Bacterial microbiota from the gut of Rhodnius ecuadoriensis, a vector of Chagas disease in Ecuador's Central Coast and Southern Andes.

Introduction: Chagas disease is a neglected tropical disease caused by the parasite Trypanosoma cruzi that is transmitted mainly by the feces of infected Triatomines. In Ecuador the main vector is Rhodnius ecuadoriensis which is distributed in several provinces of the country. More than 40% of these insects in the wild have T. cruzi as part of their intestinal microbiota. For this reason, the objective of this research was to characterize the intestinal bacterial microbiota of R. ecuadoriensis. Methods: The methodology used was based on the DNA extraction of the intestinal contents from the wild collected insects (adults and nymphs V), as well as the insects maintained at the insectary of the CISeAL. Finally, the samples were analyzed by metagenomics extensions based on the different selected criteria. Results: The intestinal microbiota of R. ecuadoriensis presented a marked divergence between laboratory-raised and wild collected insects. This difference was observed in all stages and was similar between insects from Loja and Manabí. A large loss of microbial symbionts was observed in laboratory-raised insects. Discussion: This study is a crucial first step in investigating microbiota interactions and advancing new methodologies.

Biogenesis of EVs in Trypanosomatids.

Trypanosomes are protozoan parasites responsible for human diseases such as Chagas disease, African trypanosomiasis, and leishmaniasis. These organisms' growth in various environments and exhibit multiple morphological stages, while adapting their surface components. They acquire and release materials extensively to get nutrients and manage interactions with the extracellular environment. They acquire and utilize proteins, lipids, and carbohydrates for growth via using membrane transport and endocytosis. Endocytosis takes place through distinct membrane areas known as the flagellar pocket and cytostome, depending on the parasite species and its developmental stage. Some forms establish a complex endocytic system to either store or break down the absorbed materials. In contrast, membrane transport facilitates the uptake of small molecules like amino acids, carbohydrates, and iron via particular receptors on the plasma membrane. Concurrently, these parasites secrete various molecules such as proteins, enzymes, nucleic acids, and glycoconjugates either in soluble form or enclosed in extracellular vesicles, which significantly contribute to their parasitic behavior. These activities require exocytosis through a secretory pathway in certain membrane domains such as the flagellum, flagellar pocket, and plasma membrane, which are controlled at various developmental stages. The main features of the endocytic and exocytic mechanisms, as well as the organelles involved, are discussed in this chapter along with their connection to the formation of exosomes and extracellular vesicles in the Tritryp species.

Trypanosoma cruzi infection enhances olfactory response in Triatoma pallidipennis Stål (Hemiptera: Triatominae) to compounds potentially useful for insect control.

In Mexico, Triatoma pallidipennis is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease. Current efforts are focused on developing attractants to control these vectors, using volatile substances derived from vertebrate hosts or compounds known to attract hematophagous insects. However, the efficacy of these compounds in attracting parasite-infected triatomines remains to be evaluated. In this study, we assessed the attractant activity of octenol (1-octen-3-ol), nonanal and a mixture of odorants consisting of ammonium hydroxide, lactic acid and hexanoic acid (in a ratio of 1:0.2:0.4 respectively), at concentrations of 1, 10 and 100 ng on the N3, N4 and N5 nymphal stages of T. pallidipennis, both infected and non-infected with T. cruzi. We also evaluated the synergistic effect of the most effective compounds and doses. All experiments were performed in a laboratory using a Y-type glass olfactometer. We found that both infected and non-infected N3 and N4 nymphs were attracted to low doses of octenol, nonanal and the odorant mixture. Particularly noteworthy was the synergistic effect observed between the odorant mixture and nonanal, which significantly increased attraction of T. cruzi-infected individuals. These findings contribute to the development of baited traps utilising these compounds for monitoring triatomines in epidemiological studies or for mass trapping to control these vectors.

Detection of Bartonella henselae DNA in Triatoma sordida collected in peridomiciliary environments.

Bartonelloses represent a group of potentially fatal diseases associated with various clinical manifestations including endocarditis. Caused by bacteria belonging to the genus Bartonella, these microorganisms have a remarkable ability to infect mammals, and their transmission is commonly associated with hematophagous vectors such as fleas, lice, mosquitoes, and ticks. The aim of this study was to evaluate the occurrence of Bartonella sp. DNA in 81 triatomines of the species Triatoma sordida collected in the field in peri­domiciliary areas of the Brazilian city of Seabra, located in the state of Bahia. Nested PCR was conducted targeting the ftsZ gene and real-time PCR targeting the gltA gene, both representing specific reactions for Bartonella henselae. Additionally, conventional PCR targeting kDNA was employed to evaluate the presence of Trypanosoma cruzi. Of the samples tested, 23/81 (28.39 %) bugs showed positive PCR for B. henselae. No sample showed positive PCR for T. cruzi. The high prevalence of triatomines with a positive PCR for B. henselae emphasizes the close relationship between these insects and the bacteria, indicating the need for further studies to investigate the vectorial potential of these kissing bugs.

Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites.

Recently, we published that the monoclonal antibody (D12 mAb) recognizes gp63 of L. mexicana, and it is responsible for COX activity. This D12 mAb exhibited cross-reactivity with Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. COX activity assays performed in these parasites suggested the potential presence of such enzymatic activity. In our investigation, we confirmed that wild-type recombinant gp63 exhibits COX-like activity, in contrast to a mutated recombinant gp63 variant. Consequently, our objective was to identify sequences orthologous to gp63 and subsequently analyze the binding of arachidonic acid (AA) to the putative active sites of these proteins. Given the absence of a crystallized structure for this protein in the Protein Data Bank (PDB), it was imperative to first obtain a three-dimensional structure by homology modeling, using leishmanolysin from Leishmania major (PDB ID: LML1) as a template in the Swiss model database. The results obtained through molecular docking simulations revealed the primary interactions of AA close to the Zinc atom present in the catalytic site of gp63-like molecules of several parasites, predominantly mediated by hydrogen bonds with HIS264, HIS268 and HIS334. Furthermore, COX activity was evaluated in commensal species such as E. dispar and during the encystment process of E. invadens.

Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP.

Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases.

New Drugs and Promising Drug Combinations in the Treatment of Chagas Disease in Brazil: A Systematic Review and Meta-Analysis.

Chagas disease (CD) is a parasitic infection caused by the protozoan Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae). Benznidazole (Bz) has a limited ability to interfere with the pathogenicity of the parasite, which manages to overcome host defenses. This study aimed to conduct a systematic literature review and meta-analysis to understand and describe the drugs and their combinations, as well as new promising compounds used in the treatment of CD in Brazil. This study was registered in the Open Science Framework (OSF) and the International Prospective Register of Systematic Reviews, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Searches were performed in the electronic scientific databases PubMed, LILACS, SciELO, and BVS. Searches were conducted using descriptors cataloged in the Health Sciences Descriptors (DeCS) and Medical Subject Headings (MeSH), in Portuguese, English, and Spanish. Of the 26 articles included in this systematic review and meta-analysis, 16 were related to drug combinations, and nine described new inhibitors of parasitic molecules. Despite high heterogeneity (I² = 92%), studies that evaluated the combination of Bz with other treatments for CD had an overall grouped cure rate of 74% (95% CI 54-94%). Only one study presented drug repositioning by monotherapy. Thus, drug combinations offer quick and accessible solutions for CD treatment, acting against resistant strains of T. cruzi. Certainly, the introduction of these promising compounds into the pharmaceutical market is distant, and the adoption of prophylactic measures is recommended as a barrier to the increasing number of CD cases.

Exploring Trypanosoma cruzi transmission dynamics in an acute Chagas disease outbreak using next-generation sequencing.

BACKGROUND: Chagas disease (CD), caused by Trypanosoma cruzi, poses a major global public health challenge. Although vector-borne transmission is the primary mode of infection, oral transmission is increasingly concerning. METHODS: This study utilized long-amplicon-based sequencing (long-ABS), focusing on the 18S rRNA gene, to explore T. cruzi's genetic diversity and transmission dynamics during an acute CD outbreak in Colombia, an area without domestic infestation. RESULTS: Analyzing samples from five patients and five T. cruzi-positive marsupial samples, we identified coinfections between T. cruzi and Trypanosoma rangeli, mixed T. cruzi DTUs, suggesting possible links between human and marsupial T. cruzi infections. Coexistence of TcI, TcIV and T. rangeli suggests marsupial secretions as the possible source of T. cruzi transmission. Our investigation revealed diversity loss in DTUs TcIV and T. rangeli in humans after infection and in marsupial samples after culture. CONCLUSION: These findings provide significant insights into T. cruzi dynamics, crucial for implementing control and prevention strategies.

Acute Chagas disease outbreaks in Colombia in 2019.

This study seeks to address the critical knowledge gap surrounding the acute phase of Chagas disease in Colombia, with a specific focus on cases reported in 2019. The acute phase of Chagas disease is a pivotal period for intervention, yet it remains poorly understood, particularly in regions where oral transmission is presumed to be a significant factor. By analyzing these recent cases, our research aims to provide a deeper understanding of the dynamics of Chagas disease during its acute phase in Colombia in 2019. This understanding is essential not only for improving disease management and treatment strategies but also for enhancing public health responses to this neglected tropical disease. In particular, our study highlights the importance of identifying and addressing the unique challenges posed by oral transmission routes, which have been increasingly recognized within Colombia's Chagas disease landscape.

First record of a possible trypanotolerant cattle breed in Latin America: Parasitological, serological, and clinical aspects.

Trypanosoma vivax infections are endemic in Africa, where they provoke trypanosomosis against which some local taurine breeds are tolerant and are thus named trypanotolerant. In Latin America, T. vivax was imported in 1919, since when it has been responsible for periodic outbreaks of the disease. This study assessed whether a South American taurine breed resilient to several parasitic and infectious diseases (Curraleiro Pé-Duro-CPD) can meet trypanotolerant criteria (control parasite proliferation, prevent anemia, survive without treatment, and maintain productivity). Three groups were established, each consisting of six animals (Group 1: CPD-infected; Group 2: Holstein/Gyr-infected; Group 3: Holstein/Gyr-uninfected, negative control). Groups 1 and 2 were infected with T. vivax on Day 0 and evaluated until day 532. Throughout the experimental period, parasitological (Woo and Brener), molecular (cPCR), serological (enzyme-linked immunosorbent assay - ELISA, indirect fluorescent antibody test - IFAT, immunochromatographic assay - IA), and clinical (hemogram, fever, weight loss) aspects were evaluated. During the acute phase of the disease, T. vivax was initially detected in Holstein/Gyr. Notably, the CPD animals restored their packed cell volume (PCV) values to the normal range 74 days after inoculations. In the chronic phase, two of the six CPD animals were positive by cPCR until D + 522 following immunosuppression with dexamethasone. Regarding serological aspects, the two CPD animals had positive tests until D + 532. The absence of T. vivax in blood during the chronic phase did not correspond to "self-cure". Holstein/Gyr animals exhibited fever on more evaluation days than CPD animals. Both breeds experienced weight loss, with Holstein/Gyr animals losing significantly more weight. On D + 25, the Holstein/Gyr group required treatment. During the 532 days, none of the CPD animals required treatment, even after being sensitized with dexamethasone. Animals from Group 3 tested negative for T. vivax throughout the experiment. This study demonstrated that CPD cattle fulfill the mentioned trypanotolerant criteria.

Triatomine (Hemiptera: Reduviidae) populations and Trypanosoma cruzi genotyping in peridomestic and sylvatic environments in the semiarid region of Sergipe, Northeastern, Brazil.

We assessed the diversity of triatomines, the rates of natural infection, and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them in two municipalities in the state of Sergipe, Brazil. Active searches for triatomines were conducted in the peridomicily and wild enviroments of 10 villages within the two municipalities. Triatomines were taxonomically identified and their feces were extracted using the abdominal compression method. Parasite detection was performed using optical microscopy. For Trypanosoma cruzi genotyping via PCR-FFLB, 151 samples of the subspecies Triatoma brasiliensis macromelasoma and Triatoma brasiliensis were isolated from both municipalities. In total, 505 triatomines were collected, with Triatoma brasiliensis macromelasoma being the most frequent species (58.81 %). Triatoma b. brasiliensis was the only species in both peridomestic and wild environments. Regarding the other species, T. pseudomaculata was found only in the peridomestic environment; and T. b. macromelasoma and Psammolestes tertius were found in the wild environment. Three Discrete Typing Units were identified: TcI (87.51 %) detected in T. b. brasiliensis and T. b. macromelasoma, TcI+TcIII (10.41 %) in T. b. macromelasoma, and TcI+Trypanosoma rangeli (2.08 %) in T. b. macromelasoma. It is concluded that T. b. macromelasoma is the species collected most frequently in the studied region and the one that presents the highest rates of natural infection, highlighting its epidemiological importance for the vectorial transmission of Chagas disease in Sergipe.

Prevalence analysis of Chagas disease by age group in an endemic region of Brazil: possible scenario of active vectorial transmission.

Objectives: Chagas disease (CD) is an infectious disease that predominantly affects poor and vulnerable populations. The last estimate conducted by the World Health Organization in Latin America regarding the prevalence of CD occurred more than 10 years ago. However, there is a scarcity of data assessing the magnitude of CD in populations residing in considered high-risk regions. Therefore, this study aimed to assess the seroprevalence of CD in an endemic region in Northern Minas Gerais through serologic screening. Methods: This is a prevalence study conducted in the municipalities of Catuti, Mato Verde, Mirabela, Montes Azul, and São Francisco, Minas Gerais, Brazil. Data collection occurred between December 2021 and December 2022, involving a questionnaire with closed-ended questions. The variables analyzed included serologic test results, stratified age groups, health indicators, and housing conditions. Results: Of the 2978 participants, 272 individuals (9.1%) tested positive for CD serology. In the age group of 4 to 14 years, 15 to 49 years, and 50 years or older, the prevalence of positive serology was 0.8% (95% confidence interval [CI] 0.16-1.43), 5.5% (95% CI 4.20-6.83), and 18.8% (95% CI 16.48-21.11), respectively. Among the participating municipalities, Mato Verde had the highest prevalence of positive serology for CD (17%). For participants aged 4 to 14 years with positive serology for CD, first-degree relatives were invited to undergo serologic testing. It was possible to collect samples from relatives of all participants in this age group. However, none of the relatives tested positive. Conclusion: This study identified a 9.1% prevalence of individuals affected by CD who were unaware of their condition. In addition, having infected children in the 4 to 14 age group with mothers with negative serology would rule out congenital transmission of the disease.

Enfermedad de Chagas: prevalencia de infección por Trypanosoma cruzi en pacientes de un hospital polivalente / Chagas disease: prevalence of Trypanosoma cruzi infection in patients of a general hospital

La enfermedad de Chagas afecta a más de 7 millones de personas a nivel mundial, la mayoría de en América Latina. En la Argentina se calcula existen 2 millones de infectados. Objetivos: Determinar la prevalencia de infec-ción por Trypanosoma cruzi en pacientes que se atienden en un servicio de infectología de adultos y caracterizar dicha población. Población: pacientes seguidos con diag-nóstico de infección por Trypanosoma cruzi, realizado por serología, considerando infección en aquellos con al menos dos de tres técnicas positivas (HAI/ELISA/IFI) de una misma muestra. Se obtuvieron los datos de mane-ra retrospectiva de 90 historias clínicas. Resultados: La prevalencia de infección fue del 3%; de ellos, 50% eran argentinos. El 85,5% eran mujeres, en rango etario entre 31-45 años. El nivel educativo fue bajo. El diagnóstico se realizó en contexto de embarazo en 53%. Predominó la presentación Chagas crónico sin patología evidente (62%). El 94,5% cumplía criterio de tratamiento; lo reci-bió el 39%. Conclusiones: La prevalencia se asemeja a la publicada por otros autores. Predominó la nacionalidad argentina, en mujeres de edad fértil con diagnóstico en contexto de embarazo. Un gran porcentaje se encontraba en fase clínica sin lesión de órgano blanco, algo previsible dadas las características epidemiológicas del grupo. Casi la totalidad de ellos con criterio de recibir tratamiento, aunque menos de la mitad lo recibió. Esto puede deberse a la dificultad para realizar el seguimiento luego del diag-nóstico. Enfatizamos la importancia de concientizar a los pacientes sobre adherir a la consulta con la posibilidad de cura con tratamiento oportuno

Chagas disease affects more than 7 million people world-wide, the majority in Latin America. In Argentina it is es-timated that there are 2 million infected. Objectives: De-termine the prevalence of Trypanosoma cruzi infection in patients treated in an adult infectious disease service and characterize said population. Population: patients fol-lowed with a diagnosis of Trypanosoma cruzi infection, made by serology, considering infection in those with at least 2 of 3 positive techniques (HAI/ELISA/IFI) from the same sample. Data were obtained retrospectively from 90 medical records. Results: The prevalence of infection was 3%, of which 50% were Argentine. 85.5% were women in the age range between 31-45 years. The educational level was low. The diagnosis was made in the context of pregnancy in 53%. Chronic Chagas clinical presentation without obvious pathology predominated (62%). 94.5% with treatment criteria, 39% received it. Conclusions: The prevalence is similar to that published by other authors. Argentine nationality predominated in women of child-bearing age diagnosed in the context of pregnancy. A large percentage was in the clinical phase without target organ injury, something predictable given the epidemio-logical characteristics of the group. Almost all of them were eligible for treatment, although less than half re-ceived it. This may be due to the difficulty in following up after diagnosis. We emphasize the importance of raising patients' awareness about adhering to the consultation with the possibility of cure with timely treatment

Tumor-Suppressive Cross-Linking of Anti-T. cruzi Antibodies in Acute Lymphoblastic Leukemia.

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

In Vitro Antiprotozoal Activity of Schinus molle Extract, Partitions, and Fractions against Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, represents an important and worldwide public health issue, particularly in Latin America. Limitations of conventional treatment with benznidazole and nifurtimox underscore the urgent need for new therapeutic strategies for this disease. Schinus molle, a tree used in traditional medicine for various ailments, has demonstrated promising antiparasitic activity. The in vitro anti-T. cruzi activity of Schinus molle crude methanol extract, partitions, and fractions, as well as their cytotoxicity in Vero cells and Artemia salina, and hemolytic activity in human erythrocytes were assessed. Most of the extracts possessed anti-T. cruzi effects, with Sm-CF3 being the fraction with the highest activity (IC50 = 19 µg/mL; SI = 6.8). Gas chromatography-mass spectrometry analysis identified 20 compounds, with fatty acyls comprising the predominant chemical class (55%). We also identified the antiparasitic compounds cis-5,8,11,14,17-eicosapentaenoic acid and trans-Z-α-bisabolene epoxide, suggesting their potential contribution to the observed anti-T. cruzi activity. In conclusion, our findings support the therapeutic potential of S. molle as a source of novel antiparasitic agents against T. cruzi.

Immune signaling pathways in Rhodnius prolixus in the context of Trypanosoma rangeli infection: cellular and humoral immune responses and microbiota modulation.

Introduction: Rhodnius prolixus is a hematophagous insect and one of the main vectors for Trypanosoma cruzi and Trypanosoma rangeli parasites in Latin America. Gut microbiota and insect immune responses affect T. cruzi and T. rangeli infection within triatomines. Particularly the Toll and IMD signaling pathways activations and how they orchestrate the antimicrobial peptides (AMPs) expressions in R. prolixus, especially when infected by T. rangeli. Objectives: Examine how T. rangeli infection modulates R. prolixus cellular and humoral immunity and its impacts on insect microbiota. Methods: R. prolixus was fed on blood containing epimastigotes of T. rangeli, and infection was quantified in insect tissues. The gene expression of dorsal, cactus, relish, PGRP, and AMPs was examined in the midgut, fat body, and salivary glands by quantitative real-time PCR. Microbiota composition was analyzed using RT-q PCR targeting specific bacterial species. Hemocyte numbers and phenoloxidase activity were quantified to assess cellular immune responses. Results: T. rangeli infection modulated triatomine immunity in midgut and hemocoel, activating the expression of the NF-kB gene dorsal, associated with the Toll pathway; increasing expression of the gene encoding PGRP receptor, a component involved in the IMD pathway, both in the intestine and fat body; repressing the expression of the relish transcription factor, mainly in salivary glands. Among the R. prolixus AMPs studied, T. rangeli infection repressed all AMP gene expression, other than defensin C which increased mRNA levels. The PO activity was enhanced in the hemolymph of infected insects. T. rangeli infection did not induce hemocyte number alterations compared to control insects. However, an increase in hemocyte microaggregation was detected in infected insects. Discussion: R. prolixus recognizes T. rangeli infection and triggers humoral and cellular immune responses involving Toll pathway activation, defensin C synthesis, increased phenoloxidase activity, and enhanced hemocyte aggregation. On the other hand, T. rangeli infection suppressed some IMD pathway components, suggesting that, in R. prolixus, this pathway is involved in defensins A and B gene regulation. Importantly, these immune responses altered the bacterial microbiota composition, potentially favoring T. rangeli establishment in the insect vector.

Cardioembolic stroke in Chagas disease: unraveling the underexplored connection through a systematic review.

BACKGROUND: Chagas disease (CD), triggered by the Trypanosoma cruzi parasite, is originally endemic across Latin America, affecting millions. While cardiac complications are widely recognized, the association between CD and stroke remains underexplored. This systematic review aims to elucidate the relationship between CD and stroke, highlighting the cardioembolic origins of stroke in CD patients and assessing the elevated stroke risk compared to non-infected individuals. METHODOLOGY: Adhering to the PRISMA guidelines, we conducted a comprehensive search in PubMed and Scopus databases without date restrictions, including articles in both Spanish and English. This approach enabled the identification and analysis of relevant studies to understand the interplay between CD and stroke risk. RESULTS: Our analysis of 25 selected studies indicates that strokes in CD patients predominantly arise from cardioembolic sources. The data underscore a significant increase in stroke risk among individuals infected with T. cruzi compared to uninfected counterparts. Additionally, CD patients face a higher stroke and mortality risk than those with other heart failure etiologies, irrespective of disease severity. CONCLUSION: The review establishes CD as a critical contributor to stroke incidence, emphasizing the need for heightened awareness and diagnosis of CD in stroke patients, particularly in regions with high CD prevalence. Recognizing the increased stroke risk associated with T. cruzi infection is crucial for developing targeted educational and preventive strategies in endemic areas.

Organic solvent-free benznidazole nanosuspension as an approach to a novel pediatric formulation for Chagas disease.

Aim: Benznidazole (BNZ), a class-II drug, is the primary treatment for Chagas disease, but its low aqueous solubility presents challenges in formulation and efficacy. Nanosuspensions (NS) could potentially address these issues.Methods: BNZ-NS were prepared using a simple, organic solvents-free nano-milling approach. Physicochemical characterizations were conducted on both NS and lyophilized solid-state BNZ-nanocrystals (NC).Results: BNZ-NS exhibited particle size <500 nm, an acceptable polydispersity index (0.23), high Z-potential, and physical stability for at least 90 days. BNZ-NC showed tenfold higher solubility than pure BNZ. Dissolution assays revealed rapid BNZ-NS dissolution. BNZ-NC demonstrated biocompatibility on an eukaryotic cell and enhanced BNZ efficacy against trypomastigotes of Trypanosoma cruzi.Conclusion: BNZ-NS offers a promising alternative, overcoming limitations associated with BNZ for optimized pharmacotherapy.

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Adverse events associated with benznidazole treatment for Chagas disease in children and adults.

AIMS: Chagas disease (ChD) affects approximately 7 million people in Latin America, with benznidazole being the most commonly used treatment. METHODS: Data from a retrospective cohort study in Argentina, covering January 1980 to July 2019, was reanalysed to identify and characterize benznidazole-related adverse drug reactions (ADRs). RESULTS: The study included 518 patients: 449 children and 69 adults (median age in children: 4 years; adults: 25 years; age ranges: 1 month-17.75 years and 18-59 years, respectively). The median benznidazole doses received were 6.6 mg/kg/day for at least 60 days in children and 5.6 mg/kg/day for a median of 31 days in adults. Overall, 29.34% (152/518) of patients developed benznidazole-related ADRs, with an incidence of 25.83% (116/449) in children and 52.17% (36/69) in adults (odds ratio [OR] = 0.32, 95% confidence interval [CI] = 0.19-0.54, P < .001). The incidence rate was 177 cases per 1000 person-years (95% CI = 145-214) in children and 537 per 1000 person-years (95% CI = 360-771) in adults. There were 240 ADRs identified, primarily mild to moderate. Severe ADRs occurred in 1.11% (5/449) of children and 1.45% (1/69) of adults. The skin was the most affected system. A total of 10.23% (53/518) of patients discontinued treatment. More adults than children discontinued treatment (OR = 3.36, 95% CI = 1.7-6.4, P < .001). CONCLUSIONS: Although 29.34% of patients experienced ADRs, most were mild to moderate, indicating a manageable safety profile for benznidazole. While optimized dosing schedules and new drugs are needed, avoiding benznidazole solely due to safety concerns is not justified.

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.