas. While improved surveillance and control have reduced human cases in recent decades, the pathogen’s zoonotic nature and potential for rapid spread in a changing climate underscore its enduring public health relevance. Memórias do Instituto Oswaldo Cruz (MIOC), one of Latin America’s foremost tropical medicine journals, has been instrumental in documenting and shaping the course of plague research in Brazil. Its archives provide a unique chronological record, from pioneering studies on serum production and reservoir ecology to modern molecular analyses of bacterial virulence. This perspective synthesises the seminal contributions published in Memórias that have defined our understanding of plague in Brazil, identifies critical knowledge gaps that persist, and discusses emerging challenges in an era of climate change and shifting zoonotic disease dynamics.

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Wolbachia-based vector control has emerged as one of the most innovative biological interventions in contemporary dengue prevention, offering an ecologically grounded alternative to insecticide-dependent strategies. By exploiting cytoplasmic incompatibility (CI) and strain-specific viral interference, Wolbachia pipientis-transinfected Aedes aegypti (Linnaeus, 1762) mosquitoes can reduce viral transmission, as demonstrated by the Wolbachia-based field trials demonstrating proof of principle. However, the long-term sustainability of this approach remains uncertain. Accumulating field evidence indicates that Wolbachia persistence and epidemiological impact are highly context-dependent, shaped by mosquito fitness, strain-host compatibility, climatic sensitivity, urban ecology and operational continuity. Experiences from Vietnam, Thailand and parts of Brazil illustrate that stable introgression cannot be assumed and may decline when releases are interrupted or ecological conditions are unfavourable. Operational constraints — including mass-rearing quality, dispersal limitations, surveillance intensity and financial costs — pose additional scalability challenges. This perspective critically evaluates whether Wolbachia can function as a sustainable dengue control strategy across diverse endemic settings. We argue that Wolbachia should not be framed as a self-sustaining or stand-alone solution, but rather as a maintenance-dependent, context-sensitive intervention whose effectiveness relies on ecological tailoring, thermotolerant strains, robust post-release surveillance and integration within adaptive integrated vector management (IVM) frameworks. When deployed judiciously alongside complementary control measures, Wolbachia has the potential to become an important — though not exclusive — pillar of long-term dengue control.

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BACKGROUND In the Holocene, South American humans transformed large extensions of the continent, especially in cave ecosystems. Such transformations produced predictable eutrophic habitats that could have attracted foxes and further favored the adaptation of Lutzomyia longipalpis, insect vector of American Visceral Leishmaniosis (AVL), to human-contaminated habitats. OBJECTIVES Here we present spatial analyses on the distribution of caves, Holocene human populations, the present-days main wild reservoirs of Leishmania infantum, Cerdocyon thous and Lycalopex vetulus, and the vector Lu. longipalpis in Brazil. METHODS The presence or absence of Lu. longipalpis in function of cave abundance, based on coordinates of all recorded samples (Fiocruz and GBIF database and literature), and cave locations taken from ICMBio/CECAV database, were tested by contingency table. The overlap in the distribution of Lu. longipalpis, C. thous and L. vetulus with humans from Holocene was tested by permutational multivariate analysis of variance (PERMANOVA) from a nonmetric multidimensional scaling (NMDS), using published archaeological data on human demography and ICMBio’s data on caves and foxes. FINDINGS Caves are present in 18% of Brazilian municipalities, and Lu. longipalpis were significantly more frequent in these places than in municipalities without caves. Native humans and foxes have broader distributions than caves but co-occurred with Lu. longipalpis in cave-ecosystems. MAIN CONCLUSION The most relevant implication of our findings is that visceral leishmaniasis should be considered a long-term human related disease, associated with few sandfly species well adapted to our modified, and heavily contaminated, environments.

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BACKGROUND Chronic Trypanosoma cruzi infection causes significant liver pathology, and current antiparasitic treatments often worsen hepatic damage. Hookworm-derived proteins have shown immunomodulatory effects in inflammatory diseases, including T. cruzi-induced myocarditis. OBJECTIVE This study evaluates recombinant hookworm proteins AIP-1 and AIP-2 for treating liver inflammation in a murine model of chronic Chagas disease (CD). METHODS Female BALB/c mice infected with T. cruzi were treated with AIP-1 or AIP-2 (1 mg/kg) for seven days. Controls were untreated or received aspirin (25 mg/kg) for 14 days. Liver tissues were analyzed for parasite burden (quantitative polymerase chain reaction - qPCR), histopathology (H&E, Picrosirius Red), and cytokines (multiplex assay). Splenocytes were assessed by flow cytometry, and serum was tested for liver enzyme levels. FINDINGS AIP-1 and AIP-2 increased hepatic interferon gamma (IFN-γ) and interleukin 10 (IL-10), decreased Nfκ-B and Stat-1, and elevated Arg1 and Nos2 expression. AIP-1 uniquely upregulated Mmp9 and Btg2. Increased splenic CD11b⁺CD11c⁺ and CD11b⁺Ly6GloLy6C⁺ cells were observed. Despite increased immune cell infiltration, parasite load and fibrosis remained unchanged, and liver enzyme levels were stable. MAIN CONCLUSION AIP-1 and AIP-2 reduce hepatic inflammation and promote a balanced TH1/TH2 response, likely mediated by regulatory dendritic and myeloid-derived suppressor cells, supporting their potential as immunotherapeutic for T. cruzi-induced liver pathology.

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BACKGROUND Extracellular vesicles (EVs) are involved in the pathogenesis of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. OBJECTIVES We analysed the concentration, size, cellular origin, and capacity for carrying viral components in plasma samples from patients with Coronavirus disease 2019 (COVID-19) and sepsis. METHODS Plasma samples from COVID-19 patients admitted to the intensive care unit (ICU) with sepsis (N = 42) and healthy individuals (N = 19) were analysed. EVs were characterised by size and concentration using nanoparticle tracking analysis (NTA), polymerase chain reaction (RT-qPCR) for SARS-CoV-2 components, and flow cytometry for immunophenotyping. EVs were marked with phosphatidylserine and tetraspanins. Cellular origin markers were used for neutrophils, endothelial cells, T lymphocytes and platelets. Cryo-EM was used to assess EV size and integrity. FINDINGS NTA showed an increased concentration of microparticles in patients. RT-qPCR analysis of EVs detected the virus in 14 samples, two of which were consistent with the Gamma variant. EVs predominantly derived from T cells and platelets and demonstrated an increased expression of CD81 in individuals who died. Cryo-EM revealed EVs with an average size of 200 nm. MAIN CONCLUSIONS Our findings suggest that patients’ EVs likely harboured viral components, suggesting their potential role as carriers of SARS-CoV-2. In addition, EVs from deceased patients demonstrated elevated levels of CD81 expression.

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BACKGROUND Culex (Melanoconion) species are known to act as vectors for different arboviruses, and little is known about the mitochondrial genome of these species. OBJECTIVES Aiming to expand the genetic knowledge of this subgenus, a 12Kb fragment of the mitochondrial genome was sequenced from 23 specimens belonging to the Atratus and Educator Groups of the subgenus Melanoconion of Culex. METHODS The sequenced specimens were morphologically identified as Culex dunni, Culex ensiformis, Culex theobaldi, Culex trigeminatus, Culex eknomios, Culex zeteki, Culex near commevynenis, Culex angularis, Culex longistriatus, and Culex near vaxus. The reads were assembled with the reference genome of Culex quinquefasciatus and MITOS2 was used for gene annotation. Values of guanine-cytosine (GC) and adenine-thymine (AT) skews, nucleotide diversity, ratio between non-synonymous (Ka) and synonymous (Ks) substitution, and nucleotide composition were calculated, and phylogenetic analysis was performed. FINDINGS As in other Culex mitogenomes, the partial mitochondrial genomes include 12 protein coding genes (PCGs), 15 tRNA, and 1 rRNA (rrnL). The PCGs showed no length variation between the species studied. ND5 gene presented one less base in the new sequences, which generated a stop codon and, consequently, a shorter length in relation to the Cx. quinquefasciatus sequence reference. All specimens showed a positive value for AT-skew and negative for GC-skew. Nucleotide diversity varied between 0.00407 and 0.12085. Ka/Ks values ranged from 0.0 to 2.775. MAIN CONCLUSIONS Leucine and Serine were the most abundant amino acids. Phylogenetic analysis suggested three putative species.

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BACKGROUND While Chagas disease (CD) has been controlled in many South American regions, the Amazon basin has emerged as a new focus of transmission. Metropolitan Iquitos (Loreto, Peru), has recently shown signs of potential disease emergence. OBJECTIVE To assess the risk of CD transmission by evaluating triatomine presence and infection rates in households across Iquitos and nearby communities. METHODS Entomological surveillance was conducted in domestic and peridomestic environments, following blood donor screenings (2011-2018) that confirmed local Trypanosoma cruzi cases. Triatomines were collected manually, with traps, and through community reporting. Specimens were identified, epidemiological indices calculated, and infestation risk factors analysed using penalised logistic regression, receiver operating characteristic (ROC)/area under the curve (AUC) metrics, and exploratory principal component analysis (PCA). FINDINGS Of 142 houses visited, 113 were inspected, yielding a density index of 0.26. Nine houses were infested, mostly in Loboyacu, with 29 adult triatomines collected — by Rhodnius robustus (89.7%) and Panstrongylus geniculatus. T. cruzi infection was confirmed, and palm roofs emerged as the strongest predictor of infestation [odds ratio (OR) > 16, p < 0.001]. MAIN CONCLUSIONS This first evidence of T. cruzi circulation in sylvatic triatomines within Metropolitan Iquitos highlights an emerging risk of CD. Although vectors remain scarce, palm-roofed houses, deforestation, and urban expansion may facilitate future transmission.

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BACKGROUND Inflammatory bowel disease (IBD) is an increasingly prevalent disease, affecting over seven million people worldwide and imposes a heavy burden on public health. The rising prevalence of IBD may be attributed to the hygiene hypothesis, which suggests that reduced exposure to parasites and microbes may weaken the immune system, thereby increasing susceptibility to developing IBD. Studies suggest helminths and their secretory products can modulate the host immunity and attenuate IBD. Our previous research also demonstrated that intestinal schistosomiasis can mitigate chronic IBD symptoms by restoring intestinal immune balance and dysbiosis. OBJECTIVES While the primary pathology of schistosomiasis results from egg entrapment, we hypothesised that soluble egg antigen (SEA), known for its strong immunomodulatory effect, may contribute to the improvement of IBD. Given that SEA comprises multiple different proteins, identifying the role of individual components may clarify the therapeutic potential of SEA in IBD. METHODS BALB/c mice were induced with dextran sodium sulphate (DSS) to develop IBD. Throughout the experiment, mice were intraperitoneally injected with 250 μg/mL crude SEA extract or recombinant egg antigen proteins, including SM14, GST28, and SMP40, three times a week. Colonic histopathology was assessed by H&E staining, and the immune response was evaluated through periodic acid-Schiff (PAS) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), western blot, and quantitative polymerase chain reaction (qPCR). FINDINGS Both SEA and Smp40 alleviated DSS-induced IBD, whereas SM14 exacerbated the disease and led to colonic dysplasia. In contrast, GST28 showed no significant effect on IBD. Further investigation revealed that all tested proteins modulated the immune response in mice, though each did so in different ways. These differences in immune modulation may underlie the varying disease outcomes observed. MAIN CONCLUSIONS While SEA has shown therapeutic promise in IBD, it is also important to investigate the safety and mechanisms of individual antigens before considering their clinical application in the future.

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BACKGROUND Yellow fever virus (YFV) re-emerged among non-human primates (NHPs) in Rio Grande do Sul in early 2021, more than a decade after its last detection in the state. The spread of the virus was accompanied by increased mortality among NHPs. OBJECTIVES To conduct entomological surveillance and molecular detection of YFV and other Orthoflavivirus species in mosquito samples collected from affected and potentially receptive areas. METHODS Mosquitoes were collected during epizootics using human landing catches, BG-Pro traps, and ovitraps. Virus detection was performed using reverse transcription real-time polymerase chain reaction (RT-qPCR) assays targeting YFV and pan-Orthoflavivirus sequences. FINDINGS A total of 1,210 mosquitoes, representing 26 taxa, were collected across 17 municipalities. Psorophora ferox was the most abundant species, followed by Culex (Culex) spp., accounting for 27% and 12% of the specimens, respectively. Haemagogus leucocelaenus, the primary YFV vector in the region, was also among the most frequently captured species, representing 7%. In total, 203 mosquito pools were assembled by species, location, and date of collection. RT-qPCR analysis did not detect YFV or other Orthoflavivirus RNA in any of the samples. MAIN CONCLUSIONS Although mosquitoes were collected during a period of active YFV circulation, the absence of virus detection suggests that arboviral circulation in vector populations may occur at low frequencies, even during outbreaks.

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BACKGROUND Egg detection still has a role in schistosomiasis control, as a screening strategy or to provide a reference standard for the assessment of the accuracy of other diagnostic tools. The Helmintex method is highly sensitive but laborious, and several improvements of it, including automated egg detection, are currently under development. OBJECTIVE We conducted a preliminary evaluation of Schistosoma mansoni eggs’ autofluorescence as a distinctive marker amid very complex fecal sediments. METHODS Eggs from mouse livers and human feces were examined under a fluorescence microscope. FINDINGS More intense green fluorescence (greater for miracidia than for eggshell) was consistently detected using a B-2A filter (FITC, 420-495 nm). MAIN CONCLUSIONS These findings may help to improve diagnostic methods, especially with automated egg detection systems. Besides access to safe water and adequate sanitation, as well as health education and the treatment of infected individuals, laboratory diagnosis is a key measure that can help eliminate schistosomiasis as a public health problem.

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BACKGROUND Long non-coding RNAs (lncRNAs) are important regulatory molecules that can be considered potential biomarkers for schistosomiasis. However, the identification and characterisation of these molecules in such complex organism as Schistosoma mansoni, remains very challenging. OBJECTIVES This study aimed to identify novel lncRNAs in S. mansoni using RNA-seq libraries representative of six developmental stages (eggs, miracidia, sporocysts, cercariae, schistosomula, and juveniles). METHODS A pipeline for lncRNAs identification was defined and applied to 41 RNA-seq libraries of eggs, miracidia, 1-day, 5-days, 32-days sporocysts, cercariae, schistosomula, and juveniles of S. mansoni. Novel identified lncRNAs and annotated mRNAs were submitted to a weighted co-expression network analysis (WGCNA) to establish lncRNA-mRNAs correlation. FINDINGS We identified 1,082 novel lncRNAs, mostly presenting stage-specific expression. Co-expression analysis demonstrate that MSTRG.5305.1 can potentially target MEGs and tetraspanin, essential for juvenile worm survival, making it a promising candidate for further functional studies. MAIN CONCLUSIONS These findings expand the current catalog of S. mansoni lncRNAs and provide new insights into their potential roles in parasite development and host adaptation. Further functional validation could reveal new molecular targets for schistosomiasis control.

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BACKGROUND Malaria remains a major public health concern in Brazil, with the Amazon region accounting for 99.9% of the country’s cases. Indoor residual spraying (IRS) using Etofenprox 20% PM is a core vector control strategy. However, inefficiencies in the insecticide custody chain, including planning, storage, and distribution, may compromise intervention effectiveness. OBJECTIVES This study aimed to evaluate the insecticide custody chain from a process perspective, identifying logistical bottlenecks, while also exploring potential impact by examining associations between insecticide allocation and malaria burden in Brazil and in three high-incidence municipalities in the state of Amazonas (Barcelos, Tefé, and São Gabriel da Cachoeira) from 2019 to 2023. The underlying hypothesis is that in a well-functioning system, insecticide distribution should be correlated with malaria risk determinants, such as epidemiological and environmental variables, rather than merely responding to reported case counts. METHODS A mixed-methods approach was used. Quantitative analysis applied Pearson correlation, simple and multiple linear regressions (with time-lag), and ARIMA models to evaluate associations between insecticide volume and malaria cases, incorporating environmental and demographic covariates. A complementary qualitative assessment, based on a structured risk matrix, examined failures across four stages of the custody chain: planning, storage, application, and monitoring. FINDINGS At the national level, insecticide volume was significantly associated with malaria cases (β = 0.161; p = 0.038; R² = 0.74) and deforestation (β = 0.626; p = 0.034). Time-series analysis revealed a reactive pattern, with insecticide allocation often lagging behind malaria incidence peaks. In the municipalities studied, models lacked statistical significance, but trends suggested weak local planning and disconnects from risk-based forecasting. The risk matrix revealed systemic weaknesses, including limited data interoperability, insufficient integration of environmental indicators, and poor federal coordination. MAIN CONCLUSIONS The current custody chain functions reactively and lacks integration with surveillance and predictive environmental data, contradicting World Health Organization (WHO) recommendations. Structural reforms are urgently needed. These include: (i) risk-based planning incorporating environmental variables, (ii) interoperable information systems, (iii) improved surveillance of vector resistance, and (iv) intergovernmental agreements for equitable and efficient resource allocation. Findings highlight the need for strategic reorientation of IRS logistics toward anticipatory and data-driven planning. Strengthening the custody chain through intersectoral coordination and environmental intelligence is essential not only to improve operational efficiency but also to increase the cost-effectiveness and epidemiological impact of malaria control interventions in the Amazon. Lessons learned may inform broader efforts in other endemic regions aiming for malaria elimination.

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BACKGROUND Paracoccidioidomycosis (PCM) is a systemic infection that is endemic to Latin America, caused by thermodimorphic fungi from the Paracoccidioides genus. These fungi are facultative intracellular parasites of macrophages. LC3-associated phagocytosis (LAP), a non-canonical form of autophagy, plays a critical role in the response of these phagocytes to similar pathogens. OBJECTIVES In this study, we investigated the role of LAP in the macrophage responses to Paracoccidioides spp. METHODS We detected LAP in macrophages infected with Paracoccidioides spp by immunofluorescence microscopy with antibodies to LC3. Piceatannol and diphenyleneiodonium chloride (DPI), respectively Syk and nicotinamide adenine dinucleotide phosphate oxidase (NADPH) inhibitors, were used to understand the role their pathways played. To determine the function of LAP, we targeted ATG5, a key autophagy gene, by RNA interference. FINDINGS We observed LC3 recruitment to phagosomes containing Paracoccidioides spp. in RAW264.7 and J774.16 cell lines and in bone marrow-derived macrophages. ATG5 RNA interference reduced the antifungal activity of J774.16 cells, highlighting the importance of LC3 recruitment for effective fungal control. Interestingly, pharmacological inhibition of Syk kinase and NADPH oxidase pathways, essential for LAP against Aspergillus fumigatus and Candida albicans, did not impair LAP against P. brasiliensis. MAIN CONCLUSIONS This suggests distinct triggering mechanisms, possibly due to differences in the fungal cell surface composition. These findings suggest that LAP plays a significant role in the host defense against Paracoccidioides spp. and may represent a promising target for host-directed PCM therapies.

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BACKGROUND The life cycle of the parasitic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), includes two well-recognised insect-dwelling stages: the replicative non-infective epimastigotes and the non-replicative infective metacyclic trypomastigotes. Nonetheless, the existence of multiple intermediate forms has been reported. Since nutrient restriction is considered one of the main factors driving metacyclogenesis and is very frequent due to the long-term starvation periods that the insect vectors commonly undergo, we have studied the transcriptomic effects of nutrient restriction on long-lasting epimastigote cultures. We previously reported that in these conditions, we observed a long stationary phase characterised by an RNA content per cell three times smaller than the epimastigote’s and a distinctive transcriptomic profile. Remarkably, our study identified gene expression changes that distincty characterise transitional parasite forms enriched by nutrient restriction. OBJECTIVES In this work we focused on pathogenic genes to further characterise the transcriptomic dynamics accompanying the nutrient restriction within the insect-dwelling parasite stage. METHODS The alterations of morphology, growth rate and complement resistance of parasite population on long-lasting epimastigote cultures as well as the transcriptomic dynamics was studied. FINDINGS We found a gene expression early rise of surface proteins (such as trans-sialidase and GP63) and even a rise of TcTASV and δ-amastin, which is not accompanied by increased expression of metacyclic transcript markers. In addition, we found increased expression of genes coding for proteins involved in two other processes activated during the differentiation of epimastigotes to the infective form of the parasite: autophagy (Atg4, Atg7, Atg8.2) and complement resistance (TcCRP and T-DAF). MAIN CONCLUSIONS Altogether, these results, plus our previous identification of transcriptomic markers for transitional parasites, further support earlier proposals of a specific parasite stage that morphologically resembles epimastigotes but exhibits distinctive biological characteristics, including key features related to infectivity.

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BACKGROUND Triatoma dimidiata is a widely distributed vector of Trypanosoma cruzi in Mesoamerica, but its epidemiological role in most regions of Panamá remains poorly understood. OBJECTIVES To investigate the presence, infection status, and feeding behaviour of T. dimidiata populations in peridomestic areas of Palmira Arriba, western Panamá. METHODS Entomological surveys were conducted in five peridomestic sites of a rural highland community. Thirty-seven triatomines (13 adults and 24 nymphs) were collected from wooden piles and construction materials in contact with the ground. DNA from 30 specimens was analysed by polymerase chain reaction (PCR) for T. cruzi detection, genotyping [discrete typing unit (DTU) and haplotype identification], and blood meal source determination through cytochrome b amplification. FINDINGS Twenty-one insects (70.0%) were positive for T. cruzi. Sixteen infections (76.2%) belonged to DTU I (TcI), including 13 TcIDOM and 14 TcIa genotypes, both linked to domestic and sylvatic cycles. Blood meal analysis revealed one mammalian and two avian feedings, indicating opportunistic behaviour. MAIN CONCLUSIONS This study provides the first molecular confirmation of T. cruzi infection in T. dimidiata from Palmira Arriba. The combination of high infection prevalence, multiple developmental stages, and recent feeding suggests active local transmission favoured by humid and cool ecological conditions. Expanded surveillance and integrative One Health approaches are needed to elucidate transmission dynamics in highland rural Panamá.

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BACKGROUND Plasmodium vivax rosetting is a cytoadhesion phenomenon associated with parasite virulence and clinical manifestations of malaria. However, the molecular mechanisms underlying this process remain poorly understood. Comparative transcriptomic analysis between isolates with different rosetting capacities may provide insights into the molecular base and clinical outcome of parasite populations with distinct rosetting characteristics. OBJECTIVES Our study aims to identify and describe the transcription profile of P. vivax isolates with high and low rosetting rates. METHODS We used RNA-seq to compare the transcriptomes of 10 field P. vivax isolates from the Brazilian Amazon. FINDINGS Among the 492 differentially expressed genes of P. vivax isolates with high rosetting (HR) versus low rosetting (LR) formation, 172 (34,96%) are annotated as genes conserved within Plasmodium and of unknown function. The expression profiles of the other 320 genes (65,04%) highlight the importance of integral membrane proteins and membrane-associated proteins with adhesive or adhesin-like properties, representing 10% of the transcribed genes (53 genes), such as Plasmodium Helical Interspersed Sub-telomeric (PHIST) proteins in rosetting phenotypes. Transcriptomic analyses revealed that approximately 4% (19 genes) of differentially expressed genes were kinases and 50% (248 genes) other proteins. Among cell surface proteins and integral/membrane-associated proteins, differentiated expression and positive regulation of representative 6-cysteine gene family were observed in HR formation group, which includes a tryptophan-rich protein (TRAG16), the 41K blood stage antigen precursor 41-3 protein, and merozoite surface protein 7-like (MSP7-like). MAIN CONCLUSIONS These results contribute to understanding the molecular basis of P. vivax rosetting.

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BACKGROUND In previous studies, we identified a deficiency of γδ T cells and an increased prevalence of anti-Anisakis simplex antibodies in patients with Crohn’s disease (CD). Additionally, decreased gene expression of the interleukin 2 (IL-2) receptor γ subunit (CD132) was observed in tissues from CD patients. OBJECTIVE To analyse the gene expression of IL-7 and its receptors in tissues from CD patients and to explore its relationship with anti-A. simplex antibodies. METHODS 52 patients diagnosed with CD were compared with a control group of 52 healthy individuals. Peripheral blood samples were analysed to assess levels of anti-A. simplex antibodies and IL-7. In addition, intestinal tissue samples from 20 subjects in each group were examined to evaluate IL-7 gene expression, IL-7 protein levels, the IL-2 receptor γ subunit (CD132), the IL-7 receptor α subunit (CD127), and caspase-3 expression. FINDINGS Anti-A. simplex antibody levels were elevated in patients with CD. Caspase-3 expression was significantly reduced in the tissues of CD patients with anti-A. simplex IgA, and this reduction extended to IgG and IgE in healthy individuals. A negative correlation was observed between caspase-3 levels and serum anti-A. simplex IgA, as well as IL-7 levels in the tissues of CD patients. In healthy subjects, tissue IL-7 levels were lower in those positive for anti-A. simplex IgA, while serum IL-7 levels were higher in individuals positive for anti-A. simplex IgG. MAIN CONCLUSIONS This study revealed the interplay between IL-7 signalling, γδ T cell deficiency, and immune responses to A. simplex in CD. Our findings underscored a cause-effect relationship between CD132 deficiency, γδ T cell depletion, and defective mucosal immunity, which may drive both CD inflammation and susceptibility to parasitic infections like A. simplex.

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BACKGROUND In light of the biotechnological potential demonstrated by Aspergillus species and, considering the great need for further research into the search for new sources of active molecules and the biodiversity of these microorganisms occurring in the Amazon region. OBJECTIVES This research aimed to investigate the biotechnological potential of the fungus Aspergillus japonicus Amazon Fungi Collection (CFAM) 0234, a fungal strain isolated from Amazonian soil and stored in the CFAM. METHODS For this purpose, the Aspergillus species was investigated through comparative genomic analysis and antimicrobial activity assays. FINDINGS Genome sequencing revealed a fragmented assembly (72.67 Mbp, N50 = 152 kbp) containing 106 biosynthetic clusters (BGCs), surpassing the reference strain CBS 114.51 (57 BGCs). Among the clusters identified, NRPS, PKS type I and hybrid NRPS-PKS systems stood out, including clusters exclusive to betalactones and isocyanides, potentially involved in the synthesis of β-lactam antibiotics and innovative metabolites. BiG-SCAPE analysis identified 63 BGC families unique to CFAM 0234, suggesting evolutionary adaptations to the competitive environment of the Amazon. Biological assays demonstrated selective antimicrobial activity of the ethyl acetate extract against Escherichia coli, Shigella sonnei and Sthapylococcus aureus (MRSA), with inhibition halos ranging from 8 mm to 6 mm in diameter, pathogens classified as priorities for research into new antibiotics. The correlation between predicted BGCs and antimicrobial activity reinforces the strain’s biotechnological potential. Despite the fragmentation of the genome, the high completeness assessed by BUSCO (98.5%) confirms the quality of the assembly, while the detection of single nucleotide polymorphisms (SNPs) in regulatory regions and rearrangements close to BGCs suggests evolutionary pressure for metabolic diversification. The lack of correspondence with the minimum information about a biosynthetic gene cluster (MIBiG) bank and the limitations of crude extracts highlight the need for complementary techniques, such as long-read sequencing (Oxford Nanopore) and metabolomic analysis [liquid chromatography-mass espectrometry (LC-MS)], to link clusters to active metabolites. MAIN CONCLUSIONS Aspergillus japonicus CFAM 0234 represents a promising microbial resource for bioprospecting in the Amazon, offering relevant genomic and chemical insights for the development of new antimicrobial agents. Future studies will focus on the purification of compounds and activation of silent BGCs, aiming at sustainable pharmaceutical applications.

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BACKGROUND The Mayaro virus (MAYV) is an alphavirus endemic to Central and South America, primarily transmitted by mosquitoes of the Haemagogus genus. Human infection causes "Mayaro fever," characterized by symptoms similar to dengue and chikungunya, including debilitating arthralgia. Despite its potential for urbanisation, many aspects of MAYV-host interactions, particularly the role of host microRNAs (miRNAs), remain poorly understood. OBJECTIVES This study aimed to investigate the expression profile of miRNAs in Vero cells infected with MAYV and to predict their potential biological targets and associated pathways. METHODS Infection was performed using the MAYV strain (BeAr 20290), and small RNA libraries were prepared from infected and control cells. Initial experiments were conducted to evaluate viral replication, cell viability, and small RNA expression. Based on these parameters, the 24-h post-infection time point was selected for small RNA sequencing. Bioinformatic tools were used to identify differentially expressed miRNAs and predict their targets in Homo sapiens and the MAYV genome. FINDINGS Among the 348 miRNAs identified, 46 were differentially expressed at 24 h (42 upregulated and four downregulated). Principal component analysis (PCA) indicated a clear separation between infected and control groups. In silico predictions of the targets of these miRNAs suggest potential associations with biological processes that may be relevant to virus-host interactions, such as immune response, programmed cell death pathways, viral replication, and persistence. Additionally, one miRNA detected in Vero cells was predicted to target a viral non-structural protein. MAIN CONCLUSIONS Our findings indicate a potential dual role for host miRNAs during MAYV infection, involving both the modulation of host responses by the virus to enhance replication and a possible antiviral effect. While these interactions underscore the prospective relevance of miRNAs as biomarkers and therapeutic targets in arboviral infections, it is important to note that these conclusions are based solely on computational analyses. Therefore, they should be interpreted with caution until they are supported by further experimental validation.

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BACKGROUND Arboviruses represent a potential threat to global public health due to their ability to infect various vertebrate hosts and vectors, as well as their adaptability to diverse ecosystems, allowing them to expand geographically across continents. OBJECTIVE The present study aimed to describe the molecular epidemiology of encephalitic arboviruses, including Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Toscana virus (TOSV), and Rift Valley fever virus (RVFV) in nervous tissue samples from domestic and wild animals from the Northern region of Brazil, between February 2023 and June 2024. METHODS Samples negative for rabies virus were analysed by reverse transcription real-time polymerase chain reaction (RT-qPCR) targeting Alphavirus, Orthoflavivirus and Phlebovirus species. Positive samples were subjected to viral isolation in cell culture and whole-genome sequencing using next-generation sequencing. Phylogenetic and molecular clock analyses were performed to characterise viral lineages and evolutionary relationships. FINDINGS Two samples tested positive for arboviruses by RT-qPCR: one SLEV sample from the state of Amazonas, which showed a low viral load, preventing virus isolation and sequencing, and one Madariaga virus (MADV) sample from the state of Pará, which could be isolated and sequenced. The isolated agent belongs to lineage III of EEEV, showing most similarity to strains from Guyana and Argentina. MAIN CONCLUSIONS The present study detected two arboviruses in animals, suggesting its circulation in the study area.

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BACKGROUND Amblyomma sculptum is the main vector implicated in the transmission of Rickettsia rickettsii in southeastern Brazil, where capybaras are known to be the main amplifying hosts of this pathogen. Interestingly, many areas inhabited by large populations of capybaras and A. sculptum remain free of R. rickettsii, suggesting that other drivers may be involved in the pathogen perpetuation. A. sculptum frequently occurs in sympatry with Amblyomma dubitatum ticks, sharing the same hosts with them. Considering that A. dubitatum ticks are frequently infected with Rickettsia bellii, a possible interference of R. bellii in the reservoir competence of capybaras for R. rickettsii could explain the heterogeneous distribution of R. rickettsii in A. sculptum populations in southeastern Brazil. OBJECTIVES The present study aimed to evaluate the effect of primary exposure to R. bellii-infected A. dubitatum on the reservoir competence for R. rickettsii using guinea pigs as experimental models. METHODS Three guinea pigs were infested with R. bellii-infected A. dubitatum (Group GB), four guinea pigs with uninfected A. dubitatum (Group GD), and three guinea pigs were not exposed to A. dubitatum (Group GC). After infestation with A. dubitatum, all guinea pigs were exposed to one single R. rickettsii-infected A. sculptum female and then were infested with uninfected A. sculptum larvae. After ecdysis, nymphs were tested for detection of spotted fever group rickettsiae. FINDINGS Five out of six guinea pigs from GD and GC groups died, while one out of three guinea pigs in the GB group had a fatal outcome. R. rickettsii infection rate among ticks fed on animals from GB group was 21% (17/80), significantly lower than the rate of 54% (60/111) recorded in ticks fed on animals from GD and GC groups. MAIN CONCLUSIONS Prior exposure of guinea pigs to R. bellii-infected A. dubitatum ticks reduced their reservoir competence for R. rickettsii.

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BACKGROUND Malaria remains a major public health challenge in the Brazilian Amazon, where ecological heterogeneity and socio-economic vulnerability hinder the effectiveness of vector elimination. The primary vector, Nyssorhynchus darlingi, shows substantial spatiotemporal variation influenced by land-use changes and local microclimates. OBJECTIVES Investigate how land cover and microclimatic factors affect Ny. darlingi human biting rate (HBR) across different Amazonian ecoregions. METHODS HBR was measured through human landing catches at 70 sites in four ecoregions, with simultaneous temperature and humidity recordings. Landscape composition was quantified within 100-, 250-, and 500-m buffers. Data were analyzed using linear regressions, mixed-effects models, and principal component analysis. FINDINGS We recorded 14,689 mosquito bites across 384 events. HBR was highest in areas with intermediate forest degradation and was positively associated with temperature and negatively with humidity. Floodable vegetation was the strongest land cover predictor, especially at the 500-m scale. Intrazonal variation exceeded interzonal differences. MAIN CONCLUSIONS Local environmental conditions, particularly floodable vegetation near human settlements, strongly influence Ny. darlingi activity. Vector elimination in the Amazon should adopt climate-sensitive, spatially targeted strategies informed by land cover and microclimate data.

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BACKGROUND Giardia duodenalis is an intestinal protozoan parasite that infects a wide range of vertebrate hosts, including humans, and is the etiological agent of giardiasis. Within this species, assemblage A is further subdivided into three sub-assemblages: AI, AII, and AIII. OBJECTIVES To apply the high-resolution multilocus sequence typing (MLST) to perform molecular epidemiology of G. duodenalis assemblage A in humans and animals in the State of Amazonas, Brazil. METHODS We performed parasitological analyses of faecal samples from animals and collected Giardia-positive samples from humans in the State of Amazonas, Brazil. Giardia genotyping was carried out using an MLST scheme based on six genes (CID1, NEK15411, DIS3, HCMP22547, HCMP6372, RHP26), as well as the three conventional loci (tpi, bg and gdh), followed by phylogenetic analysis. FINDINGS Using an extended MLST scheme for assemblage A, we identified a new genotype infecting two non-human primate (NHP) species from the Amazon region. Additionally, Giardia from sub-assemblage AII infecting humans in this region shows a close phylogenetic relationship with isolates from other countries. Furthermore, a sub-assemblage AI isolate from an anteater (Tamandua tetradactyla) belongs to a lineage infecting diverse animals and humans, highlighting its zoonotic and cosmopolitan nature. MAIN CONCLUSIONS These findings reveal a new, genetically distinct lineage within G. duodenalis assemblage A, likely representing a new sub-assemblage (AIV) associated with NHPs in the Amazon region, and reinforce the usefulness of high-resolution MLST for detecting fine-scale genetic diversity and potential zoonotic transmission pathways.

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BACKGROUND Triatoma brasiliensis, the primary Chagas disease (CD) vector in the north-east of Brazil, poses a significant challenge for control due to its adaptability and ability to colonise anthropic environments. The limited number of previous studies on the population dynamics of T. brasiliensis hinders the development of effective control strategies. OBJECTIVES This study characterises the genetic variability of T. brasiliensis populations in Jaguaruana using microsatellite markers, in order to understand the population processes of triatomine infestation and reinfestation. METHODS We analysed the genetic structure of 229 T. brasiliensis specimens collected in the municipality of Jaguaruana in the north-east Brazilian State of Ceará using microsatellite markers. FINDINGS Hardy-Weinberg disequilibrium prevailed, with substantial genetic variability (67.2%) among individuals and inbreeding, but genetic differentiation lacked correlation with geographical distance (Mantel’s test). MAIN CONCLUSIONS The complex population dynamics in Jaguaruana revealed diverse sources of anthropogenic colonisation, impacting regional control. This study underscores the necessity of comprehending intricate infestation processes for planning effective vector surveillance and control strategies.

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BACKGROUND Oropouche virus (OROV), an arbovirus endemic to the Amazon region, has recently expanded into non-endemic areas including Rio de Janeiro State, Brazil. OBJECTIVE To characterise the spatio-temporal dynamics and ecological factors associated with OROV transmission in Rio de Janeiro during 2024-2025. METHODS We analysed OROV case-associated ecological factors and performed a phylodynamic analysis on 40 viral genomes, comprising 35 new and five published sequences, sampled from 15 municipalities across the state during 2024-2025. FINDINGS OROV cases showed significant positive correlations with forest area (r = 0.50, p < 0.0001), banana harvest area (r = 0.39, p < 0.01), and cassava harvest area (r = 0.29, p < 0.05); but these factors were autocorrelated, suggesting a confounded relationship. We identified two OROV sub-clades circulating in the Rio de Janeiro State. The OROVRJ/ES sub-clade was likely introduced into the Southern Fluminense region around January 2024, spread primarily by short (> 2 km, 50% of events) and mid-distance movements (2-9 km; 30%) with a mean dispersal rate of 0.3 km/day, and seed outbreaks in Metropolitan and Northwest Fluminense regions in 2025. The OROVES-I clade was likely introduced into Central Fluminense and Coastal Lowlands regions, later spreading to the Northern Fluminense region. MAIN CONCLUSIONS Following its introduction in early 2024, OROV persisted in Rio de Janeiro State by spreading through short-distance movements among municipalities with high forest cover and agricultural areas. The sustained multi-year OROV transmission in the Atlantic Forest biome highlights the potential for establishment of endemic cycles beyond the Amazon region and the need for enhanced surveillance in extra-Amazonian areas, where OROV will evolve in a different ecosystem.

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BACKGROUND Ocular toxoplasmosis is a leading cause of infectious posterior uveitis worldwide. The retinal pigment epithelium (RPE), a key barrier and immunomodulatory layer in the eye, is directly targeted by Toxoplasma gondii during infection. However, its role in orchestrating the local immune response remains unclear. OBJECTIVES To investigate whether RPE cells actively drive macrophage migration during T. gondii infection in vitro, and to identify associated cytokine profiles. METHODS Adult retinal pigment epithelial cells (ARPE)-19 and primary RPE cells were exposed to tachyzoites, soluble antigens or conditioned supernatants. Macrophage migration was assessed using Transwell® and under-agar assays. Cytokines were quantified by cytometric bead array. FINDINGS Both ARPE-19 and primary RPE exhibited chemotaxis toward parasite antigens (0.12 - 0.5 μg), and enhanced interleukin-6 (IL-6), IL-10 and tumor necrosis factor-α (TNF-α) secretion. Co-culture with RAW 264.7 macrophages further amplified cytokine production. Primary RPE from infected animals occluded 90% of Transwell® pores within 24h. IL-6 and IL-10 levels strongly correlated with migratory activity (r = 0.82 and 0.77, respectively). MAIN CONCLUSIONS RPE cells are not passive targets but active participants in the ocular immune response to T. gondii. By secreting IL-6 and IL-10, they establish a chemotactic environment that recruits macrophages. These insights identify the RPE-cytokine-macrophage axis as a potential therapeutic target in ocular toxoplasmosis.

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BACKGROUND Coxiella burnetii, the etiological agent of coxiellosis in animals and Q fever in humans, is a zoonotic pathogen of global relevance that can infect a wide range of species. Although several domestic and wild animals are involved in the natural cycle, the role of wildlife hosts remains poorly understood. OBJECTIVES Our study aimed to investigate the presence of C. burnetii in feral pigs hunted in Brazilian Pantanal wetland. METHODS In this study, 36 free-living feral pigs legally hunted in Mato Grosso State, Brazil, were sampled. Sera were tested by enzyme-linked immunosorbent assay (ELISA), and spleen, liver, sera and tick samples were analysed by polymerase chain reaction (PCR). FINDINGS Serological evidence of exposure was detected in 22.2% [8/36; 95% confidence interval (CI): 11.7% - 38.1%], while C. burnetii DNA was found in one spleen sample (1/36 - 2.8%; 95% CI: 0.1% - 14.5%). Only Coxiella-like endosymbiont was detected in Amblyomma sculptum ticks (9/23 - 39.13%; 95% CI: 22.2% - 59.2%). MAIN CONCLUSIONS These results represent the first detection of C. burnetii in free-living feral pigs in Brazil and suggest potential exposure of this invasive mammal species to the pathogen. The findings underscore the need for broader surveillance of C. burnetii at the wildlife-livestock-human interface in Brazil.

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BACKGROUND Giardia lamblia is a parasite that infects humans. To date, there is no vaccine available for human giardiasis. Thus, discovering new immunogenic antigens is crucial for the rational design of a vaccine. OBJECTIVES This study aimed to identify the main immunogenic antigens of G. lamblia from its entire proteome using immunoinformatic and data science techniques. To our knowledge, this is the first study to systematically identify immunogenic antigens of G. lamblia across its complete proteome, providing a comprehensive map of potential immunogenic antigens. METHODS Briefly, FASTA sequences of G. lamblia isolates WB and GS were submitted to the NetMHCII 2.3 predictor. The analysis was conducted for five murine major histocompatibility complex (MHC)-II molecules: I-Ab, I-Ad, I-Ed, I-Ak, and I-Ek. Python 3.9 was used to develop custom code for data processing and analysis. FINDINGS We identified 414 potential immunogenic polypeptides for isolate WB and 350 for isolate GS. For both isolates, most polypeptides contained peptides with high affinity for I-Ab and I-Ek. Notably, no polypeptides with high affinity for I-Ak were detected. Homologous potential immunogenic antigens (129 polypeptides) were identified in both isolates. The analysis revealed that 12 potential immunogenic polypeptides from isolate WB and 10 from isolate GS are part of the Giardia secretome. Additionally, promiscuous polypeptides that bind to at least two different MHC-II molecules were found in both isolates. MAIN CONCLUSIONS These findings lay a valuable foundation for the rational development of a vaccine against human giardiasis and show a computational strategy that can be applied to the study of other pathogens.

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BACKGROUND Mosquito saliva contains bioactive molecules that modulate host immunity and may influence arboviral infection. The contribution of Aedes aegypti salivary gland extract (SGE) to viral replication and immune regulation during Zika virus (ZIKV) infection remains poorly understood. OBJECTIVES To investigate the immunomodulatory effects of Ae. aegypti SGE during ZIKV infection. METHODS Peripheral blood mononuclear cells (PBMCs) and murine antigen-presenting cell lines were exposed to ZIKV with or without SGE. Viral replication was measured by quantitative polymerase chain reaction (qPCR), cell death and immune subsets by flow cytometry, oxidative stress markers by biochemical assays, and cytokine production by enzyme-linked immunosorbent assay (ELISA). FINDINGS SGE enhanced ZIKV replication, particularly in PBMCs, with increased RNA copies (median Δ = +1,779), reduced late apoptosis of CD4⁺ T cells (p = 0.0055), dendritic cell death (p < 0.01), and impaired T-cell proliferation. SGE attenuated ZIKV-induced oxidative damage by restoring glutathione levels, reducing lipid and protein oxidation (p < 0.001), and increasing nitric oxide (NO) production. Cytokine profiling revealed suppression of interferon-y (IFN-γ) (p < 0.001) and induction of interleukin-4 (IL-4) (p < 0.0001), indicating a Th2-skewed response. Murine cell lines confirmed SGE-driven cytokine modulation. MAIN CONCLUSIONS Ae. aegypti SGE alters host immune homeostasis, favouring ZIKV infection by weakening antiviral defences and redirecting immune and redox pathways, thereby facilitating viral expansion.

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BACKGROUND Phlebotomine sand flies are hematophagous vectors of major human pathogens, including Leishmania spp., with blood ingestion essential for reproduction and vector competence. Accurate quantification of blood meal volume is crucial for understanding physiological processes and transmission dynamics. OBJECTIVES Here, we introduce a novel, non-destructive image-based method to estimate blood intake in Lutzomyia longipalpis, the principal vector of Leishmania infantum in the Americas. METHODS High-resolution images of unfed and blood-fed females were analysed using Fiji ImageJ (open-source software) when several morphometric parameters were measured and validated against biochemical haemoglobin (Hb) quantification. FINDINGS Blood-fed females exhibited a 56.6% increase in abdominal width and a shift toward a rounded body shape, which was strongly correlated with a visible transilluminated abdominal area (R² = 0.92). Some parameters, such as mean grey value and abdominal length, showed a low to moderate correlation with Hb content (R < 0.60). However, the correlation with abdominal area and width was R ≈ 0.90, indicating those are reliable parameters that can be used to estimate blood intake by Lu. longipalpis females. MAIN CONCLUSIONS Unlike spectrophotometric methods, this approach preserves specimen integrity, which, in theory, enables longitudinal studies on physiology and host-parasite interactions. This methodology offers a reliable, scalable, and cost-effective alternative for estimating blood meal.

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BACKGROUND Parastrongyloides trichosuri Mackerras, 1959 (Nematoda: Strongyloididae) is a facultatively parasitic nematode infecting the common brushtail possum. A previously reported mitochondrial genome (mitogenome) from a Kruger National Park (KNP) isolate (GenBank: NC_028620) is incomplete, lacking the nad3 gene and the noncoding region (NCR), limiting its utility for comparative and phylogenetic studies. OBJECTIVES This study aimed to reconstruct, annotate, and validate a complete mitogenome of P. trichosuri (KNP strain) to enhance genomic accuracy and phylogenetic resolution within Strongyloididae. METHODS Whole-genome sequencing data (SRA: ERS056619) were reanalysed. Missing genes, including tRNA-Glu and atp6, were manually curated using BLASTn searches against Rfam v15, while cox3 and nad3 were confirmed through transmembrane topology analysis. FINDINGS The reconstructed mitogenome was 13,809 bp long, comprising 12 protein-coding genes, 22 tRNAs, 2 rRNAs, and a 539-bp tandem-repeat NCR. Gene order and structure were consistent with other Strongyloididae mitogenomes. Phylogenetic analysis supported P. trichosuri as a distinct lineage within the family. The annotated sequence has been deposited in the Third Party Annotation database in GenBank (accession No. BK075097). MAIN CONCLUSIONS This improved mitogenome fills an existing genomic gaps and provides a reliable reference for future comparative, phylogenetic, and evolutionary studies of Strongyloididae.

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BACKGROUND Sand flies (Diptera: Psychodidae) are well-known vectors of Leishmania, yet their associations with other trypanosomatids remain poorly understood. Expanding knowledge on these interactions is essential to elucidate the ecological diversity of parasites circulating in natural and periurban environments. OBJECTIVE To characterise sand fly species composition and assess the diversity of trypanosomatids naturally infecting sand flies in the Serra do Cipó district, Minas Gerais, Brazil. METHODS Sand flies were collected between 2023 and 2025 in the Mata da Tapera and nine surrounding peridomestic sites using Centre for Disease Control light trap (CDC-LT) and a Shannon trap. Females were examined by midgut dissection and screened individually for trypanosomatids using nested polymerase chain reaction (PCR) targeting the 18S rRNA V7-V8 region, followed by sequencing and phylogenetic analyses. FINDINGS A total of 1,460 sand flies representing 21 species were collected, with Pintomyia pessoai (35.5%) being the most abundant. No flagellates were observed in 105 dissected females. Molecular screening of 730 females revealed 12 positives (overall positivity = 1.6%), including Leishmania infantum in Pi. pessoai, Pi. christenseni, and Pa. barretoi; L. braziliensis in Pi. monticola; and non-Leishmania taxa such as Herpetomonas samuelpessoai, Novymonas esmeraldas, a representative of Strigomonadinae, Trypanosoma sp. (Anura clade), and a lineage related to Sergeia. MAIN CONCLUSION These findings confirm the circulation of Leishmania in Serra do Cipó while revealing hidden trypanosomatid diversity spanning at least five genera. The results suggest that sand flies may act as ecological “hubs”, transiently interacting with multiple parasite lineages beyond the classical Leishmania cycle, highlighting the need to broaden the ecological perspective in sand fly-parasite studies.

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BACKGROUND Visceral leishmaniasis (VL), caused by Leishmania infantum, is the most severe form of leishmaniasis, prevalent in many countries, but still with limitations in diagnosis for both human and canine hosts. Serological assays based on recombinant proteins are the most efficient diagnostic alternatives, with the rapid diagnostic test (RDT) being the most cost-effective. The previously described chimeric Q5 is a recombinant protein derived from three native L. infantum antigens, which is potentially useful for both human and canine VL diagnosis, through preliminary enzyme-linked immunosorbent assay (ELISA), but which was not evaluated within an RDT setting. OBJECTIVES To evaluate the diagnostic performance of the chimeric recombinant protein Q5 in both ELISA and RDT formats for the detection of human and canine VL, and to compare its performance with RDTs based on Lci2 and Lci13 antigens. METHODS Here, we first expanded the Q5 evaluation through ELISA with a larger set of human and canine VL-positive sera from multiple origins. We confirmed a sensitivity ranging between 80% and 90% for the human VL and greater than 90% with the canine VL sera. A new RDT-Q5 was then set up and tested with multiple batches of human and canine sera. FINDINGS An improved performance was seen for the human VL diagnosis (94% sensitivity), but it was reduced with canine sera (86% sensitivity). Specificity values for both the ELISA-Q5 and RDT-Q5 were generally greater than 95%, with limited (8%) or no false-positive results with human sera from individuals with cutaneous leishmaniasis (CL) and Chagas disease (CD), respectively. The RDT-Q5 performance was compared with RDTs based on two other recombinant proteins, Lci2 and Lci13, tested respectively for the human and canine VL diagnosis. MAIN CONCLUSIONS Despite an equivalent performance for the human VL diagnosis, the RDT-Lci2 led to a much greater incidence of false-positive results with the CL and CD sera. In contrast, no setup for the RDT-Lci13 was effective with the canine sera. Our results confirm the RDT-Q5 as an efficient alternative for the VL diagnosis in the field, particularly for the human form of the disease.

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BACKGROUND Chagas disease (CD) and Leishmaniasis, caused by Trypanosoma cruzi and Leishmania species, are treated with outdated drugs that are toxic and have limited efficacy. New therapeutic alternatives are therefore needed. Natural products are valuable scaffolds for drug discovery, and Asteraceae species exhibit microbicidal activity. OBJECTIVES This study evaluated the antiparasitic activity and selectivity of two Asteraceae species, Chromolaena hookeriana and Campuloclinium macrocephalum, against trypanosomatids. METHODS C. hookeriana and C. macrocephalum extracts were profiled by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), and their cytotoxicity and antiparasitic activity were evaluated against multiple forms and strains of T. cruzi and Leishmania amazonensis (L. amazonensis) in 2D and 3D cultures. FINDINGS UHPLC-MS/MS revealed predominantly pentacyclic triterpenoids in C. macrocephalum, including ursolic and oleanolic acids (negative mode) and lupeol derivatives (positive mode). C. macrocephalum was more effective than Benznidazole (Bz) (EC50 = 1.17 vs 4.47 µg/mL) against epimastigotes and nine times more potent against trypomastigotes (EC50 = 0.38 vs 3.5 µg/mL). Both extracts matched Bz activity on intracellular T. cruzi but exhibited time-dependent cytotoxicity. They also showed similar effectiveness to miltefosine (Mt) against L. amazonensis amastigotes (EC50 = 0.36–0.70 µg/mL). MAIN CONCLUSIONS The chemical profile supports the stronger antiparasitic activity of C. macrocephalum, likely linked to triterpenoids, and highlights Asteraceae extracts as promising candidates for drug discovery against neglected tropical diseases (NTDs).

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BACKGROUND Dengue has long been considered a neglected disease since its emergence. However, in recent years, the number of cases has increased significantly, particularly in tropical countries across Latin America. OBJECTIVE This study aims to describe the evolution of dengue during the 2023 outbreak in Peru. METHODS Here, we describe the analysis of 245 positive samples for dengue carried out by the National Institute of Health (INS) of Peru through genomic surveillance protocols, and analyse the evolution of serotypes and genotypes during the outbreak. FINDINGS We analysed 245 dengue-positive samples sequenced by the INS of Peru during the 2023 outbreak to describe the evolutionary dynamics of circulating serotypes. We identified dengue virus (DENV)-1 genotype V (lineages D.1 and D.2), DENV-2 genotype II (Cosmopolitan, lineage F.1.2), and DENV-3 genotype III (lineage B.3). Phylogenetic and population-structure analyses revealed multiple introductions, rapid diversification, and wide dispersal of DENV-1 and DENV-2 across Peru, while DENV-3III showed a localised introduction in Lima with phylogenetic links to the Caribbean. Our findings provide the most comprehensive genomic characterisation to date of dengue circulation in Peru in 2023 year, and highlight the need for sustained genomic surveillance. MAIN CONCLUSION To summarise, the three serotypes of dengue in Peru show a dynamic diversity that becomes necessary to understand, to improve public health decisions by authorities.

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BACKGROUND Hepatitis delta virus (HDV) is associated with the worst prognosis among viral hepatitis infections; however, it remains largely underdiagnosed, particularly in endemic developing regions, underscoring the need for new, accessible serological methods for large-scale screening. OBJECTIVES To develop HDV-mimetic molecules using phage display for application in immunodiagnostic platforms. METHODS HDV-mimetic peptides were selected via phage display biopanning, sequenced and screened by phage-enzyme-linked immunosorbent assay (ELISA). Based on the sequences of these peptides, a recombinant protein (rHDV) was constructed and employed in an ELISA. The test was validated using 87 anti-HDV-positive samples and 93 hepatitis B virus (HBV) control samples collected from a public diagnostic laboratory in the Amazon region. Statistical analyses were performed to evaluate the diagnostic performance of the synthetic peptides and the recombinant protein. FINDINGS Overall, rHDV exhibits a sensitivity of 74.71%, specificity of 97.85%, and area under the curve (AUC) of 0.8906. In HDV ribonucleic acid (HDV RNA)-positive patients, diagnostic performance improved, with a sensitivity of 88.0%, specificity of 98.92%, and AUC of 0.96. MAIN CONCLUSIONS These findings highlight that the rHDV protein used in ELISA effectively discriminated HDV-infected individuals from patients monoinfected with hepatitis B. This demonstrates the potential of rHDV as an effective, rapid, and low-cost tool for HDV detection for broader HDV screening.

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BACKGROUND Tuberculosis/human immunodeficiency virus (TB/HIV) coinfection is associated with advanced HIV disease and variable responses to antiretroviral therapy (ART). OBJECTIVES We examined whether baseline HIV severity markers, ART regimes, or human genetic variants influenced HIV-1 virologic suppression in HIV-associated TB. METHODS We included TB/HIV participants from Regional Prospective Observational Research in Tuberculosis (RePORT)-Brazil study, who received standard TB therapy and antiretroviral treatment. The primary endpoint was HIV-1 virologic suppression (≤ 1,000 copies/mL); Baseline characteristics, viral load (VL), CD4 cell count, timing of ART initiation, and ART regimens were included. We genotyped UGT1A1 (rs887829; integrase strand transfer inhibitor-related) and CYP2B6 [rs3745274, rs28399499, rs4803419; efavirenz (EFZ)-related]; all have defined normal, intermediate, and slow genotypes. Genotyping was performed by MassARRAY iPLEX Gold; Kaplan-Meier curves compared time-to-suppression with log-rank tests; Cox proportional hazards models estimated hazard ratios. FINDINGS Among 194 participants, 68% (n = 132) achieved virologic suppression (≤ 1,000 copies/mL). Median time-to-suppression: 84 days [95% confidence interval (CI): 42-125]. Participants with higher baseline viral load (BVL) (≥ 5 log10 copies/mL) had delayed suppression compared with those with lower VL (< 5 log10; log-rank χ² = 75.9; p < 0.001). Individuals with CD4 ≤ 200 cells/µL suppressed more slowly than those with CD4 > 200 cells/µL (log-rank χ² = 29.6; p < 0.001). Participants starting ART before TB treatment achieved suppression faster than ART-naïve individuals (32 vs. 147 days; log-rank χ² = 48.5; p < 0.001). Higher BVL was associated with reduced hazard of suppression [adjusted hazard ratio (aHR) = 0.67; 95% CI: 0.61-0.75], while higher baseline CD4 count increased the hazard of suppression (per 100 cells/µL: aHR = 1.11; 95% CI: 1.01-1.21). ART-naïve status was associated with lower hazard of suppression in univariate analysis (Hazard Ratio = 0.51; 95% CI: 0.36-0.72) but not after adjustment. ART regimen class and pharmacogenetic metabolizer profiles were not significantly associated with virologic suppression. MAIN CONCLUSIONS BVL and CD4 count were the strongest determinants of virologic suppression in TB/HIV patients. Suppression rates were low, and neither ART regimen nor pharmacogenetic profiles significantly influenced the likelihood of suppression.

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BACKGROUND The Leishmania (Viannia) subgenus contains important pathogens that cause a variety of different clinical forms of cutaneous leishmaniasis in the Americas. Their response to antimonial chemotherapy differs according to species. Having high-quality genomic resources of these species is a significant step towards investigating and understanding these factors. OBJECTIVES This study aims to characterise the main genomic features of L. (V.) guyanensis strain MHOM/BR/75/M4147 and L. (V.) shawi strain MCEB/BR/84/M8408. METHODS Genomes were sequenced combining short- and long-read sequencing platforms and assembled, scaffolded, and polished using Flye2, Ragtag, and Pilon, respectively. Annotations were performed using mainly similarity and profile search methods, and phylogenetic analyses were performed using the maximum likelihood (ML) and Bayesian inference approaches, using IQ-TREE and MrBayes, respectively. FINDINGS De novo assembly produced genome sizes of 32.27 Mb for L. guyanensis and 32.41 Mb for L. shawi, and predicted 8,505 and 8,592 protein-coding genes, respectively. Phylogenetic analysis based on these assemblies confidently places L. guyanensis and L. shawi as the closest known relatives to L. panamensis within the Viannia clade. MAIN CONCLUSIONS These genomes will increase the knowledge about the subgenus L. (Viannia) in the Americas and also represent valuable information for future comparative studies with other human pathogenic Leishmania spp.

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BACKGROUND Persistent reinfestation of Triatoma infestans in the Gran Chaco undermines Chagas disease (CD) control. While insecticide resistance is a known factor, the fine-scale social and demographic structure of vector colonies remains poorly understood. OBJECTIVES To analyse the micro-geographic genetic structure, mating systems, and demographic history of T. infestans in a rural community in the Bolivian Chaco. METHODS We genotyped 381 individuals from three neighbouring structures (domiciliary and peridomestic) using eight highly polymorphic microsatellite loci (expected heterozygosity, HE = 0.675). Genealogical relationships were reconstructed using maximum likelihood (ML) analysis (COLONY), and demographic history was inferred through heterozygosity tests. FINDINGS The infestation comprised 42 distinct full-sibling families nested within a single colony. We observed a stark dichotomy: two dominant families (> 80 individuals) exhibited monogamy and signs of distinct demographic histories (one in expansion, one recovering from a bottleneck), while 40 minor families showed high promiscuity (polygyny/polyandry). Finite growth rates varied significantly (λ = 1.03 vs 1.22), evidencing active intra-colonial competition. MAIN CONCLUSIONS Domestic populations are not random aggregates, but complex mosaics of coexisting lineages with different adaptive strategies. The dominance of monogamous families suggests a priority effect advantage, while the "tail" of promiscuous minor families represents a cryptic reservoir. Effective control must account for this structural resilience, as surviving minor lineages could rapidly recolonise the niche.

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BACKGROUND Research on Leishmania pathogenesis, as well as drug and vaccine discovery, primarily relies on complex mammalian models that raise ethical concerns. To overcome these limitations, we tested a simpler and more accessible experimental system. In this study, we examined the interaction between Leishmania amazonensis and Acanthamoeba, a widespread free-living protozoan that interacts with various microorganisms. OBJECTIVES To provide a deeper morphological and kinetic characterisation of the interaction between L. amazonensis and Acanthamoeba. METHODS The parasite interaction was characterised using light microscopy, fluorescence microscopy, scanning electron microscopy (SEM), confocal microscopy, and live-cell imaging. FINDINGS Co-culture was most optimal in RPMI medium at 26ºC. L. amazonensis promastigotes invaded Acanthamoeba trophozoites via their flagellum; trophozoites phagocytosed parasites through acanthopodia. Inside the amoeba, L. amazonensis became rounded and shortened, with no visible flagellum. These forms, isolated after 3 h of co-culture, differentiated back into promastigotes and were viable. The percentage of amoebas with L. amazonensis decreased over time. MAIN CONCLUSIONS Acanthamoeba trophozoites can interact and clear out L. amazonensis. This model is unsuited for sustained infection studies necessary for drug screening. However, it could be an effective model for exploring cellular leishmanicidal mechanisms.

Resumo em Inglês:

BACKGROUND Dengue, the disease caused by dengue virus (DENV), is responsible for over 300 million cases worldwide. Although DENV-4 usually presents with milder symptoms, some cases can result in death. OBJECTIVES The present study aims to characterise the immunohistopathological profile of hepatic, pulmonary, cardiac and renal involvement in fatal cases of DENV-4 that occurred between 2011 and 2013 in Brazil. METHODS To this end, tissue samples from the patients were subjected to histopathological analysis using bright-field microscopy and cytokine detection by immunohistochemistry. FINDINGS Cells stained for tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were observed in the liver, lungs and kidneys. In the liver, the most common finding was periportal necrosis, along with steatosis and mononuclear cell infiltration. In the lungs, pneumocyte necrosis was only observed in areas of alveolar oedema. Septal and alveolar haemorrhage were present, along with thickening of the alveolar walls. In the kidneys, glomerular capillary congestion was observed in most of the analysed cases. Inflammatory cell infiltration was observed in the cortical region, along with proximal and distal tubule necrosis. Finally, in the heart, despite limited local cytokine expression, the cardiac tissue remained morphologically preserved. MAIN CONCLUSIONS Although the findings reported in this study are consistent with those described in fatal dengue cases, the absence of cardiac alterations indicates that DENV-4 may cause a milder infection in this organ.

Resumo em Inglês:

BACKGROUND Single-nucleotide polymorphisms (SNPs) can influence the hepatitis C virus (HCV) infection and progression. ABCB1-gene SNPs - c.1236C>T, c.2677G>T and c.3435C>T - are associated with drug efficacy, hepatotoxicity, and liver injury. ABCB11 c.1331T>C is associated with cholestasis and altered bilirubin levels, potentially worsening liver disease. OBJECTIVE To investigate the impact of ABCB1 and ABCB11 SNPs on disease progression in chronic HCV patients. METHODS A total of 232 HCV patients unresponsive to conventional therapy were analysed. Serum samples were genotyped by quantitative polymerase chain reaction (qPCR), and the genotype and allele-based analysis was performed using RStudio. FINDINGS Cirrhosis was present in 59.1% of patients, along with diabetes (28.4%) and hepatic steatosis (46.1%). The most frequent ABCB1 variant allele was c.3435C>T (36.8%), followed by c.1236C>T (30.8%) and c.2677G>T (26.7%). The ABCB11 c.1331CC genotype was observed in 31.3% of the cohort. Genotypes 1236TT and 2677TT and their alleles were associated with lower total cholesterol. 2677TT genotype and 2677T allele were associated with lower high-density lipoprotein. Patients with 1331CC genotype had higher aspartate aminotransferase levels, and the 1331CC genotype was a risk factor for cirrhosis. A fully variant combined allele (1236T and 2677T and 3435T and 1331C) was associated with higher alpha-fetoprotein and lower cholesterol. MAIN CONCLUSIONS ABCB1 and ABCB11 SNPs are associated with worse clinical outcomes in HCV, underscoring their relevance in disease monitoring.

Resumo em Inglês:

BACKGROUND Children experience Coronavirus disease 2019 (COVID-19) often with milder symptoms but severe outcomes. OBJECTIVES To compare clinical, epidemiological and viral load profiles of hospitalised paediatric COVID-19 cases before and after the emergence of the Omicron variant in Espírito Santo, Brazil, and to explore factors associated with hospitalisation duration. METHODS This cross-sectional observational study included 54 hospitalised children with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, grouped according to epidemiological period: G1 (2020-2021) and G2 (2022). Viral load was quantified by reverse transcription real-time polymerase chain reaction (RT-qPCR), and whole-genome sequencing was performed using Nanopore technology. Group comparisons used chi-square/Fisher's exact tests and Mann-Whitney U tests. Cox regression was applied in an exploratory manner to identify factors associated with hospitalisation duration. FINDINGS The median age was 19.5 months and 68.5% were male. Comorbidities were present in 65% of the cohort, with no significant differences between groups. Children in G1 were more often hospitalised for respiratory syndromes, while G2 had a longer intensive care unit (ICU) stay. More than half of the patients required oxygen support, and those needing oxygen at admission were younger. Viral load was higher in G1 than in G2. In the multivariable Cox model, comorbidities [aHR = 0.47; 95% confidence interval (CI): 0.26-0.86] and male sex (aHR = 0.47; 95% CI: 0.25-0.89) were associated with prolonged hospitalisation. Sequencing identified five SARS-CoV-2 lineages (P.1., B.1.1., BA.1*, BQ.1.1, and BE.9), reflecting the transition from pre-Omicron to Omicron periods. MAIN CONCLUSIONS The pre-Omicron period was marked by higher viral loads and distinct clinical patterns compared with the Omicron period. Comorbidities and male sex were associated with prolonged hospitalisation. These findings should be interpreted as exploratory and hypothesis-generating given the limited cohort size.

Resumo em Inglês:

BACKGROUND Dengue outbreaks pose a significant public health risk in Suriname, with challenges in mosquito surveillance and limited data on insecticide resistance hampering control efforts. OBJECTIVES To investigate the resistance status and involved mechanisms of Aedes aegypti mosquitoes for malathion and λ-cyhalothrin insecticides. METHODS The Centres for Disease Control and Prevention (CDC) bottle bioassay was used to test the resistance phenotypic status of Ae. aegypti while the occurrence and frequency of knockdown resistance (kdr) mutations were accessed by TaqMan genotype assays for the sites 410L (Valine/Leucine), 1016I (Valine/Isoleucine), and 1534C (Phenylalanine/Cysteine). FINDINGS Results showed resistance to malathion in the Blauwgrond population, with other regions exhibiting reduced susceptibility based on mortality rates between 89% and 94%. Very low mortality rates indicate resistance to λ-cyhalothrin in all tested areas. Knockdown resistance (kdr) mutations were detected at high frequencies. The triple homozygous kdr genotype leucine/leucine, isoleucine/isoleucine, cysteine/cysteine (LL/II/CC) predominated (84.2%), while the wild-type genotype was found only in 1.8% of the samples. MAIN CONCLUSION This study reports the first detection of malathion resistance in Ae. aegypti from Suriname and confirms high levels of resistance to λ-cyhalothrin, possibly driven by kdr gene mutations. The results emphasise the importance of sustained surveillance and continued research on resistance mechanisms to guide effective and evidence-based vector control strategies.