Slow recovery rates and spatial aggregation of Triatoma infestans populations in an area with high pyrethroid resistance in the Argentine Chaco.

BACKGROUND: The emergence of pyrethroid resistance has threatened the elimination of Triatoma infestans from the Gran Chaco ecoregion. We investigated the status and spatial distribution of house infestation with T. infestans and its main determinants in Castelli, a municipality of the Argentine Chaco with record levels of triatomine pyrethroid resistance, persistent infestation over 2005-2014, and limited or no control actions over 2015-2020. METHODS: We conducted a 2-year longitudinal survey to assess triatomine infestation by timed manual searches in a well-defined rural section of Castelli including 14 villages and 234 inhabited houses in 2018 (baseline) and 2020, collected housing and sociodemographic data by on-site inspection and a tailored questionnaire, and synthetized these data into three indices generated by multiple correspondence analysis. RESULTS: The overall prevalence of house infestation in 2018 (33.8%) and 2020 (31.6%) virtually matched the historical estimates for the period 2005-2014 (33.7%) under recurrent pyrethroid sprays. While mean peridomestic infestation remained the same (26.4-26.7%) between 2018 and 2020, domestic infestation slightly decreased from 12.2 to 8.3%. Key triatomine habitats were storerooms, domiciles, kitchens, and structures occupied by chickens. Local spatial analysis showed significant aggregation of infestation and bug abundance in five villages, four of which had very high pyrethroid resistance approximately over 2010-2013, suggesting persistent infestations over space-time. House bug abundance within the hotspots consistently exceeded the estimates recorded in other villages. Multiple regression analysis revealed that the presence and relative abundance of T. infestans in domiciles were strongly and negatively associated with indices for household preventive practices (pesticide use) and housing quality. Questionnaire-derived information showed extensive use of pyrethroids associated with livestock raising and concomitant spillover treatment of dogs and (peri) domestic premises. CONCLUSIONS: Triatoma infestans populations in an area with high pyrethroid resistance showed slow recovery and propagation rates despite limited or marginal control actions over a 5-year period. Consistent with these patterns, independent experiments confirmed the lower fitness of pyrethroid-resistant triatomines in Castelli compared with susceptible conspecifics. Targeting hotspots and pyrethroid-resistant foci with appropriate house modification measures and judicious application of alternative insecticides with adequate toxicity profiles are needed to suppress resistant triatomine populations and prevent their eventual regional spread.

Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models.

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Clinician Knowledge of Chagas Disease After an Educational Intervention.

This survey study describes the self-reported medical knowledge among physicians before and after attending a lecture on Chagas disease.

From proteome to candidate vaccines: target discovery and molecular dynamics-guided multi-epitope vaccine engineering against kissing bug.

Introduction: Trypanosoma cruzi is a protozoan parasite that causes the tropical ailment known as Chagas disease, which has its origins in South America. Globally, it has a major impact on health and is transported by insect vector that serves as a parasite. Given the scarcity of vaccines and the limited treatment choices, we conducted a comprehensive investigation of core proteomics to explore a potential reverse vaccine candidate with high antigenicity. Methods: To identify the immunodominant epitopes, T. cruzi core proteomics was initially explored. Consequently, the vaccine sequence was engineered to possess characteristics of non-allergenicity, antigenicity, immunogenicity, and enhanced solubility. After modeling the tertiary structure of the human TLR4 receptor, the binding affinities were assessed employing molecular docking and molecular dynamics simulations (MDS). Results: Docking of the final vaccine design with TLR4 receptors revealed substantial hydrogen bond interactions. A server-based methodology for immunological simulation was developed to forecast the effectiveness against antibodies (IgM + IgG) and interferons (IFN-g). The MDS analysis revealed notable levels of structural compactness and binding stability with average RMSD of 5.03 Aring;, beta-factor 1.09e+5 Å, Rg is 44.7 Aring; and RMSF of 49.50 Aring;. This is followed by binding free energies calculation. The system stability was compromised by the complexes, as evidenced by their corresponding Gibbs free energies of -54.6 kcal/mol. Discussion: Subtractive proteomics approach was applied to determine the antigenic regions of the T cruzi. Our study utilized computational techniques to identify B- and T-cell epitopes in the T. cruzi core proteome. In current study the developed vaccine candidate exhibits immunodominant features. Our findings suggest that formulating a vaccine targeting the causative agent of Chagas disease should be the initial step in its development.

Circulation of Trypanosoma cruzi in triatomines and Didelphis sp. in urban areas: Transmission risk assessment in the Metropolitan Region.

The presence of Trypanosoma cruzi vectors in urban areas has been frequent, with colonization of homes and associated with reservoir animals that increase risk to humans, with simultaneous circulation of vectors and T. cruzi. The study aimed to describe the circulation of triatomines and T. cruzi in the Metropolitan Region of São Paulo, as well as evaluate risk situations. For analysis purposes, the triatomine notification information from January 2016 to July 2023 was used. While for Didelphis sp. collection with the aid of traps, notification information used was from 2019 to 2023. Information about triatomines came from spontaneous demand by the population and notification services were carried out by state field teams following defined protocols. 202 notifications were received with the capture of 448 triatomines. The positivity for T. cruzi observed was 60.5%. Regarding Didelphis sp., 416 animals were collected, 5.3% of which were positive for T. cruzi. There was overlapping areas of presence of infected triatomines and Didelphis sp., whose Discrete Typing Unit (DTU) was T. cruzi I. This work indicates the presence of infected vectors in urban areas, and the presence of a wild cycle of T. cruzi in didelphiids, reaffirming the need for and importance of vector surveillance work, through actions that can prevent the transmission of Chagas disease.

A novel prolixicin identified in common bed bugs with activity against both bacteria and parasites.

The hematophagous common bed bug, Cimex lectularius, is not known to transmit human pathogens outside laboratory settings, having evolved various immune defense mechanisms including the expression of antimicrobial peptides (AMPs). We unveil three novel prolixicin AMPs in bed bugs, exhibiting strong homology to the prolixicin of kissing bugs, Rhodnius prolixus, and to diptericin/attacin AMPs. We demonstrate for the first time sex-specific and immune mode-specific upregulation of these prolixicins in immune organs, the midgut and rest of body, following injection and ingestion of Gr+ (Bacillus subtilis) and Gr- (Escherichia coli) bacteria. Synthetic CL-prolixicin2 significantly inhibited growth of E. coli strains and killed or impeded Trypanosoma cruzi, the Chagas disease agent. Our findings suggest that prolixicins are regulated by both IMD and Toll immune pathways, supporting cross-talk and blurred functional differentiation between major immune pathways. The efficacy of CL-prolixicin2 against T. cruzi underscores the potential of AMPs in Chagas disease management.

INFLUENCE OF ESOPHAGEAL MOTILITY IMPAIRMENT ON UPPER AND LOWER ESOPHAGEAL SPHINCTER PRESSURE IN CHAGAS DISEASE.

BACKGROUND: Chagas disease causes digestive anatomic and functional changes, including the loss of the myenteric plexus and abnormal esophageal radiologic and manometric findings. OBJECTIVE: To evaluate the association of abnormal esophageal radiologic findings, cardiac changes, distal esophageal contractions, and complaints of dysphagia and constipation in upper (UES) and lower (LES) esophageal sphincter basal pressure in Chagas disease patients. METHODS: The study evaluated 99 patients with Chagas disease and 40 asymptomatic normal volunteers. The patients had normal esophageal radiologic examination (n=61) or esophageal retention without an increase in esophageal diameter (n=38). UES and LES pressure was measured with the rapid pull-through method in a 4-channel water-perfused round catheter. Before manometry, the patients were asked about dysphagia and constipation and submitted to electrocardiography and chest radiography. RESULTS: The amplitude of esophageal distal contraction decreased from controls to chagasic patients with esophageal retention. The proportion of failed and simultaneous contractions increased in patients with abnormal radiologic examination (P<0.01). There were no significant differences in UES and LES pressure between the groups. UES pressure was similar between Chagas disease patients with cardiomegaly (n=27, 126.5±62.7 mmHg) and those without it (n=72, 144.2±51.6 mmHg, P=0.26). Patients with constipation had lower LES pressure (n=23, 34.7±20.3 mmHg) than those without it (n=76, 42.9±20.5 mmHg, P<0.03). CONCLUSION: Chagas disease patients with absent or mild esophageal radiologic involvement had no significant changes in UES and LES basal pressure. Constipation complaints are associated with decreased LES basal pressure.

Profile of interstitial cells of Cajal in a murine model of chagasic megacolon.

Disorders of gastrointestinal motility are the major physiologic problem in chagasic megacolon. The contraction mechanism is complex and controlled by different cell types such as enteric neurons, smooth muscle, telocytes, and an important pacemaker of the intestine, the interstitial cells of Cajal (ICCs). The role of ICCs in the progression of acute and chronic Chagas disease remains unclear. In the present work, we investigate the aspects of ICCs in a long-term model of Chagas disease that mimics the pathological aspects of human megacolon. Different subsets of ICCs isolated from Auerbach's myenteric plexuses and muscle layers of control and Trypanosoma cruzi infected animals were determined by analysis of CD117, CD44, and CD34 expression by flow cytometer. Compared with the respective controls, the results showed a reduced frequency of mature ICCs in the acute phase and three months after infection. These results demonstrate for the first time the phenotypic distribution of ICCs associated with functional dysfunction in a murine model of chagasic megacolon. This murine model proved valuable for studying the profile of ICCs as an integrative system in the gut and as a platform for understanding the mechanism of chagasic megacolon development.

Assessment of the Activity of Nitroisoxazole Derivatives against Trypanosoma cruzi.

The development of new compounds to treat Chagas disease is imperative due to the adverse effects of current drugs and their low efficacy in the chronic phase. This study aims to investigate nitroisoxazole derivatives that produce oxidative stress while modifying the compounds' lipophilicity, affecting their ability to fight trypanosomes. The results indicate that these compounds are more effective against the epimastigote form of T. cruzi, with a 52 ± 4% trypanocidal effect for compound 9. However, they are less effective against the trypomastigote form, with a 15 ± 3% trypanocidal effect. Additionally, compound 11 interacts with a higher number of amino acid residues within the active site of the enzyme cruzipain. Furthermore, it was also found that the presence of a nitro group allows for the generation of free radicals; likewise, the large size of the compound enables increased interaction with aminoacidic residues in the active site of cruzipain, contributing to trypanocidal activity. This activity depends on the size and lipophilicity of the compounds. The study recommends exploring new compounds based on the nitroisoxazole skeleton, with larger substituents and lipophilicity to enhance their trypanocidal activity.

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Dynamics of Trypanosoma cruzi infection in hamsters and novel association with progressive motor dysfunction.

Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Clinical outcomes range from long-term asymptomatic carriage to cardiac, digestive, neurological and composite presentations that can be fatal in both acute and chronic stages of the disease. Studies of T. cruzi in animal models, principally mice, have informed our understanding of the biological basis of this variability and its relationship to infection and host response dynamics. Hamsters have higher translational value for many human infectious diseases, but they have not been well developed as models of Chagas disease. We transposed a real-time bioluminescence imaging system for T. cruzi infection from mice into female Syrian hamsters (Mesocricetus auratus). This enabled us to study chronic tissue pathology in the context of spatiotemporal infection dynamics. Acute infections were widely disseminated, whereas chronic infections were almost entirely restricted to the skin and subcutaneous adipose tissue. Neither cardiac nor digestive tract disease were reproducible features of the model. Skeletal muscle had only sporadic parasitism in the chronic phase, but nevertheless displayed significant inflammation and fibrosis, features also seen in mouse models. Whereas mice had normal locomotion, all chronically infected hamsters developed hindlimb muscle hypertonia and a gait dysfunction resembling spastic diplegia. With further development, this model may therefore prove valuable in studies of peripheral nervous system involvement in Chagas disease.

Trypanosoma cruzi Infection in Pregnancies without Congenital Transmission Is Associated with Reduced Fetal Growth: A Cross-Sectional Study in Argentina, Honduras, and Mexico.

We aimed to measure the association between Trypanosoma cruzi infection in pregnancy and reduced fetal growth in the absence of T. cruzi congenital transmission. We conducted a cross-sectional study of secondary data of all singleton live births between 2011 and 2013 in five hospitals from Argentina, Honduras, and Mexico. We excluded newborns with T. cruzi infection. Noninfected pregnant people were those without any positive rapid tests. The main study outcomes were birth weight, head circumference, and length for gestational age and sex. Logistic regression models were adjusted for country, age, education level, and obstetric history. Of the 26,544 deliveries, 459 (1.7%) pregnant people were found by rapid tests to be positive for T. cruzi. Of these, 320 were positive by enzyme-linked immunosorbent assay and 231 had a positive polymerase chain reaction (PCR) test. Uninfected newborns from T. cruzi-infected pregnant people were more likely to have birth weights below the 5th and 10th percentiles and head circumferences below the 3rd and 10th percentiles. Among T. cruzi-infected pregnant people diagnosed by PCR, the odds ratios were 1.58 for birth weight below the 10th percentile (95% CI, 1.12-2.23) and 1.57 for birth weight below the 5th percentile (95% CI, 1.02-2.42). Higher T. cruzi parasitic loads in pregnancy had a stronger association with reduced fetal growth (both in birth weight and head circumference), with an odds ratio of 2.31 (95% CI, 1.36-3.91) for a birth weight below the 5th percentile. The association shows, irrespective of causality, that newborns of pregnancies with T. cruzi have an increased risk of reduced fetal growth. We recommend further studies to assess other potential confounders and the causality of these associations.

Listen to what the animals say: a systematic review and meta-analysis of sterol 14-demethylase inhibitor efficacy for in vivo models of Trypanosoma cruzi infection.

Sterol 14-demethylase (CYP51) inhibitors, encompassing new chemical entities and repurposed drugs, have emerged as promising candidates for Chagas disease treatment, based on preclinical studies reporting anti-Trypanosoma cruzi activity. Triazoles like ravuconazole (RAV) and posaconazole (POS) progressed to clinical trials. Unexpectedly, their efficacy was transient in chronic Chagas disease patients, and their activity was not superior to benznidazole (BZ) treatment. This paper aims to summarize evidence on the global activity of CYP51 inhibitors against T. cruzi by applying systematic review strategies, risk of bias assessment, and meta-analysis from in vivo studies. PubMed and Embase databases were searched for original articles, obtaining fifty-six relevant papers meeting inclusion criteria. Characteristics of animal models, parasite strain, treatment schemes, and cure rates were extracted. Primary outcomes such as maximum parasitaemia values, survival, and parasitological cure were recorded for meta-analysis, when possible. The risk of bias was uncertain in most studies. Animals treated with itraconazole, RAV, or POS survived significantly longer than the infected non-treated groups (RR = 4.85 [3.62, 6.49], P < 0.00001), and they showed no differences with animals treated with positive control drugs (RR = 1.01 [0.98, 1.04], P = 0.54). Furthermore, the overall analysis showed that RAV or POS was not likely to achieve parasitological cure when compared with BZ or NFX treatment (OD = 0.49 [0.31, 0.77], P = 0.002). This systematic review contributes to understanding why the azoles had failed in clinical trials and, more importantly, how to improve the animal models of T. cruzi infection by filling the gaps between basic, translational, and clinical research.

Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naïve rhesus macaques.

BACKGROUND: A vaccine against Trypanosoma cruzi, the agent of Chagas disease, would be an excellent additional tool for disease control. A recombinant vaccine based on Tc24 and TSA1 parasite antigens was found to be safe and immunogenic in naïve macaques. METHODS: We used RNA-sequencing and performed a transcriptomic analysis of PBMC responses to vaccination of naïve macaques after each vaccine dose, to shed light on the immunogenicity of this vaccine and guide the optimization of doses and formulation. We identified differentially expressed genes and pathways and characterized immunoglobulin and T cell receptor repertoires. RESULTS: RNA-sequencing analysis indicated a clear transcriptomic response of PBMCs after three vaccine doses, with the up-regulation of several immune cell activation pathways and a broad non-polarized immune profile. Analysis of the IgG repertoire showed that it had a rapid turnover with novel IgGs produced following each vaccine dose, while the TCR repertoire presented several persisting clones that were expanded after each vaccine dose. CONCLUSIONS: These data suggest that three vaccine doses may be needed for optimum immunogenicity and support the further evaluation of the protective efficacy of this vaccine.

Trajectory-driven computational analysis for element characterization in Trypanosoma cruzi video microscopy.

Optical microscopy videos enable experts to analyze the motion of several biological elements. Particularly in blood samples infected with Trypanosoma cruzi (T. cruzi), microscopy videos reveal a dynamic scenario where the parasites' motions are conspicuous. While parasites have self-motion, cells are inert and may assume some displacement under dynamic events, such as fluids and microscope focus adjustments. This paper analyzes the trajectory of T. cruzi and blood cells to discriminate between these elements by identifying the following motion patterns: collateral, fluctuating, and pan-tilt-zoom (PTZ). We consider two approaches: i) classification experiments for discrimination between parasites and cells; and ii) clustering experiments to identify the cell motion. We propose the trajectory step dispersion (TSD) descriptor based on standard deviation to characterize these elements, outperforming state-of-the-art descriptors. Our results confirm motion is valuable in discriminating T. cruzi of the cells. Since the parasites perform the collateral motion, their trajectory steps tend to randomness. The cells may assume fluctuating motion following a homogeneous and directional path or PTZ motion with trajectory steps in a restricted area. Thus, our findings may contribute to developing new computational tools focused on trajectory analysis, which can advance the study and medical diagnosis of Chagas disease.

The risk of vector transmission of Trypanosoma cruzi remains high in the State of Paraná.

BACKGROUND: Monitoring and analysing the infection rates of the vector of Trypanosoma cruzi, that causes Chagas disease, helps assess the risk of transmission. OBJECTIVES: A study was carried out on triatomine in the State of Paraná, Brazil, between 2012 and 2021 and a comparison was made with a previous study. This was done to assess the risk of disease transmission. METHODS: Ecological niche models based on climate and landscape variables were developed to predict habitat suitability for the vectors as a proxy for risk of occurrence. FINDINGS: A total of 1,750 specimens of triatomines were recorded, of which six species were identified. The overall infection rate was 22.7%. The areas with the highest risk transmission of T. cruzi are consistent with previous predictions in municipalities. New data shows that climate models are more accurate than landscape models. This is likely because climate suitability was higher in the previous period. MAIN CONCLUSION: Regardless of uneven sampling and potential biases, risk remains high due to the wide presence of infected vectors and high environmental suitability for vector species throughout the state and, therefore, improvements in public policies aimed at wide dissemination of knowledge about the disease are recommended to ensure the State remains free of Chagas disease.

Preclinical evaluation of combined therapy with amiodarone and low-dose benznidazole in a mouse model of chronic Trypanosoma cruzi infection.

Chagasic chronic cardiomyopathy (CCC) is the primary clinical manifestation of Chagas disease (CD), caused by Trypanosoma cruzi. Current therapeutic options for CD are limited to benznidazole (Bz) and nifurtimox. Amiodarone (AMD) has emerged as most effective drug for treating the arrhythmic form of CCC. To address the effects of Bz and AMD we used a preclinical model of CCC. Female C57BL/6 mice were infected with T. cruzi and subjected to oral treatment for 30 consecutive days, either as monotherapy or in combination. AMD in monotherapy decreased the prolonged QTc interval, the incidence of atrioventricular conduction disorders and cardiac hypertrophy. However, AMD monotherapy did not impact parasitemia, parasite load, TNF concentration and production of reactive oxygen species (ROS) in cardiac tissue. Alike Bz therapy, the combination of Bz and AMD (Bz/AMD), improved cardiac electric abnormalities detected T. cruzi-infected mice such as decrease in heart rates, enlargement of PR and QTc intervals and increased incidence of atrioventricular block and sinus arrhythmia. Further, Bz/AMD therapy ameliorated the ventricular function and reduced parasite burden in the cardiac tissue and parasitemia to a degree comparable to Bz monotherapy. Importantly, Bz/AMD treatment efficiently reduced TNF concentration in the cardiac tissue and plasma and had beneficial effects on immunological abnormalities. Moreover, in the cardiac tissue Bz/AMD therapy reduced fibronectin and collagen deposition, mitochondrial damage and production of ROS, and improved sarcomeric and gap junction integrity. Our study underlines the potential of the Bz/AMD therapy, as we have shown that combination increased efficacy in the treatment of CCC.

A panel of phenotypically and genotypically diverse bioluminescent:fluorescent Trypanosoma cruzi strains as a resource for Chagas disease research.

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that displays considerable genetic diversity. Infections result in a range of pathological outcomes, and different strains can exhibit a wide spectrum of anti-parasitic drug tolerance. The genetic determinants of infectivity, virulence and therapeutic susceptibility remain largely unknown. As experimental tools to address these issues, we have generated a panel of bioluminescent:fluorescent parasite strains that cover the diversity of the T. cruzi species. These reporters allow spatio-temporal infection dynamics in murine models to be monitored in a non-invasive manner by in vivo imaging, provide a capability to detect rare infection foci at single-cell resolution, and represent a valuable resource for investigating virulence and host:parasite interactions at a mechanistic level. Importantly, these parasite reporter strains can also contribute to the Chagas disease drug screening cascade by ensuring that candidate compounds have pan-species in vivo activity prior to being advanced into clinical testing. The parasite strains described in this paper are available on request.

The beneficial effect of fluoxetine on behavioral and cognitive changes in chronic experimental Chagas disease unveils the role of serotonin fueling astrocyte infection by Trypanosoma cruzi.

BACKGROUND: In Chagas disease (CD), a neglected tropical disease caused by the parasite Trypanosoma cruzi, the development of mental disorders such as anxiety, depression, and memory loss may be underpinned by social, psychological, and biological stressors. Here, we investigated biological factors underlying behavioral changes in a preclinical model of CD. METHODOLOGY/PRINCIPAL FINDINGS: In T. cruzi-infected C57BL/6 mice, a kinetic study (5 to 150 days postinfection, dpi) using standardized methods revealed a sequential onset of behavioral changes: reduced innate compulsive behavior, followed by anxiety and depressive-like behavior, ending with progressive memory impairments. Hence, T. cruzi-infected mice were treated (120 to 150 dpi) with 10 mg/Kg/day of the selective serotonin reuptake inhibitor fluoxetine (Fx), an antidepressant that favors neuroplasticity. Fx therapy reversed the innate compulsive behavior loss, anxiety, and depressive-like behavior while preventing or reversing memory deficits. Biochemical, histological, and parasitological analyses of the brain tissue showed increased levels of the neurotransmitters GABA/glutamate and lipid peroxidation products and decreased expression of brain-derived neurotrophic factor in the absence of neuroinflammation at 150 dpi. Fx therapy ameliorated the neurochemical changes and reduced parasite load in the brain tissue. Next, using the human U-87 MG astroglioma cell line, we found no direct effect of Fx on parasite load. Crucially, serotonin/5-HT (Ser/5-HT) promoted parasite uptake, an effect increased by prior stimulation with IFNγ and TNF but abrogated by Fx. Also, Fx blocked the cytokine-driven Ser/5-HT-promoted increase of nitric oxide and glutamate levels in infected cells. CONCLUSION/SIGNIFICANCE: We bring the first evidence of a sequential onset of behavioral changes in T. cruzi-infected mice. Fx therapy improves behavioral and biological changes and parasite control in the brain tissue. Moreover, in the central nervous system, cytokine-driven Ser/5-HT consumption may favor parasite persistence, disrupting neurotransmitter balance and promoting a neurotoxic environment likely contributing to behavioral and cognitive disorders.

Molecular tools to regulate gene expression in Trypanosoma cruzi.

Developing molecular strategies to manipulate gene expression in trypanosomatids is challenging, particularly with respect to the unique gene expression mechanisms adopted by these unicellular parasites, such as polycistronic mRNA transcription and multi-gene families. In the case of Trypanosoma cruzi (T. cruzi), the causative agent of Chagas Disease, the lack of RNA interference machinery further complicated functional genetic studies important for understanding parasitic biology and developing biomarkers and potential therapeutic targets. Therefore, alternative methods of performing knockout and/or endogenous labelling experiments were developed to identify and understand the function of proteins for survival and interaction with the host. In this review, we present the main tools for the genetic manipulation of T. cruzi, focusing on the Clustered Regularly Interspaced Short Palindromic Repeats Cas9-associated system technique widely used in this organism. Moreover, we highlight the importance of using these tools to elucidate the function of uncharacterized and glycosylated proteins. Further developments of these technologies will allow the identification of new biomarkers, therapeutic targets and potential vaccines against Chagas disease with greater efficiency and speed.