Hospital case fatality and mortality related to Chagas disease in Brazil over two decades.

OBJECTIVE: To analyse hospital case fatality and mortality related to Chagas disease (CD) in Brazil, 2000-2019. METHOD: This is a mixed ecological study with spatial and temporal trends, based on national population data from the Brazilian Ministry of Health - hospital admissions (HA) and death certificates (DC). Records with CD as a primary or secondary cause of death in HA and/or as an underlying or associated cause of death in DC were evaluated. Temporal trends were analysed by Joinpoint regression and the spatial distribution of age- and gender-adjusted rates, spatial moving averages, and standardized morbidity ratios. RESULTS: There were a total of 4,376 HA due to CD resulting in death in Brazil, with a hospital case fatality rate of 0.11/100,000 inhabitants. The Southeast region had the highest rate (63.9%, n = 2,796; 0.17/100,000 inhabitants). The general trend for this indicator in Brazil is upwards (average annual percentage change [AAPC] 7.5; 95% confidence interval [CI] 5.3 to 9.9), with increases in the North, Northeast and Southeast regions. During the same period 122,275 deaths from CD were registered in DC, with a mortality rate of 3.14/100,000 inhabitants. The highest risk of CD-related death was found among men (relative risk [RR] 1.27) and Afro-Brazilians (RR 1.63). There was a downward trend in CD mortality in the country (AAPC - 0.7%, 95%CI -0.9 to -0.5), with an increase in the Northeast region (AAPC 1.1%, 95%CI 0.6 to 1.6). Municipalities with a very high Brazilian Deprivation Index tended to show an increase in mortality (AAPC 2.1%, 95%CI 1.6 to 2.7), while the others showed a decrease. CONCLUSION: Hospital case fatality and mortality due to CD are a relevant public health problem in Brazil. Differences related to gender, ethnicity, and social vulnerability reinforce the need for comprehensive care, and to ensure equity in access to health in the country. Municipalities, states, and regions with indicators that reveal higher morbidity and mortality need to be prioritized.

Factors related to the discontinuation and mortality rates of a cardiac rehabilitation programme in patients with Chagas disease: a 6-year experience in a Brazilian tertiary centre.

OBJECTIVES: To describe the clinical and sociodemographic characteristics of participants as well as discontinuation and mortality rates in a cardiac rehabilitation programme (CRP) tailored to Chagas disease (CD). METHODS: Participants underwent functional capacity, anthropometry and cardiac function evaluations before beginning a CRP. Univariate and multivariate Cox proportional hazards models were performed to investigate the associations between clinical and sociodemographic characteristics at baseline with discontinuation rates and deaths. RESULTS: Forty-two patients were enrolled in the CRP (61.9% men, mean age of 58.1 ± 11.8 years). During a median follow-up period of 10.8 months, 74% discontinued and 14% died while enrolled in CRP. 34% of the patients who discontinued CRP died during follow-up. White race (HR = 0.09; 95% CI 0.01-1.00), right ventricular systolic dysfunction (HR = 10.54; 95% CI 1.24-89.50) and oxygen pulse (HR = 0.69; 95% CI 0.48-0.99) were independently associated with death while enrolled in CRP. Married status (HR = 0.44; 95% CI 0.21-0.95) was independently associated with discontinuation rates from CRP. VO2 peak (HR = 0.85; 95% CI 0.74-0.98) and CRP discontinuation due to CD-related reasons (HR = 8.33; 95% CI 1.91-36.27) were the variables independently associated with death after discontinuation of CRP. CONCLUSION: In this population, sociodemographic aspects and severity of CD were important determinants of CRP discontinuation and mortality.

OBJECTIFS: Décrire les caractéristiques cliniques et sociodémographiques des participants ainsi que les taux d'abandon et de décès dans un programme de réadaptation cardiaque (PRC) adapté à la maladie de Chagas (MC). MÉTHODES: Les participants ont subi des évaluations de la capacité fonctionnelle, de l'anthropométrie et de la fonction cardiaque avant de commencer un PRC. Des modèles de risques proportionnels de Cox univariés et multivariés ont été appliqués pour étudier les associations entre les caractéristiques cliniques et sociodémographiques au départ avec les taux d'abandon et les décès. RÉSULTATS: 42 patients ont été enrôlés dans le PRC (61,9% d'hommes, âge moyen de 58,1 ± 11,8 ans). Au cours d'une période médiane de suivi de 10,8 mois, 74% ont abandonné et 14% sont décédés durant leur enrôlement au PRC. 34% des patients qui ont arrêté le PRC sont décédés au cours du suivi. La race blanche (HR = 0,09; IC95%: 0,01-1,00), le dysfonctionnement systolique ventriculaire droite (HR = 10,54; IC95%: 1,24-89,50) et le pouls d'oxygène (HR = 0,69; IC95%: 0,48-0,99) étaient indépendamment associés avec le décès lors de l'enrôlement au PRC. Le statut marié (HR = 0,44; IC95%: 0,21-0,95) était indépendamment associé aux taux d'abandon de la CRP. Le pic de VO2 (HR = 0,85; IC95%: 0,74-0,98) et l'arrêt du PRC pour des raisons liées à la MC (HR = 8,33; IC95%: 1,91 à 36,27) étaient les variables indépendamment associées au décès après l'arrêt du PRC. CONCLUSION: Dans cette population, les aspects sociodémographiques et la sévérité de la MC étaient des déterminants importants de l'arrêt du PRC et du décès.

MicroRNA-155 Deficiency Exacerbates Trypanosoma cruzi Infection.

Chagas disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a public health problem affecting 6 to 8 million people, mainly in Latin America. The role of microRNAs in the pathogenesis of Chagas disease has not been well described. Here, we investigate the role of microRNA-155 (miR-155), a proinflammatory host innate immune regulator responsible for T helper type 1 and type 17 (Th1 and Th17) development and macrophage responses during T. cruzi infection. For this, we compared the survival and parasite growth and distribution in miR-155-/- and wild-type (WT) C57BL/6 mice. The lack of miR-155 caused robust parasite infection and diminished survival of infected mice, while WT mice were resistant to infection. Immunological analysis of infected mice indicated that, in the absence of miR-155, there was decreased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. In addition, we found that there was a significant reduction of CD8-positive (CD8+) T cells, natural killer (NK) cells, and NK-T cells and increased accumulation of neutrophils and inflammatory monocytes in miR-155-/- mice. Collectively, these data indicate that miR-155 is an important immune regulatory molecule critical for the control of T. cruzi infection.

Resolvin D1 Administration Is Beneficial in Trypanosoma cruzi Infection.

Chagas disease is a major public health issue, affecting ∼10 million people worldwide. Transmitted by a protozoan named Trypanosoma cruzi, this infection triggers a chronic inflammatory process that can lead to cardiomyopathy (Chagas disease). Resolvin D1 (RvD1) is a novel proresolution lipid mediator whose effects on inflammatory diseases dampens pathological inflammatory responses and can restore tissue homeostasis. Current therapies are not effective in altering the outcome of T. cruzi infection, and as RvD1 has been evaluated as a therapeutic agent in various inflammatory diseases, we examined if exogenous RvD1 could modulate the pathogenesis of Chagas disease in a murine model. CD-1 mice infected with the T. cruzi Brazil strain were treated with RvD1. Mice were administered 3 µg/kg of body weight RvD1 intraperitoneally on days 5, 10, and 15 to examine the effect of RvD1 on acute disease or administered the same dose on days 60, 65, and 70 to examine its effects on chronic infection. RvD1 therapy increased the survival rate and controlled parasite replication in mice with acute infection and reduced the levels of interferon gamma and transforming growth factor ß (TGF-ß) in mice with chronic infection. In addition, there was an increase in interleukin-10 levels with RvD1 therapy in both mice with acute infection and mice with chronic infection and a decrease in TGF-ß levels and collagen content in cardiac tissue. Together, these data indicate that RvD1 therapy can dampen the inflammatory response, promote the resolution of T. cruzi infection, and prevent cardiac fibrosis.

Spatial patterns and temporal tendency of mortality related to Chagas disease in an endemic area of northeastern Brazil.

OBJECTIVE: To analyse spatial patterns and the temporal tendency of mortality related to Chagas disease, in order to identify priority control areas in the state of Sergipe, Northeast Brazil. METHODS: We conducted an ecological and time-series study with spatial analysis techniques on deaths from Chagas disease in the state of Sergipe (1996-2016). We used data from the Mortality Information System (SIM). The temporal analysis was performed using a statistical technique capable of describing changes in the trend pattern for the period. Thematic maps were elaborated from point and polygonal analyses. RESULTS: There were 247 deaths related to Chagas disease, with a mean of 11.7 deaths/year, most of them male (64%), and aged 50-59 years (21%) and 60-69 years (26%). Two segments with increasing, non-constant and significant trends were identified: 1996-2005 (APC = 21.6%; P = 0.01) and 2005-2016 (APC = 4.4%; P = 0.01), with APPC = 11.8% (P = 0.01). A positive and significant spatial autocorrelation with areas of higher risk of death was found in the southern region of the state. CONCLUSIONS: The trend of mortality related to Chagas disease in the state of Sergipe was increasing during the period analysed, with a heterogeneous distribution of cases. A main risk area was identified in the southern region of the state.

OBJECTIF: Analyser les profils spatiaux et la tendance temporelle de la mortalité liée à la maladie de Chagas, afin d'identifier les domaines de priorité de lutte dans l'Etat de Sergipe, dans le nord-est du Brésil. MÉTHODES: Nous avons mené une étude écologique et de séries chronologiques avec des techniques d'analyse spatiale sur les décès dus à la maladie de Chagas dans l'état de Sergipe (1996-2016). Nous avons utilisé les données du système d'information sur la mortalité (SIM). L'analyse temporelle a été réalisée à l'aide d'une technique statistique capable de décrire les changements dans le profil de tendance pour la période. Des cartes thématiques ont été élaborées à partir d'analyses ponctuelles et polygonales. RÉSULTATS: Il y a eu 247 décès liés à la maladie de Chagas, avec une moyenne de 11,7 décès/an, pour la plupart de sexe masculin (64%), et âgés de 50 à 59 ans (21%) et de 60 à 69 ans (26%). Deux segments avec des tendances à la hausse, non constantes et significatives ont été identifiés: 1996-2005 (APC = 21,6%; p = 0,01) et 2005-2016 (APC = 4,4%; p = 0,01), avec APPC = 11,8% (p = 0,01). Une autocorrélation spatiale positive et significative avec des zones à haut risque de décès a été trouvée dans la région sud de l'Etat. CONCLUSIONS: La tendance de la mortalité liée à la maladie de Chagas dans l'état de Sergipe a augmenté au cours de la période analysée , avec une répartition hétérogène des cas. Une principale zone à risque a été identifiée dans la région sud de l'Etat.

Spatiotemporal trends of Chagas disease-related mortality in the Northeast of Brazil, 2007-2017.

Chagas disease (CD) is a neglected disease and endemic in Brazil. In the Brazilian Northeast Region, it affects millions of people. Therefore, it is necessary to identify the spatiotemporal trends of CD mortality in the Northeast of Brazil. This ecological study was designed, in which the unit of analysis was the municipality of the Brazilian northeast. The data source was the Information System of Mortality. It was calculated relative risk from socioeconomic characteristics. Mortality rates were smoothed by the Local Empirical Bayes method. Spatial dependency was analysed by the Global and Local Moran Index. Scan spatial statistics were also used. A total of 11 287 deaths by CD were notified in the study. An expressive parcel of this number was observed among 70-year-olds or more (n = 4381; 38.8%), no schooling (n = 4381; 38.8%), mixed-race (n = 4381; 62.3%), male (n = 6875; 60.9%). It was observed positive spatial autocorrelation, mostly in municipalities of the state of Bahia, Piauí (with high-high clusters), and Maranhão (with low-low clusters). The spatial scan statistics has presented a risk of mortality in 24 purely spatial clusters (P < 0.05). The study has identified the spatial pattern of CD mortality mostly in Bahia and Piauí, highlighting priority areas in planning and control strategies of the health services.

Histopathological changes in the gastrointestinal tract and systemic alterations triggered by experimental oral infection with Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in almost all countries of Latin America. In Brazil, oral infection is becoming the most important mechanism of transmission of the disease in several regions of the country. The gastrointestinal tract is the gateway for the parasite through this route of infection, however, little is known about the involvement of these organs related to oral route. In this sense, the present study evaluated the impact of oral infection on the digestive tract in mice infected by Berenice-78 (Be-78) T. cruzi strain, in comparison with the intraperitoneal route of infection. In this work, the intraperitoneal route group showed a peak of parasitemia similar to the oral route group, however the mortality rate among the orally infected animals was higher when compared to intraperitoneal route. By analyzing the frequency of blood cell populations, differences were mainly observed in CD4+ T lymphocytes, and not in CD8+, presenting an earlier reduction in the number of CD4+ T cells, which persisted for a longer period, in the animals of the oral group when compared with the intraperitoneal group. Animals infected by oral route presented a higher tissue parasitism and inflammatory infiltrate in stomach, duodenum and colon on the 28th day after infection. Therefore, these data suggest that oral infection has a different profile of parasitological and immune responses compared to intraperitoneal route, being the oral route more virulent and with greater tissue parasitism in organs of the gastrointestinal tract evaluated during the acute phase.

Galectin-3 mediates survival and apoptosis pathways during Trypanosoma cruzi-host cell interplay.

Neglected tropical diseases, such as Chagas disease caused by the protozoa Trypanosoma cruzi, affect millions of people worldwide but lack effective treatments that are accessible to the entire population, especially patients with the debilitating chronic phase. The recognition of host cells, invasion and its intracellular replicative success are essential stages for progression of the parasite life cycle and the development of Chagas disease. It is predicted that programmed cell death pathways (apoptosis) would be activated in infected cells, either via autocrine secretion or mediated by cytotoxic immune cells. This process should play a key role in resolving infections by hindering the evolutionary success of the parasite. In this research, we performed assays to investigate the role of the lectin galectin-3 (Gal3) in parasite-host signaling pathways. Using cells with endogenous levels of Gal3 compared to Gal3-deficient cells (induced by RNA interference), we demonstrated that T. cruzi mediated the survival pathways and the subverted apoptosis through Gal3 promoting a pro-survival state in infected cells. Infected Gal3-depleted cells showed increased activation of caspase 3 and pro-apoptotic targets, such as poly (ADP-ribose) polymerase (PARP), and lower accumulation of anti-apoptotic proteins, such as c-IAP1, survivin and XIAP. During the early stages of infection, Gal3 translocates from the cytoplasm to the nucleus and must act in survival pathways. In a murine model of experimental infection, Gal3 knockout macrophages showed lower infectivity and viability. In vivo infection revealed a lower parasitemia and longer survival and an increased spleen cellularity in Gal3 knockout mice with consequences on the percentage of T lymphocytes (CD4+ CD11b+) and macrophages. In addition, cytokines such as IL-2, IL-4, IL-6 and TNF-α are increased in Gal3 knockout mice when compared to wild type genotype. These data demonstrate a Gal3-mediated complex interplay in the host cell, keeping infected cells alive long enough for infection and intracellular proliferation of new parasites. However, a continuous knowledge of these signaling pathways should contribute to a better understanding the mechanisms of cell death subversion that are promoted by protozoans in the pathophysiology of neglected diseases such as Chagas disease.

Mortality and Stroke Recurrence in a Rehabilitation Cohort of Patients with Cerebral Infarcts and Chagas Disease.

BACKGROUND: Chagas disease is related to ischemic stroke (IS), although few epidemiological studies have evaluated the associated mortality and recurrence. Our objective is to determine factors associated with mortality and recurrence of IS in patients with IS and Chagas disease. METHODS: We retrospectively studied data obtained from electronic medical records of patients admitted at SARAH Hospitals across Brazil between 2009 and 2013. Using Cox regression analysis for mortality and logistic regression for recurrence, we assessed primary population characteristics and statistical associations between risk factors and outcomes. RESULTS: We analyzed 279 patients who were followed up until 2016. The mean age at stroke onset was 61 with a 10% frequency of death. Multivariate analysis assessing mortality demonstrated that the associated factors were age at stroke (hazard ratio [HR] 1.04), initial modified Rankin Scale (mRS; HR 20.91), bladder dysfunction (HR 2.51), diabetes mellitus (DM; HR 3.64), and alcoholism (HR 3.37). Multivariate analysis assessing recurrence demonstrated that the associated factors were age at ictus (OR 0.96), cognitive deficit (OR 0.44), initial mRS (OR 1.84), cardioembolic etiology (OR 2.47), and female sex (OR 2.73). CONCLUSIONS: Cardiac conditions did not correlate with mortality or recurrence. Age was a protective factor against recurrence, probably due to cumulative risk of IS over time, while initial mRS was associated with both outcomes. Treating diseases such as DM and bladder dysfunction, and early treatment to reduce the initial mRS could potentially prevent both outcomes; also, establishing a correct etiological diagnosis is important.

Protective effect of aspirin treatment on mouse behavior in the acute phase of experimental infection with Trypanosoma cruzi.

Chagas disease is a potentially fatal disease caused by the parasite Trypanosoma cruzi, which can in some cases affect the central nervous system. The objective was to evaluate the effect of aspirin (ASA) in the behavior of mice infected with T. cruzi during the acute phase. This was an experimental study with random assignation. Twenty four BALB/c mice were divided into four groups of six animals each as follows: only ASA (OA), ASA before infection (BI), ASA after infection (AI) and only infection (OI). The strain used for infection was M/HOM/Bra/53/Y. An ASA dose of 100 mg/kg per day was administered 72 h before infection to BI group and the same dose 48 h after infection to AI group. Mice behavior in the open field test, mortality, and brain histopathology was evaluated. Data were analyzed using ANOVA, chi square test, and Kaplan-Meier with long-rank for survival analysis. In the open field test, the OA group has similar results with the BI group, in the variables of immobility and escape. Also, the OA group displayed significantly higher rates of micturition (p < 0.001) and defecation (p < 0.001) compared to infected groups. Mortality was higher in BI group (p = 0.02). The presence of T. cruzi amastigotes were higher in brain tissues of the AI and OI groups (p = 0.008). In conclusion, the administration of ASA before infection seemed to prevent behavioral changes induced by the acute infection, but it led to accelerated mortality. The study highlighted the potential importance of the pathways inhibited by ASA in the early hours of acute infection with T. cruzi.

Development and in vitro/in vivo evaluation of a novel benznidazole liquid dosage form using a quality-by-design approach.

OBJECTIVES: To develop an alcohol-free solution suitable for children of benznidazole, the drug of choice for treatment of Chagas disease. METHODS: In a quality-by-design approach, a systematic optimisation procedure was carried out to estimate the values of the factors leading to the maximum drug concentration. The formulations were analysed in terms of chemical and physical stability and drug content. The final preparation was subjected to an in vivo palatability assay. Mice were infected and treated orally in a murine model. RESULTS: The results showed that benznidazole solubility increased up to 18.38 mg/ml in the optimised co-solvent system. The final formulation remained stable at all three temperatures tested, with suitable drug content and no significant variability. Palatability of the preparation was improved by taste masking of BZL. In vivo studies showed that both parasitaemia and mortality diminished, particularly at a dose of 40 mg/kg/day. CONCLUSION: Quality by design was a suitable approach to formulate a co-solvent system of benznidazole. The in vivo studies confirmed the suitability of the optimised such solutions to diminish both parasitaemia and mortality. Thus, this novel alternative should be taken into account for further clinical evaluation in all age ranges.

Effects of IFN-γ coding plasmid supplementation in the immune response and protection elicited by Trypanosoma cruzi attenuated parasites.

BACKGROUND: Previous studies showed that a naturally attenuated strain from Trypanosoma cruzi triggers an immune response mainly related to a Th2-type profile. Albeit this, a strong protection against virulent challenge was obtained after priming mice with this attenuated strain. However, this protection is not enough to completely clear parasites from the host. In T. cruzi infection, early Interferon-gamma (IFN-γ) is critical to lead type 1 responses able to control intracellular parasites. Therefore we evaluated whether the co-administration of a plasmid encoding murine IFN-γ could modify the immune response induced by infection with attenuated parasites and improve protection against further infections. METHODS: C57BL/6J mice were infected intraperitoneally with three doses of live attenuated parasites in combination with plasmid pVXVR-mIFN-γ. Before each infection dose, sera samples were collected for parasite specific antibodies determination and cytokine quantification. To evaluate the recall response to T. cruzi, mice were challenged with virulent parasites 30 days after the last dose and parasite load in peripheral blood and heart was evaluated. RESULTS: As determined by ELISA, significantly increase in T. cruzi specific antibodies response was detected in the group in which pVXVR-mIFN-γ was incorporated, with a higher predominance of IgG2a subtype in comparison to the group of mice only inoculated with attenuated parasites. At our limit of detection, serum levels of IFN-γ were not detected, however a slight decrease in IL-10 concentrations was observed in groups in which pVXVR-mIFN-γ was supplemented. To analyze if the administration of pVXVR-mIFN-γ has any beneficial effect in protection against subsequent infections, all experimental groups were submitted to a lethal challenge with virulent bloodstream trypomastigotes. Similar levels of challenge parasites were detected in peripheral blood and heart of mice primed with attenuated parasites alone or combined with plasmid DNA. Expansion of IgG antibodies was not significant in TCC+ pVXVR-mIFN-γ; however, the overall tendency to sustain a Th2 profile was maintained. CONCLUSIONS: Overall, these results suggest that administration of plasmid pVXVR-mIFN-γ could have beneficial effects on host specific antibody production in response to T. cruzi attenuated infection; however, this outcome is not reflected in an improved protection against further virulent infections.

Trypanosoma cruzi Infection Imparts a Regulatory Program in Dendritic Cells and T Cells via Galectin-1-Dependent Mechanisms.

Galectin-1 (Gal-1), an endogenous glycan-binding protein, is widely distributed at sites of inflammation and microbial invasion. Despite considerable progress regarding the immunoregulatory activity of this lectin, the role of endogenous Gal-1 during acute parasite infections is uncertain. In this study, we show that Gal-1 functions as a negative regulator to limit host-protective immunity following intradermal infection with Trypanosoma cruzi. Concomitant with the upregulation of immune inhibitory mediators, including IL-10, TGF-ß1, IDO, and programmed death ligand 2, T. cruzi infection induced an early increase of Gal-1 expression in vivo. Compared to their wild-type (WT) counterpart, Gal-1-deficient (Lgals1(-/-)) mice exhibited reduced mortality and lower parasite load in muscle tissue. Resistance of Lgals1(-/-) mice to T. cruzi infection was associated with a failure in the activation of Gal-1-driven tolerogenic circuits, otherwise orchestrated by WT dendritic cells, leading to secondary dysfunction in the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells. This effect was accompanied by an increased number of CD8(+) T cells and higher frequency of IFN-γ-producing CD4(+) T cells in muscle tissues and draining lymph nodes as well as reduced parasite burden in heart and hindlimb skeletal muscle. Moreover, dendritic cells lacking Gal-1 interrupted the Gal-1-mediated tolerogenic circuit and reinforced T cell-dependent anti-parasite immunity when adoptively transferred into WT mice. Thus, endogenous Gal-1 may influence T. cruzi infection by fueling tolerogenic circuits that hinder anti-parasite immunity.

Antiprotozoal drug nitazoxanide enhances parasitemia, tissue lesions and mortality caused by Trypanosoma cruzi in murine model.

Chagas' disease is caused by unicellular parasite Trypanosoma cruzi (T. cruzi). It is endemic throughout Latin America, but nowadays has become a global challenge due to tourism and migration. Non-treated infection may result in health-threatening complications and lead to death. Current medications for this infection are nifurtimox (NFT) and benznidazol. Both drugs may cause side effects and are ineffective in the chronic phase. Therefore, new antichagasic compounds are urgently required. Nitazoxanide (NTZ) is a broad spectrum antiparasitic drug, proposed recently as a potential candidate to be added to the list of essential medicines for integrated neglected tropical disease control and elimination. Although the effect of NTZ against T. cruzi epimastigotes in vitro was reported, the corresponding experiments in animal models of T. cruzi infection have never been undertaken. The present work was designed to fill this gap and evaluate the effect of NTZ on experimental murine trypanosomiasis, in comparison with classical antichagasic agent NFT. Highly sensitive to T. cruzi BALB/c mice were infected using Albarrada T. cruzi strain, recently isolated in Mexico. Experimental groups were either left untreated, or otherwise treated with NFT, NTZ (100 and 1000 mg/kg), or with both drugs simultaneously. The severity of the infection was estimated based on criteria such as parasitemia, lesions in target tissues (heart, muscles and lungs) and mortality. Despite the expected protective effect, NTZ drastically aggravates the course of T. cruzi infection. Namely, parasitemia, tissue lesions and mortality caused by T. cruzi infection were significantly higher in NTZ-treated mice groups, even in comparison with untreated infected animals. NTZ by itself no produced mortality o tissue damage, and NFT showed an expected protective effect. Our results indicate that NTZ cannot be considered for Chagas' disease treatment. Moreover, NTZ should be used with caution in patients positive for T. cruzi infection.

In Vitro and In Vivo Trypanosomicidal Action of Novel Arylimidamides against Trypanosoma cruzi.

Arylimidamides (AIAs) have been shown to have considerable biological activity against intracellular pathogens, includingTrypanosoma cruzi, which causes Chagas disease. In the present study, the activities of 12 novel bis-AIAs and 2 mono-AIAs against different strains ofT. cruziin vitroandin vivowere analyzed. The most active wasm-terphenyl bis-AIA (35DAP073), which had a 50% effective concentration (EC50) of 0.5 µM for trypomastigotes (Y strain), which made it 26-fold more effective than benznidazole (Bz; 13 µM). It was also active against the Colombiana strain (EC50= 3.8 µM). Analysis of the activity against intracellular forms of the Tulahuen strain showed that this bis-AIA (EC50= 0.04 µM) was about 100-fold more active than Bz (2 µM). The trypanocidal effect was dissociated from the ability to trigger intracellular lipid bodies within host cells, detected by oil red labeling. Both an active compound (35DAP073) and an inactive compound (26SMB060) displayed similar activation profiles. Due to their high selectivity indexes, two AIAs (35DAP073 and 35DAP081) were moved toin vivostudies, but because of the results of acute toxicity assays, 35DAP081 was excluded from the subsequent tests. The findings obtained with 35DAP073 treatment of infections caused by the Y strain revealed that 2 days of therapy induced a dose-dependent action, leading to 96 to 46% reductions in the level of parasitemia. However, the administration of 10 daily doses in animals infected with the Colombiana strain resulted in toxicity, preventing longer periods of treatment. The activity of the combination of 0.5 mg/kg of body weight/day 35DAP073 with 100 mg/kg/day Bz for 10 consecutive days was then assayed. Treatment with the combination resulted in the suppression of parasitemia, the elimination of neurological toxic effects, and survival of 100% of the animals. Quantitative PCR showed a considerable reduction in the parasite load (60%) compared to that achieved with Bz or the amidine alone. Our results support further investigations of this class with the aim of developing novel alternatives for the treatment of Chagas disease.

Efficacy of Lychnopholide Polymeric Nanocapsules after Oral and Intravenous Administration in Murine Experimental Chagas Disease.

The etiological treatment of Chagas disease remains neglected. The compounds available show several limitations, mainly during the chronic phase. Lychnopholide encapsulated in polymeric nanocapsules (LYC-NC) was efficacious in mice infected with Trypanosoma cruzi and treated by intravenous administration during the acute phase (AP). As the oral route is preferred for treatment of chronic infections, such as Chagas disease, this study evaluated the use of oral LYC-NC in the AP and also compared it with LYC-NC administered to mice by the oral and intravenous routes during the chronic phase (CP). The therapeutic efficacy was evaluated by fresh blood examination, hemoculture, PCR, and enzyme-linked immunosorbent assay (ELISA). The cure rates in the AP and CP were 62.5% and 55.6%, respectively, upon oral administration of LYC-poly(d,l-lactide)-polyethylene glycol nanocapsules (LYC-PLA-PEG-NC) and 57.0% and 30.0%, respectively, with LYC-poly-ε-caprolactone nanocapsules (LYC-PCL-NC). These cure rates were significantly higher than that of free LYC, which did not cure any animals. LYC-NC formulations administered orally during the AP showed cure rates similar to that of benznidazole, but only LYC-NC cured mice in the CP. Similar results were achieved with intravenous treatment during the CP. The higher cure rates obtained with LYC loaded in PLA-PEG-NC may be due to the smaller particle size of these NC and the presence of PEG, which influence tissue diffusion and the controlled release of LYC. Furthermore, PLA-PEG-NC may improve the stability of the drug in the gastrointestinal tract. This work is the first report of cure of experimental Chagas disease via oral administration during the CP. These findings represent a new and important perspective for oral treatment of Chagas disease.

Concomitant Benznidazole and Suramin Chemotherapy in Mice Infected with a Virulent Strain of Trypanosoma cruzi.

Although suramin (Sur) is suggested as a potential drug candidate in the management of Chagas disease, this issue has not been objectively tested. In this study, we examined the applicability of concomitant treatment with benznidazole (Bz) and suramin in mice infected with a virulent strain of Trypanosoma cruzi. Eighty 12-week-old male C57BL/6 mice were equally randomized in eight groups: (i) noninfected mice (negative control) and mice infected with T. cruzi Y strain receiving (ii) no treatment (positive control), (iii) Bz, 100 mg/kg of body weight per day, (iv) Sur, 20 mg/kg/day, and (v to viii) Sur, 20 mg/kg/day, combined with Bz, 100, 50, 25, or 5 mg/kg/day. Bz was administered by gavage, and Sur was administered intraperitoneally. Sur dramatically increased the parasitemia, cardiac content of parasite DNA, inflammation, oxidative tissue damage, and mortality. In response to high parasitic load in cardiac tissue, Sur stimulated the immune system in a manner typical of the acute phase of Chagas disease, increasing tissue levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and inducing a preferential IgG2a anti-T. cruzi serum pattern. When Sur and Bz were combined, the infection severity was attenuated, showing a dose-dependent Bz response. Sur therapy had a more harmful effect on the host than on the parasite and reduced the efficacy of Bz against T. cruzi infection. Considering that Sur drastically reinforced the infection evolution, potentiating the inflammatory process and the severity of cardiac lesions, the in vivo findings contradicted the in vitro anti-T. cruzi potential described for this drug.

In vitro and in vivo studies of the biological activity of novel arylimidamides against Trypanosoma cruzi.

Fifteen novel arylimidamides (AIAs) (6 bis-amidino and 9 mono-amidino analogues) were assayed against Trypanosoma cruzi in vitro and in vivo. All the bis-AIAs were more effective than the mono-AIAs, and two analogues, DB1967 and DB1989, were further evaluated in vivo. Although both of them reduced parasitemia, protection against mortality was not achieved. Our results show that the number of amidino-terminal units affects the efficacy of arylimidamides against T. cruzi.

Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease.

This study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole-treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole.

Reversible cysteine protease inhibitors show promise for a Chagas disease cure.

The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile "warhead" were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 µM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 µM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg.